Application of 16S rDNA Ribosomal RNA Approach for the Analysis and Identification of Bacterium in Granular Sludge

Both Eubacteria and Archaea community diversity and dynamicity were studied during PCP-degrading micro-aerobic granular sludge cultivation using 16S rDNA ribosomal RNA approach. Main bands of DGGE were compared with sequences. The results showed that Eubacteria and Archaea community were closely related to uncultured microorganisms. There were aerobic, micro-aerobic and anaerobic bacterium in micro-aerobic granular sludge simultaneously. After granular sludge was domesticated by PCP, the dominant Eubacteria and Archaea for PCP degradation were related toProteobacteria, Sphingomonas, Methanogenic bacteriumand so on.

Isolation of a methanogenic bacterium, Methanosarcina sp. strain FR, for its ability to degrade high concentrations of perchloroethylene

Tetrachloroethylene (PCE) is a toxic compound essentially used as a degreasing and dry-cleaning solvent. A methanogenic and sulfate-reducing consortium that dechlorinates and mineralizes high concentrations of PCE was derived from anaerobically digested sludge obtained from a waste water treatment plant (Bourg-en-Bresse, France). A methanogenic bacterium, strain FR, was isolated from this acclimated consortium. On the basis of morphological and physiological characteristics, strain FR was classified in the genus of Methanosarcina. Phylogeny analysis with the 16S rRNA gene sequence revealed that strain FR is highly related to Methanosarcina mazei and Methanosarcina frisia (99.6 and 99.5% identity, respectively). High concentrations (50-87 μM) of PCE were completely dechlorinated by strain FR cultures at the rate of 76 nM·mg protein-1·day-1. PCE dechlorination produced a nonidentified compound. The tracer experiments with [13C]PCE revealed that the product was nonchlorinated. Dechlorination of PCE to trichloroethylene was still active in the presence of boiled cell extract of the strain FR. However, no further dechlorination was observed. This result suggests that a cofactor rather than an enzymatic system is responsible for the first dechlorination of PCE. Dechlorination-active fractions purified from cell extracts on a XAD-4 column revealed the presence of F420, F430, and cobamides cofactors. This is the first report of the isolation of a methanogenic bacterium with the ability to dechlorinate high concentrations of PCE to a nonchlorinated product.Key words: dechlorination, Methanosarcina, tetrachloroethylene.

Localization of carbon monoxide dehydrogenase in acetate-adapted Methanosarcina barkeri

Immunogold labeling techniques were utilized to determine the cellular location of carbon monoxide dehydrogenase in the methanogenic bacterium Methanosarcina barkeri grown on acetate to enhance levels of the enzyme. Formaldehyde fixation followed by dehydration in methanol and embedment in Lowicryl K4M resin preserved the antigens. Sections were labelled with primary polyclonal rabbit antibody followed by protein A - gold. Low levels of labeling were improved by use of an antibody to protein A followed by a second incubation on protein A - gold. The enzyme is primarily located in the cytoplasm, which agrees with biochemical results showing that it resides in the soluble cellular fraction.Key words: Methanosarcina barkeri, methanogen, methanogenesis, enzyme localization, immunogold.