Freeze- and Thaw-Based Procedures for Extracting DNA from Activated Sludge

Activated sludge treatment is one of the most popular biological processes for wastewater treatment. Detection of activated sludge structures from complex substrates such as soil and water is not easy. A rapid and effective method for extracting high molecular weight amplifiable DNA from activated sludge was modified. Five nanogram quantities of DNA per milligram activated sludge were recovered with SDS-based and freeze-and-thaw procedures. The ratio of absorbance at 260 nm to absorbance at 280 nm varies between 1.6 and 2, which is an indicator of the purity of DNA. Gel electrophoresis of the isolated DNA further showed intact genomic DNA bands of high molecular weight (greater than 12,000 base pairs) with no RNA contamination. The quality of obtained DNA was verified by PCR amplification reactions and restriction enzyme digestion. A comparison of the optimized protocol with commercial dBioZol DNA isolation kit suggested that the method described in this report would be more efficient in removing PCR inhibitors from activated sludge samples. The random amplified polymorphic DNA (RAPD) patterns demonstrated the genetic diversity of activated sludge samples. A 542-bp fragment of the 16S rRNA gene of the bacterial isolates was amplified. PCR using primers targeting 16S rDNA shows promise in the enumeration of gram-positive bacteria in activated sludge samples. The current protocol can be used to efficiently monitor the presence of microorganisms in sludge from effluent treatment plants.