Methanogenesis

Methanogenesis is an anaerobic respiration process catalyzed by strictly anaerobic obligately methanogenicArchaea [6, 92]. Methanogens belong to the Euryarchaeota [184] and form 6 orders (Methanobacteriales,Methanocellales,Methanococcales, Me-thanomicrobiales,Methanopyrales,Methanosarcinales) including 32 genera [51, 144, 169].

Methanogens have a limited substrate range and produce CH₄ hydrogenotrop-ically, acetoclastically or methylotrophically [51, 92] (Figure 6, Table 5). Hydro-genotrophic methanogens reduce CO2 with H2 to form CH4 [6, 92]. Many hy-drogenotrophic methanogens also utilize formate, while some can utilize secondary alcohols like 2-propanol or 2-butanol, ethanol, or CO [92]. Hydrogenotrophic methano-genesis is widespread and occurs in all methanogenic orders [6]. Acetoclastic metha-nogens belong to the orderMethanosarcinales(generaMethanosarcinaand Methano-saeta) and utilize acetate by oxidizing the carboxyl-group to CO2 and reducing the methyl-group to CH₄ [92]. While Methanosarcina produce CH₄ hydrogenotrophi-cally, methylotrophically and acetoclastihydrogenotrophi-cally, Methanosaeta are strictly acetoclas-tic [51, 92]. Methylotrophic methanogens utilize methylated compounds such as methanol, methylamines, and methylated sulfides and occur only within the Methano-sarcinales (with exception of Methanosaeta) and within the genus Methanosphaera (Methanobacteriales) [6, 92]. The methyl-coenzyme M reductases Mcr and Mrt (catalytical subunits encoded by mcrA or mrtA) are central enzymes involved in all types of methanogenic pathways (Figure 6; [34]), and mcrA is frequently used as a structural genemarker to assess the community composition of methanogens in environmental samples [47, 55, 101, 191].

1.3.2.1 Hydrogenotrophic methanogenesis

In hydrogenotrophic methanogenesis, CO₂ is reduced with H₂ or formate as a

pri-1 General introduction

Figure 6: Schematic overview of the three methanogenic pathways. Methyl-Coenzyme M reductase functions as a key enzyme in all three methanogenic pathways. Further enzymes, cofactors and compounds are not shown. Dashed lines indicate that more than one step is needed for the conversion. R=e.g., -SH, -OH, or NH2. Mcr=Methyl M reductase I; Mrt=Methyl coenzyme-M reductase II. Based on [34].

Table 5: Types of methanogenesis.

Type Reaction ∆G0’ (kJ)¹ Organisms²

Hydrogenotrophic 4 H2+ CO2→CH4+ 2 H2O -35 Most methanogens

Acetoclastic CH3COOH→CH4+ CO2 -33 Methanosarcina,Methanosaeta Methylotrophic 4 CH3OH→3 CH4+ CO2+ H2O -105 Methanosarcinaand others

1Gibbs free energy under standard conditions (Temperature: 25^◦C, Pressure: 101.3 kPA, pH 7.0).

2Examples of secondary fermenters involved in this type of syntrophic reaction. [92]

mary electron donor to CH4 [92]. When formate is used as an electron donor, 4 molecule of formate are oxidized to CO₂ and the obtained reduction equivalents are used to reduced 1 molecule of CO2 [92]. In a first step, CO2 binds to methanofu-ran (MF) and is reduced by ferredoxin to a formyl-group, the ferrodoxin in turn is reduced by H2 (Figure 7; [169]). The formyl-group is transfered to tetrahy-dromethanopterin (H4MPT), dehydrated to a methenyl-group and subsequently re-duced to methylene-H4MPT and methyl-H4MPT by reduced F420 [92, 169]. The methyl-group is then transfered to coenzyme-M (CoM) and reduced to CH4 in a final step by the methy CoM reductase [92, 169].

1.3 Processes involved in greenhouse gas production in anoxic peatland soils

Figure 7: Reactions involved in hydrogenotrophic methanogene-sis. F420=coenzyme F420; Fd=ferredoxin; MF=methanofuran;

H₄MPT=tetrahydromethanopterin; HS-CoB=coenzyme B; HS-CoM=coenzyme M; based on [92, 169]

1.3.2.2 Acetoclastic methanogenesis

In acetoclastic methanogenesis, acetate is split, the carboxyl-group is oxidized to CO2, while the methyl-group is reduced to CH4 [92]. Acetate is first activated with ATP and transfered to coenzyme A by acetate kinase-phospotransacetylase, form-ing acetyl-CoA. Acetyl-CoA is cleaved to methyl-H₄MPT and CO-CoA by the CO

1 General introduction

dehydrogenase/acetyl-CoA synthase system. CO-CoA is further oxidized to CO2, electrons are first transfered to oxidized ferredoxin, then to H2 in a hydrogenase reaction. The methyl-group is transfered to CoM and reduced to CH₄ as in hy-drogenotrophic methanogenesis [92].

1.3.2.3 Methylotrophic methanogenesis

In methylotrophic methanogenesis, the methyl-groups of methylated compounds like methanol are transfered to CoM to form methyl-CoM. Methyl-CoM is reduced to CH4 by the methyl CoM reductase. The reduction equivalents for this reduc-tion are obtained by oxidareduc-tion of addireduc-tional methyl-groups to CO2 via a reversed hydrogenotrophic pathway (i.e. via methyl-H4MPT, methylene-H4MPT, methenyl-H₄MPT and formyl-MFR) [92].

1.3.2.4 Factors influencing methanogenesis in soils

Methanogenesis is affected by pH, temperature, groundwater level, the amout of available organic carbon, and the amount of available alternative electron acceptors [23, 148].

Most cultured methanogens show optimal growth at near-neutral pH, and methano-genesis is generally inhibited at ph <5 [148]. However, acid-tolerant methanogenic strains have been isolated from peatlands [183]. Indeed, methanogenesis is active in many acidic wetland systems [15, 14, 54, 81]. CH4 production rates of methanogenic communities from acidic wetlands are higher at acidic than at neutral pH, indicating an adaptation of the methanogens to in situ pH [14, 81]

Most wetland methanogenic communities are mesophilic and show optimum CH4

production capacities at 20-35^◦C [14, 81, 102, 103]. Increased soil temperatures result in increased CH4 production rates [23]. However, methanogenesis in wetland soils occurs also at lower temperatures and is still comparably high at 4^◦C [81, 102,

1.3 Processes involved in greenhouse gas production in anoxic peatland soils

103] .Moreover, the contribution of the methanogenic precursors differs according to temperature. At lower temperatures, CH4 is mainly derived from acetate [23].

However, this is likely attributed to a change in the fermentation pathways that occur prior to methanogenesis than to the methanogenic processes themselves, as e.g. the production of H2 is reduced at low temperatures [23]. On the other hand, an increased contribution of hydrogenotrophic methanogenesis at low temperatures is observed in acidic wetland soils, and is attributed to the occurence of further sinks for acetate or H2 in those soils [54, 82].

The groundwater level affect the degree of soil aeration and determines the po-sition of the oxic/anoxic interface [72]. A lower water table results in higher soil aeration and a concomitant rise of the redox potential [70]. Oxygen (O₂) inhibits methanogenesis, and increased soil aeration leads to a decrease in CH4 emissions from soil as O2 inactivates the methyl-coenzyme M reductase [67]. Even though methanogens are detected in more oxic systems such as desert soils, CH4 produc-tion in these systems is negligible [64, 127, 186]. Decreased CH4 production in more oxic systems is attributed to a combination of O2 inhibition and desiccation [35]. Methanogens can survive or even grow under oxic conditions, however, the exact mechanisms is not yet resolved, since there are no known resting stages of methanogens [35]. However, methanogens show enzymatic protection against oxida-tive stress, e.g. by the enzymes superoxide dismutase and catalase [69, 162].

1 General introduction