Indirect Extracellular Electron Transfer Research Articles

Accelerating cell division of Shewanella oneidensis to promote extracellular electron transfer rate for efficient pollution treatment

The slow rate of extracellular electron transfer (EET) of electroactive microorganisms (EAMs) remains a predominate bottleneck that restricts practical applications of bio-electrochemical systems. Cell division has significant effects on cell cycle, morphology, growth and metabolism. However, the relation between cell division and the EET rate of Shewanella oneidensis has not been established. Here, we employed modular engineering strategy to accelerate DNA replication in the C period and divisome formation in the D period of cell cycle, which decreased cellular volume and enhanced the EET efficiency. Assembly of the C and D period modules further decreased the cell volume by 82.0 % and enhanced power density by 3.12-fold. Electrophysiological and transcriptomic analyses synergistically revealed that the programmed cell volume decrease facilitated lactate uptake and cellular metabolism due to the increased specific surface area (SSA), which consequently reinforced intracellular electron generation. Moreover, the reduced cell size facilitated electroactive biofilm formation. Finally, programmed increase in riboflavin biosynthesis and transport further strengthened indirect EET and boosted output power density to 1537.8 ± 116.9 mW m−2, 21.1-fold of that of the WT. The engineered strains exhibited superior abilities for Cr6+ reduction and azo dyes degradation. This study shed light on the underlying mechanism how reduced cell size impacts electrophysiology of EAMs, and indicated accelerating cell division is a promising avenue to increase the EET of EAMs for efficient environmental pollution treatment.

Electrochemical analysis of extracellular electron transfer process of Pseudomonas aeruginosa NEJ07R using pyocyanin on a carbon electrode

Indirect extracellular electron transfer (IEET), or mediated electron transfer (MET), is a process that has attracted attention for its application in electrochemical-microbial technologies for power generation, wastewater treatment, or electrofermentation. Pseudomonas aeruginosa is a bacterium known to carry out IEET because it produces its own redox mediator (RM), pyocyanin (PYO), which has electrochemical properties such as a high electron transfers rate and low resistance to charge transfer. However, other physical processes, such as PYO adsorption on the electrode, can alter that function. In this work, interaction parameters and adsorption-free energy values were determined to obtain an adsorption mechanism on a carbon-based electrode. Furthermore, the effect of PYO addition and its adsorption were studied in the IEET process of P. aeruginosa NEJ07R. The electrochemical profiling of PYO by cyclic voltammetry (CV) and chronocoulometry (CC) measurements allowed the calculation of the Laviron interaction parameter (ν'gθT). Since the values of ν'gθT > 0, it was determined that PYO maintains attractive interactions with the surface in cathodic and anodic reaction. Moreover, the low interaction free energies (ΔG0' ≈ −2.91 ×10−5 ± 1.28 ×10−8 kJ/cm3 and -−2.92 ×10−5 ± 2.74 ×10−8 kJ/cm3 for reduction and oxidation, respectively) suggests that the interactions are weak. As for the IEET of P. aeruginosa NEJ07R, the microbial catalysis process on a carbon cloth electrode was confirmed by adding PYO to the electrochemical cell. Electrochemical and FT-IR characterization confirmed the adsorption of PYO on the surface, so it is proposed that P. aeruginosa NEJ07R takes advantage of PYO adsorption to carry out IEET.

Efficient power recovery from aromatic compounds by a novel electroactive bacterium Pseudomonas putida B6-2 in microbial fuel cells

Environmental pollution caused by aromatic compounds (ACs) has long stimulated the emergence of creative bioremediation strategies. Pseudomonas putida B6–2, an aerobic ACs degrading bacterium, successfully coupled ACs degradation with power generation in microbial fuel cells (MFCs) when a nickel-coated sponge with a three-dimensional macroporous structure served as the anode. The simultaneous removal of coexisting ACs pollutants including biphenyl (BP), carbazole (CA), and dibenzothiophene (DBT) were achieved in MFCs, accompanied by the efficient power densities of 469 mW m−2, 499 mW m−2, 541 mW m−2, and 587 mW m−2 for single (BP), binary (BP+DBT or BP+CA), and ternary (BP+DBT+CA) ACs fueled MFCs, respectively. Degradation dynamics analysis indicated that the degradation of BP, CA, and DBT was consistent with the pseudo-first order kinetic model in the ternary ACs fueled MFC, and BP had a higher degradation rate (kobs=0.0337 h–1) than DBT (kobs=0.0201 h–1) and CA (kobs=0.0149 h–1). It is the first time that P. putida B6–2 was discovered as an electroactive bacterium with high extracellular electron transfer (EET) capability. The EET preliminary analysis showed that P. putida B6–2 might transfer extracellular electrons by redox mediators and microbial nanowires. The mediators may play a key role in indirect EET at the start-up period of the MFCs, while the nanowires may involve in direct EET during the whole discharge process of the MFCs. The interesting findings may lay a foundation for further exploration of the EET of P. putida B6–2 and bioremediation of ACs pollutants.

