Immobilizing electro-active microbes within polymer matrices (thereby forming biohybrids) is a promising approach to accelerate microbial attachment to electrodes and increase the biofilm robustness. However, little is known on the fine scale chemical environment that develops within the electro-active biohybrids. Herein, we develop a biohybrid by immobilizing a culture of Shewanella oneidensis MR1 in agar matrix on the surface of a graphite electrode poised at +0.25 V. The resulting bioanode (3-6 mm thick) was grown under anoxic conditions and produced a steady current of 40 μA. Oxygen and pH distribution within the biohybrid were characterized in-situ using microsensors. As Shewanella is a facultative aerobe, it will halt the current production in the presence of oxygen. Thus, in addition, we investigated the alteration of the microenvironment during and after aeration of the medium to evaluate the oxygen tolerance of the system. During aeration, oxygen was effectively consumed in the top layers of the biofilm, leaving a 400-900 μm thick anoxic zone on the anode surface, that sustained >60% of the initial current. Current production recovered to pre-oxic condition within 5 h after the aeration was stopped, showing that immobilization can promote both high resistance and resilience of the system. Despite the absence of strong buffering conditions, pH profiles indicated a maximum drop of 0.2 units across the biohybrid. Characterizing the chemical microenvironment helps to elucidate the mechanistic functioning of artificial biofilms and hold a great potential for the designing of future, more effective biohybrid electrodes.
Keywords: Biohybrids; Chemical gradients; Electroactive microbes; Microsensors; Oxygen.
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