Soluble Electron Acceptors Research Articles

Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. Experiments were conducted by adding along with glycerol, acetate and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. Ametabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Interdisciplinary Research on Electrons and Biofilms

Interdisciplinary research on electrons and biofilms is being carried out by Assistant Professor Dr Yoshihide Tokunou, Faculty of Life and Environmental Sciences, University of Tsukuba, Japan. His work is aimed at gaining a deeper understanding of the role of electrons in bacterial society and exploring the process of bacterial extracellular electron transfer (EET) in biofilms. By combining microbiology and electrochemistry, Dr Tokunou is developing EET-based biotechnologies and improving our understanding of the physiological importance of EET in bacterial society. To achieve this, he is collaborating with various experts such as microbiologists, chemists, and engineers, including Professor Dr Nobuhiko Nomura. His current interest is in the EET process that terminates with soluble electron acceptors. By regulating the EET in biofilms respiring with oxygen, he is trying to control various biofilm-related phenomena such as host-bacteria interaction, antibiotic resistance of biofilms, biochemical production from biofilms, etc.

Electron transport bifurcation in bioanode with the metabolic shift to nitrate reduction

Electron transport bifurcation in bioanode determines the performance of microbial electrochemical technologies with the presence of an alternative electron acceptor. Here, the bioanode responses including electron transfer efficiency, microbial community, and microbial structure are investigated with the metabolic shift from current production to denitrification. Electrochemical measurements including cyclic voltammetry and electrochemical impedance spectra are performed to identify the change of electron transfer pathways in bioanode. Electron transfer efficiency for electrode reduction decreases ∼17 % with nitrate reduction. Biofilm resistance and charge transfer resistance increase from 23.3 Ω and 22.5 Ω to 36.6 Ω and 61.4 Ω with the metabolic shift, respectively. These results are mainly due to the loss of exoelectrogens inhabited in bioanode. Confocal imaging results indicate the elevated proportion of inactive cells in bioanode as the denitrification. Our results propose a possible mechanism for electron transfer bifurcation in bioanode with the metabolic shift from electrode reduction to soluble electron acceptor reduction.

The bidirectional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition.

Bidirectional extracellular electron transfer (EET) is crucial to upholding microbial metabolism with insoluble electron acceptors or donors in anoxic environments. Investigating bidirectional EET-capable microorganisms is desired to understand the cell-cell and microbe-mineral interactions and their role in mineral cycling besides leveraging their energy generation and conversion, biosensing, and bio-battery applications. Here, we report on iron cycling by haloalkaliphilic Geoalkalibacter halelectricus via bidirectional EET under haloalkaline conditions. It efficiently reduces Fe3+ oxide (Fe2O3) to Fe0 at a 0.75 ± 0.08 mM/mgprotein/d rate linked to acetate oxidation via outward EET and oxidizes Fe0 to Fe3+ at a 0.24 ± 0.03 mM/mgprotein/d rate via inward EET to reduce fumarate. Bioelectrochemical cultivation confirmed its outward and inward EET capabilities. It produced 895 ± 23 µA/cm2 current by linking acetate oxidation to anode reduction via outward EET and reduced fumarate by drawing electrons from the cathode (‒2.5 ± 0.3 µA/cm2) via inward EET. The cyclic voltammograms of G. halelectricus biofilms revealed redox moieties with different formal potentials, suggesting the involvement of different membrane components in bidirectional EET. The cyclic voltammetry and GC-MS analysis of the cell-free spent medium revealed the lack of soluble redox mediators, suggesting direct electron transfer by G. halelecctricus in achieving bidirectional EET. By reporting on the first haloalkaliphilic bacterium capable of oxidizing and reducing insoluble Fe0 and Fe3+ oxide, respectively, this study advances the limited understanding of the metabolic capabilities of extremophiles to respire on insoluble electron acceptors or donors via bidirectional EET and invokes the possible role of G. halelectricus in iron cycling in barely studied haloalkaline environments. IMPORTANCE Bidirectional extracellular electron transfer (EET) appears to be a key microbial metabolic process in anoxic environments that are depleted in soluble electron donor and acceptor molecules. Though it is an ecologically important and applied microbial phenomenon, it has been reported with a few microorganisms, mostly from nonextreme environments. Moreover, direct electron transfer-based bidirectional EET is studied for very few microorganisms with electrodes in engineered systems and barely with the natural insoluble electron acceptor and donor molecules in anoxic conditions. This study advances the understanding of extremophilic microbial taxa capable of bidirectional EET and its role in barely investigated Fe cycling in highly saline-alkaline environments. It also offers research opportunities for understanding the membrane components involved in the bidirectional EET of G. halelectricus. The high rate of Fe3+ oxide reduction activity by G. halelectricus suggests its possible use as a biocatalyst in the anaerobic iron bioleaching process under neutral-alkaline pH conditions.

