Latex was applied to two different types of anodes, carbon paper (without wet proofing; E-Tek) or graphite blocks (Grade GM-10; GraphiteStore.com Inc.), in two different types of BESs in order to evaluate the immobilization method under different conditions. Carbon paper was used as the anode in a single-chamber 28-mL microbial fuel cell (MFC) reactor with a platinum-catalyzed air cathode (Cheng et al. 2006
; Liu and Logan 2004
) (both electrodes with projected surface area of 7 cm2
). Graphite blocks (projected surface area of 4.6 cm2
) were used as anodes for a single-chamber 5-mL microbial electrolysis cells (MECs) with a 1.0 × 1.5 cm2
304 stainless steel 90 × 90 mesh cathode (Call and Logan 2011
). Carbon paper (projected surface area of 3.0 cm2
) was also used as anode material in some 5-mL MECs. All reactors were inoculated using cell suspensions from pre-acclimated MFCs that were originally inoculated with domestic wastewater and acetate. A multimeter (2700, Keithley Instruments, Inc.) was used to monitor the voltage across an external resistor (Rex
= 10 Ω, MEC; 1000 Ω, MFC). A power source (3645A, Circuit Specialists, Inc.) was connected to the MEC circuit to add -0.7 V to the cathode. All BESs were maintained at 30°C.
MFC medium was 100 mM phosphate buffer with 17 mM acetate as the substrate (per L: 0.62 g NH4
Cl, 4.9 g NaH2
O, 9.15 g Na2
, 0.26 g KCl, 1.4 g sodium acetate, and Wolfe's vitamins and minerals) (Lovley and Phillips 1988
). MEC medium was 30-mM bicarbonate buffer with 10-mM acetate as the substrate, based on the ATCC recipe for G. sulfurreducens
, #1957 (per L: 1.5 g NH4
Cl, 0.6 g NaH2
, 0.1 g KCl, 2.5 g NaHCO3
, 0.82 g sodium acetate, and Wolfe's vitamins and minerals), without the addition of the electron acceptor. MFC and MEC reactors were operated in fed-batch mode until they successively produced at least 3 equivalent batch cycles, indicating a well-established anodic biofilm.
A monodisperse latex emulsion (SF-091; Rohm & Haas) was amended with 5% glycerol to optimize the degree of coalescence and subsequent diffusivity of the film to the substrate (Lyngberg et al. 2001
; Gosse et al. 2007
). This solution was applied in two different ways to well-established biofilms in the different BESs by removing the anodes temporarily from the reactors. Glycerol-amended latex (referred to simply as "latex") was applied to the carbon paper biofilm from the MFC using an air brush (Paache, BearAir, S. Easton, MA; 4.5 L/min of airflow). One, three, or five layers were applied, allowing 15 minutes between each layer, and one hour after the final layer, for drying at room temperature. For the graphite blocks and carbon paper anodes from the MEC a simpler application procedure was used, where the latex was applied by dipping the blocks or paper into the latex, and excess solution was drawn off the anode with a laboratory wipe. In other experiments, the glycerol-amended latex was diluted in water to 30% to see if performance improved with a thinner layer of latex.
The effectiveness of the latex to immobilize bacteria on the anode materials was examined using several different techniques. Direct observation of individual bacteria on an electrode, when bacteria were stained using acridine orange, was not possible due to high levels of background fluorescence. Therefore, application of individual microbes on an electrode was simulated by applying droplets of fluorescent microspheres (Fluoresbrite spheres, 4.1-μm diameter, Invitrogen) to graphite electrodes. Latex was applied by the dipping method described above. After drying, the latex-coated electrode was immersed in MFC media to simulate the electrode in a BES. The droplets were observed with fluorescence microscopy before and after latex application and MFC simulation.
For SEM visualization, small sections of carbon paper anodes with exoelectrogenic biofilms with and without latex coating were mounted in cryo-matrix and frozen. Thin slices were removed from the cross-section with a microtome until a smooth surface was obtained. The surface was etched with the cryo-SEM electron beam to remove ice crystals before viewing.