An experimental design was performed to study the combined effects of the parameters used for bacteria selective biofilm formation. The parameters were the potential applied, the time of the biofilm formation with electroactive bacteria or the biofilm formation, and the initial substrate concentration in the medium. The performance of the biofilms formed was compared by their kinetic parameter the apparent electron transfer rate constant (K app ), which was obtained from cyclic voltammetry analysis. The results from the experimental design showed that the highest K app of 0.56 s −1 was with the potential applied of −0.45 V for 5 h and 12 mM of the initial substrate concentration in the medium. New values of these parameters were calculated from the statistical analysis of the experimental design results and successfully higher value of K app of 0.64 s −1 can be calculated and obtained. The electroactivity and capability of the biofilm for hydrogen production was also proved in a single chamber microbial electrolysis cell, and a hydrogen production yield of 0.24 m 3 H 2 /m 3 d was obtained. The biofilm formed with these last parameters was also identified with molecular techniques as Exiguobacterium which has barely been reported as electroactive bacteria and used for hydrogen production.

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