Bidirectional Extracellular Electron Transfer Research Articles

Retraction for Yadav et al., "The bidirectional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition".

In our paper, we reported the bidirectional extracellular electron transfer capability of Geoalkalibacter halelectricus based on biochemical (i.e., with insoluble Fe-oxide and Fe(0)) and bioelectrochemical (i.e., with electrodes) experimental approaches. We noticed some issues and limitations of the methods and techniques that were used to analyze Fe species, particularly the reduced Fe ions in insoluble and precipitated Fe(II) minerals that led to incorrect interpretation of the results, specifically the reduction of Fe(III) to Fe(0) in this study. We were made aware of thermodynamic constraints that would make the biological reduction of Fe(III) to Fe(0) implausible. Hence, the conclusion about microbial Fe(III)-oxide reduction to Fe(0) is invalid. We also noticed errors in estimating the protein-based iron reduction/oxidation rates and faradic efficiencies, besides the limitations of the methods used for Fe analysis. For these reasons, we retract this article and sincerely apologize for the inconvenience it may have caused to the readers.

Bidirectional extracellular electron transfer pathways of Geobacter sulfurreducens biofilms: Molecular insights into extracellular polymeric substances

The basis for bioelectrochemical technology is the capability of electroactive bacteria (EAB) to perform bidirectional extracellular electron transfer (EET) with electrodes, i.e. outward- and inward-EET. Extracellular polymeric substances (EPS) surrounding EAB are the necessary media for EET, but the biochemical and molecular analysis of EPS of Geobacter biofilms on electrode surface is largely lacked. This study constructed Geobacter sulfurreducens-biofilms performing bidirectional EET to explore the bidirectional EET mechanisms through EPS characterization using electrochemical, spectroscopic fingerprinting and proteomic techniques. Results showed that the inward-EET required extracellular redox proteins with lower formal potentials relative to outward-EET. Comparing to the EPS extracted from anodic biofilm (A-EPS), the EPS extracted from cathodic biofilm (C-EPS) exhibited a lower redox activity, mainly due to a decrease of protein/polysaccharide ratio and α-helix content of proteins. Furthermore, less cytochromes and more tyrosine- and tryptophan-protein like substances were detected in C-EPS than in A-EPS, indicating a diminished role of cytochromes and a possible role of other redox proteins in inward-EET. Proteomic analysis identified a variety of redox proteins including cytochrome, iron-sulfur clusters-containing protein, flavoprotein and hydrogenase in EPS, which might serve as an extracellular redox network for bidirectional EET. Those redox proteins that were significantly stimulated in A-EPS and C-EPS might be essential for outward- and inward-EET and warranted further research. This work sheds light on the mechanism of bidirectional EET of G. sulfurreducens biofilms and has implications in improving the performance of bioelectrochemical technology.

Carbon source priority and availability limit bidirectional electron transfer in freshwater mixed culture electrochemically active bacterial biofilms

This study investigated, if a mixed electroactive bacterial (EAB) culture cultivated heterotrophically at a positive applied potential could be adapted from oxidative to reductive or bidirectional extracellular electron transfer (EET). To this end, a periodic potential reversal regime between − 0.5 and 0.2 V vs. Ag/AgCl was applied. This yielded biofilm detachment and mediated electroautotrophic EET in combination with carbonate, i.e., dissolved CO2, as the sole carbon source, whereby the emerged mixed culture (S1) contained previously unknown EAB. Using acetate (S2) as well as a mixture of acetate and carbonate (S3) as the main carbon sources yielded primarily alternating electrogenic organoheterotropic metabolism with the higher maximum oxidation current densities recorded for mixed carbon media, exceeding on average 1 mA cm−2. More frequent periodic polarization reversal resulted in the increase of maximum oxidative current densities by about 50% for S2-BES and 80% for S3-BES, in comparison to half-batch polarization. The EAB mixed cultures developed accordingly, with S1 represented by mostly aerobes (84.8%) and being very different in composition to S2 and S3, dominated by anaerobes (96.9 and 96.5%, respectively). S2 and S3 biofilms remained attached to the electrodes. There was only minor evidence of fully reversible bidirectional EET. In conclusion the three triplicates fed with organic and/or inorganic carbon sources demonstrated two forms of diauxie: Firstly, S1-BES showed a preference for the electrode as the electron donor via mediated EET. Secondly, S2-BES and S3-BES showed a preference for acetate as electron donor and c-source, as long as this was available, switching to CO2 reduction, when acetate was depleted.Graphical

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.

