Molecular monitoring of microbial populations during bioremediation of contaminated soils

Microbes are being used to help clean up polluted sites around the world. Yet, the current knowledge of the complex assemblages of microbes in natural or polluted environments is limited due to inability to culture so many of the organisms by conventional microbiological methods. Many questions about what impact a pollutant or a treatment regime may have on the microbial community still remain to be answered. Molecular techniques have rapidly become valid methods for examining microbial communities in situ and are helping to answer many of these questions. Digesting nucleic acids with restriction endonucleases cuts DNA into different length fragments. Subsequent electrophoretic separation of the fragments will produce banding profiles (DNA fingerprints) that can be used to discriminate between populations. Variations of this restriction fragment length polymorphism (RFLP) method have successfully been used to monitor microbial community dynamics during bioremediation. The restriction fragment-based techniques (e.g., RFLP, T-RFLP) give some indication of the complexities of the communities but still underestimate the true diversity or dynamics of the microbial community. Amplicon length heterogeneity-PCR (LH-PCR) is a molecular technique that discriminates between different bacteria based on the natural sequence variations in the lengths of the 16S ribosomal DNA (rDNA) genes not on restriction enzyme recognition sites. Soils contaminated with petroleum hydrocarbons were treated in bench-scale bioreactors. Whole community DNA extracted from the bioreactor samples and the polymerase chain reaction (PCR) was used to amplify the 16S rDNA genes. The three DNA fingerprinting methods were compared to each other and to non-molecular techniques to ascertain which of the techniques could be used to monitor whole community dynamics. The LH-PCR method proved to be the most robust, reproducible and diagnostic of all the techniques tested. The LH-PCR technique was able to detect greater genetic diversity within the microbial communities. It also profiled significant differences in community structure between the bioreactor microbial communities undergoing different bioremediation treatment regimes. The LH-PCR technique has great potential as a monitoring tool that can significantly contribute to the basic understanding of the bioremediation processes.