Effect of Inhibition of Acetoclastic Methanogenesis on Growth of Archaeal Populations in an Anoxic Model Environment

Abstract

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H 2 /CO 2 to total CH 4 production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH 3 F, measured the activity and Gibbs free energy (⌬G) of CH 4 production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH 3 F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of Methanosarcina spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH 3 F seem to be a suitable method for quantifying acetoclastic CH 4 production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.Atmospheric CH 4 significantly contributes to global warming (4, 35). Among the dominant sources of CH 4 are wetlands and flooded rice fields, which are responsible for a third of the atmospheric CH 4 budget (4). To understand the processes responsible for methane production and consumption in anoxic environments, it is useful to study the flow of carbon and electrons. For this purpose, metabolic inhibitors are frequently used to allow quantification of single processes (31). Methyl fluoride (CH 3 F), when applied at the appropriate concentration, was found to be a rather specific inhibitor of acetoclastic methanogenesis, while the operation of CH 4 production from H 2 /CO 2 was unaffected (8, 16). Consequently, CH 3 F has been used to monitor changes in carbon flow in methanogenic systems (9). However, a crucial requirement for unbiased application of inhibitors is specificity, meaning that they must not influence any other process or reaction, while the target reaction or process must be completely inactivated. Methyl fluoride is also known to affect processes other than acetoclastic methanogenesis. For example, it also inhibits aerobic oxidation of CH 4 and, to a lesser extent, oxidation of ammonia (32, 33). With respect to CH 4 production, the optimized concentration of CH 3 F for complete inhibition of acetoclastic methanogenesis must not be exceeded, since at too-high concentrations hydrogenotrophic methanogenesis can also be partially inhibited (16). On the other hand, CH 3 F is apparently not inhibitory for acetotrophic sulfate reducers (e.g., Desulfotomaculum spp.) and for acetogenic fermenting bacteria (e.g., Acetobacterium spp.) (16). However, many archaeal and bacterial phylogenetic clusters that are found in natural environments are yet unc...

Wetland restoration and methanogenesis: the activity of microbial populations and competition for substrates at different temperatures

“…respectively. It is suggested that the T-RFs of 185 bp represents members of Methanosarcinaceae or RC-VI (Kemnitz et al 2004), 382 bp members of the euryarchaeal RC-III, and the T-RF of 394 bp members of the methanogenic RC-I, (Kemnitz et al, 2004;Penning and Conrad, 2006). This affiliation is consistent with the clone library: archaeal 16S rRNA gene clone sequences from the original soil belonged exclusively to RC-VI (Fig.…”

“…The tubes were closed with butyl rubber stoppers and flushed with N 2 . In inhibition experiments, CH 3 F, a specific inhibitor of acetoclastic methanogenesis, was added to the gas phase to a final concentration of 1% (Frenzel and Bosse 1996;Penning and Conrad 2006).…”

Different Bacterial Populations Associated with the Roots and Rhizosphere of Rice Incorporate Plant-Derived Carbon

“…Vercelli soil contains a wide diversity of methanogenic archaea, including acetoclastic Methanosarcinaceae and Methanosaetaceae as well as hydrogenotrophic Methanocellales, Methanomicrobiales, and Methanobacteriales (11,20). CH 4 is formed by both pathways in Vercelli soil (50), but on rice roots, CH 4 is formed mostly by the reduction of CO 2 (51). Furthermore, Lu and Conrad (18) showed that in particular Methanocellales incorporate rice plant carbon in the rhizosphere, but Methanosaetaceae and Methanomicrobiaceae are also active (52).…”

Biogas process parameters—energetics and kinetics of secondary fermentations in methanogenic biomass degradation

“…Later, we corrected this value to −12 to −15 kJ per mol reaction, based on new insights into the stoichiometry of the ATPase reactions and considerations on the energy charge of syntrophically fermenting bacteria (Schink and Stams 2013). Energy gains in this range were reported before for syntrophic oxidation of butyrate (Adams et al 2006;Wang et al 2013), propionate (Krylova and Conrad 1998;Scholten and Conrad 2000), and acetate (Hao et al 2010;Schnürer et al 1997), as well as for homoacetogenic fermentation (Conrad and Wetter 1990;Fey and Conrad 2000;Seitz et al 1990), aceticlastic methanogenesis (Fey and Conrad 2000;Penning and Conrad 2006), and hydrogen-dependent methanogenesis (Dwyer et al 1988;Seitz (Müller 2015;Spahn et al 2015, Lim et al 2014. In any case, we have to keep in mind that measurements of bulk concentrations both in the gas phase and in the liquid phase give only a rough estimate of the actual in situ concentrations of the intermediates within the microstructure of microbial aggregates (Pauss et al 1990).…”