Disentangling extracellular current of electroactive bacteria with oblique-incidence reflection difference imaging

Abstract

Understanding the extracellular electron transfer (EET) process of electroactive bacteria is of great significance. It is critical yet challenging to differentiate the partial currents from direct (DET) and mediated electron transfer (MET) pathways in the integrated EET current. Herein the EET current of model exoelectrogen is successfully disentangled by using spatiotemporally-resolved oblique-incidence reflection difference (OIRD) technique coupled with polyaniline (PANI)-based dual electrode. The PANI film serves as an electron acceptor to translate the charge information into OIRD signals, enabling mapping of EET current. Upon complete reduction of PANI, the local EET current is switched off, and the soluble mediators are forced to discharge on the nearby PANI electrode, enabling measuring of MET current. In such a way, the DET and MET currents are measured and the average currents from each bacterium are quantified. As-reported method enables successful disentangling the EET current and may offer valuable insights to related research.

Fig. 1. Principle of OIRD imaging of EET current.

a Schematic illustration of the OIRD setup coupled with a PANI-underlying bioanode, where the EET electron injection in PANI film results in OIRD response to enable quantitative imaging of the EET current; b LSV curve and in situ OIRD response of PANI-FTO electrode from 0.1 to −0.4 V at 1 mV s⁻¹ in M9 buffer with 18 mM lactate. The PANI-FTO electrode was pre-conditioned at 0.1 V for 5 min before sweeping; c Correlation between averaged ∆I and passed charge density (Q) derived from (b) and its fitting curve. The applied potential was also plotted against Q for reference. Data were presented as mean ± standard deviation. Error bar indicates the standard deviation, n = 1000.

Fig. 2. OIRD responses on dual electrode upon EET of adherent Shewanella putrefaciens CN32.

a Schematic illustration showing the dual electrode setup; b OIRD image of the dual electrode at the potential of 0.1 V; c In situ ΔI signals collected along the dashed line as indicated in (b) on the dual electrode in 140 min discharge in M9 buffer containing 18 mM nutrient. At t₀ = 0 the potential of both W₁ and W₂ were removed from 0.1 V to open circuit state; d Representative in situ ΔI signals collected at W₁ (top) and W₂ (bottom), and the OCP evolution of W₁ (middle) during 140 min discharge; e OIRD image of the dual electrode after 140 min discharge; f Charge distribution and intensity profile along the marked line of the dual electrode during 140 min discharge, obtained from the differential OIRD image (Supplementary Fig. 8). The scale bars in (b, e, f) are 3 mm.

Fig. 3. Electron-injection induced switch off of PANI film.

a Schematic illustration showing the reduction-induced switch off of EET on PANI-FTO electrode and the interplay of EET electrons on the dual electrode; b Discharge curve recorded on W₁ (top) and in situ ΔI signals collected on W₁ and W₂, respectively (bottom) in M9 buffer containing 18 mM nutrient; W₁ with bacteria at 0.1 V while W₂ w/o bacteria at OCP; c Discharge curves of W₁ and W₂, both at 0.1 V in M9 buffer containing 18 mM nutrient; W₁ with bacteria while W₂ w/o bacteria; d Discharge curve recorded on Bacteria-PANI-FTO at 0.1 V and −0.3 V in M9 buffer containing 18 mM nutrient; e CV curves of CC, sterile PANI-FTO and PANI-FTO pre-conditioned at −0.3 V in M9 buffer containing 2 μM of FMN, scan rate: 1 mV s⁻¹.

Fig. 4. Measuring DET and MET currents of Shewanella putrefaciens CN32 bioanode by controlling on/off state of PANI film.

a Schematic illustration of strategy for measuring the DET and MET currents; b Representative in situ ΔI signals collected at W₁ (top) and W₂ (bottom), and the OCP evolution of W₁ (middle) during 147 min discharge; at t = 0, the potential of 0.1 V was simultaneously removed on W₁ and W₂; at t = 147 min and 184 min, the potential of W₁ was stepped to −0.5 and −0.3 V, respectively while W₂ was kept at open circuit state; c ΔI vs. time (t) plots collected on W₁ and W₂, respectively. Data were presented as mean ± standard deviation. Error bar indicates the standard deviation, n = 1000; d Corresponding EET, DET, and MET currents; e Corresponding DET and MET currents of individual bacterium.

Fig. 5. Validation and applicability of as-reported method.

a In situ OIRD responses on W₁ and W₂ upon adding 2 μM FMN mediator into the supernatant after the switch off of W₁; b Plots of measured integrated DET/MET currents of various bioanodes; c corresponding DET/MET currents from individual bacterium.

Fig. 6. Measuring the partial currents from adherent and suspended Shewanella putrefaciens CN32.

a Schematic illustration showing the bioanode setup; b Representative in situ ΔI signal collected at W₁ (top) and W₂ (bottom); at t = 0, the potential of 0.1 V was removed on W₁ and W₂; c ΔI vs. time (t) plots collected on W₁ and W₂, respectively. Data were presented as mean ± standard deviation. Error bar indicates the standard deviation, n = 1000; d Corresponding DET and MET currents from adherent bacteria (a-DET, a-MET) and MET current from suspended bacteria (s-MET); e Percentage of corresponding current densities from a-DET, a-MET and s-MET pathways; f Corresponding a-DET, a-MET currents of individual adherent bacterium and s-MET current of individual suspended bacterium.