Designed and synthesized de novo ANTPABA-PDI nanomaterial as an acceptor in inverted solar cell at ambient atmosphere

In this work, a novel soluble and air-stable electron acceptor containing perylenediimide moiety named ANTPABA-PDI was designed and synthesized with band gap 1.78eV and that was used as non-fullerene acceptor material. ANTPABA-PDI possess not only good solubility but also much lower LUMO (lowest unoccupied molecular orbital) energy level. Furthermore, its excellent electron acceptor capability also supported by density functional theory calculation which validates the experimental observations. Inverted organic solar cell has been fabricated using ANTPABA-PDI along with P3HT as standard donor material in ambient atmosphere. The device, after characterization in open air, exhibited a power conversion efficiency of 1.70%. This is the first ever PDI based organic solar cell that has been fabricated completely in ambient atmosphere. The characterizations of the device have also been performed in ambient atmosphere. This kind of stable organic material can easily be used in fabricating organic solar cell and therefore it can be used as the best alternative as non-fullerene acceptor materials.

Extracellular Electron Transfer of Weak Electricigens in the Presence of a Competing Electron Acceptor

To maximise microbial electroactivity in bioelectrochemical systems, soluble electron acceptors are typically omitted as they compete with the electrode. While practical, this approach provides engineered conditions that do not reflect the natural environment of electroactive microorganisms, which may contain both soluble and insoluble electron acceptors. This study investigates the behaviour of weak electricigens, a relatively understudied category of microorganisms whose members switch between non-electroactive and electroactive states. Enrichments were performed in microbial fuel cells containing both an electrode and the soluble alternative fumarate to probe extracellular electron transfer of weak electricigens. Using fluorescence spectroscopy, chromatography and voltammetry, the electron shuttle riboflavin was not found in these conditions but was found in controls in which only the electrode was available to reduce. Despite this dichotomy in ability to perform riboflavin-based mediated electron transfer, communities of weak electricigens were similarly electroactive in each condition (19.36 ± 0.9 mW m−2 vs 20.25 ± 2.0 mW m−2). 16S rRNA gene sequencing revealed similar communities enriched in each condition, but with differing abundance. Understanding extracellular electron transfer in natural environments is of both fundamental and applied interest, as it can inform the design of real-world bioelectrochemical systems whose influents are likely to contain competing electron acceptors.

Pharmaceutical Attenuation Differs within Woodchip-Based Lignocellulose Bioreactors across Nitrate- and Sulfate-Reducing Conditions

Lignocellulosic bioreactors use solid-phase substrates such as woodchips to sustain microbial respiration and have been applied to treat agricultural runoff, stormwater, and other impaired waters. Here, we query how respiration of different soluble electron acceptors impacts the degradation of environmentally relevant pharmaceuticals associated with treated municipal wastewater discharge. Laboratory-scale columns containing a mixture of woodchips and alfalfa were manipulated across nitrate- and sulfate-reducing conditions using residence time and influent composition. Under steady-state conditions, bioreactors dominated by nitrate reduction harbored a distinct phylogenetic profile containing the genera Denitratisoma and increases in the denitrification gene nirS. In contrast, bioreactors where sulfate reduction dominated exhibited increased relative abundance of fermenters (e.g., Obscuribacteriales) and putative sulfate reducers (e.g., Desulfobulbus). Atenolol attenuation and biotransformation to carboxy-metoprolol accelerated under nitrate-reducing conditions; in contrast, trimethoprim attenuation and biotransformation to desmethyl trimethoprim was nearly an order of magnitude faster under sulfate-reducing conditions. Modest sulfamethoxazole attenuation occurred under all tested conditions. Denitrification-associated rate constants for atenolol were comparable to those reported in constructed wetlands and aquifer recharge, suggesting commonality in biotransformation mechanisms. Collectively, results suggest that manipulation of biogeochemical gradients during nature-based treatment can be applied to attenuate nitrate and trace quantities of pharmaceuticals.