Microbial electrosynthetic nitrate reduction to ammonia by reversing the typical electron transfer pathway in Shewanella oneidensis

Ammonia production is a critical industrial process, and mild routes to recycle nitrates in wastewater could be a promising route to ammonia synthesis. In this study, ammonia production is demonstrated in a microbial electrosynthesis system with nitrate and an electrode as electron acceptors and donors, respectively. Based on the bidirectional extracellular electron transfer capability of Shewanella oneidensis MR-1, our microbial electrosynthetic system achieves a maximum ammonia production rate of 24.3 μg h−1·mg protein−1 with 82.5% selectivity and 33.1% cathodic efficiency and functions for several cycles over 30 days. Electrochemical analysis suggests that cytochromes c, flavins, and the flavin/c-cytochrome combination play a pivotal role. Charge transfer resistance weakens over the course of weeks, resulting in easier electron transfer. Parallel reaction monitoring proteomics suggest that reversing a typical “Mtr pathway” plays a role, and a dissimilatory nitrate to ammonia pathway is used. This work proposes a progressive route to carry out ammonia synthesis under mild conditions.

Enhanced bidirectional extracellular electron transfer based on biointerface interaction of conjugated polymers-bacteria biohybrid system

The low bacteria loading capacity and low extracellular electron transfer (EET) efficiency are two major bottlenecks restricting the performance of the bioelectrochemical systems from practical applications. Herein, we demonstrated that conjugated polymers (CPs) could enhance the bidirectional EET efficiency through the intimate biointerface interactions of CPs-bacteria biohybrid system. Upon the formation of CPs/bacteria biohybrid, thick and intact CPs-biofilm formed which ensured close biointerface interactions between bacteria-to-bacteria and bacteria-to-electrode. CPs could promote the transmembrane electron transfer through intercalating into the cell membrane of bacteria. Utilizing the CPs-biofilm biohybrid electrode as anode in microbial fuel cell (MFC), the power generation and lifetime of MFC had greatly improved based on accelerated outward EET. Moreover, using the CPs-biofilm biohybrid electrode as cathode in electrochemical cell, the current density was increased due to the enhanced inward EET. Therefore, the intimate biointerface interaction between CPs and bacteria greatly enhanced the bidirectional EET, indicating that CPs exhibit promising applications in both MFC and microbial electrosynthesis.

A Microbial Cell Coating Based on a Conjugated Polyelectrolyte with Broad Reduction Potential Increases Inward and Outward Extracellular Electron Transfer

AbstractBioelectrochemical systems hold the promise of enabling sustainable microbial‐mediated energy interconversion between electrical and chemical energy. Herein, it is demonstrated how a single conjugated polymer can be used to enhance bidirectional extracellular electron transfer through forming self‐assembled coatings on individual cells. Specifically, the n‐type conjugated polyelectrolyte p(cNDI‐gT2) exhibits a reduction potential window between −0.1 and −0.8 V (vs Ag/AgCl), thereby driving thermodynamically favored electron transfer in both directions across the abiotic‐biotic interface that involves the outer membrane cytochromes and flavins of Shewanella oneidensis MR‐1. Electrochemical tests show that injection from an external electrode into Shewanella oneidensis MR‐1 is enabled at negative potentials (−0.6 V), while electron extraction is possible at positive potentials (0.2 V). Relative to controls, the biohybrid shows a sixfold increase in biocurrent generation and a 35‐fold increase in current uptake for the bioelectrosynthesis of succinate from fumarate. This demonstrated abiotic‐biotic synergy provides new strategies for designing multifunctional biohybrids.

