Anaerobic benzene-degrading enrichment cultures performing methanogenesis were obtained from non-contaminated lotus field soil. Stable isotope probing with C-13 benzene was used to detect the bacteria that were involved in benzene degradation. Denaturing gradient gel electrophoresis (DGGE) of fractionated samples exhibited an obvious shift of some DGGE bands to a heavier DNA fraction. An almost full-length 16S rRNA gene sequence corresponding to the DCGE band, namely Hasda-A, was obtained by constructing a clone library of the heavier fraction. The Hasda-A sequence showed only 85.1% identity with the closest identified bacterium, Syntrophus gentianae. Hasda-A may be an important bacterium involved in the initial steps of benzene degradation under methanogenic conditions, as it was the most prominent bacterium that assimilated labeled benzene early in the process of benzene degradation. A primer set was designed to quantify the gene copies of Hasda-A by quantitative PCR. Hasda-A was present at a concentration of (3.5 +/- 0.8) x 10(6) copies/mL and represented 8.4% of gene copies among bacteria in the enrichment culture. The enrichment culture consisted of three dominant bacterial groups: Hasda-A and both aceticlastic and hydrogenotrophic methanogens. Methane is believed to be produced from benzene by the sequential degradation of benzene by fermenting bacteria, hydrogen-producing acetogens, and methanogens. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.