Electron shuttle-dependent biofilm formation and biocurrent generation: Concentration effects and mechanistic insights.

Electron shuttles (ESs) play a key role in extracellular electron transfer (EET) in Shewanella oneidensis MR-1. However, the quantification relationship between ES concentration, biofilm formation, and biocurrent generation has not been clarified. In this study, 9,10-anthraquinone-2-sulfonic acid (AQS)-mediated EET and biofilm formation were evaluated at different AQS concentrations in bioelectrochemical systems (BESs) with S. oneidensis MR-1. Both the biofilm biomass (9- to 17-fold) and biocurrent (21- to 80-fold) were substantially enhanced by exogenous AQS, suggesting the dual ability of AQS to promote both biofilm formation and electron shuttling. Nevertheless, biofilms barely grew without the addition of exogenous AQS, revealing that biofilm formation by S. oneidensis MR-1 is highly dependent on electron shuttling. The biofilm growth was delayed in a BES of 2,000 μM AQS, which is probably because the redundant AQS in the bulk solution acted as a soluble electron acceptor and delayed biofilm formation. In addition, the maximum biocurrent density in BESs with different concentrations of AQS was fitted to the Michaelis-Menten equation (R 2 = 0.97), demonstrating that microbial-catalyzed ES bio-reduction is the key limiting factor of the maximum biocurrent density in BESs. This study provided a fundamental understanding of ES-mediated EET, which could be beneficial for the enrichment of electroactive biofilms, the rapid start-up of microbial fuel cells (MFCs), and the design of BESs for wastewater treatment.

Geobacter sulfurreducens' Unique Metabolism Results in Cells with a High Iron and Lipid Content.

Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.

Geoalkalibacter halelectricus SAP-1 sp. nov. possessing extracellular electron transfer and mineral-reducing capabilities from a haloalkaline environment.

The extracellular electron transfer (EET)-capable electroactive microorganisms (EAMs) play crucial roles in mineral cycling and interspecies electron transfer in different environments and are used as biocatalysts in microbial electrochemical technologies. Studying EAMs from extreme environments is desired to advance the electromicrobiology discipline, understanding their unique metabolic traits with implications to extreme microbiology, and develop specific bioelectrochemical applications. Here, we present a novel haloalkaliphilic bacterium named Geoalkalibacter halelectricus SAP-1, isolated from a microbial electroactive biofilm enriched from the haloalkaline lake sediments. It is a rod-shaped Gram-negative heterotrophic anaerobe that uses various carbon and energy sources and respires on soluble and insoluble terminal electron acceptors. Besides 16S-rRNA and whole-genome sequence-based phylogeny, the GGDC values of 21.7%, ANI of 78.5%, and 2.77% genomic DNA GC content difference with the closest validly named species Geoalkalibacter ferrihydriticus (DSM 17813T ) confirmed its novelty. When grown with the solid-state electrode as the only electron acceptor, it produced 460 ± 23 μA/cm2 bioelectrocatalytic current, thereby confirming its electroactivity. Further electrochemical analysis revealed the presence of membrane redox components with a high formal potential, putatively involved in the direct mode of EET. These are distinct from EET components reported for any known electroactive microorganisms, including well-studied Geobacter spp., Shewanella spp., and Desulfuromonas acetexigens. The capabilities of G. halelectricus SAP-1 to respire on soluble and insoluble electron acceptors including fumarate, SO4 2- , Fe3+ , and Mn4+ suggests its role in cycling these elements in haloalkaline environments.

Engineering Extracellular Electron Transfer in Escherichia coli for Microbial Electrochemical Devices

Microbes hold great potential for green electrochemical synthesis as well as bio-electricity generation from organic waste. The key to the successful development of microbial electrochemical devices lies with the ability of the microbes to transport electrons across their outer membranes. These devices thus often rely on the use of microbes known as exoelectrogens, which include bacteria that have evolved metabolic pathways that enable extracellular electron transfer (EET) to solid substrates for cellular respiration. While exoelectrogens demonstrate facilitated electron transfer with the electrodes of these devices, natural exoelectrogens are often restricted to utilizing a narrow range of substrates that limits their versatility for processing different organic wastes and electrosynthesizing a rich diversity of high-value chemicals. By contrast, non-exoelectrogenic microbes such as Escherichia coli that lack efficient EET pathways are host to rich metabolic reaction networks that can be more readily tailored for various applications using synthetic biology.In this work, we biologically engineer E. coli for enhanced EET. To this end, we expressed different combinations of proteins from the natural exoelectrogen S. oneidensis MR-1, as well as E. coli native proteins to optimize EET of the bioengineered strain. The bioengineered strains show significant improvements in electron transfer rates, as confirmed through colorimetric reduction assays of soluble electron acceptors and electrochemical characterizations following electrode reduction. The improved EET demonstrated in this work paves the way for increasing the efficiency of existing E. c oli - based electrochemical devices while opening the doors to new applications that benefit from the broad chemical repertoire of these microbes.