Mechanism and applications of bidirectional extracellular electron transfer of Shewanella.

Electrochemically active microorganisms (EAMs) play an important role in the fields of environment and energy. Shewanella is the most common EAM. Research into Shewanella contributes to a deeper comprehension of EAMs and expands practical applications. In this review, the outward and inward extracellular electron transfer (EET) mechanisms of Shewanella are summarized and the roles of riboflavin in outward and inward EET are compared. Then, four methods for the enhancement of EET performance are discussed, focusing on riboflavin, intracellular reducing force, biofilm formation and substrate spectrum, respectively. Finally, the applications of Shewanella in the environment are classified, and the restrictions are discussed. Potential solutions and promising prospects for Shewanella are also provided.

Two polarity reversal modes lead to different nitrate reduction pathways in bioelectrochemical systems

Polarity reversal is one of the effective strategies to rapidly start up denitrifying BESs，but the long-term performances of the denitrifying BESs operated under polarity reversal receive little attention. This study investigated the effects of periodic polarity reversal (PPR) and polarity reversal once only (PRO) on the long-term performances of denitrifying BESs. Repeatable oxidative and reductive currents were observed in the BESs obtained by PPR (PPR-BESs). The peak reductive currents of the PPR-BESs reached 0.95 A/m2, and nitrate was mainly removed by dissimilatory nitrate reduction to ammonium pathway with removal rates higher than 95 %. In contrast, the peak reductive currents of the BESs obtained by PRO (PRO-BESs) progressively decreased from 1.01 A/m2 to 0.12 A/m2. The nitrate removal rates of the PRO-BESs were <50 %, and the product of nitrate reduction turned to N2 instead of ammonium. 16S rDNA sequencing and metatranscriptomic analysis revealed that Geobacter capable of bidirectional extracellular electron transfer (EET) and Afipia capable of autotrophic growth were the dominant genera in the two types of BESs. Outer membrane cytochrome c and formate dehydrogenase were potentially involved in the cathodic electron uptake. These findings contribute to a better understanding of the EET mechanisms of electroautotrophic denitrifiers.

Microbiologically influenced corrosion of stainless steels by Bacillus subtilis via bidirectional extracellular electron transfer

The corrosion of 304 stainless steel by Bacillus subtilis was investigated under different glycerol (electron donor) and nitrate (electron acceptor) concentrations. When nitrate was deficient, the passive film served as an alternative electron acceptor to complete outward extracellular electron transfer (EET) of B. subtilis, inducing more severe corrosion especially in the early stage. When glycerol concentration was deficient, the inward EET of B. subtilis was significantly enhanced, resulting in accelerated corrosion in the late stage. These results demonstrated that bidirectional EET was involved in the corrosion of stainless steel by B. subtilis and depended on the electron donor/acceptor concentrations.

Autotrophic nitrate reduction to ammonium via reverse electron transfer in Geobacter dominated biofilm

Geobacter dominated electroactive biofilms (EABs) have been demonstrated to perform bidirectional extracellular electron transfer (EET) in bioelectrochemical systems, but it is largely unknown when nitrate is the electron acceptor at the cathode. If reverse EET occurs on biocathode, this EAB has to perform dissimilatory nitrate reduction to ammonia (DNRA) rather than denitrification according to genomes. Here, we have proven the feasibility of reverse bioelectron transfer in EAB, achieving a DNRA efficiency up to 93 ± 3% and high Faraday efficiency of 74 ± 1%. Constant current was found to be more effective than constant potential to maintain Geobacter on the cathode, which highly determines this electrotrophic respiration. The prevalent DNRA at constant current surpassed denitrification, demonstrated by the reverse tendencies of DNRA (nrfA) and denitrification (nirS and nirK) gene transcription. Metatranscriptomics further revealed the possible electron uptake mechanisms by which the outer membrane (OmcZ and OmcB) and periplasmic cytochromes (PpcB and PpcD) may be involved. These findings extend our understanding of the bidirectional electron transfer and advance the applications of EABs.

