title: Comparison of six methods for the recovery of PCR-compatible microbial DNA from an agricultural biogas plant.
abstract: Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal library.
authors.affiliation: Department of Sustainable Crop Production, Faculty of Agricultural, Food and Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122, Piacenza, Italy. lorenzo.stagnati@unicatt.it.
authors.forename: L
authors.identifier: http://orcid.org/0000-0002-4924-7309
authors.initials: L
authors.lastname: Stagnati
authors.affiliation: Department of Sustainable Crop Production, Faculty of Agricultural, Food and Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122, Piacenza, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Soffritti
authors.affiliation: Department of Sustainable Crop Production, Faculty of Agricultural, Food and Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122, Piacenza, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Lanubile
authors.affiliation: Department of Sustainable Crop Production, Faculty of Agricultural, Food and Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122, Piacenza, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Busconi
mesh_terms: D001674:Bioelectric Energy Sources; D056804:Biofuels / Q000382:microbiology*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005057:Eukaryotic Cells; D008828:Microbiological Techniques / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D011387:Prokaryotic Cells
chemical_list: D056804:Biofuels; D004247:DNA
keywords: DNA extraction methods comparison; Inhibitors; Metagenomics; PCR-grade DNA; Prokaryotic/eukaryotic DNA ratio