Extracellular Electron Transfer Activity Research Articles

Predicting microbial extracellular electron transfer activity in paddy soils with soil physicochemical properties using machine learning

Predicting microbial extracellular electron transfer activity in paddy soils with soil physicochemical properties using machine learning

Electroactive microorganisms synthesizing iron sulfide nanoparticles for enhanced hexavalent chromium removal in microbial fuel cells

Microbial fuel cells (MFCs) have been considered a promising technology for Cr6+ removal, but they are limited by Cr6+-reducing biocathodes with low extracellular electron transfer (EET) and poor microbial activity. In this study, three kinds of nano-FeS hybridized electrode biofilms, obtained through synchronous biosynthesis (Sy-FeS), sequential biosynthesis (Se-FeS) and cathode biosynthesis (Ca-FeS), were applied as biocathodes for Cr6+ removal in MFCs. The Ca-FeS biocathode exhibited the best performance due to the superior properties of biogenic nano-FeS (e.g., more synthetic amount, smaller particle size, better dispersion). The MFC with the Ca-FeS biocathode achieved the highest power density (42.08 ± 1.42 mW/m2) and Cr6+ removal efficiency (99.18 ± 0.1 %), which were 1.42 and 2.08 times as high as those of the MFC with the normal biocathode, respectively. The synergistic effects of nano-FeS and microorganisms enhanced the bioelectrochemical reduction of Cr6+, first realizing deep reduction of Cr6+ to Cr0 in biocathode MFCs. This significantly alleviated the cathode passivation caused by Cr3+ deposition. In addition, the hybridized nano-FeS as “armor” layers protected the microbes from toxic attack by Cr6+, improving the biofilm physiological activity and extracellular polymeric substances (EPS) secretion. The hybridized nano-FeS as “electron bridges” facilitated the microbial community to form a balanced, stable and syntrophic ecological structure. This study proposes a novel strategy through the cathode in-situ biosynthesis of nanomaterials to fabricate hybridized electrode biofilms with enhanced EET and microbial activity for toxic pollutant treatment in bioelectrochemical systems.

Reconstructing electron transfer components from an Fe(II) oxidizing bacterium.

Neutrophilic Fe(II) oxidizing bacteria play an important role in biogeochemical processes and have also received attention for multiple technological applications. These micro-organisms are thought to couple their metabolism with extracellular electron transfer (EET) while oxidizing Fe(II) as electron donor outside the cell. Sideroxydans lithotrophicus ES-1 is a freshwater chemolithoautotrophic Fe(II) oxidizing bacterium that is challenging to culture and not yet genetically tractable. Analysis of the S. lithotrophicus ES-1 genome predicts multiple EET pathways, which are proposed to be involved in Fe(II) oxidation, but not yet validated. Here we expressed components of two of the proposed EET pathways, including the Mto and Slit_0867-0870 PCC3 pathways, from S. lithotrophicus ES-1 into Aeromonas hydrophila, an established model EET organism. We demonstrate that combinations of putative inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways partially complemented EET activity in Aeromonas mutants lacking native components. Our results provide evidence for electron transfer functionality and interactions of inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways. Based on these findings, we suggest that EET in S. lithotrophicus ES-1 could be more complicated than previously considered and raises questions regarding directionality of these electron transfer pathways.

Simultaneous boost of anodic electron transfer and exoelectrogens enrichment by decorating electrospinning carbon nanofibers in microbial fuel cell

Microbial fuel cell (MFC) is a promising technology in wastewater recovery driven by microbial metabolism. However, the low power output resulting from the sluggish extracellular electron transfer (EET) between the anode surface and exoelectrogens dramatically restricted the further application. This study fabricated a high-performance anode by decorating porous and conductive electrospinning carbon nanofibers (CNFs). The maximum power density in MFC modified with 14 wt% polyacrylonitrile CNFs (M-CNF14, 9.6 ± 0.2 W m−3) was 1.9 and 2.7 times higher than carbon black modified MFC (M-CB, 5.1 ± 0.1 W m−3) and the blank (M-BA, 3.6 ± 0.1 W m−3), respectively. Denser biofilm and more microbial nanowires were observed in the M-CNF14 anode than in other conditions. Furthermore, the redox peak current of c-type cytochrome was 1.7–21 times higher in M-CNF14 than in the blank control, verifying the preferable EET activity. Several exoelectrogens like Petrimonas and Comamonas were enriched in M-CNF14 and showed a positive correlation to power generation. Besides, more simplified and modular interrelations among exoelectrogens and other bacteria were obtained in M-CNF14. This study revealed the microbial-related mechanism for simultaneously improving EET and exoelectrogens enrichment by CNFs modified anode, providing guidelines for high-performance wastewater recovery.

