Denaturing gradient gel electrophoresis and cloning

Methanogenic communities associated with hyporheic sediments at two different depths (0­25 cm and 25-50 cm) along the longitudinal stream profile were compared based on the DGGE patterns. As shown in Fig. 9, the DGGE patterns varied highly among study localities (Fig. 9A), irrespective of the depth (Fig. 9B). However, presence of the bands in all samples indicates that methanogens may occur up to 50 cm of the sediment depth. The number of DGGE bands of the methanogenic archaeal communities was compared either among localites or among different sediment depths. A total of 22 different bands were observed in the DGGE image ranging from 4 (locality II) to 16 (locality IV) in the samples (Fig. 9A).

The number of DGGE bands also ranged from 2 to 10 for the samples from upper layer (0-25 cm) and from 2 to 11 for the samples from deeper layer (25-50 cm), respectively (Fig. 9B). We found no clear trend in the number of DGGE bands with increasing depth (Fig. 9B). Locality IV appears to be the richest in number of DGGE bands. We suppose that this might be due to most favorable conditions prevailing for the methanogens life as indicated by a relatively low grain median size, lower dissolved oxygen concentration or higher concentration of the ferrous iron compared to other localities (cf. Table 2).

The methanogenic community diversity in hyporheic sediment of Sitka stream was also analysed by PCR amplification, cloning and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 60 mcrA gene sequences revealed 26 different mcrA gene clones.


‘-“""’V Locility

Figure 9. Number of DGGE bands associated with hyporheic sediments at two different depths along the longitudinal stream profile. A — Total number of all bands detected at each locality; B — number of bands found at different sediment depths

Most of the clones showed low affiliation with known species (< 97% nucleotide identity) and probably represented genes of novel methanogenic archeal genera/species, but all of them were closely related to uncultured methanogens from environmental samples (> 97% similarity) retrieved from BLAST. The 25 clones were clustered to four groups and were confirmed to be affiliated to Methanosarcinales, Methanomicrobiales and Methanobacteriales orders and other unclassified methanogens. The members of all three orders and novel methanogenic cluster were detected to occur in a whole bottom sediment irrespective of a depth, nevertheless, the richness of methanogenic archaea in the sediment was slightly higher in the upper sediment layer 0-25 cm (15 clones) than in the deeper sediment layer 25­50 cm (11 clones)(Buriankova et al. in review). The clones affiliated with Methanomicrobiales predominated in the deeper layer while Methanosarcinales clones dominated in the upper sediment layer. This prevalence of Methanosarcinales in the upper sediment layer was also confirmed by our FISH analyses as has been mentioned above.

4. Discussion