Adaptive bidirectional extracellular electron transfer during accelerated microbiologically influenced corrosion of stainless steel

Microbiologically influenced corrosion of metals is prevalent in both natural and industrial environments, causing enormous structural damage and economic loss. Exactly how microbes influence corrosion remains controversial. Here, we show that the pitting corrosion of stainless steel is accelerated in the presence of Shewanella oneidensis MR-1 biofilm by extracellular electron transfer between the bacterial cells and the steel electrode, mediated by a riboflavin electron shuttle. From pitting measurements, X-ray photoelectron spectroscopy and Mott-Schottky analyses, the addition of an increased amount of riboflavin is found to induce a more defective passive film on the stainless steel. Electrochemical impedance spectroscopy reveals that enhanced bioanodic and biocathodic process can both promote the corrosion of the stainless steel. Using in situ scanning electrochemical microscopy, we observe that extracellular electron transfer between the bacterium and the stainless steel is bidirectional in nature and switchable depending on the passive or active state of the steel surface.

Long-distance electron transfer in a filamentous Gram-positive bacterium

Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.

Rechargeable microbial fuel cell based on bidirectional extracellular electron transfer

Rechargeable microbial electrochemical systems can be used as renewable energy storage systems or as potable bioelectronics devices. In this study, a bioelectrode capable of bidirectional extracellular electron transfer was firstly introduced to construct the rechargeable microbial fuel cell (MFC). The performance of rechargeable MFC was enhanced with the increase of charge/discharge cycles, and a maximum energy efficiency of 4.5 ± 0.2% and Coulombic efficiency of 29.4 ± 4.1% were obtained. H2 was the main charge carrier, while the accumulated acetate was only about 10 mg L−1. The charge time under constant current mode largely affected the energy recovery. A decreased abundance of Mycobacteria, Geobacter, and Azospirillum, accompanied by an increase of Azonexus and Rhodococcus was observed in the rechargeable MFC, compared to control tests fueled with acetate. This study demonstrates the potential of bioelectrode for energy storage and recovery.

Different mechanisms for riboflavin to improve the outward and inward extracellular electron transfer of Shewanella loihica

Exploring extracellular electron transfer (EET) is important to promote the applications of electrochemically active bacteria (EAB). Shewanella loihica PV-4, which is a model EAB capable of bidirectional EET, has shown promising application prospects in water quality monitoring and nitrogen removal. However, its bidirectional EET mechanism is not clear yet. This study focused on the electrochemical characteristics and electron transfer processes of S. loihica PV-4 bidirectional EET. Results revealed that riboflavin (RF) facilitates both the outward and inward EET, and the enhancement mechanism depends on EET direction. When S. loihica PV-4 performs outward EET, free RF acts as a redox mediator to promote electricity production. When the EET direction is reversed, bound RF is involved in electricity consumption. These results elucidated the effect of RF on S. loihica PV-4 bidirectional EET, providing a basis for the enhancement of S. loihica PV-4 EET.

The mechanism and application of bidirectional extracellular electron transport in the field of energy and environment

Bidirectional extracellular electron transfer (EET) is mediated by back and forth electron delivery between microorganisms and extracellular substances. This enables the exchange of biochemical information and energy with the surrounding environments. As a novel bioenergy strategy, bidirectional EET provides low-cost opportunities for the production of clean energy sources and carriers (e.g., hydrogen and methane) as well as the production of value-added chemicals from carbon dioxide. Electrochemically active bacteria (EAB) can also transform pollutants to less toxic or benign substances in contaminated environments, and therefore they have been widely applied in bioremediation studies. Among all the available EAB, Geobacter and Shewanella are well-known for their versatility to accept/donate electrons from/to external environments. In this review, we focus on how these model EAB generate or harvest energy through bidirectional EET, as well as recent advances in the application of EET in bioelectrochemical technology and environmental bioremediation. Finally, the challenges, perspectives and new directions in the bidirectional EET studies are discussed.

