We developed an amperometric acetate microbiosensor capable of measuring in situ sub-micromolar concentration of acetate inside biofilms. The operating principle of the microbiosensor utilized electrochemically active biofilms (EABs) to convert acetate to electrons on the microelectrode tip. The acetate microbiosensor consisted of a 30 µm glass-covered carbon wire working electrode with G.sulfurreducens biofilm on the tip recessed into a glass outer case approximately 200 μm and a Ag/AgCl reference electrode, as shown in Figure 1. The glass outer case terminated to a 30 µm tip diameter where acetate could flow into and diffuse up to the active tip. Acetate-free G.sulfurreducens growth medium served as electrolyte. The microbiosensor showed a linear response between 50 µM and 1.6 mM acetate concentration with a 79 ± 8 µM limit of detection (S/N=2). The operating potential of the microbiosensor was selected as +300 mV_Ag/AgCl satisfying the limiting current condition. The microbiosensor also demonstrated a higher specificity to acetate over other possible metabolites that could interfere with acetate measurements.

To test the microbiosensor, we measured local acetate concentration inside G. sulfurreducens biofilms grown on a 1 in² electrode in a three-electrode bioreactor operated in fed-batch mode. Measuring acetate profiles in G.sulfurreducens biofilms is critical to understand acetate limitations and acetate diffusion and to test the developed microbiosensor in G.sulfurreducens biofilms which utilizes acetate as the electron donor. The microbiosensor was used to measure acetate depth profiles within ~225 µm thick G.sulfurreducens biofilm under no current generation (open circuit potential) and current generation (at +300 mV_Ag/AgCl) conditions. For depth profile measurements, bulk acetate concentration in the bioreactor was set to 1.5 mM by considering the linear calibration range of the microbiosensor. The acetate concentration remained at bulk values throughout the biofilm at open circuit potential when the biofilm was not respiring. However, the acetate concentration decreased from the bulk values to below the detection limit within 200 μm when current was allowed to pass at +300 mV_Ag/AgCl. This showed that the biofilm was acetate-limited due to the zero acetate concentration at the bottom of the biofilm.