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Abstract:
Methane is a final product of the anaerobic degradation of organic matter and an important greenhouse gas. With the carbon isotope signature of atmospheric methane it is possible to quantify the different methane sources. Most important terrestrial sources are wetlands and rice fields, where methane is almost exclusively formed from CO2 and acetate. Methanogenesis pathways differ in their isotope fractionation, which allows the quantification of the amounts of methane formed from acetate and CO2. Here, I determined isotope fractionation factors (a) of different anaerobic processes, which are required for modelling of the methane formation pathways. The isotope signature of acetate (dac) and the fractionation factor of hydrogenotrophic methanogenesis (aCO2/CH4) are mandatory parameters for the above described modelling. Experimentally they can be determined by inhibition of acetoclastic methanogenesis with the inhibitor methyl fluoride (CH3F). Here I tested, if CH3F also unspecifically influenced other processes or archaeal groups. For the rice root model system it could be shown by methods of geochemistry and molecular biology, that only the target process, the acetoclastic methanogenesis, and the respective target organisms, the family of Methanosarcinaceae, were directly inhibited. This indicated that incubation experiments with CH3F are suitable to determine dac and aCO2/CH4. Acetate is almost exclusively formed by fermentation, which allows the calculation of dac via the isotope fractionation factor of fermentation. Here, the fractionation behavior of Clostridium papyrosolvens for the formation of acetate, ethanol, lactate, formate and CO2 was elucidated. In the linear part of the fermentation pathway from the saccharide to the intermediate pyruvate, 12C was preferred in soluble saccharides, whereas with the polysaccharide cellulose no fractionation occurred. The branch points pyruvate and acetyl-CoA showed characteristic fractionation patterns, which allowed to predict the isotope signature of the final products. Since polysaccharides are the main substrate of fermentation in nature and therefore no fractionation to pyruvate exists, the isotope signatures of final products can be predicted only by the isotope signature of organic matter and the carbon flow of fermentation. The aCO2/CH4 is also an essential parameter, which however has been shown to vary strongly. The underlying factors for this variation were so far unknown and were investigated in pure and cocultures of hydrogenotrophic archaea of the families Methanomicrobiaceae and Methanobacteriaceae. The isotope fractionation correlated with the Gibbs free energy change (DG) of hydrogenotrophic methanogenesis. In soils and sediments, where due to H2 concentration gradients DG cannot be determined correctly, the aCO2/CH4-ΔG relation allowed an unbiased determination of DG of the hydrogenotrophic methanogenesis in situ. It could be shown that in environments influenced by H2 gradients more free energy was available to hydrogenotrophic methanogens than previously assumed. In the rice root model system the determined parameters were used to quantify the relative carbon flow to methane by the different pathways. The calculated values agreed with geochemical data and confirmed that such a quantification solely based on stable carbon isotope signatures is indeed feasible. In conclusion, this work deepened the knowledge on the 12C/13C isotope flow in single processes and in the reaction network of the anaerobic degradation of organic matter and therefore simplified the modelling of carbon flow in this system.
