Within the last two decades biogas plants in agricultural environments have developed to produce energy from organic wastes and, mainly, from devoted biomass. The complex biotransformations taking place within the reactor are carried out by a diverse consortium of fungi, anaerobic bacteria and archaea that, mostly, is still not-well characterized. These microorganisms working together are able to degrade complex molecules like cellulose, lignin and xenobiotics producing methane. This unknown biodiversity is an important reserve of genes and new enzymatic activities, as cellulases, having a huge range of applications in various commercial fields. The opportunity to discover, investigate and apply these new genes is based on our ability to isolate DNA of good quality from the original matrix. The main limits of DNA purification from complex matrices like soil, biogas digestate and ruminal liquid are: the preferential isolation of some taxonomic groups, depending on the type of cellular lysis step, and the co-isolation of humic acids, polyphenols and other aromatic compounds that can inhibit subsequent PCRs and enzymatic applications. Because of this, it is necessary to have rapid and cheap strategies to avoid or solve these problems. In this work we have tested the ability of different commercial Genomic DNA extraction kits and a CTAB-based method to provide high quantity and quality amplifiable DNA from an agricultural corn-fed mesophilic biogas plant. Amplificability was assessed on the recovered DNA and on different DNA dilutions. The addition of polyvinylpyrrolidone (PVP) in the PCR was also considered in order to improve the DNA amplificability. To verify the specificity of the employed methods in recovering DNA from the three domains, a set of real-time quantitative (q) PCR was carried out using bacteria, archaea and eukaryotic universal primers. The contemporary absence of plant DNA was verified using universal chloroplast primers. While some methods are able to provide directly amplifiable DNA, generally by properly combining DNA dilution with the addition of inhibitors removal substances like PVP is sufficient to obtain amplification in all the tested methods. Further, real-time qPCR analyses demonstrate that the different methods have the same specificity in recovering DNA from the different domains, thus supporting that the extracted DNA is representative of the biodiversity of the initial substrate.

Stagnati, L., Lanubile, A., Marudelli, M., Marocco, A., Busconi, M., Comparison of methods for the recovery of PCR-compatible genomic DNA from an agricultural biogas plant., Abstract de <<Joint Congress SIBV-SIGA>>, (Milano, 08-11 September 2015 ), Joint Congress SIBV-SIGA, Milano 2015: 428-428 [http://hdl.handle.net/10807/69138]

Comparison of methods for the recovery of PCR-compatible genomic DNA from an agricultural biogas plant.

Stagnati, Lorenzo;Lanubile, Alessandra;Marudelli, Mariangela;Marocco, Adriano;Busconi, Matteo
2015

Abstract

Within the last two decades biogas plants in agricultural environments have developed to produce energy from organic wastes and, mainly, from devoted biomass. The complex biotransformations taking place within the reactor are carried out by a diverse consortium of fungi, anaerobic bacteria and archaea that, mostly, is still not-well characterized. These microorganisms working together are able to degrade complex molecules like cellulose, lignin and xenobiotics producing methane. This unknown biodiversity is an important reserve of genes and new enzymatic activities, as cellulases, having a huge range of applications in various commercial fields. The opportunity to discover, investigate and apply these new genes is based on our ability to isolate DNA of good quality from the original matrix. The main limits of DNA purification from complex matrices like soil, biogas digestate and ruminal liquid are: the preferential isolation of some taxonomic groups, depending on the type of cellular lysis step, and the co-isolation of humic acids, polyphenols and other aromatic compounds that can inhibit subsequent PCRs and enzymatic applications. Because of this, it is necessary to have rapid and cheap strategies to avoid or solve these problems. In this work we have tested the ability of different commercial Genomic DNA extraction kits and a CTAB-based method to provide high quantity and quality amplifiable DNA from an agricultural corn-fed mesophilic biogas plant. Amplificability was assessed on the recovered DNA and on different DNA dilutions. The addition of polyvinylpyrrolidone (PVP) in the PCR was also considered in order to improve the DNA amplificability. To verify the specificity of the employed methods in recovering DNA from the three domains, a set of real-time quantitative (q) PCR was carried out using bacteria, archaea and eukaryotic universal primers. The contemporary absence of plant DNA was verified using universal chloroplast primers. While some methods are able to provide directly amplifiable DNA, generally by properly combining DNA dilution with the addition of inhibitors removal substances like PVP is sufficient to obtain amplification in all the tested methods. Further, real-time qPCR analyses demonstrate that the different methods have the same specificity in recovering DNA from the different domains, thus supporting that the extracted DNA is representative of the biodiversity of the initial substrate.
Inglese
Proceedings of the Joint Congress SIBV-SIGA
Joint Congress SIBV-SIGA
Milano
8-set-2015
11-set-2015
978-88-904570-5-0
Stagnati, L., Lanubile, A., Marudelli, M., Marocco, A., Busconi, M., Comparison of methods for the recovery of PCR-compatible genomic DNA from an agricultural biogas plant., Abstract de <>, (Milano, 08-11 September 2015 ), Joint Congress SIBV-SIGA, Milano 2015: 428-428 [http://hdl.handle.net/10807/69138]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/69138
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