RESUMO
In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firmicutes, Tenericutes or Thermotogae. According to 16S rRNA gene sequences, most isolates were related to Clostridium sporosphaeroides, Defluviitoga tunisiensis and Dendrosporobacter quercicolus. Acetic, propionic or butyric acid were produced in cultures of isolates affiliated to Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, C. sporosphaeroides, D. quercicolus, Proteiniborus ethanoligenes, Selenomonas bovis and Tepidanaerobacter sp. Isolates related to Thermoanaerobacterium thermosaccharolyticum produced acetic, butyric and lactic acid, and isolates related to D. tunisiensis formed acetic acid. Specific primer sets targeting 16S rRNA gene sequences were designed and used for real-time quantitative PCR (qPCR). The isolates were physiologically characterized and their role in BGP discussed.
Assuntos
Ácido Acético/metabolismo , Bactérias/isolamento & purificação , Biocombustíveis/microbiologia , Ácido Butírico/metabolismo , Propionatos/metabolismo , Animais , Bacillus/genética , Bactérias/genética , Bactérias/metabolismo , Sequência de Bases , Reatores Biológicos/microbiologia , Bovinos , Clostridium/genética , Primers do DNA , DNA Ribossômico , Fermentação , Genoma Bacteriano , Esterco/microbiologia , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Silagem/microbiologia , Suínos , Zea mays/microbiologiaRESUMO
To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-labeled specific oligonucleotides for fluorescence in situ hybridization (FISH). These probes are partial complementary to each other. This feature promotes the accessibility to the target sequence within the ribosome and enhances the fluorescence signal. For the rapid identification of Oenococci both the 5S rRNA gene and the ITS-2 region are useful targets.