Structure and core taxa of bacterial communities involved in extracellular electron transfer in paddy soils across China

Microbial communities with extracellular electron transfer (EET) activity are capable of driving geochemical changes and cycles, but a comprehensive understanding of the key microbiota responsible for EET in complex soil matrices is still lacking. Herein, the EET activities, in terms of maximum current density (jmax) and accumulated charge output (Cout), of 41 paddy soils across China were evaluated from the exoelectrogenic properties with a conventional bioelectrochemical system (BES). The jmax with a range of 8.85 × 10−4 to 0.41 A/m2 and Cout with a range of 0.27 to 172.21C were obtained from these soil-based BESs. The bacterial community analyses revealed that the most abundant phylum, order, and genus were Firmicutes, Clostridiales, and Clostridum-sensus-stricto 10, respectively. Bacterial network analysis displayed the positive correlations between the majority of electroactive bacteria-containing genera and multiple other genera, indicating their underlying cooperation for the EET. Partial least squares regression (PLSR) model showed remarkable performance in describing the EET activity with 75 most abundant genera as input variables, identified that 32 genera were very important for governing the EET activities. Multiple linear regression (MLR) analyses further prioritized that the genera norank-c-Berkelbacteria and Fonticella were the key contributors, while the genus Paenibacillus was the key competitor against bacterial exoelectrogenesis in paddy soils. Moreover, the spearman analysis showed that the abundance of these keystone taxa was mainly influenced by the carbon content and pH. This approach provides a promising avenue to monitor the microbial activities in paddy soils as well as the links between microbial community composition and ecological function.

Order-of-magnitude enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation

Biophotovoltaics (BPV) generates electricity from reducing equivalent(s) produced by photosynthetic organisms by exploiting a phenomenon called extracellular electron transfer (EET), where reducing equivalent(s) is transferred to external electron acceptors. Although cyanobacteria have been extensively studied for BPV because of their high photosynthetic activity and ease of handling, their low EET activity poses a limitation. Here, we show an order-of-magnitude enhancement in photocurrent generation of the cyanobacterium Synechocystis sp. PCC 6803 by deprivation of the outer membrane, where electrons are suggested to stem from pathway(s) downstream of photosystem I. A marked enhancement of EET activity itself is verified by rapid reduction of exogenous electron acceptor, ferricyanide. The extracellular organic substances, including reducing equivalent(s), produced by this cyanobacterium serve as respiratory substrates for other heterotrophic bacteria. These findings demonstrate that the outer membrane is a barrier that limits EET. Therefore, depriving this membrane is an effective approach to exploit the cyanobacterial reducing equivalent(s).

Label-Free Probing of Electron Transfer Kinetics of Single Microbial Cells on a Single-Layer Graphene via Structural Color Microscopy.

Studies of electron transfer at the population level veil the nature of the cell itself; however, in situ probing of the electron transfer dynamics of individual cells is still challenging. Here we propose label-free structural color microscopy for this aim. We demonstrate that Shewanella oneidensis MR-1 cells show unique structural color scattering, changing with the redox state of cytochrome complexes in the outer membrane. It enables quantitatively and noninvasive studies of electron transfer in single microbial cells during bioelectrochemical activities, such as extracellular electron transfer (EET) on a transparent single-layer graphene electrode. Increasing the applied potential leads to the associated EET current, accompanied by more oxidized cytochromes. The high spatiotemporal resolution of the proposed method not only demonstrates the large diversity in EET activity among microbial cells but also reveals the subcellular asymmetric distribution of active cytochromes in a single cell. We anticipate that it provides a potential platform for further exploring the electron transfer mechanism of subcellular structure.

