Shewanella secretes flavins that mediate extracellular electron transfer

Abstract

Bacteria able to transfer electrons to metals are key agents in biogeochemical metal cycling, subsurface bioremediation, and corrosion processes. More recently, these bacteria have gained attention as the transfer of electrons from the cell surface to conductive materials can be used in multiple applications. In this work, we adapted electrochemical techniques to probe intact biofilms of Shewanella oneidensis MR-1 and Shewanella sp. MR-4 grown by using a poised electrode as an electron acceptor. This approach detected redox-active molecules within biofilms, which were involved in electron transfer to the electrode. A combination of methods identified a mixture of riboflavin and riboflavin-5'-phosphate in supernatants from biofilm reactors, with riboflavin representing the dominant component during sustained incubations (>72 h). Removal of riboflavin from biofilms reduced the rate of electron transfer to electrodes by >70%, consistent with a role as a soluble redox shuttle carrying electrons from the cell surface to external acceptors. Differential pulse voltammetry and cyclic voltammetry revealed a layer of flavins adsorbed to electrodes, even after soluble components were removed, especially in older biofilms. Riboflavin adsorbed quickly to other surfaces of geochemical interest, such as Fe(III) and Mn(IV) oxy(hydr)oxides. This in situ demonstration of flavin production, and sequestration at surfaces, requires the paradigm of soluble redox shuttles in geochemistry to be adjusted to include binding and modification of surfaces. Moreover, the known ability of isoalloxazine rings to act as metal chelators, along with their electron shuttling capacity, suggests that extracellular respiration of minerals by Shewanella is more complex than originally conceived.

Fig. 1.

Evidence for riboflavin secretion by electrode-attached bacteria. (A) Oxidation current by an established Shewanella MR-4 biofilm (black trace), decline after addition of fresh medium (red trace), and recovery after replacement of original medium found to contain secreted riboflavin (blue trace). Gaps indicate CV and DPV analysis. (B) LC-MS identification of microbially produced riboflavin from biofilm chambers after 96 h of incubation. The LC peak at 5.65 min (Upper) corresponds to compound with a m/z of 377.2, whereas analysis of the 377.2 peak yielded an ion with a m/z ratio of 243 (Lower).

Fig. 2.

Evidence for flavins controlling the rate of electron transfer to electrodes. (A) CV of Shewanella MR-4 in the presence of electron donor (lactate), showing current-voltage relationships for established biofilms (black trace), the same biofilms washed in medium free of redox mediators (red trace), and reimmersed in the original medium (blue trace). The green trace shows the effect of adding 250 nM riboflavin, to approximately double the concentration. (B) Nonturnover CV, showing a peak centered at −0.21 V. Raw data are shown in black, baseline-subtracted data are in red. (C) CV of riboflavin at identical sterile carbon electrodes.

Fig. 3.

DPV (from negative to positive potentials) of sterile carbon electrodes in growth medium after different times of exposure to riboflavin. (Inset) Increase in baseline-subtracted peak height over time.

Fig. 4.

Relationship between peak potential (A) or peak height (B) and scan rate for sterile carbon electrodes in Shewanella growth medium containing riboflavin.

Fig. 5.

DPV of carbon electrodes colonized by Shewanella MR-4 (from negative to positive potentials). (A) Increase in peak height (at −0.21 V) with growth of culture. (B) Changes in peak height after addition of medium, showing retention in older biofilms. Black trace shows DPV of a young biofilm in presence (solid line) and absence (dotted trace) of soluble riboflavin. Red trace shows DPV of an older biofilm in presence (solid line) and absence (dotted trace) of soluble riboflavin. (Inset) Change in peak height (at −0.21 V) in each treatment.

Fig. 6.

Combined electron shuttling and chelator (shelator) activity by FMN or riboflavin (abbreviated as vitamin B2). (A) Model of interactions with metal oxides. (B) Electron-accepting abilities of solubilized metal.