Methanogenic archaea

Methanogens are microorganisms that produce CH4. They are strictly anaerobic and belong to the archaea. They are a phylogenetically diverse group, classified into five established orders: Methanobacteriales,

Figure 2.1 General scheme of the anaerobic digestion process

Methanococcales, Methanomicrobiales, Methanosarcinales and Methano-pyrales, and further divided into 10 families and 31 genera (Liu and Whitman, 2008). Methanogens have been isolated from a wide variety of anaerobic environments, including marine and freshwater sediments, human and animal gastrointestinal tracts, anaerobic digestors and landfills and geothermal and polar systems. The habitats of methanogens differ largely in temperature, salinity and pH. Although methanogens are very diverse phylogenetically, they are physiologically very restricted. They can grow on a number of simple organic molecules and hydrogen (Table 2.1). Methanogenic substrates can be divided into three major types (Liu and Whitman, 2008; Thauer et al, 2008):

1 H2 (hydrogen)/CO 2, formate and carbon monoxide (CO);

2 methanol and methylated compounds;

3 acetate.

The general pathway of methanogenesis is presented in Figure 2.2. More complex organic substances are not degraded by methanogens, though a few species can use ethanol and pyruvate.

Most methanogens can reduce CO2 to CH4 with H2 as electron donor. Many of these hydrogenotrophic methanogens can also use formate or CO as electron donor. In hydrogenotrophic methanogenesis CO2 is reduced successively to CH4 through formyl, methylene and methyl levels. The Q moiety is carried by special coenzymes, methanofuran (MFR),

formate

formate

1„

CO2

„ 1

CO

CHO-MFR

CHO-H4MPT

O-H4

CHO-H4MPT

O-H4

Figure 2.2 Combined pathways of methanogenesis from H2/CO2 (CO, formate), methanol and acetate

Note: MFR = methanofuran; H4MPT = tetrahydromethanopterin; HS-CoM = coenzyme M; HS-CoB = coenzyme B.

Figure 2.2 Combined pathways of methanogenesis from H2/CO2 (CO, formate), methanol and acetate

Note: MFR = methanofuran; H4MPT = tetrahydromethanopterin; HS-CoM = coenzyme M; HS-CoB = coenzyme B.

tetrahydromethanopterin (H4MPT) and coenzyme M (HS-CoM). In the first step, CO2 binds to MFR and is reduced to the formyl level. In this reduction step, ferredoxin (Fd), which is reduced with H2, is the electron donor. The formation of formyl-MFR is an endergonic conversion, which is driven by an ion gradient. The formyl group is then transferred to H4MPT, forming formyl-H4MPT. The formyl group is dehydrated to methenyl group, which is subsequently reduced to methylene-H4MPT and then to methyl-H4MPT. In these two reduction steps, reduced factor F420 (F420H2) is the electron donor. The methyl group is transferred to CoM, forming methyl-CoM. In the final

Table 2.1 Energy conserving reactions of methanogenic archaea

Table 2.1 Energy conserving reactions of methanogenic archaea

4H2 + CO2

^ CH4 + 2H2O

-131

4 formate- + 4H+

^ CH4 + 3CO2+ 2H2O

-145

4CO + 2H2O

^ CH4 + 3CO2

-211

Acetate- + H+

^ CH4 + CO2

-36

4 methanol

^ 3CH4 + CO2 + 2H2O

-106

H2 + methanol

^ CH4 + H2O

-113

Source: Gibbs free energy changes from Thauer et al (1977)

Source: Gibbs free energy changes from Thauer et al (1977)

reduction methyl-CoM is reduced to CH4 by methyl coenzyme M reductase. Methyl-CoM reductase is the key enzyme in methanogenesis. Coenzyme B (HS-CoB) is the electron donor in this reduction, after oxidation a heterodisulphide is formed with HS-CoM (CoM-S-S-CoB). The heterodisulphide is reduced to HS-CoB and HS-CoM. The methyl transfer from H4MPT to HS-CoM and the reduction of CoM-S-S-CoB are the steps in which energy conservation takes place (Liu and Whitman 2008; Thauer et al, 2008).

The second substrate type is methyl-containing compounds, including methanol, methylated amines and methylated sulphides. Methanogens of the order Methanosarcinales and Methanosphaera convert methylated compounds. The methyl group is first transferred to a corrinoid protein and then to HS-CoM, involving methyltransferases. Methyl-CoM enters the methanogenesis pathway and is reduced to CH4. The electrons required for this reduction are obtained from the oxidation of methyl-CoM to CO2, which proceeds via a reverse of the described hydrogenotrophic methanogenesis pathway. Methylotrophic growth of some methanogens (Methanomicrococcus blatticola and Methanosphaera spp) is H2-dependent (Sprenger et al, 2005; Liu and Whitman 2008).

Acetate is the major intermediate in the anaerobic food chain; about two thirds of biologically generated CH4 is derived from acetate. Surprisingly, only two genera are known to use acetate for methanogenesis: Methanosarcina and Methanosaeta (Jetten et al, 1992). Acetate is split into CH4 and CO2 after activation to acetyl-CoA and then split into methyl-CoM and CO. Methyl-CoM is reduced to CH4, while CO is oxidized to CO2. Methanosarcina is a versatile methanogen. It shows fast growth on methanol and methylamine, but growth on acetate is slower. Many species also utilize H2/CO2 but not formate. Methanosaeta is a specialist that uses only acetate. Methanosaeta can use acetate at concentrations as low as 5-20 micromolar (pM), while Methanosarcina requires a minimum concentration of about 1 millimolar (mM). The difference in acetate affinity is due to the different acetate activation mechanism. Methanosarcina uses the low-affinity acetate kinase/phosphotransacetylase system to form acetyl-CoA, while Methanosaeta uses the high-affinity acetyl-CoA synthase, which requires a higher energy investment than acetate kinase (Jetten et al, 1992).

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