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Open AccessArticle

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters

1
Chair of Microbiology, Technical University of Munich, Emil-Ramann-Str. 4, 85354 Freising, Germany
2
Bavarian State Research Center for Agriculture, Central Department for Quality Assurance and Analytics, Lange Point 6, 85354 Freising, Germany
3
ZIEL—Core Facility Microbiome, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany
4
Aspratis GmbH. Munich, Germany, Hübnerstr. 11, 80637 Munich, Germany
5
Institute of Molecular Genetics of National Research Centre (Kurchatov Institute), Kurchatov Sq. 2, 123182 Moscow, Russia
*
Author to whom correspondence should be addressed.
Microorganisms 2020, 8(9), 1297; https://doi.org/10.3390/microorganisms8091297
Received: 13 July 2020 / Revised: 21 August 2020 / Accepted: 23 August 2020 / Published: 25 August 2020
(This article belongs to the Section Environmental Microbiology)
Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50–99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium dominated with 60–91% relative abundance. The novel primer combinations enabled detection and relative quantification of a wide spectrum of GH48 genes in cellulolytic microbial communities. Deep phylogenetic correlation analysis and a simplified taxonomic identification with the novel database facilitate identification of cellulolytic organisms, including the detection of novel taxa in biogas fermenters. View Full-Text
Keywords: amplicon sequencing; qPCR; biomass hydrolysis; taxonomy; DNA; RNA amplicon sequencing; qPCR; biomass hydrolysis; taxonomy; DNA; RNA
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MDPI and ACS Style

Rettenmaier, R.; Lo, Y.K.; Schmidt, L.; Munk, B.; Lagkouvardos, I.; Neuhaus, K.; Schwarz, W.; Liebl, W.; Zverlov, V. A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters. Microorganisms 2020, 8, 1297. https://doi.org/10.3390/microorganisms8091297

AMA Style

Rettenmaier R, Lo YK, Schmidt L, Munk B, Lagkouvardos I, Neuhaus K, Schwarz W, Liebl W, Zverlov V. A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters. Microorganisms. 2020; 8(9):1297. https://doi.org/10.3390/microorganisms8091297

Chicago/Turabian Style

Rettenmaier, Regina; Lo, Yat K.; Schmidt, Larissa; Munk, Bernhard; Lagkouvardos, Ilias; Neuhaus, Klaus; Schwarz, Wolfgang; Liebl, Wolfgang; Zverlov, Vladimir. 2020. "A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters" Microorganisms 8, no. 9: 1297. https://doi.org/10.3390/microorganisms8091297

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