Principals of AOM

Methane oxidation

Sulphate reduction is an anaerobic microbial process in which sulphate is converted to sulphide. Research has indicated that sulphate reduction can be coupled with methane oxidation. In order to reduce sulphate an electron donor is needed. Common electron donors are hydrogen and a variety of organic compounds, like organic acids, alcohols, and methane. Sulphate reducing bacteria (SRB) are well studied and are able to reduce sulphate under anaerobic and micro-aerobic conditions. Also a large amount of substrates and electron donors can be used for growth and sulphate reduction. In vitro oxidation of methane by SRB has not been found, but first clear evidence for anaerobic oxidation of methane (AOM) came from geochemical in situ studies of marine sediments. These studies revealed that methane diffusing upwards from deep sites of sediments often disappears long before any contact with oxygen is possible. In such anoxic zones of sediments, sulphate is the only electron acceptor that can account for methane oxidation. The zone of AOM in depth profiles is usually evident from a concave-up curvature of the methane concentration coinciding with an increased sulphate reduction rate. In zones where biogenic methane with its naturally low ¹³C:¹²C ratio disappears, isotopically light dissolved inorganic carbon and precipitated carbonates have been detected. This finding as well as the formation of radiolabelled CO₂ upon injection of [¹⁴C]-methane into anoxic marine sediments further provided evidence for AOM. AMO has been detected at temperatures from 4 to over 30°C and at different locations like lakes and seashores. The depth of the methane-sulfate transition zone ranges from centimetres to meters under the seafloor surface depending on the local conditions.

If radiolabelled methane was added to pure cultures of actively methane-producing archaea, a partial conversion to CO₂ (without net consumption of methane) was observed. This suggested that reactions of methanogenesis are reversible to a certain extent, and that AOM may be catalysed by methanogens themselves if an electron sink is available.

Stable isotope analysis of lipid biomarkers and rRNA gene surveys have implicated specific microbes in the anaerobic oxidation of methane. Fluorescent in situ hybridisation (FISH) combined with secondary ion mass spectrometry analyses, has provided direct evidence for the involvement of at least two distinct archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps and some sequences can be affiliated with a third seep-specific clad, ANME-3. The ANME-1 archaeal group more frequently existed in monospecific aggregations or as single filaments, apparently without a bacterial partner. Bacteria associated with both archaeal groups included relatives of Desulfosarcina and Desulfococcus species. The metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. Isotopic analyses suggest that monospecific archaeal cells and cell aggregates were active in anaerobic methanotrophy, as were multispecies consortia. Data indicate that the microbial species and biotic interactions mediating anaerobic methanotrophy are diverse and complex.

The pathways of methane oxidation with sulphate reduction are not known. Different intermediates are proposed which could act in the electron transfer from methane oxidizing archaea to sulphate reducing bacteria. Hydrogen, acetate, formate and methanol are theoretically the most probable intermediates as can be seen in the proposed reaction mechanisms.

Proposed mechanisms for sulphate-dependent methane oxidation:

The biochemical pathway of anaerobic methane oxidation is not well characterized, It is suggested that methane-consuming archaea use essentially the same biochemical processes as methane producers, but in reverse order. This hypothetical process is called, "reverse methanogenesis." However, running the reactions in reverse would require the microbes to expend more energy than they got out of the process and that would not be a good strategy for survival. Recent research with environmental genomics provided more arguments for the ‘reverse methanogenesis’ hypothesis. The unfavourable thermodynamics of methane activation in AOM might be overcome if the energy conservation reactions driven by the F₄₂₀-dependent respiratory chain are coupled to the methane oxidation reactions.

Suggested intermediates in the anaerobic degradation pathway of methane oxidizing communities are hydrogen, acetate, formate, alcohols, fatty acids, carbondioxide and an unknown, thermodynamically tuned electron shuttle (such as a cofactor). Addition of fluoroacetate (known inhibitor for acetogens) caused no change in methane oxidation rate and therefore acetate cannot be an intermediate in AMO.

Research of Nauhaus et al. (2002) proved that in an anoxic medium sulphide was produced from sulphate if methane was added. The same sediment samples did not produce sulphide at the same amount if hydrogen, formate, acetate or methanol were added instead of methane. These compounds are therefore not clear intermediates during AOM. In the presence of methane these compounds neither stimulated nor inhibited sulphate reduction.

Tor et al. (2003) on the other hand found that sediments from a shallow marine hydrothermal vent system accumulated acetate and hydrogen over time after addition of molybdate. Molybdate inhibits sulphate reduction in the sediments. Tor concluded that acetate is an important extracellular intermediate in the anaerobic degradation of organic matter in hot microbial ecosystems.

Reverse methanogenesis yields CO₂ that is extremely ¹³C depleted compared to normal marine dissolved CO₂. Assimilation of this CO₂ produced by the methane –oxidizing archaea could yield ¹³C-depleted sulphate-reducing bacterial biomass.

A recent thermodynamic and kinetic study by Sørensen et al. (2001) suggest that hydrogen, acetate, and methanol are all excluded as potential electron shuttles in coupled methane oxidation/sulphate reduction. This reduces the likelihood that acetate is an important intermediate in AOM. However Sørensen et al. did not consider the possibility of high methane levels where acetate becomes a favourable intermediate, nor did they consider the specific mechanisms proposed by Valentine and Reeburgh (2000) involving the transformation of two molecules methane into acetate and hydrogen. Therefore, acetate and acetic acid as reactive intermediates in net AMO remain distinct possibilities that would adequately explain the transfer of ¹³C depleted methane-derived carbon into SRB biomass.

A range of different sulphate-reducing bacterial cultures and archaea has been tested, but none of the strains were able to grow on methane as the main electron donor.

The driving force for research on this process is that microbial communities intercept and consume methane from anoxic environments, which would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments.

Sulphate reduction has also an important function in the environmental technology. The sulphate reduction process is able to remove heavy metals from wastewater because of the formation of heavy metal sulphides. Heavy metal sulphides are insoluble and consequently precipitate. Some biological zinc removal processes work with sulphate as reductor and hydrogen as electron donor but methane as an electron donor would economically be more favourable if it is used in biological sulphate and zinc removal.

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