Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples

Abstract

The anaerobic digestion (AD) of organic waste for biogas production has received much attention in recent years due to the increasing need for renewable energy and environmentally friendly waste management systems. Identification of the microbial community involved in AD aids in better understanding and optimising of the process. The choice of DNA extraction method is an integral step in any molecular biodiversity study. In the present study, potential biases introduced by DNA extraction methods were examined by comparing quality, quantity and representability of DNA extracted from AD samples using various extraction methods. In spite of the non-kit based method (cetyltrimethylammonium bromide) yielding the largest quantity of DNA (approximately 44 µg DNA per gram dry weight), the extracted DNA contained PCR inhibitors. Furthermore, the quantity of extracted DNA was not proportional to species diversity. Diversity, determined using denaturing gradient gel electrophoresis (DGGE), was strongly linked to the type of extraction method used. The spin-column filter-based kit that incorporated mechanical and chemical lysis (Macherey-Nagel kit) gave the best results in terms of bacterial and archaeal diversity (Shannon–Wiener indices: average 2.5 and 2.6, respectively). Furthermore, this kit was the most effective at lysing hard-to-lyse bacterial and archaeal cells. The choice of DNA extraction method significantly influences the reliability and comparability of results obtained during AD microbial ecology investigations. Moreover, the careful selection of the DNA extraction method is of particular importance when analysing AD samples since these samples are rich in PCR inhibitors and hard-to-lyse cells such as archaea and gram-positive bacteria