A simple and rapid method of DNA extraction from soil was developed and DNA was made suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key features of the extraction and purification were cold lysozyme-and SDS-assisted lysis with either freezing-thawing or bead beating, cold phenol extraction of the resulting soil suspension, CsCl and KAc precipitation and, finally, spermine-HC1 or glass milk purification of DNA. Crude DNA preparations contained 4-20 p g DNA per g of soil extracted, and at least 50% of this was recovered in the final purified DNA preparations. The resulting DNA was pure enough to be restricted by various enzymes, and was amplifiable at concentrations of up to 20 ng of soil-derived DNA per 50 p1 reaction mix.Amplification of a 683 bp target sequence, put, was performed with different Taq DNA polymerases. Application of the protocol enabled us to detect target DNA derived from roughly lo3 introduced Pseudomonaspuorescens (RP4 : : put) cfu per g of soil. The fate of an introduced population in the soil could be followed to this limit with PCR-assisted detection of target DNA. In addition, target DNA was detected in soil 5 months after release, when the introduced organism was no longer detectable on selective agar plates.The extraction and purification protocol applied to various different soil types resulted in DNA of sufficient purity to permit amplification by PCR.

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This paper reports on continuing research at Coventry University into the improvement of highway water quality following flow through a permeable pavement. Such pavements have been shown elsewhere to be efficient in-situ bio-reactors, capable of degrading large quantities of clean motor oil. Further laboratory research, reported here, demonstrates that a commercially obtained oil degrading, microbial mixture was not significantly better at degrading clean motor oil than the indigenous microbial biomass established within the pavement over a 4-year period, when provided with an adequate nutrient supply. Scanning electron microscopy has been used to monitor biofilm development, which has also identified that the pavement has developed a complex community structure with high bio-diversity.

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