Tracing the slow growth of anaerobic methane-oxidizing communities by (15)N-labelling techniques.

The anaerobic oxidation of methane (AOM) is an important methane sink in marine ecosystems mediated by still uncultured Archaea. We established an experimental system to grow AOM communities in different sediment samples. Approaches to show growth of the slow-growing anaerobic methanotrophs have been either via nucleic acids (quantitative PCR) or required long-term incubations. Previous long-term experiments with (13)C-labelled methane led to an unspecific distribution of the (13)C-label. Although quantitative PCR is a sensitive technique to detect small changes in community composition, it does not determine growth yield. Therefore, we tested an alternative method to detect a biomass increase of AOM microorganisms with (15)N-labelled ammonium as N-source. After only 3 weeks, significant (15)N-labelling became apparent in amino acids as major structural units of microbial proteins. This was especially evident in methane-containing incubations, showing the methane-dependent uptake of the (15)N-labelled ammonium by microorganisms. Cell counts demonstrated a two- and fourfold increase at ambient or elevated methane concentrations. With denaturing gradient gel electrophoresis, over 6 months incubation no changes in community composition of sulphate-reducing bacteria and archaea were detected. These data indicate doubling times for AOM microorganisms between 2 and 3.4 months. In conclusion, the (15)N-labelling approach proved to be a sensitive and fast way to show growth of extremely slow-growing microorganisms.