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MEMBRANE PROTEINS OF ELECTROACTIVE BACTERIA PROBED AT LIPID LAYERS ONTO MODIFIED ELECTRODES

Periodic Reporting for period 1 - MELBA (MEMBRANE PROTEINS OF ELECTROACTIVE BACTERIA PROBED AT LIPID LAYERS ONTO MODIFIED ELECTRODES)

Okres sprawozdawczy: 2017-04-01 do 2019-03-31

Electroactive bacteria are living micro-organisms that are able to directly connect their respiratory metabolism to their extracellular environment by transferring electrons across biological membranes to, or from solids like metal oxides or electrodes. These bacteria are organized at conducting surfaces as biofilms and can indeed shuttle electrons via periplasmic and membrane proteins to/from electrodes. Hence, electroactive bacteria represent living, stable, self-replicating, and low-cost electrode catalysts. This unique property leads to potential “green” biotechnology applications such as microbial fuel cells, microbial electrosynthesis cells, wastewater treatment, desalination and biosensors. In order to permit the advent of these promising microbial electrochemical technologies it is crucial to progress toward the fundamental knowledge of these electroactive biofilms. Electroactive microorganisms such as Gram negative Geobacter sulfurreducens directly connect and transfer electrons to anodes via outer-membrane c-type cytochrome redox proteins as ultimate redox relays. In the absence of an organic substrate like acetate, at least four redox proteins may be detected electrochemically in the biofilm, while in the presence of an organic substrate extracellular electron release from bacterial catabolism is easily followed by recording a catalytic anodic current. The anolyte then becomes more acidic consistent with the oxidation of the organic substrate to carbon dioxide concomitantly with proton release. Acidification of the anolyte, of the biofilm and/or of the biofilm/electrode interface compromises the performance and viability of the catalytic biofilms. In fact, the extracellular electron transfer may be coupled to proton transfer in outer-membrane c-type cytochromes or, alternatively, non-redox proton transport proteins may be responsible for the acidification of the anolyte. Although extracellular electron and proton transfers are known to occur in electroactive bacteria, the fundamental understanding of these processes is still in its infancy and precludes the optimization of microbial electrochemical technologies.
The overall objective of MELBA is to develop an efficient and versatile electrochemical platform for probing electroactive bacteria membrane proteins incorporated in artificial lipid deposits supported on modified carbon electrode. In a first approach, the electrodes are modified with pH-responsive electrophores such as quinone units and then covered by lipid layers or deposits creating an artificial supported lipid membrane in which membrane proteins are incorporated and electrochemically probed. Indeed, the intrinsic electroactivity of the protein (if any) can be probed directly with the lipid modified electrode and possible proton transport occurring across the lipid layer by the membrane protein can in principle be detected with the grafted pH-responsive redox probe.
Concerning the career objectives of the fellow, the goal of holding a permanent academic position in France has been reached because the experienced researcher will be hired as an assistant professor at the University of Nantes (France) on September 2019.
The first proof of concept of the electrochemical platform of MELBA was presented using surface grafted catechol moieties as pH-responsive electrophores and cytochrome c as the model membrane-associated redox protein. Cytochrome c was immobilized into the supported lipid deposit at the catechol modified glassy carbon electrode surface. We focused on the detection of both the pH-dependent electrophore and the redox protein reversible redox systems at glassy carbon successively modified by covalent catechol grafting, then by an optimized lipid deposit and finally by immobilization of cytochrome c. In summary, the electrochemical detection of both grafted catechol and immobilized cytochrome c onto lipid deposit-modified glassy carbon electrode was achieved at the same electrode. First, a glassy carbon electrode was functionalized by a catechol covalent grafting procedure following the one-pot/three steps electrochemical reduction then, an optimized lipid film was deposited by solvent evaporation on the catechol-modified glassy carbon electrode surface for promoting the cytochrome c electrochemical activity. The cytochrome c immobilization onto the catechol/lipid deposit-modified electrode was achieved following a simple and efficient strategy consisting of recurrent cyclic voltammetry in a cytochrome c solution. The resulting catechol/lipid deposit/cytochrome c-modified glassy carbon electrode was finally transferred into a protein-free phosphate buffer where its electrochemical behavior was studied by cyclic voltammetry. The electrochemical detection of both grafted catechol and immobilized cytochrome c reversible redox systems was achieved while maintaining intact their native electrochemical properties, namely the pH-dependent reversible system of the grafted quinone/catechol redox couple and the pH-independent electroactivity of the redox protein.
This work was then extended periplasmic proteins from electroactive bacteria such as flavocytochrome c3, a tetraheme FAD-containing periplasmic flavoenzyme isolated from the bacterium Shewanella putrefaciens, taken as a model pH-dependent redox protein from electroactive bacteria. The production and purification of the flavocytochrome c3 enzyme from Shewanella putrefaciens DSM 9451 was carried out in the lab partner (ITQB, Lisbon) of Secondment. In summary, the electrochemical detection of adsorbed flavocytochrome c3 onto edge plane pyrolytic graphite electrode was achieved before and after surface grafting of a catechol pH probe. The periplasmic flavocytochrome c3 redox enzyme from Shewanella putrefaciens was successfully immobilized onto electrode with a high surface coverage, following a simple and efficient strategy consisting of consecutive cyclic voltammetry in a flavocytochrome c3 solution containing polymyxin as co-adsorbate until a steady state current is reached. The electrochemical detection of both grafted catechol and adsorbed flavocytochrome c3 redox systems was achieved while maintaining intact their native electrochemical properties such as the pH-dependent electroactivity of the redox enzyme and its fumarate reductase activity. The pH-dependent redox properties of the grafted catechol were evaluated in the presence of the immobilized enzyme onto electrode and demonstrated the effectiveness of the catechol pH sensor in the presence of the immobilized protein.
MELBA opens the opportunity to investigate larger and more complex redox proteins from electroactive bacteria with not only pH-dependent redox potentials but also extracellular proton transport ability, which is essential for viability of electroactive microorganisms and biofilms.
In a future work, the electrochemical platform of MELBA could be extended to probe ionophore activity by immobilizing appropriate ion sensor onto electrode surface.
In addition, covalent grafted molecules with quinone-like moieties as suitable probes to evaluate the pH changes at biofilm/electrode interface in microbial fuel cells as a promising biotechnology.
A personal impact of MELBA for the experienced researcher will be to submit a personal proposal for an ERC Starting grant on October 2019.
Electrochemical response of a catechol/lipid deposit/cytochrome c-modified glassy carbon electrode
Electrochemical response of a catechol/Fcc3-modified pyrolytic graphite electrode