Extracellular electron transfer influences the transport and retention of ferrihydrite nanoparticles in quartz sand coated with Shewanella oneidensis biofilm

Microbial biofilm has been found to impact the mobility of nanoparticles in saturated porous media by altering physicochemical properties of collector surface. However, little is known about the influence of biofilm’s biological activity on nanoparticle transport and retention. Here, the transport of ferrihydrite nanoparticles (FhNPs) was studied in quartz sands coated with biofilm of Shewanella oneidensis MR-1 that is capable of reducing Fe(III) through extracellular electron transfer (EET). It was found that MR-1 biofilm coating enhanced FhNPs’ deposition under different pH/ionic strength conditions and humic acid concentrations. More importantly, when the influent electron donor (glucose) concentration was increased to promote biofilm’s EET activity, the breakthrough of FhNPs in biofilm-coated sands was inhibited. A lack of continuous and stable supply of electron donor, on the contrary, led to remobilization and release of the originally retained FhNPs. Column experiments with biofilm of EET-deficient MR-1 mutants (ΔomcA/ΔmtrC and ΔcymA) further indicated that the impairment of EET activity decreased the retention of FhNPs. It is proposed that the effective surface binding and adhesion of FhNPs that is required by direct EET cannot be neglected when evaluating the transport of FhNPs in sands coated with electroactive biofilm.

Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates

Extracellular electron transfer (EET) pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging because a functional understanding of its EET pathway proteins and their effect on engineering parametrizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures that predictably turn on and turn off EET flux, respectively, in response to isopropyl β-D-1-thiogalactopyranoside (IPTG). Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

Using Cathodic Poised Potential Experiments to Investigate Extracellular Electron Transport in the Crustal Deep Biosphere of North Pond, Mid-Atlantic Ridge

The crustal sub-seafloor covers a large portion of the Earth’s surface but is poorly understood as a habitat for life. It is unclear what metabolisms support the microscopic cells that have been observed, and how they survive under resource limitation. As the deep crustal subsurface represents a significant portion of the Earth’s surface, microbially-mediated reactions may therefore be significant contributors to biogeochemical cycling. In the present study, we used electrochemical techniques to investigate the possibility that crustal subsurface microbial groups can use the solid rock matrix (basalts, etc.) as a source of electrons for redox reactions via extracellular electron transfer (EET). Subsurface crustal fluids and mineral colonization experiments from the cool and oxic basaltic crustal subsurface at the North Pond site on the western flank of the Mid-Atlantic Ridge were used as inocula in cathodic poised potential experiments. Electrodes in oxic microbial fuel cells were poised at -200 mV versus a standard hydrogen electrode to mimic the delivery of electrons in an energy range equivalent to iron oxidation. In this way, microbes that use reduced iron in solid minerals for energy were selected for from the general community onto the electrode surface for interrogation of extracellular electron transfer activity, and potential identification by scanning electron microscopy and DNA sequencing. The results document that there are cathodic EET-capable microbial groups in the low biomass crustal subsurface at this site. The patterns of current generation in the experiments indicate that these microbial groups are active but likely not growing under the low-resource condition of the experiments, consistent with other studies of activity versus growth in the deep biosphere. Lack of growth stymied attempts to determine the phylogeny of EET-capable microbial groups from this habitat, but the results indicate that these microbial groups are a small part of the overall crustal deep biosphere community. This first demonstration of using electromicrobiology techniques to investigate microbial metabolic potential in the crustal deep biosphere reveals the challenges and opportunities for studying EET in the crustal deep biosphere.

In situ Electrochemical Studies of the Terrestrial Deep Subsurface Biosphere at the Sanford Underground Research Facility, South Dakota, USA

The terrestrial deep subsurface is host to significant and diverse microbial populations. However, these microbial populations remain poorly characterized, partially due to the inherent difficulty of sampling, in situ studies, and isolating of the in situ microbes. Motivated by the ability of microbes to gain energy from redox reactions at mineral interfaces, we here present in situ electrochemical colonization (ISEC) reactor as a method to directly study microbial electron transfer activity and to enable the capture and isolation of electrochemically active microbes. We installed a potentiostatically controlled ISEC reactor containing four working electrodes 1500 m below the surface at the Sanford Underground Research Facility. The working electrodes were poised at different redox potentials to mimic energy-yielding mineral reducing and oxidizing reactions predicted to occur at this site. We present a 16S rRNA analysis of the in situ electrode-associated microbial communities, revealing the dominance of novel bacterial lineages under cathodic conditions. We also demonstrate that the in situ electrodes can be further used for downstream electrochemical laboratory enrichment and isolation of novel strains. Using this workflow, we isolated Bacillus, Anaerospora, Comamonas, Cupriavidus, and Azonexus strains from the electrode-attached biomass. Finally, the extracellular electron transfer activity of Comamonas strain (isolated at -0.19 V vs. SHE and designated WE1-1D1) and Bacillus strain (isolated at +0.53 V vs. SHE and designated WE4-1A1-BC) from and to a poised electrode, respectively, were confirmed in electrochemical reactors. Our study highlights the utility of in situ electrodes and electrochemical enrichment workflows to shed light on microbial activity in the deep terrestrial subsurface.

