Functional Electrodes for Enzymatic and Microbial Electrochemical Systems, pp. 563-597 (2017) No AccessChapter 16: Spectroelectrochemistry of Microbial BiofilmsDiego Millo and Bernardino VirdisDiego MilloBiomolecular Spectroscopy/LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands and Bernardino VirdisThe University of Queensland, Advanced Water Management Centre (AWMC) Level 4, Gehrmann Building (60), Brisbane, QLD 4072, Australiahttps://doi.org/10.1142/9781786343543_0016Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The following sections are included: Introduction and Motivation Spectroscopic Techniques Equipment for Spectroelectrochemical Analyses Achievements in Spectroelectrochemistry of Microbial Biofilms Conclusions and Future Prospects List of Abbreviations References FiguresReferencesRelatedDetails Functional Electrodes for Enzymatic and Microbial Electrochemical SystemsMetrics History PDF download

Electron transport within living cells is essential for energy conservation in all respiring and photosynthetic organisms. While a few bacteria transport electrons over micrometer distances to their surroundings, filaments of cable bacteria are hypothesized to conduct electric currents over centimeter distances. We used resonance Raman microscopy to analyze cytochrome redox states in living cable bacteria. Cable-bacteria filaments were placed in microscope chambers with sulfide as electron source and oxygen as electron sink at opposite ends. Along individual filaments a gradient in cytochrome redox potential was detected, which immediately broke down upon removal of oxygen or laser cutting of the filaments. Without access to oxygen, a rapid shift toward more reduced cytochromes was observed, as electrons were no longer drained from the filament but accumulated in the cellular cytochromes. These results provide direct evidence for long-distance electron transport in living multicellular bacteria.

The high quantum efficiency of photosynthetic reaction centers (RCs) makes them attractive for bioelectronic and biophotovoltaic applications. However, much of the native RC efficiency is lost in communication between surface-bound RCs and electrode materials. The state-of-the-art biophotoelectrodes utilizing cytochrome c (cyt c) as a biological wiring agent have at best approached 32% retained RC quantum efficiency. However, bottlenecks in cyt c-mediated electron transfer have not yet been fully elucidated. In this work, protein film voltammetry in conjunction with photoelectrochemistry is used to show that cyt c acts as an electron-funneling antennae that shuttle electrons from a functionalized rough silver electrode to surface-immobilized RCs. The arrangement of the two proteins on the electrode surface is characterized, revealing that RCs attached directly to the electrode via hydrophobic interactions and that a film of six cyt c per RC electrostatically bound to the electrode. We show that the additional electrical connectivity within a film of cyt c improves the high turnover demands of surface-bound RCs. This results in larger photocurrent onset potentials, positively shifted half-wave reduction potentials, and higher photocurrent densities reaching 100 μA cm-2. These findings are fundamental for the optimization of bioelectronics that utilize the ubiquitous cyt c redox proteins as biological wires to exploit electrode-bound enzymes.

The highest photocurrent to date on a bare metal electrode is reported by R. N. Frese and co-workers. Plasmon-enhanced light harvesting in a photosynthetic pigment protein on a nanoporous silver substrate results in record photocurrents up to 416 μA cm−2, representing a breakthrough in the field of biohybrid photoelectrodes. This is significant for the development of potential bioelectronic devices such as biohybrid solar cells and biosensors.

Electrochemically active microbial biofilms are capable to produce electric current when grown onto electrodes. This work investigates the dynamics of electron transfer inside the biofilm as well as at the biofilm/electrode interface.

The study of electroactive microbial biofilms often requires knowledge of the biofilm thickness. Unfortunately, this parameter is, nowadays, only accessible through expensive microscopic techniques. This work overcomes this limitation by presenting a new strategy, exploiting the use of chronoamperometry (CA) alone. A mixed-culture biofilm is exposed to an O2-saturated solution during anode respiration to suppress its catalytic activity. Assuming that inactivation of the electrocatalytic process is caused by O2 diffusion through the biofilm, a simple relation allows the use of the time constant extracted from the fitting of the curve of the CA trace during inactivation for the straightforward and quantitative determination of biofilm thickness. The biofilm thickness obtained with this method obeys the expected trend reported for biofilm growth and is in agreement with optical measurements. Contrary to the techniques usually employed to determine biofilm thickness, this new strategy is very rapid, nondisruptive, inexpensive, and may become a convenient alternative with respect to expensive and time-consuming microscopic techniques.

