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              <p>Fermentative biohydrogen technologies are environment-friendly ways for producing renewable hydrogen from a wide range of organic sources, and at a low cost. The main restriction of such bioprocesses remains their high instability when operated with mixed cultures. The role of minority bacteria has often been suspected to explain metabolic instability in hydrogen fermentation, but has never really been investigated. The trophic competition of lactic bacteria with hydrogen-producing bacteria for substrate is well documented in literature. The consequence is a loss of hydrogen production due to shift towards unwanted fermentative metabolisms. However, experimental studies on non-trophic interactions in engineered ecosystems are scarce. In this work, two complex hydrogen-producing microbial ecosystems deriving from anaerobic sludge treating industrial waste were used. The molecular characterization of mixed cultures was done using CE-SSP fingerprinting patterns. The first ecosystem (EcoA) presented a low hydrogen production (440 mL.H2.L-1) but was metabolically stable (CV=8%), and the second ecosystem (EcoB) produced more hydrogen (2157 mL.H2.L-1) but was metabolically unstable (CV=36%). The two ecosystems chosen corresponded to the lower and the upper range of hydrogen productivity found in the literature. The impact of a contamination with nine various exogenous bacterial strains (Clostridium acetobutylicum, Clostridium pasteurianum, Lactobacillus bulgaris, Enterobacter cloacae, Escherichia coli, Enterococcus casseliflavus, Desulfovibrio vulgaris, Pseudomonas fluorescens, Ralstonia eutropha) exhibiting distinct metabolic abilities and belonging to diverse genera was investigated. In EcoA, a positive effect on the hydrogen yield was observed after addition of two hydrogen-producing facultative anaerobic bacteria, Escherichia coli (1.5-fold increase, p=0.05) and Enterobacter cloacae (2.5-fold increase, p=0.04). Interestingly, Ralstonia eutropha led to a four-fold increase in hydrogen production (p&lt;0.001), although Ralstonia eutropha did not produce hydrogen alone. In EcoB, the added strains reduced the metabolic instability. This better reproducibility of hydrogen fermentation was linked with the establishment of a stable bacterial consortium, as shown by fingerprinting patterns. Taken together, these results were interpreted as cooperative trophic interactions. In contrast, microbial competition did not increase hydrogen production. Thus, this study showed the potentiality of using exogenic bacteria as biotic factors to stabilize microbial metabolisms and/or increase their hydrogen production performances.</p>
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