Monitoring the Physiological State in the Dark Fermentation of Maize/Grass Silage Using Flow Cytometry and Electrooptic
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Monitoring the Physiological State in the Dark Fermentation of Maize/Grass Silage Using Flow Cytometry and Electrooptic Polarizability Measurements Carlos E. Gómez-Camacho 1 Bernardo Ruggeri 1
&
Klaus Pellicer Alborch 2 & Anika Bockisch 2 & Peter Neubauer 2 & Stefan Junne 2 &
Received: 25 February 2020 / Accepted: 18 August 2020 # The Author(s) 2020
Abstract Dark fermentation (DF), a key biohydrogen-producing process, is generally operated as a black-box, by monitoring different operative macroscopic process parameters without evaluating or tracking the physiology of the biotic phase. The biotic phase in DF is constituted by a large variety of microorganisms, mainly fermentative bacteria. The present study uses two (electro)optical techniques, flow cytometry (FC) and frequency-dependent polarizability anisotropy (FDPA) measurements, to gain insights into the physiology of open mixed consortia throughout the DF process. The mixed consortia for DF were obtained from a methanogenic sludge, selecting spore-forming bacteria by means of an acid treatment. Then, DF systems with and without pH control were studied, using as substrate a mixture of maize and grass silage (9:1 w/w). Over the course of fermentation, the butyric pathway was dominant in both systems, and relevant titers of acetate, formate, and ethanol were detected; while hydrogen yields amounted to 20.80 ± 0.05 and 17.08 ± 0.05 NmL/gVS under pH-regulated and non-regulated conditions, respectively. The cytometric pattern analysis of the culture together with microscopic observations made it possible, over the course of fermentation, to identify and track the predominant morphologies in play (i.e., free spore, rod-shaped, and endospore, which are typical of Clostridium spp.). Furthermore, the use of the fluorescent dye DiBAC4(3) in FC and FDPA measurements provided similar information regarding the physiological state (PS) of the mixed consortia during the different phases of the culture. Keywords Dark fermentation . Biohydrogen . Physiological state . Flow cytometry . Frequency-dependent polarizability anisotropy
Introduction The shift from oil-based economies to circular bioeconomic models is a growing concern of the biotechnological industry. In particular, the industrially feasible exploitation of different biomass resources and the vast microbial diversity is an Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12155-020-10184-x) contains supplementary material, which is available to authorized users. * Carlos E. Gómez-Camacho [email protected] 1
DISAT, Department of Applied Science and Technology, Politecnico di Torino, C/so Duca degli Abruzzi 24, 10129 Torino, Italy
2
Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Ackerstrasse 76 ACK 24, 13355 Berlin, Germany
important pillar for the production of bioenergy and biochemicals. Europe accounts for 224 biorefineries [1] that use various feedstock, such as bio-waste, lignocellulosic material, waste fa
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