1. I NTRODUCTION
1.3. Methanogenesis as the main biotic source of CH 4
anaerobic methanogenic Archaea belonging to the orders Methanobacteriales, Methanocellales, Methanococcales, Methanomicrobiales, Methanopyrales, and Methanosarcinales within the Euryarchaeota (Bapteste et al. 2005, Hedderich & Whitman 2006, Liu & Whitman 2008, Sakai et al. 2008, Thauer et al. 2008). Methanogens have a limited substrate range, i.e., CH4 is produced via hydrogenotrophic, acetoclastic, and metholytrophic Archaea (Hedderich & Whitman 2006, Liu & Whitman 2008). Only members of the genus Methanocarcina (order Methanosarcinales) are capable of all three CH4-forming pathways (Liu & Whitman 2008). Methanogens cannot use most organic substances as carbohydrates, long-chain fatty acids, and alcohols, but rely on anaerobic microorganisms to produce the substrates needed for methanogenesis (Liu & Whitman 2008).
Equation 2: Exemplary reactions of hydrogenotrophic (a), acetoclastic (b), and methylotropic methanogenesis (c) (Liu & Whitman 2008, Thauer et al. 2008)
(a) 4 H2 + CO2 → CH4 + 2 H2O (b) CH3COOH → CH4 + CO2
(c) 4 CH3OH → 3 CH4 + CO2 + 2 H2O
Hydrogenotrophic methanogens form CH4 via the reduction of CO2 with H2 (Bapteste et al. 2005, Liu & Whitman 2008), but are often also able to utilize formate, with some species utilizing CO, ethanol, or 2-butanol (Liu & Whitman 2008). All six methanogenic orders of Archaea harbor hydrogenotrophic methanogens (Bapteste et al. 2005). In the hydrogenotrophic pathway (Equation 2a), CO2 binds to methanofuran (MF) first and gets reduced to a formyl-group via ferredoxin (Fd) that is reduced by H2 (Thauer et al. 2008) (Figure 4). The formyl-group in transferred to tetrahydromethanopterin (H4MPT) and MF is released. In Methanosarcina, a modified H4MPT is prevalent named tetrahydrosarcinapterin (H4SPT) (Liu & Whitman 2008, Thauer et al. 2008). The formyl-group is dehydrated resulting in the formation of a methenyl-group that is subsequently reduced to a methylene-group and then to a methyl-group. These reductions are performed by the coenzyme F420 that is reduced by H2 (Liu & Whitman 2008, Thauer et al. 2008). Next to methanogens, the fluorescent F420 appears only sporadically and sparsely among prokaryotes but was detected in the Mycobacterium smegmatis (Actinobacteria) in high abundances (Selengut & Haft 2010). The methyl-group is then transferred to a reduced coenzyme M (HS-CoM) and H4MPT/H4MSP is released. The final reduction and subsequent release of CH4 is catalyzed by the methyl-CoM reductase with reduced coenzyme B (HS-CoB) (Liu & Whitman 2008,
Thauer et al. 2008) (Figure 4). With formate as substrate for hydrogenotrophic methanogens, four molecules of formate are oxidized resulting in the creation of reduction equivalents that are required to reduce CO2 to CH4 (Liu & Whitman 2008) as described avove.
Acetoclastic methanogens (Equation 2b) use acetate and belong to the order Methanosarcinales (genera Methanosaeta and Methanosarcina) (Hedderich & Whitman 2006, Liu & Whitman 2008). They split acetate and reduce the methyl-group to CH4 whereas the carboxyl-group is oxidized to CO2 (Liu & Whitman 2008). Other than species of Methanosarcina, those of Methanosaeta are strictly acetoclastic (Hedderich & Whitman 2006, Liu & Whitman 2008). Initially, acetate is activated to acetyl phosphate with ATP and then synthesized to acetyl-CoA with HS-CoA (Figure 4). With H4MPT/H4SPT, acetyl-CoA is split into methyl-H4MPT/H4SPT and CO-CoA by the CO dehydrogenase/acetyl-CoA synthase enzyme complex. The methyl-H4MPT/H4SPT is reduced to CH4 as in the hydrogenotrophic pathway; CO-CoA is oxidized to CO2 with electrons being transferred to oxidized Fd and later to protons with the creation of H2 in a hydrogenase reaction (Liu & Whitman 2008, Thauer et al. 2008).
