Dissimilatory sulfur metabolism coupled to anaerobic oxidation of methane

The seafloor and its microbial inhabitants play an important role in the biogeochemical cycling of elements. These environments are generally anoxic but contain high concentrations of sulfate penetrating from the overlying seawater. The main carbon mineralization processes such as the anaerobic oxidation of methane (AOM; Eq. 1) are therefore generally coupled to sulfate reduction. CH4 SO42 → HCO3 HS H2O (Eq. 1) AOM plays a crucial role in both carbon and sulfur cycling. It oxidizes the majority of the methane a potent greenhouse gas diffusing from the seafloor and prevents its escape to the atmosphere. Methane oxidation also returns the carbon trapped in the form of recalcitrant methane back to the carbon cycle as carbon dioxide. The AOM-coupled sulfate reduction consumes a large portion of the downwards sulfate flux and forms sulfide, which diffuses upwards towards the seafloor where it supports free-living sulfide- and sulfur-oxidizers but also gutless worms, clams and mussels that rely for their nutrition on the thiotrophic symbionts. Despite the pronounced effect of AOM on the sediment geochemistry little is known about its biology. The organisms responsible for AOM a consortium of methanotrophic archaea and Deltaproteobacteria have been identified in situ but their slow metabolism complicates growing them in pure cultures and renders the physiological investigations challenging. So far, AOM research has predominantly focused on the C1 metabolism of the methanotrophic archaea. The investigations presented in this thesis address the dissimilatory sulfur metabolism of the organisms involved in AOM and the mechanisms of its coupling to methane oxidation. Chapters 2 and 3 describe the purification and characterization of the three known enzymes involved in dissimilatory sulfate reduction (SR enzymes; ATP sulfurylase, APS reductase, sulfite reductase). The enzymes were purified from a naturally enriched microbial mat using liquid chromatography. The identity of the SR enzymes was confirmed by N-terminal amino acid sequencing and their activity in total cell extracts as well as in individual chromatography fractions was quantified by corresponding enzyme essays. Our aim was to assign these enzymes to a particular organism in the mat sample. For this purpose, polyclonal antibodies against the purified ATP sulfurylase and sulfite reductase were used APS reductase could not be sufficiently purified for antibody generation in situ in the original environmental sample as well as in our other enrichment cultures. This combination of environmental proteomics and immunolocalization allowed us to unambiguously assign the isolated SR enzymes exclusively to the bacterial partner. The archaea did not express detectable amounts of the identified SR enzymes themselves and therefore likely depend on their bacterial partners to perform the sulfate reduction. These results are presented as manuscripts in revision (Manuscript 1) and in preparation (Manuscript 2). The following Chapter 4 introduces experiments that were performed in order to elucidate sulfur transfer and speciation in AOM consortia. We used stable and radioactive sulfur isotopes to follow sulfur exchange between the medium and biomass and on a single cell level among individual cells. Based on our results and thermodynamic consideration we propose a model, in which DSS bacteria reduce sulfate to a zerovalent sulfur compound (probably polysulfide) that might be utilized by ANME as an electron acceptor for methane oxidation. Thus, unexpectedly, ANME participate in the dissimilatory sulfur metabolism coupled to AOM. Our combined data suggest that ANME obtain this compound from the associated bacteria. Such sulfur shuttling between two organisms not only represents a unique mechanism for a syntrophic relationship but also has significant implications for our understanding of sulfur transformations in the AOM zones in marine sediments. These results are presented as a manuscript in preparation (Manuscript 3)