Microbial ecology, physiology and genome analysis of microbes mediating the anaerobic oxidation of methane (AOM, “MUMM II” project)

After eight years of intensive studies of the microbial diversity and community structure in seep sediments, sulfate methane transition zones in shallow, subsurface and deep biosphere sediments, and microbial mats, a picture of global distribution of the archaea catalyzing AOM (= ANME types) is evolving. All ANME groups were detected at almost all seep sites (Figure 1). However, always one group was shown to dominate a certain habitat. Furthermore, ANME- 2a/DSS (DSS = Desulfosarcina/Desulfococcus cluster) and ANME-2c/DSS aggregates differed significantly in morphology (mixed type vs. shell type aggregates, Figure 1).

Figure 1: Overview of 16S rRNA based ANME diversity.

Seep sites are characterized by extremely high microbial biomass mainly build from ANME-2 organisms and its sulphate-reducing partners, for example 2x1010 cell cm-3 at HMMV (Niemann et al. 2006; Lösekann et al. 2007), and 3x1011 cells cm-3 in microbial mats from the Black Sea (Knittel, unpublished data) accounting for more than 90% of the total microbial biomass in these habitats. ANME-1 archaea was found in lower abundance in all seep systems but dominate main parts of massive microbial mats, which produce large carbonate columns and platforms in the permanently anoxic Black Sea (Knittel et al. 2005, Treude et al. 2005, Treude et al. 2007)

Unpublished data of the Ph.D. student Julia Arnds suggest high heterogeneity within Black Sea microbial reef structures. When samples were analyzed from different reef zones, ANME-1 was found in all samples. Samples from the top had highest detection rates, and were dominated by ANME-2, and relatives of Desulfosarcina/Desulfococcus. In addition, the top zone showed the lowest bacterial diversity. In the exterior there was a predominance of ANME-1, Epsilonproteobacteria, and Crenarchaeota. In the center we found twice as many ANME-1 compared to top and exterior samples, and also many empty sheets of ANME-1. Our hypothesis is that the most active microbial growth zone of the reef is the top, whereas the center seems to be oldest. The reef’s exterior obviously provides various microniches. Reefs are highly heterogeneous structures with significant differences between different reef sites, and reef zones. New FISH probe were developed for SRB (SEEP-SRB2, -3, -4, ANME-3 SRB partner group) at AOM sites. Initial results using these probes on sediments from a Gulf of Mexico cold seep (cruise OTEGA SO174, station 161, 0-10 cm) showed high abundances. Except for SEEP-SRB4 all groups were detected in numbers of 2-6 % of total cells (Ritz et al. unpublished). Flow cytometry was optimized for sorting of FISH-stained ANME archaea from enrichment cultures. A high sorting purity of more than 99% could be achieved for ANME-3 cells. Using sorted ANME-3 aggregates, the sulphate-reducing partner of ANME-3 could be identified and the methyl-coenzyme m reductase (mcr) of ANME-3 cloned (Lösekann et al. 2007). Sorted single ANME-3 cells are currently subjected to genome amplification and pyrosequencing.