Anaerobic ammonium oxidation (anammox) promoted by pyrogenic biochar: Deciphering the interaction with extracellular polymeric substances (EPS)

Efficient biological nitrogen removal (BNR) by anaerobic ammonium oxidation (anammox) can be achieved with presence of redox-active pyrogenic biochar that potentially acting as an insoluble electron acceptor. Anammox bacteria and other symbiotic consortia are surrounded by extracellular polymeric substances (EPS) forming aggregate architecture, which also contains electrochemical-active biomolecules such as aromatic proteins and humic substances. Therefore, understanding the role of EPS is necessary in biochar-promoting anammox process. Herein, we investigated the influence of biochar with granular-sized (GP) and micrometer-sized (MP) particle sizes on microbiology and characteristics of EPS in anammox sludge. Addition of GP and MP biochar not only improved the BNR efficiency by 17.5% and 34.6%, respectively, but also increased the relative abundance of Candidatus Brocadia. The bulk and bound EPS contents substantially decreased in biochar-amended groups, while more slime EPS was produced. Spectroscopic (FTIR, Raman, and circular dichroism) and electrochemical (voltammetry and impedance spectrum) analyses revealed that biochar addition enhanced the structural integrity and electron-transfer capability of anammox sludge. EPS depletion led to a steep decrease in BNR efficiency (21.5% vs 83.0% with EPS-retained sludge), whereas it resumed to 42.1% in the presence of MP biochar. Electron transport system activity data showed that biochar replenished the loss of anaerobic respiration metabolism due to EPS depletion. In summary, these results suggested that EPS possibly work as transient mediator for shuttling electrons from ammonium oxidation to soluble (nitrite) and insoluble electron acceptors (redox-active biochar).

Tuning the LUMO Levels of Z-Shaped Perylene Diimide via Stepwise Cyanation.

The central dogma in constructing organic electron acceptors is to attach electron-withdrawing groups to polycyclic aromatic hydrocarbons. Yet, the full potentials of many organic acceptors were never realized due to synthetic obstacles. By combining the Wittig-Knoevenagel benzannulation, the Pd(0)-catalyzed cyanation, and nucleophilic addition/oxidation cyanation, six polynitrile Z-shaped perylene diimide were synthesized. These stable and soluble electron acceptors possess LUMO energy levels comparable with those of benchmark compounds. Electrochemical investigation reveals that each additional nitrile group reduces the LUMO energy by 0.2 eV.

The role of chemotaxis and efflux pumps on nitrate reduction in the toxic regions of a ciprofloxacin concentration gradient.

Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.

Developing microbial communities containing a high abundance of exoelectrogenic microorganisms using activated carbon granules

Microorganisms that can transfer electrons outside their cells are useful in a range of wastewater treatment and remediation technologies. Conventional methods of enriching exoelectrogens are cost-prohibitive (e.g., controlled-potential electrodes) or lack specificity (e.g., soluble electron acceptors). In this study a low-cost and simple approach to enrich exoelectrogens from a mixed microbial inoculum was investigated. After the method was validated using the exoelectrogen Geobacter sulfurreducens, microorganisms from a pilot-scale biological activated carbon (BAC) filter were subjected to incubations in which acetate was provided as the electron donor and granular activated carbon (GAC) as the electron acceptor. The BAC-derived community oxidized acetate and reduced GAC at a capacity of 1.0 mmol e− (g GAC)−1. After three transfers to new bottles, acetate oxidation rates increased 4.3-fold, and microbial morphologies and GAC surface coverage became homogenous. Although present at <0.01% in the inoculum, Geobacter species were significantly enriched in the incubations (up to 96% abundance), suggesting they were responsible for reducing the GAC. The ability to quickly and effectively develop an exoelectrogenic microbial community using GAC may help initiate and/or maintain environmental systems that benefit from the unique metabolic capabilities of these microorganisms.

