Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Gene libraries of anaerobic methanotrophs, sulphate reducers and methanogens found in Skagarrak, Aarhus Bay, Tommeliten, Gullfaks and the Black Sea

The purpose of this work was to investigate if active seeps of the Central (Tommeliten) and Northern North Sea (Gullfaks), the Skagarrak, the Baltic Sea (Aarhus Bay) and the Black Sea host communities of methanotrophs controlling the methane efflux. The hypothesis was that anaerobic methane-oxidizing communities associated with methane seepage in the North Sea are similar to those known from deep-water seeps.

Researchers have not yet been able to isolate microorganisms capable of oxidizing methane under anoxic conditions. Thus, no pure cultures are currently available for physiological and biochemical characterization and the present knowledge about microbes mediating the anaerobic oxidation of methane (AOM) is therefore mostly derived from cultivation-independent techniques. To assess the microbial diversity and community structure at METROL sites, we mainly worked according to the rRNA approach (Amann et al., 1995). DNA is extracted directly from the environmental sample. The sequences of interest are amplified by general archaeal or bacterial specific primers from the bulk DNA, followed by cloning and sequencing. By comparative sequence analysis with existing 16S rRNA gene databases it is possible to identify whether the sequences found in this environment belong to known or to not yet described species. All sequences which have been published in scientific literatrue, were made available to the public by submitting the sequence data to one of the three public databases (GenBank, EBI, DDBJ) maintained by the National Centre for Biotechnology Information (NCBI,, the European Molecular Biological Laboratory EMBL,, and the DNA Data Bank of Japan (DDJB, The databases are produced in an international collaboration and each of the three groups collects a portion of the total sequence data reported worldwide, and all new and updated database entries are exchanged between the groups on a daily basis. Currently, there are more than 250000 16S rRNA gene sequences available.

Thus the potential users, which include scientists working in the field of molecular biology or bioinformatics but also scientists studying sulphate reducing bacteria (SRB) and anaerobic methanotrophic archaea (ANME) have an direct access to sequencing data from the METROL project.

Sequences were aligned and phylogenetically analysed using the ARB software package ( Prior to phylogenetic analysis environmental sequences were checked for chimeras.

Current status:
To study the microbial diversity at different METROL seep sites six 16S rRNA gene libraries (three libraries for archaea and three for bacteria) have been constructed for three sites: Tommeliten seep areas (He180- 1904, 160 cm), Gullfaks field (He208, MUC766, 0-10cm) and Aarhus Bay (HeIV_04, 173 cm). The work on Skagarrak and Aarhus Bay Sediments and the Black Sea sediments and mats is in progress. All samples were taken from communities associated with the sulfate-methane transition zone (SMTZ) of methane seeps. For the evaluation of sequences retrieved during METROL, existing information of the BMBF/DFG project MUMM were utilized (Knittel et al. 2003 and 2005).

The most abundant bacterial clone group consisted of different sulfate-reducing bacteria within the Deltaproteobacteria. Several different SRB groups were retrieved from the samples: The four seep-specific clades SEEP-SRB1-4, which are Desulfobacterium anilini relatives, other Desulfobacterium relatives and two unaffiliated groups. Furthermore, one group affiliated with Desulfuromonas, which are sulfur-reducing organisms, was detected. Organisms of group SEEP-SRB1 were shown to be the sulfate-reducing partners for ANME-2 cells in AOM mediating consortia (Boetius et al., 2000).

Archaeal diversity in SMTZ sediments was as high as for bacteria indicated by the varying presence of six different phylogenetic groups of archaea (ANME-1, Marine benthic group B, C, D, and E, Marine Group 1, and one unaffiliated group). Furthermore, the intra group diversity was high among the archaea with often less than 95% sequence similarity.

The results from gene libraries generally matched the biomarker studies of the SMTZ of all investigated sediments. The development of CARD FISH protocols as a method to detect single cells with low RNA content in subsurface sediments has resulted in the first images and quantification of coastal methanotrophs both in North Sea and Baltic Sea sediments, confirming the clone library results.

Very similar groups as those known from deep water gas hydrate sites and cold seeps have been found, which supports the idea of a cosmopolitan presence of AOM-mediating consortia in SMTZs. However, their biomass is highly variable, spanning a difference of 5 orders of magnitude from low methane flux, subsurface sediments to highly advective deep water seeps.

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Alfred-Wegener-Institute Bremerhaven
Celsiusstr. 1
28359 Bremen
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