Direct Extraction of Microbial DNA from Wastewater using Optimized Treatment Method

Abstract

Microbes that can be cultivated under standard laboratory conditions were detected as limited to only 1-4%. Culture independent direct microbial DNA extraction methods from environmental samples were identified as effective in D unbiased microbial diversity analysis. A DNA extraction method using two different treatments including the supplementation of different aeration periods (5h, lOh, 12h, 14h and 20h) together with different glucose concentrations (5g/l, 10g/l, 15g/l and 20g/l) was tested on wastewater taken from five contaminated environments. The DNA extraction protocol was also optimized by adjusting the speed and time period of centrifugation facilitating the formation of the pellet. Extracted genomic DNA was evaluated using agarose gel electrophoresis and PCR amplification of 16S ribosomal RNA gene. It was identified that the optimized DNA extraction protocol supplied high quality DNA of sufficient quantity. The absence of differential amplifications, artificial PCR products and lack of problems such as inhibition of PCR amplification by co-extracted contaminants indicated the quality of DNA. Supplementation of glucose along with aeration increased the DNA yield and optimum conditions were identified as 12hr aeration with 10g/L glucose. The developed method could be effectively used in DNA extractions from different environmental samples for the detection of diversity of microbial species.