Abstract
The predominant methanogens in an anaerobic wstewater treatment plant were studied by using a molecular technique without isolation and cultivation. The 16S rRNA genes from the bulk DNA of the anaerobic sludge were amplified by PCR, cloned, and reamplified. Of 30 clones isolated and tested, 24 clones contained a 16S rDNA insert. These positive clones were divided into four ribotypes on the basis of restriction fragment length polymorphism (RFLP) patterns with Hae III. The RFLP pattern of the most abundant ribotype (designated ribotype 1) was almost the same as that of Methanosaeta soehngenii which was computer-predicted and/or experimentally obtained. This was also supported by the results of RFLP analyses with HhaI and SmaI. Phylogenetic trees for methanogens was constructed on the basis of 16S rDNA sequences and 16S rDNA-RFLP patterns. Although there were some differences in branching patterns between the two trees, the RFLP-based tree was suggested to be used roughly as a model showing natural phylogenetic relationships among methanogens. The results of this studyshow that 16S rDNA-KELP analysis with PCR cloning has a promise to identify the predominant methanogens in anaerobic wastewater treatment systems.
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