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Applied and Environmental Microbiology, May 2005, p. 2325-2330, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2325-2330.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities

Shabir A. Dar, J. Gijs Kuenen, and Gerard Muyzer*

Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands

Received 24 September 2004/ Accepted 30 November 2004

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.

* Corresponding author. Mailing address: Dept. of Biotechnology, Julianalaan 67, NL-2628 BC Delft, The Netherlands. Phone: 31 15 2781193. Fax: 31 15 282355. E-mail: g.muyzer{at}tnw.tudelft.nl.

Applied and Environmental Microbiology, May 2005, p. 2325-2330, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2325-2330.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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