A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria.

Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell.1 Mineral-respiring bacteria employ elaborate heme-based electron transfer mechanisms,2–4 but the existence or basis of other EETs remains largely unknown. In this study, we show that the foodborne pathogen Listeria monocytogenes utilizes a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. A forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity led to the characterization of an 8-gene locus responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channeling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit compatible with the single-membrane gram-positive cell structure. Activation of EET supports growth on non-fermentable carbon sources and a EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologs of the identified EET genes are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a surprising prevalence of EET-based growth capabilities and establish new relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.

Comparative metatranscriptomics reveals extracellular electron transfer pathways conferring microbial adaptivity to surface redox potential changes

Some microbes can capture energy through redox reactions with electron flow to solid-phase electron acceptors, such as metal-oxides or poised electrodes, via extracellular electron transfer (EET). While diverse oxide minerals, exhibiting different surface redox potentials, are widely distributed on Earth, little is known about how microbes sense and use the minerals. Here we show electrochemical, metabolic, and transcriptional responses of EET-active microbial communities established on poised electrodes to changes in the surface redox potentials (as electron acceptors) and surrounding substrates (as electron donors). Combination of genome-centric stimulus-induced metatranscriptomics and metabolic pathway investigation revealed that nine Geobacter/Pelobacter microbes performed EET activity differently according to their preferable surface potentials and substrates. While the Geobacter/Pelobacter microbes coded numerous numbers of multi-heme c-type cytochromes and conductive e-pili, wide variations in gene expression were seen in response to altering surrounding substrates and surface potentials, accelerating EET via poised electrode or limiting EET via an open circuit system. These flexible responses suggest that a wide variety of EET-active microbes utilizing diverse EET mechanisms may work together to provide such EET-active communities with an impressive ability to handle major changes in surface potential and carbon source availability.

Facile One-Step Strategy for Highly Boosted Microbial Extracellular Electron Transfer of the Genus Shewanella.

High performance of bacterial extracellular electron transfer (EET) is essentially important for its practical applications in versatile bioelectric fields. We developed a facile one-step approach to dramatically boost the bacterial EET activity 75-fold by exogenous addition of ethylenediamine tetraacetic acid disodium salt (EDTA-2Na, 1 mM) into the electrochemical cells, where the anodic process of microbial EET was monitored. We propose that EDTA-2Na enables both the alternation of the local environment around the c-type cytochromes located on the outer membrane channels (OMCs), which therefore changes the redox behavior of OMCs in mediating the EET process, and the formation of densely packed biofilm that can further facilitate the EET process. As a synergistic effect, the highly boosted bacterial EET activity was achieved. The method shows good generality for versatile bioelectrical bacteria. We envision that the method is also applicable for constructing various bioelectric devices.

Elucidating Molecular Underpinnings of Extracellular Electron Transfer in Bioelectrochemical Systems Using Evolutionary Genomics Approaches

Microbes can be promising catalysts for the conversion of chemical energy to electrical energy in important technologies such as bioelectrochemical systems (BESs) that can support biosensing and wastewater treatment applications. The reliability of BESs depends on the ability of microbes to move electrons from various donor substrates to solid phase electron acceptors through a process known as extracellular electron transfer (EET). Although some of the genes responsible for EET have been identified in a few model prokaryotes, we lack system-wide knowledge about how key genetic interactions evolve under BES conditions and how evolution may affect the long-term operation of these systems. Because mutation and evolution together represent an inevitable aspect of any technology that uses engineered or natural strains as biocatalysts, we need to understand how the genome-wide mutational landscape changes over time under various environmental conditions in BESs. We combined the power of experimental evolution and next generation sequencing to identify the rates as well as patterns of single nucleotide polymorphisms in pure cultures of electrogenic bacteria grown under controlled laboratory conditions with or without BES stimuli. Our analysis reveals how genetic makeup of the biofilm vs. planktonic populations of electrogenic microbes diverges away from each other during evolution. Our approach also allows us to reveal previously hidden players in the genetic networks underlying the EET activity during BES operation. Lastly, our results emphasize the need to characterize evolutionary trajectories of potential biocatalysts in pure cultures as well as multi-species communities to advance the development and efficacy of bioelectrochemical systems.

