title: Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities.
abstract: Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.
authors.affiliation: Dept. of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Shabir A
authors.initials: SA
authors.lastname: Dar
authors.forename: J Gijs
authors.initials: JG
authors.lastname: Kuenen
authors.forename: Gerard
authors.initials: G
authors.lastname: Muyzer
mesh_terms: D001483:Base Sequence; D019149:Bioreactors; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016999:Sulfur-Reducing Bacteria / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers