Coupling dark fermentation and microbial electrolysis for hydrogen production: process and mechanisms occurring during formation and conservation of electroactive biofilm

Nowadays, alternative and sustainable solutions are proposed to avoid the use of fossil fuel. Hydrogen, which constitutes a promising energy vector, is essentially produced by fossil fuel reforming (95%). Environmentally friendly production systems have to be studied. Two main families of technologies are explored to produce hydrogen: 1) by thermochemical and electrochemical decomposition of water and 2) from different biomass sources. Among those last ones, microbial electrolysis cells (MEC) allow to produce hydrogen by electrolysis of organic matter. A MEC consists in a classical cathode, which provides hydrogen production by electrochemical reduction of water, associated to a bio-anode that oxidizes organic substrates into carbon dioxide. This process is only possible because of the anodic development of an electroactive microbial biofilm which constitutes an electrocatalyst. In comparison to classical water electrolysis process, a MEC requires 5 to 10 times less electrical energy and therefore reduces the energetic cost of produced hydrogen. Furthermore, classical process of dark fermentation in mixed cultures converts sugars (saccharose, glucose) to hydrogen with a limited yield of 2-3 moles of hydrogen per mole of hexose because of the coproduction of organic acids (mainly acetic and butyric acids). Fed with acetate, a MEC can produce up-to 3 moles of hydrogen per mole of acetate. Therefore, the association of these two processes could permit to produce 8 to 9 moles of hydrogen per mole of hexose, which represents a major step toward the theoretical limit of 12 moles of hydrogen per mole of hexose. Therefore, this work aims at analyzing the relationship between microbial community structures and compositions and the associated macroscopic functions (biofilm electroactive properties, hydrogen production potential) in electroactive biofilms and in dark fermentation in conditions allowing the coupling of the two processes. The originality of this study is to work in saline conditions (30-35 g_NaCl/L), which favors the charges transfer in the MEC electrolyte. First of all, feasibility of dark fermentation in saline conditions (3-75 g_NaCl/L) has been shown. This was linked to an inhibition of produced hydrogen consumption and the predominance of a new Vibrionaceae species at salt concentrations higher than 58 g_NaCl/L. Secondly, electroactive biofilm growth in conditions compatibles to dark fermentation (pH 5.5-7 and fed with different organic acids) allowed to select dominant microbial species in anodic biofilms that present promising electroactive properties (Geoalkalibacter subterraneus and Desulfuromonas acetoxidans) with maximum current densities up to 8.5 A/m². In parallel, the microbial selection occurring during iron-reducing enrichment method used to select species from a natural inoculum source and based on their capacity to transfer electrons to iron oxides (Fe(III)) has been studied. A decrease of electroactive performances of the biofilm linked to the divergence of microbial selection led to a limitation of the number of iron-enrichment steps. However, enrichment on Fe(III) presents an efficient alternative to pre-select electroactive species with an increase of coulombic efficiency from 30±4% to 99±8% in comparison with a biofilm obtained with a non-acclimated inoculum. Finally, the addition of exogenous bacteria from a dark fermenter on the electroactive biofilm revealed a decrease of electroactivity with a decrease of maximum current density produced. This diminution could be explained by a lower substrate transfer due to an apparent thickening of the biofilm. Nevertheless, the stability of microbial composition and of bacterial quantity on the anode suggests that a production of exopolymers (EPS) occurred. (author)