Microbial extracellular electron transfer and its relevance to iron corrosion

Extracellular electron transfer (EET) is a microbial metabolism that enables efficient electron transfer between microbial cells and extracellular solid materials. Microorganisms harbouring EET abilities have received considerable attention for their various biotechnological applications, including bioleaching and bioelectrochemical systems. On the other hand, recent research revealed that microbial EET potentially induces corrosion of iron structures. It has been well known that corrosion of iron occurring under anoxic conditions is mostly caused by microbial activities, which is termed as microbiologically influenced corrosion (MIC). Among diverse MIC mechanisms, microbial EET activity that enhances corrosion via direct uptake of electrons from metallic iron, specifically termed as electrical MIC (EMIC), has been regarded as one of the major causative factors. The EMIC-inducing microorganisms initially identified were certain sulfate-reducing bacteria and methanogenic archaea isolated from marine environments. Subsequently, abilities to induce EMIC were also demonstrated in diverse anaerobic microorganisms in freshwater environments and oil fields, including acetogenic bacteria and nitrate-reducing bacteria. Abilities of EET and EMIC are now regarded as microbial traits more widespread among diverse microbial clades than was thought previously. In this review, basic understandings of microbial EET and recent progresses in the EMIC research are introduced.

Influence of Humic Acid Complexation with Metal Ions on Extracellular Electron Transfer Activity.

Humic acids (HAs) can act as electron shuttles and mediate biogeochemical cycles, thereby influencing the transformation of nutrients and environmental pollutants. HAs commonly complex with metals in the environment, but few studies have focused on how these metals affect the roles of HAs in extracellular electron transfer (EET). In this study, HA-metal (HA-M) complexes (HA-Fe, HA-Cu, and HA-Al) were prepared and characterized. The electron shuttle capacities of HA-M complexes were experimentally evaluated through microbial Fe(III) reduction, biocurrent generation, and microbial azoreduction. The results show that the electron shuttle capacities of HAs were enhanced after complexation with Fe but were weakened when using Cu or Al. Density functional theory calculations were performed to explore the structural geometry of the HA-M complexes and revealed the best binding sites of the HAs to metals and the varied charge transfer rate constants (k). The EET activity of the HA-M complexes were in the order HA-Fe > HA-Cu > HA-Al. These findings have important implications for biogeochemical redox processes given the ubiquitous nature of both HAs and various metals in the environment.

Light-induced extracellular electron transport by the marine raphidophyte Chattonella marina.

There is increasing interest in extracellular electron transfer (EET) from organisms to receptors, particularly in anaerobic biofilms at mineral surfaces. Less attention has been given to EET by planktonic organisms in oxic environments where extracellular electron generation and transport might be expected to be of limited consequence. In this study, the EET activity of the photosynthetic marine raphidophyte, Chattonella marina, was examined using a mediatorless photosynthetic microbial fuel cell with results showing positive light response. Electron output by organisms present in cell suspension was substantially higher than those present in biofilms at the electrode surface. Indeed, current generation under light illumination of the C. marina suspension continued even when contact between the organisms and the electrodes was prevented by dialysis membrane, suggesting that soluble electron carriers secreted by C. marina were facilitating the EET process. Cyclic voltammetry measurements of the cell-free exudate showed redox peaks in the range of 0.1-0.5 V (vs Ag/AgCl), confirming that redox active species were present in the cell suspension. Facilitation of electron transfer from the planktonic organism to the anode by endogenous redox-active exudates appears to be critical to current generation. The ability of these exudates to remain in their reduced state in the presence of oxygen is possibly a function of the spin-restricted nature of oxygen-mediated exudate oxidation. Quantification of the EET processes operating in this planktonic system assists in understanding the means and extent to which C. marina induces redox transformations in the external medium with these transformations presumably of benefit to the survival of this organism, potentially including facilitation of iron uptake and induction of toxicity to other organisms.

Wettability-regulated extracellular electron transfer from the living organism of Shewanella loihica PV-4.

C-type cytochromes located on the outer membrane (OMCs) of genus Shewanella act as the main redox-active species to mediate extracellular electron transfer (EET) from the inside of the outer membrane to the external environment: the central challenge that must be met for successful EET. The redox states of OMCs play a crucial role in dictating the rate and extent of EET. Here, we report that the surface wettability of the electrodes strongly influences the EET activity of living organisms of Shewanella loihica PV-4 at a fixed external potential: the EET activity on a hydrophilic electrode is more than five times higher than that on a hydrophobic one. We propose that the redox state of OMCs varies significantly at electrodes with different wettability, resulting in different EET activities.

Hybrid Bio-Organic Interfaces with Matchable Nanoscale Topography for Durable High Extracellular Electron Transfer Activity

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