Procaryotic community composition

The methanogenic archaea, three selected methanogen families (Methanobacteriaceae, Methanosetaceae and Methanosarcinaceae) and methanotrophic bacteria belonging to groups I and II were detected using FISH (Fluorescence in situ hybridization) with 16S rRNA — targeted oligonucleotide probe labelled with indocarbocyanine dye Cy3. The prokaryotes were hybridized according to the protocol by Pernthaler et al. (2001). Briefly, the supernatants which were used also for TCN were filtered onto polycarbonate membrane filters (0.2 pm GTTP; Millipore), filters were cut into sections and placed on glass slides. For the hybridization mixtures, 2 pL of probe-working solution was added to 16 pL of hybridization buffer in a microfuge tube. Hybridization mix was added to the samples and the slides with filter sections were incubated at 46 °C for 3 hours. After incubation, the sections were transferred into preheated washing buffer (48 °C) and incubated for 15 minutes in a water bath at the same temperature. The filter sections were washed and air — dried. The DAPI staining procedure followed as previously described. Finally, the samples were mounted in a 4:1 mix of Citifluor and Vecta Shield. The methanogens and methanotrophs were counted in three replicates from each locality and the relative proportion of bacteria, archaea, methanogens and methanotrophs to the total number of DAPI stained cells was then calculated.