Controlling the Bioluminescence Output from Shewanella woodyi using Soluble Electron Acceptors

We will present bioluminescence data from Shewanella woodyi cultures in order to correlate bioluminescence intensity with changes in the electrochemical potential in a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO3, CoCl2, or ZnCl2 to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate, Co(II), or Zn(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared to cultures lacking Co(II) or nitrate. Our presentation will show that electrochemical potentials of &lt; 0.2 V vs NHE repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicate that the luminescence intensity from S. woodyi could be systematically reduced using these three soluble electron acceptors making S. woodyi a potential model bacterium for whole‐cell luminescence bioelectrochemical sensor applications.Support or Funding InformationUniversity of Dayton Graduate Student Summer Fellowship and Dean's Summer Fellowship programs

Probing the Role of Metal Coordination and pH in Assembly and Function of Cytochrome Nanowires

Geobacter sulfurreducens produces protein nanowires for extracellular electron transfer during respiration and interspecies electron exchange to survive in environments that lack soluble electron acceptors. It was previously thought that nanowires were Type IV pili comprised by PilA monomers, but we showed via electron cryo-microscopy (cryo-EM) that nanowires instead constitute a helical polymer of the six-heme outer membrane cytochrome OmcS, yielding a periodic arrangement of hemes over the micrometer scale of the filaments (Cell, 2019, 177, 361). This 3.7 Å-resolution structure revealed several features which appear to stabilize this polymer, including close stacking of covalently bound heme ligands within ∼ 3.5-6 Å of each other, and axial coordination of a heme metal center in each subunit by a histidine residue in the adjacent subunit. These OmcS filaments represent to our knowledge the first micrometer-scale cytochrome polymer naturally evolved for electron transfer, but the mechanism of this polymerization remains unknown. Furthermore, lowering the pH of nanowire samples enhances their conductivity and improves heme stacking, yet the structural changes in OmcS underlying this conformation change are unclear. Here, we develop a method to obtain OmcS nanowires and monomers in high purity. We then combine cryo-EM with spectroscopic methods such as UV-vis and circular dichroism to examine the changes in OmcS polymers and their inter-subunit interactions as a result of changes to pH, further enabling tuning of the conductivity of purified microbial nanowires. Furthermore, we explore the spectroscopic features and biochemical properties of OmcS monomers after reversing their polymerization, to obtain a mechanistic understanding of nanowire assembly that will aid engineering of synthetic protein nanowires for bioelectronics. This research was supported by Career Award from Burroughs Welcome Fund, NIH New Innovator award NSF CAREER award and DARPA (to N.S.M.).

Soluble electron acceptors affect bioluminescence from Shewanella woodyi.

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO3 or CoCl2 to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of <0.2 V vs Normal Hydrogen Electrode (NHE) repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicated that the luminescence intensity from S. woodyi could be systematically reduced using these two soluble electron acceptors, making S. woodyi a potential model bacterium for whole-cell luminescence bioelectrochemical sensor applications.

Phenolic and non-phenolic substrates oxidation by laccase at variable oxygen concentrations: Selection of bisubstrate kinetic models from polarographic data

Using laccase as a case study, an alternative methodology for discrimination of bisubstrate mechanism types, useful for Bi-Bi reactions when water is one of the products, is presented. A purified enzyme from fungal cultures of Ganoderma resinaceum and two reducing substrates: 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) are used. Kinetic assays of ABTS or DMP oxidation using variable initial concentrations of dioxygen are performed in a closed system and depletion rates of dioxygen are measured using polarography. Seven bisubstrate models based on Michaelis-Menten Bi-Bi mechanisms are applied to the experimental data. Akaike information criterion, a penalized likelihood analysis, and Akaike weights are used for model selection. Results show that the more likely bisubstrate models for ABTS and DMP oxidation are ping-pong and Theorell-Chance, respectively. However, the catalytic efficiency of oxygen reduction to water exhibits similar values, and is compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. Finally, Michaelis-Menten constants towards dioxygen, 233 μM with ABTS and 700 μM with DMP, indicate that the soluble electron acceptor available for laccase-catalyzed reactions, is far removed from saturation.

How Thermophilic Gram-Positive Organisms Perform Extracellular Electron Transfer: Characterization of the Cell Surface Terminal Reductase OcwA

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.