Application of Denaturing High-Performance Liquid Chromatography in Microbial Ecology: Fermentor Sludge, Compost, and Soil Community Profiling

Abstract

Genetic fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), are used in microbial ecology for the analysis of mixed microbial communities but are associated with various problems. In the present study we used a new alternative method: denaturing high-performance liquid chromatography (dHPLC). This method was previously shown to work with samples from water and gut flora but had not yet been applied to complex environmental samples. In contrast to other publications dealing with dHPLC, we used a commonly available HPLC system. Samples from different origins (fermentor sludge, compost, and soil), all ecologically significant, were tested, and the 16S rRNA gene was amplified via PCR. After optimization of the HPLC elution conditions, amplicons of pure cultures and mixed microbial populations could be separated successfully. Systematic differentiation was carried out by a cloning approach, since fraction collection of the peaks did not result in satisfactory fragment separation. dHPLC was evaluated as a tool for microbial community analysis on a genetic level and demonstrated major improvements compared to gel-based fingerprinting methods, such as DGGE, that are commonly used in microbial ecology.The introduction of molecular biological methods in microbiology has changed research in microbial ecology fundamentally. The approach to characterize and classify microbial communities by cultivation methods switched to the genetic level, and the analysis of community structure became possible without any further need of cultivation for systematic analysis. This is especially important since only an estimated 1% of the naturally occurring bacteria have been isolated and characterized thus far (12,15).Genetic fingerprinting techniques, often in combination with a cloning approach, are often used. They provide a pattern or profile of the community diversity based upon the physical separation of unique nucleic acid species, while systematic assignment is often achieved by results from clone libraries. The fingerprinting methods are quite rapid, relatively easy to perform, and permit the simultaneous analysis of multiple samples. This allows comparison of the genetic diversity of microbial communities from different habitats, and in individual communities over time. The most commonly applied methods include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis, single-strand conformation polymorphism, restriction fragment length polymorphism, and terminal restriction fragment length polymorphism. All of these methods provide information on community structure, and DGGE is probably the most frequently used method for this purpose. However, in daily lab use, many problems arise with any of these methods (e.g., the lack of reliable and fast quantification, difficulties in reproducibility, labor-intensive steps, etc.), so alternatives are necessary.In the present study we describe an alternative fingerprinting method, denaturing high-performance liquid chromatography (...

Bacterial Diversity in an Amazonian Mangrove Ecosystem

“…The profiles identified in the present study were similar to those reported for complex environmental communities (Wagner et al, 2009;Barlaan et al, 2005), although the chromatogram patterns obtained in the present study showed peaks with better resolution and separation (Figure 2). By comparing the number of peaks in the DHPLC chromatograms with the number of groups observed in the taxonomic graphs there is much similarity (Figures 1 and 2).…”

Application of Denaturing High-Performance Liquid Chromatography for Monitoring Sulfate-Reducing Bacteria in Oil Fields

“…Elution of low-GC-content (45 to 52%) dsrB gene fragments before gene fragments with high GC contents (57 to 61%) in DHPLC showed that the separation was dependent on GC content, as in DGGE. A general agreement between the GC content and the behavior of gene fragments in DHPLC has also been reported in previous studies (13,16,33). Three SRB, however, produced irresolvable smears in DHPLC which could not be explained by the GC contents of the amplicons (52 to 62%).…”

“…In recent years, the technique has been successfully applied to the study of bacterial diversity in various ecosystems, such as seawater (13), the gut (14), dairy products (15), soil, fermenter sludge and compost (16), sediments (17), and human infections (18)(19)(20), and to monitor fungal communities present in air (21), wood decay (22), cheese (23), and milk (24). Similar to DGGE, DHPLC can theoretically resolve DNA fragments with sequence and/or size differences.…”

Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta crantz) fermentation

“…Similar to Wagner et al (2009), we did not obtain baseline separated peaks, but this was already expected considering the complexity of our samples.…”