Dual detection of biochemical oxygen demand and nitrate in water based on bidirectional Shewanella loihica electron transfer

This study for the first time proposed a method for simultaneously measuring BOD and nitrate in water using electrochemically active bacteria. Firstly, the bidirectional extracellular electron transfer (EET) capability of a model electricigen Shewanella loihica PV-4 was revealed. Then, based on the respective outward and inward EET, S. loihica PV-4 was utilized to detection BOD and nitrate. The results demonstrated a positive correlation between the outward EET and BOD (from 0 mg/L to 435 mg/L) while a negative correlation between the inward EET and nitrate (from 0 mg/L to 7 mg/L); both the relationships were well fitted by the combination of traditional linear model and Michaelis-Menten model (R2>0.96). Finally, a dual detection method for BOD and nitrate measurements was established based on the ano-cathodophilic capability of S. loihica PV-4 biofilm, and exhibited the characteristics of high accuracy (>80%) and fast analysis (<1h), suggesting a promising prospect in water monitoring.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.

Promoting Shewanella Bidirectional Extracellular Electron Transfer for Bioelectrocatalysis by Electropolymerized Riboflavin Interface on Carbon Electrode.

The extracellular electron transfer (EET) that connects the intracellular metabolism of electroactive microorganisms to external electron donors/acceptors, is the foundation to develop diverse microbial electrochemical technologies. For a particular microbial electrochemical device, the surface chemical property of an employed electrode material plays a crucial role in the EET process owing to the direct and intimate biotic-abiotic interaction. The functional modification of an electrode surface with redox mediators has been proposed as an effectual approach to promote EET, but the underlying mechanism remains unclear. In this work, we investigated the enhancement of electrochemically polymerized riboflavin interface on the bidirectional EET of Shewanella putrefaciens CN32 for boosting bioelectrocatalytic ability. An optimal polyriboflavin functionalized carbon cloth electrode achieved about 4.3-fold output power density (∼707 mW/m2) in microbial fuel cells and 3.7-fold cathodic current density (∼0.78 A/m2) for fumarate reduction in three-electrode cells compared to the control, showing great increases in both outward and inward EET rates. Likewise, the improvement was observed for polyriboflavin-functionalized graphene electrodes. Through comparison between wild-type strain and outer-membrane cytochrome (MtrC/UndA) mutant, the significant improvements were suggested to be attributed to the fast interfacial electron exchange between the polyriboflavin interface with flexible electrochemical activity and good biocompatibility and the outer-membrane cytochromes of the Shewanella strain. This work not only provides an effective approach to boost microbial electrocatalysis for energy conversion, but also offers a new demonstration of broadening the applications of riboflavin-functionalized interface since the widespread contribution of riboflavin in various microbial EET pathways together with the facile electropolymerization approach.

Anodic and Cathodic Extracellular Electron Transfer by the Filamentous Bacterium Ardenticatena maritima 110S.

Ardenticatena maritima strain 110S is a filamentous bacterium isolated from an iron-rich coastal hydrothermal field, and it is a unique isolate capable of dissimilatory iron or nitrate reduction among the members of the bacterial phylum Chloroflexi. Here, we report the ability of A. maritima strain 110S to utilize electrodes as a sole electron acceptor and donor when coupled with the oxidation of organic compounds and nitrate reduction, respectively. In addition, multicellular filaments with hundreds of cells arranged end-to-end increased the extracellular electron transfer (EET) ability to electrodes by organizing filaments into bundled structures, with the aid of microbially reduced iron oxide minerals on the cell surface of strain 110S. Based on these findings, together with the attempt to detect surface-localized cytochromes in the genome sequence and the demonstration of redox-dependent staining and immunostaining of the cell surface, we propose a model of bidirectional electron transport by A. maritima strain 110S, in which surface-localized multiheme cytochromes and surface-associated iron minerals serve as a conduit of bidirectional EET in multicellular filaments.