Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

The applicability of order-specific primers in minimizing the possible underestimation of microbial diversity was evaluated via denaturing gradient gel electrophoresis (DGGE) analysis of a lab-scale anaerobic digester. Initially, a population analysis with real-time quantitative PCR demonstrated the existence of three methanogenic orders-Methanobacteriales, Methanomicrobiales, and Methanosarcinales-throughout the reaction period. DGGE analyses with three pairs of order-specific primers yielded eight operational taxonomic units (OTUs), whereas DGGE analysis with two independent Archaea-specific primers identified only five. Moreover, the order-specific primers amplified at least one OTU affiliated with each order, whereas no members of Methanobacteriales or Methanomicrobiales were identified with Archaea-specific primers in most samples. These findings provide evidence that order-specific analysis can detect the diversity of methanogens in greater detail than conventional Archaea-specific analysis.