Spectroscopic Slicing to Reveal Internal Redox Gradients in Electricity‐Producing Biofilms

Robuschi, Luciana; Tomba, J. Pablo; Schrott, Germán David; Bonanni, Pablo Sebastián; Desimone, Paula Mariela; Busalmen, Juan Pablo

doi:10.1002/anie.201205440

Cited by 77 publications

(80 citation statements)

References 25 publications

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“…Spectral observations show nearly 50% of c -type cytochromes remain reduced when cells are growing in films similar to those used in this study [19], an observation also supported by confocal Raman spectroscopy [21]. Microelectrodes probing thicker (∼150 µm) G. sulfurreducens biofilms growing on electrodes showed that outer regions of the biofilm were at low redox potential, and oxidized zones were only detected in the inner portion of the film [17].…”

Section: Discussionsupporting

confidence: 78%

“…For example, conduction of electrons through active biofilms appears to become limiting at distances 10–20 µm from the electrode surface, based on microelectrode [17], spectral [18], [19], source-drain experiments [12], [20], and confocal Raman spectroscopy [21]. A pH gradient can also exist across the biofilm, where the inner layers experience a lower pH [22]–[24].…”

Section: Introductionmentioning

confidence: 99%

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Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

et al. 2014

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When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

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Section: Discussionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

et al. 2014

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“…Biofilm conductivity enabled by metallic-like mechanism is expected to be higher than that of a system where electron transfer occurs via the electron hopping mechanism [6]. In fact, in a system governed by metallic-like conduction the redox variations across long distances are expected to be hardly measurable with available technologies [8], while the accumulation of reduced cytochromes in biofilms of G. sulfurreducens have been reported by Robuschi et al ., consistently with the presence of conduction pathways at higher resistance [9].…”

Section: Introductionsupporting

confidence: 72%

“…Most likely, these heme groups belong to the outer membrane cytochromes and the periplasmic cytochromes wiring the cell metabolism to the electrode [10]. Although the bis-His heme ligation is typical of mixed-culture biofilms dominated by Geobacter species [10], [13], pure G. sulfurreducens biofilms grown on Indium tin oxide electrodes display a different ligation [9]. In fact, the cited study reports two ν 10 bands at 1620 and 1638 cm −1 , whose relative intensities vary considerably as a function of the applied potential.…”

Section: Resultsmentioning

confidence: 99%

Real-Time Measurements of the Redox States of c-Type Cytochromes in Electroactive Biofilms: A Confocal Resonance Raman Microscopy Study

et al. 2014

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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.

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“…Cells not in direct contact with the electrode can transport their respired electrons to the electrode via hopping through a proposed network of c -type cytochromes as opposed to EESs. This model is supported experimentally by measurements of biofilm electrical conductivity (12, 88, 97, 110, 113, 126–128). Much of the theory comes from models of synthetic redox polymers and proteins that contain bound redox cofactors (10, 12, 49, 91, 114).…”

Section: Eess In the Extracellular Environmentmentioning

confidence: 62%

The Colorful World of Extracellular Electron Shuttles

Glasser

Saunders

Newman

2017

Annu. Rev. Microbiol.

187

141

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Descriptions of the changeable, striking colors associated with secreted natural products date back well over a century. These molecules can serve as extracellular electron shuttles (EESs) that permit microbes to access substrates at a distance. In this review, we argue that the colorful world of EESs has been too long neglected. Rather than simply serving as a diagnostic attribute of a particular microbial strain, redox-active natural products likely play fundamental, underappreciated roles in the biology of their producers, particularly those that inhabit biofilms. Here, we describe the chemical diversity and potential distribution of EES producers and users, discuss the costs associated with their biosynthesis, and critically evaluate strategies for their economical usage. We hope this review will inspire efforts to identify and explore the importance of EES cycling by a wide range of microorganisms so that their contributions to shaping microbial communities can be better assessed and exploited.

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Spectroscopic Slicing to Reveal Internal Redox Gradients in Electricity‐Producing Biofilms

Cited by 77 publications

References 25 publications

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

Real-Time Measurements of the Redox States of c-Type Cytochromes in Electroactive Biofilms: A Confocal Resonance Raman Microscopy Study

The Colorful World of Extracellular Electron Shuttles

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