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

ABSTRACT: 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.

SUBMITTER: Rettenmaier R

PROVIDER: S-EPMC7565076 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Medium-chain carboxylates such as n-caproate and n-caprylate are valuable chemicals, which can be produced from renewable feedstock by anaerobic fermentation and lactate-based microbial chain elongation. Acidogenic microbiota involved in lactate-based chain elongation and their interplay with lactic acid bacteria have not been characterized in detail yet. Here, the metabolic and community dynamics were studied in a continuous bioreactor with xylan and lactate as sole carbon sources. Four succession stages were observed during 148 days of operation. After an adaptation period of 36 days, a relatively stable period of 28 days (stage I) was reached with n-butyrate, n-caproate and n-caprylate productivities of 7.2, 8.2 and 1.8 gCOD L-1 d-1, respectively. After a transition period, the process changed to another period (stage II), during which 46% more n-butyrate, 51% less n-caproate and 67% less n-caprylate were produced. Co-occurrence networks of species based on 16S rRNA amplicon sequences and correlations with process parameters were analyzed to infer ecological interactions and potential metabolic functions. Diverse functions including hydrolysis of xylan, primary fermentation of xylose to acids (e.g., to acetate by Syntrophococcus, to n-butyrate by Lachnospiraceae, and to lactate by Lactobacillus) and chain-elongation with lactate (by Ruminiclostridium 5 and Pseudoramibacter) were inferred from the metabolic network. In stage I, the sub-network characterized by strongest positive correlations was mainly related to the production of n-caproate and n-caprylate. Lactic acid bacteria of the genus Olsenella co-occurred with potentially chain-elongating bacteria of the genus Pseudoramibacter, and their abundance was positively correlated with n-caproate and n-caprylate concentrations. A new sub-network appeared in stage II, which was mainly related to n-butyrate production and revealed a network of different lactic acid bacteria (Bifidobacterium) and potential n-butyrate producers (Clostridium sensu stricto 12). The synergy effects between lactate-producing and lactate-consuming bacteria constitute a division of labor cooperation of mutual benefit. Besides cooperation, competition between different taxa determined the bacterial community assembly over the four succession stages in this resource-limited system. During long-term reactor operation under constant conditions, chain-elongating bacteria were outcompeted by butyrate-producing bacteria, leading to the increase of n-butyrate yield at the cost of medium-chain carboxylate yields in this closed model system.

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

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