Methylotrophic methanogens (Equation 2c) belong to the Methanosarcinales and the genus Methanosphaera within the Methanobacteriales. Their substrates are methylated compounds as methanol, methylamines, and methylated sulphides, i.e., compounds with only one carbon atom (Bapteste et al. 2005, Liu & Whitman 2008) (Figure 4). During methylotrophic methanogenesis, the methylated substrates are transferred to HS-CoM forming methyl-CoM. One fraction is further reduced to CH4 as with hydrogenotrophic and acetoclastic methanogenesis. The other fraction of methyl-groups is oxidized to CO2 via a reversal hydrogenotrophic pathway to gain reduction equivalents (Liu & Whitman 2008) (Figure 4).
Methanogenesis is influenced by environmental factors as pH, temperature, water content, and the availability of Corg and is abundant in habitats with limiting concentrations of electron acceptors as O2,nitrate, and sulphate (Conrad 1996, Segers 1998, Liu & Whitman 2008). Temperature influences fermentation processes and therefore the fermentation products H2 and acetate, resulting in an influence on hydrogenotrophic and acetoclastic methanogenesis (Conrad 1996).
The methyl-CoM reductase appears as Mcr and its isoenzyme Mrt; their catalytic subunits are encoded by mcrA and mrtA, respectively (Gunsalus et al. 1987, Springer et al.
1995). As the methyl-CoM reductase catalyzes the final step for all three methanogenic pathways, i.e., for hydrogenotrophic, acetoclastic, and metholytrophic methanogens, mcrA is a frequently used structural gene marker to analyze the community of methanogens whereas mrtA is often co-amplified in lower numbers together with mcrA with commonly used primer systems (e.g., Lueders et al. 2001, Merilä et al. 2006, Hunger et al. 2011).
Figure 4: Diagram of the hydrogenotropic (red arrows), acetoclastic (green arrows), and methylotrophic (blue arrows) methanogenesis pathway, all three prevailing in Methanosarcina.
The figure illustrates the three pathways of methanogenesis as indicated by red, green, and blue arrows for hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis, respectively.
Electrons (e-) are derived from reduction equivalents, primarily from H2. For all three pathways, the final step is the reduction of methyl-CoM resulting in the relase of CH4. This final step is catalyzed by a methyl-CoM reductase (Mcr and/or Mrt). In the hydrogenotropic pathway, CO2 is reduced by e- derived from H2. Methylotropic methanogens utilize C1 compounds, i.e., molecules with at least one methyl group. Here, one fraction is reduced to CH4 whereas the rest is oxidized to CO2 via the reversal hydrogenotrophic pathway to generate reduction equivalents. During acetoclastic methanogenesis, acetate is split into a methyl group and an enzyme-bound CO. The CO is oxidized to CO2 to provide reduction equivalents for the reduction of the methyl group to CH4. For all three pathways, an electrochemical gradient is generated for ATP synthesis. Whereas most methanogens possess only one of the three pathways, species of Methanosarcina possess all three. Abbreviations: MF, methanofuran; CoM, coenzyme M; CoA, coenzyme A; H4SPT, tetrahydrosarcinapterin; Pi, inorganic P.
Based on Bapteste et al. (2005), Welander & Metcalf (2005), and Liu & Whitman (2008).
CO2
formyl-MF
formyl-H4SPT
methenyl-H4SPT
methylene-H4SPT
methyl-H4SPT
methyl-S-CoM
CH4
2e
-2e
-2e
-2e- ATP
acetate acetyl-Pi HS-CoA
acetyl-S-CoA
2e
-[CO]
methyl-amines methanol methyl-sulfides
2e
-2e
-2e
-methyl-CoM reductase (Mcr, Mrt)
CO2