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Author: Liebl, Wolfgang ×

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    DNA and RNA extraction and quantitative real-time PCR-based assays for biogas biocenoses in an interlaboratory comparison
    (Basel : MDPI, 2016) Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael
    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.
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    Genomics and prevalence of bacterial and archaeal isolates from biogas-producing microbiomes
    (London : BioMed Central, 2017-11-13) Maus, Irena; Bremges, Andreas; Stolze, Yvonne; Hahnke, Sarah; Cibis, Katharina G.; Koeck, Daniela E.; Kim, Yong S.; Kreubel, Jana; Hassa, Julia; Wibberg, Daniel; Weimann, Aaron; Off, Sandra; Stantscheff, Robbin; Zverlov, Vladimir V.; Schwarz, Wolfgang H.; König, Helmut; Liebl, Wolfgang; Scherer, Paul; McHardy, Alice C.; Sczyrba, Alexander; Klocke, Michael; Pühler, Alfred; Schlüter, Andreas
    Background: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. Results: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. Conclusions: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members. © 2017 The Author(s).
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    Unraveling the microbiome of a thermophilic biogas plant by metagenome and metatranscriptome analysis complemented by characterization of bacterial and archaeal isolates
    (London : BioMed Central, 2016) Maus, Irena; Koeck, Daniela E.; Cibis, Katharina G.; Hahnke, Sarah; Kim, Yong S.; Langer, Thomas; Kreube, Jana; Erhard, Marcel; Bremges, Andreas; Off, Sandra; Stolze, Ivonne; Jaenicke, Sebastian; Goesmann, Alexander; Sczyrba, Alexander; Scherer, Paul; König, Helmut; Schwarz, Wolfgang H.; Zverlov, Vladimir V.; Liebl, Wolfgang; Pühler, Alfred; Schlüter, Andreas; Klocke, Michael
    One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood.