PEDOT:PSS-based Multilayer Bacterial-Composite Films for Bioelectronics

Abstract

Microbial electrochemical systems provide an environmentally-friendly means of energy conversion between chemical and electrical forms, with applications in wastewater treatment, bioelectronics, and biosensing. However, a major challenge to further development, miniaturization, and deployment of bioelectronics and biosensors is the limited thickness of biofilms, necessitating large anodes to achieve sufficient signal-to-noise ratios. Here we demonstrate a method for embedding an electroactive bacterium, Shewanella oneidensis MR-1, inside a conductive three-dimensional poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) matrix electropolymerized on a carbon felt substrate, which we call a multilayer conductive bacterial-composite film (MCBF). By mixing the bacteria with the PEDOT:PSS precursor in a flow-through method, we maintain over 90% viability of S. oneidensis during encapsulation. Microscopic analysis of the MCBFs reveal a tightly interleaved structure of bacteria and conductive PEDOT:PSS up to 80 µm thick. Electrochemical experiments indicate S. oneidensis in MCBFs can perform both direct and riboflavin-mediated electron transfer to PEDOT:PSS. When used in bioelectrochemical reactors, the MCBFs produce 20 times more steady-state current than native biofilms grown on unmodified carbon felt. This versatile approach to control the thickness of bacterial composite films and increase their current output has immediate applications in microbial electrochemical systems, including field-deployable environmental sensing and direct integration of microorganisms into miniaturized organic electronics.

Figure 1

Electrode preparation set-up for viable multilayer conductive bacterial-composite film production. (a) Schematic of the electropolymerization system, including a photograph of a single well. The electron flow in the final structure is (i) reduction of lactate to acetate by bacteria, (ii) transfer of electrons from bacteria to the PEDOT:PSS scaffold, and (iii) conduction of electrons through PEDOT:PSS scaffold to CF substrate. (b) Isometric view of the complete MCBF preparation station for parallel electropolymerization of six bio-anodes.

Figure 2

Electropolymerization greatly increases the specific capacitance in MCBFs relative to UCFs. (a) Cyclic voltammograms and (b) Nyquist plots measured for unmodified CF before (solid blue lines), and for MCBF after the electropolymerization process (red dashed lines).

Figure 3

Multilayer conductive bacterial-composite films (MCBFs) are thicker than native S. oneidensis MR-1 biofilms on unmodified CF (UCF). Confocal microscopy images of cross sections of (a and c) native biofilm on UCF and (b and d) MCBF. Red color indicates CF and blue S. oneidensis.

Figure 4

Electropolymerization of living S. oneidensis MR-1 embeds the bacteria inside a multilayer conductive bacterial-composite film. Scanning electron micrographs of a section of (a and c) an abiotic multilayer conductive film (MCF) showing smooth internal surfaces of PEDOT:PSS on CF, and (b and d) of an MCBF showing high density of bacteria within PEDOT:PSS layers. (d) One bacterium on the external surface of the MCBF is shaded in red, while two bacteria-sized voids internal to the MCBF are outlined in red.

Figure 5

S. oneidensis MR-1 transfers metabolic current through PEDOT:PSS by both direct and flavin-mediated electron transfer. (a) 5x magnification of Au/PEDOT:PSS deposited on slide. (b) SEM of gold thin film surface. (c) SEM of PEDOT:PSS film. (d, left) Average current after 12 hours for WT, ΔmtrB and Δbfe strains on gold and PEDOT:PSS films and (d, right) the Δbfe current production before and 12 hours after addition of 10 μM riboflavin (Rb).

Figure 6

MCBF bioreactors produce greater biotic current than those using a native biofilm on unmodified CF. Chronoamperometric characterization of MESs based on MCBF (red dashed line) and UCF (blue solid line) using S. oneidensis metabolizing lactate, and abiotic MCF reactors (black dotted line). Light red, blue, and black colored bands indicate the standard deviation in current from three bioreactors, respectively.