Cytochrome c (cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface. (C) 2015 Elsevier Ltd. All rights reserved.

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc–CL) immobilized at IMM. In this work, we have studied electrochemically cytc–CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc–CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc–CL in solution. In particular, immobilized cytc–CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/− coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH− states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc–CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H-2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.

In a quest to fabricate novel solar energy materials, the high quantum efficiency and long charge separated states of photosynthetic pigment‐proteins are being exploited through their direct incorporation in bioelectronic devices. In this work, a biohybrid photocathode comprised of bacterial reaction center‐light harvesting 1 (RC‐LH1) complexes self‐assembled on a nanostructured silver substrate yields a peak photocurrent of 166 μA cm −2 under 1 sun illumination, and a maximum of 416 μA cm −2 under 4 suns, the highest reported to date on a bare metal electrode. A 2.5‐fold plasmonic enhancement of light absorption per RC‐LH1 complex is observed on the rough silver substrate. This plasmonic interaction is assessed using confocal fluorescence microscopy, revealing an increase of fluorescence yield, and radiative rate of the RC‐LH1 complexes, signatures of plasmon‐enhanced fluorescence. Nanostructuring of the silver substrate also enhanced the stability of the protein under continuous illumination by almost an order of magnitude relative to a nonstructured bulk silver control. Due to its ease of construction, increased protein loading capacity, stability, and more efficient use of light, this hybrid material is an excellent candidate for further development of plasmon‐enhanced biosensors and biophotovoltaic devices.

Metal-respiring bacteria oxidize an organic substrate and transfer the liberated electrons to the electrode. Molecular oxygen interrupts the current flow by cutting the electrical circuit wiring the cell metabolism to the electrode.

Metal-respiring bacteria are microorganisms capable of oxidizing organic pollutants present in wastewater and transferring the liberated electrons to an electrode. This ability has led to their application as catalysts in bioelectrochemical systems (BESs), a sustainable technology coupling bioremediation to electricity production. Crucial for the functioning of these BESs is a complex protein architecture consisting of several surface-exposed multiheme proteins, called outer membrane cytochromes, wiring the cell metabolism to the electrode. Although the role of these proteins has been increasingly understood, little is known about the protein-electrode interactions and their impact on the performance of BESs. In this study, we used surface-enhanced resonance Raman spectroscopy in combination with electrochemical techniques to unravel the nature of the protein-electrode interaction for the outer membrane cytochrome OmcB from Desulfuromonas acetoxidans (Dace). Comparing the spectroelectrochemical properties of OmcB bound directly to the electrode surface with those of the same protein embedded inside an electroactive biofilm, we have shown that the surface-exposed cytochromes of Dace biofilms are in direct contact with the electrode surface. Even if direct binding causes protein denaturation, the biofilm possesses the ability to minimize the extent of the damage maximizing the amount of cells in direct electrical communication with the electrode.

Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208 ± 49 µA cm(-2)) and mature (57 days, j = 267 ± 73 µA cm(-2)) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190 ± 37 µA cm(-2)) with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.

SEIRA spectroscopy provides insight into the potential-induced structural changes of the anion channel hVDAC1 embedded in the tethered bilayer lipid membrane.

The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilmelectrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03s1. In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2s1. The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.

For the sulfite oxidase catalyzing the oxidation of sulfite to sulfate, it was demonstrated that the adsorption process is strongly ionic strength dependent. While under low ionic strength the flexibility of the enzyme is strongly restricted upon immobilization, it stays more mobile under high ionic strength conditions [1]. This observation is of special interest, because predicted domain motion events have a strong effect on the electron transfer [2].

We present a novel approach for determining the strength of the electric field experienced by proteins immobilised on membrane models. It is based on the vibrational Stark effect of a nitrile label introduced at different positions on engineered proteins and monitored by surface enhanced infrared absorption spectroscopy.