Biochemie und Physiologie der Sulfatreduktion in der anaeroben Oxidation von Methan

Abstract

Anaerobic oxidation of methane coupled to sulfate reduction (AOM) is the major sink for methane produced in anoxic marine sediments. AOM is catalyzed by consortia of anaerobic methanotrophic archaea (ANME) and deltaproteobacteria closely related to known sulfate-reducing bacteria (SRB). The knowledge about the biochemical reactions involved in AOM is incomplete. The most common view is that the archaea are responsible for methane oxidation, while the deltaproteobacteria are SRB scavenging reducing equivalents and producing sulfide. There is biochemical evidence that ANME catalyze the initial step of methane activation by the enzyme methyl-coenzyme M reductase as a reversal of the last step of methanogenesis. However, the pathway of sulfate reduction coupled to methane oxidation is not resolved yet. This work provides evidence that sulfate reduction with methane-derived reducing equivalents proceeds via the established sulfate reduction pathway in Black Sea microbial mats with high AOM activity. These mats represent a natural enrichment of consortia consisting of ANME-2 and deltaproteobacteria of the Desulfosarcina/Desulfococcus (DSS)-clade. They contained substantial amounts and activities of the key enzymes for sulfate reduction, ATP sulfurylase (Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). Sat as well as the small subunit of Apr and native Dsr were isolated. Their N-terminal amino acid sequences were characteristic of deltaproteobacterial enzymes. Moreover, the retrieval of the Apr-encoding genes revealed their relationship to those in SRB of the DSS clade, sustaining the hypothesis that sulfate reduction with methane-derived reducing equivalents takes place in the deltaproteobacterial cells. Different possibilities the assumed extracellular transfer of reducing equivalents from ANME to the SRB were studied. In accordance to published papers, hydrogen as well as the reduced carbon compounds formate, acetate and methanol could be excluded as transferred intermediates. Moreover, there is no transfer of reducing equivalents via methyl sulfide, which has recently been suggested as intermediate. It neither inhibited AOM nor served as electron donor for sulfate reduction in AOM cultures in the absence of methane. Additionally, the effect of diffusible redox-active electron carriers on AOM was studied. The thiols cysteine and homocysteine neither stimulated AOM nor sulfate reduction in the absence of methane. Micromolar concentrations of anthraquinones and flavins inhibited AOM, thus they are most likely not involved in the extracellular electron transfer in AOM. Finally, the possibility of an electron transfer from cell to cell via outer membrane proteins/'nanowires' was taken into account. Accordingly, an anoxic electrochemical cell was developed to study AOM. A methane-dependant current production by AOM-active Black Sea microbial mats was not observed therein, thus evidence of a direct electron transfer is still lacking.