Electrical current generation by wild type and mutant Shewanella strains

Abstract

The genus Shewanella has been reported to have the capacity to couple the transfer of electrons to insoluble metal oxides and solid carbon electrodes with cellular growth. While this process may be useful as an energy generation strategy or biotechnological tool, the electron transfer pathway by which this process occurs is not completely understood, and better techniques for studying the transfer mechanism are needed. This project used single chamber electrochemical cells to show that the current generation capabilities of Shewanella oneidensis are dependent on the ability of cells comprising a biofilm to shuttle soluble electron carriers between an electrode and its outer membrane cytochromes. The extracellular electron transfer capabilities of S. oneidensis mutant strains containing deleted or transposon-interrupted copies of genes known to be involved in extracellular electron transfer were also studied and compared to wild type. Amperometry was utilized to monitor real-time electron flow between attached anaerobic wild type and mutant cells and a poised carbon working electrode. Differential pulse voltammetry and cyclic voltammetry performed on electrode attached S. oneidensis MR-1 wild type cells detected both mediated and direct electron transfer reactions at the electrode surface. Ion exchange HPLC verified the presence of endogenously produced flavin compounds in S. oneidensis liquid cultures and confirmed the most common flavin in S. oneidensis MR-1 electrochemical cell analytes is riboflavin (vitamin B2). It was also discovered that removing the medium surrounding an electrode biofilm caused current production from the electrochemical cell to decrease. Returning the filtered original medium, or adding anaerobic riboflavin resulted in the restoration of current production. The amount of current produced at the carbon working electrode increased with biofilm development and accumulation of soluble electron mediator. A correlation was observed between the concentration of the redox shuttle in potentiostat-controlled electrochemical cells and the maximum sustainable current, as well as maximum electrode biofilm thickness. For example, wild type cultures with twice the natural amount of riboflavin approximately doubled their electrode current production and also attached to the electrode in larger numbers. Electrode phenotypes of Shewanella oneidensis MR-1 mutant strains were also observed and compared to wild type. The deletion of several genes, such as for the outer membrane cytochrome MtrC, the periplasmic cytochrome MtrA, or the membrane beta barrel protein MtrB severely impaired MR-1 cells from attaching to the carbon electrode. As a result, the deletion mutant strains were incapable of producing significant anodic current and were deficient in electrode attached biomass. However, deletion of the outer membrane cytochrome OmcA, or genes related to the formation of mature biofilms resulted in a percentage of the current production being retained. This data supports the theory that MtrC is a key component in the terminal electron transfer step for S. oneidensis MR-1 cells interacting with solid surfaces. Measurements of current production from MR-1 electrode biofilms revealed that the extracellular electron transfer process involves both cell associated enzymes and flavins acting as soluble electron transfer agents. However, these separate pathways most likely utilize many of the same, membrane proteins to accomplish their function. This study indicates it is likely that the deposition of electrons by S. oneidensis MR-1 to a poised electrode can be done both directly by MtrC while stabilized or otherwise assisted by other outer membrane elements such as OmcA or MtrB, as wells as through cycling of redox active shuttles such as flavins between MtrC and the electrode surface. As a result, for electrode attached Shewanella oneidensis MR-1 cells, a complex relationship exists between soluble flavin concentration, biofilm thickness, and electrical current production. These factors pertain greatly to the capabilities and limitations of S. oneidensis, especially while functioning as part of a electrochemical device, and must be taken into account when utilizing this organism for research or other applications.