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    Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges
    (Basel : MDPI, 2021) Azemtsop Matanfack, Georgette; Pistiki, Aikaterini; Rösch, Petra; Popp, Jürgen
    Raman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.
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    Impact of process temperature and organic loading rate on cellulolytic/hydrolytic biofilm microbiomes during biomethanation of ryegrass silage revealed by genome-centered metagenomics and metatranscriptomics
    (London : BioMed Central, 2020) Maus, Irena; Klocke, Michael; Derenkó, Jaqueline; Stolze, Yvonne; Beckstette, Michael; Jost, Carsten; Wibberg, Daniel; Blom, Jochen; Henke, Christian; Willenbücher, Katharina; Rumming, Madis; Rademacher, Antje; Pühler, Alfred; Sczyrba, Alexander; Schlüter, Andreas
    Background: Anaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage two-phase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To follow the adaptive response of the biomass-attached cellulolytic/hydrolytic biofilms at increasing ammonium/ammonia contents, genome-centered metagenomics and transcriptional profiling based on metagenome assembled genomes (MAGs) were conducted. Results: In total, 78 bacterial and archaeal MAGs representing the most abundant members of the communities, and featuring defined quality criteria were selected and characterized in detail. Determination of MAG abundances under the tested conditions by mapping of the obtained metagenome sequence reads to the MAGs revealed that MAG abundance profiles were mainly shaped by the temperature but also by the OLR. However, the OLR effect was more pronounced for the mesophilic systems as compared to the thermophilic ones. In contrast, metatranscriptome mapping to MAGs subsequently normalized to MAG abundances showed that under thermophilic conditions, MAGs respond to increased OLRs by shifting their transcriptional activities mainly without adjusting their proliferation rates. This is a clear difference compared to the behavior of the microbiome under mesophilic conditions. Here, the response to increased OLRs involved adjusting of proliferation rates and corresponding transcriptional activities. The analysis led to the identification of MAGs positively responding to increased OLRs. The most outstanding MAGs in this regard, obviously well adapted to higher OLRs and/or associated conditions, were assigned to the order Clostridiales (Acetivibrio sp.) for the mesophilic biofilm and the orders Bacteroidales (Prevotella sp. and an unknown species), Lachnospirales (Herbinix sp. and Kineothrix sp.) and Clostridiales (Clostridium sp.) for the thermophilic biofilm. Genome-based metabolic reconstruction and transcriptional profiling revealed that positively responding MAGs mainly are involved in hydrolysis of grass silage, acidogenesis and/or acetogenesis. Conclusions: An integrated-omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs. Genome-based knowledge on the metabolic potential and transcriptional activity of responsive microbiome members will contribute to the development of improved microbiological AD management strategies for biomethanation of renewable biomass. © 2020 The Author(s).