Monitoring the Physiological State in the Dark Fermentation of Maize/Grass Silage Using Flow Cytometry and Electrooptic Polarizability Measurements

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 methan- ogenic 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 fermen- tation, 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.