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Microbial Diversity in Ground and Surface Water Analyzed by Denaturing Gradient Gel Electrophoresis Using the DCode System

Bernd Eschweiler and Beate Kilb, Institute for Water Research, Schwerte, Germany


Introduction

Bacterial diversity in environmental samples is usually determined by a characterization of isolated strains. A problem for the analysis and characterization of microbial communities is the inability to culture most of the bacteria species present in the sample. Therefore isolated bacteria may account for only a minor portion of the total bacterial diversity originally present in the sample. This problem is particularly severe in oligotrophic habitats like ground water, where approximately only 0.1 to 1% of the bacterial species are culturable.1 A new approach in microbial ecology is based on the analysis of bacterial genetic information without cultivation. This culture-independent approach has greatly enhanced the ability to assess bacterial diversity in ecosystems such as ground and surface water environments. After isolation of total bacterial DNA, variable regions of the 16S rRNA gene are amplified by PCR*. The similar sized PCR-products are separated by subsequent DGGE, and the resulting diversity pattern are analyzed and compared.2, 3


Materials and Methods

DNA from surface and ground water was isolated and purified as described earlier.2, 3 Two universal bacterial 16S rDNA primers were used to amplify a 527 bp fragment from total genomic DNA. PCR was performed in a total volume of 100 l containing 1x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelantine), 200 M each dNTP, 0.5 M each primer, 0.3 mg/ml bovine serum albumin (BSA), and 2.5 U AmpliTaq DNA polymerase (Perkin
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