Au(III)-induced extracellular electron transfer by Burkholderia contaminans ZCC for the bio-recovery of gold nanoparticles

The biorecovery of gold (Au) by microbial reduction has received increasing attention, however, the biomolecules involved and the mechanisms by which they operate to produce Au nanoparticles have been not resolved. Here we report that Burkholderia contaminans ZCC is capable of reduction of Au(III) to Au nanoparticles on the cell surface. Exposure of B. contaminans ZCC to Au(III) led to significant changes in the functional group of cell proteins, with approximately 11.1% of the (C–C/C–H) bonds being converted to CO (8.1%) and C–OH (3.0%) bonds and 29.4% of the CO bonds being converted to (C–OH/C–O–C/P–O–C) bonds, respectively. In response to Au(III), B. contaminans ZCC also displayed the ability of extracellular electron transfer (EET) via membrane proteins and could produce reduced riboflavin as verified by electrochemical and liquid chromatography-mass spectrometric results, but did not do so without Au(III) being present. Addition of exogenous reduced riboflavin to the medium suggested that B. contaminans ZCC could utilize indirect EET via riboflavin to enhance the rate of reduction of Au(III). Transcriptional analysis of the riboflavin genes (ribBDEFH) supported the view of the importance of riboflavin in the reduction of Au(III) and its importance in the biorecovery of gold.

Cellular electron transfer in anaerobic photo-assisted biocathode microbial electrosynthesis systems for acetate production from inorganic carbon (HCO3–)

The crucial role played by the cellular electron transfer in the production of acetate from aqueous inorganic carbon (HCO3–, bicarbonate) in anaerobic photo-assisted microbial electrosynthesis (MES) biocathodes incorporating a WO3/MoO3/g-C3N4 Z-scheme heterojunction and either Serratia marcescens Q1 or Stenotrophomonas sp. JY6 electrotrophs is disclosed. The electron transfer inhibitor 2,4-dinitrophenol (DNP) diffusing through the bacterial cell membrane was used to alter the ratio of indirect (via H2) and direct extracellular electron transfer processes, resulting in lower physiological release of extracellular polymeric substances (EPS) and lower acetate production rates under a transitional period. DNP effectively cancelled the synergistic effects on acetate production produced by the photo-irradiation of the WO3/MoO3/g-C3N4 heterojunction biocathode. The physiological release of EPS with either a compositional diversity (Q1) or varying composition percentages (Q1 or JY6), was correlated to photo-irradiation and to the electrotrophic species, whereas the total amount of EPS was closely related with the electrotrophic DNP-based cellular electron transfer processes. Interestingly, the microorganisms adapted and overcame the impact of DNP after only 4 days of continuous operation and restored their normal physiological metabolism after 7.5 d operation with periodical replenishment of bicarbonate. This study provides a detailed theoretical basis to understand the impact of direct and indirect extracellular electron transfer in hybrid photo-assisted MES biocathodes and elucidates the behavior of non-photosynthetic electrotrophic bacteria favoring high conversion of aqueous inorganic carbon (HCO3–) to acetate.

Enhanced mechanism of extracellular electron transfer between semiconducting minerals anatase and Pseudomonas aeruginosa PAO1 in euphotic zone

Focusing the marine euphotic zone, which is the pivotal region for interaction of solar light-mineral-microorganism and the elements cycle, we have conducted the research on the mechanism of semiconducting minerals promoting extracellular electron transfer with microorganisms in depth. Therein, anatase which is one of the most representative semiconducting minerals in marine euphotic zone was selected. The mineralogical characterization of anatase was identified by ESEM, AFM, EDS, Raman, XRD, and its semiconducting characteristics was determined by UV–Vis and Mott-Schottky plots. Determined by the electrochemical measurement of I-t curves, the photocurrent density of anatase was more prominent than dark current density. Pseudomonas aeruginosa PAO1 was widely distributed in the euphotic zone, and its mutants of operons deficient in biosynthesis pyocyanin (Δphz1Δphz2) and pili deficient (ΔpilA) were employed in this study. I-t curves indicated that both direct and indirect extracellular electron transfer processes occurred between anatase and PAO1. The indirect electron transfer depending on pyocyanin secreted by PAO1 was the main electron transfer mode. This work demonstrated the light-driven extracellular electron transfer and further revealed the photo-catalyzed mechanisms between anatase and PAO1 in marine euphotic zone.

Production of bioelectricity may play an important role for the survival of Xanthomonas campestris pv. campestris (Xcc) under anaerobic conditions

The plant pathogen Xanthomonas is commonly found in biocontaminated bioreactors; however, few studies have evaluated the growth and impacts of this microorganism on bioreactors. In this study, we examined the characteristics of Xanthomonas campestris pv. campestris (Xcc). Our results showed that Xcc could reduce metal Fe (III) and decolorise methyl orange in vitro. Moreover, I-t and cyclic voltammetry curves showed that Xcc could generate bioelectricity and had two extracellular electron transfer pathways, similar to that of Shewanella. Based on the spectral analysis of intact cells and scanning electron microscopy analysis, one pathway was speculated to involve cytochrome C by direct contact with the pili or cell surface. The other pathway may involve indirect mediators, such as redox substrates, among extracellular polymeric substances. For the direct extracellular electron transfer process, the charge transfer coefficient α, electron number n, and the electron transfer rate constant ks were determined to be 0.49, 2.6, and 2.2 × 10−3 s−1, respectively. In the indirect extracellular electron transfer processes, the values of α, n, and ks were 0.52, 4, and 1.21 s−1, respectively. Of these two transfer methods, indirect electron transfer is dominant and faster than direct electron transfer. Moreover, after mutation of the dsbD gene, which is important for indirect electron transfer, the electrochemical parameters α, n, and ks decreased. Our findings reveal a new anaerobic mechanism mediating the survival of Xcc during wastewater treatment, and may help develop new strategies for preventing Xcc growth during wastewater treatment.

Surface modification of Shewanella oneidensis MR-1 with polypyrrole-dopamine coating for improvement of power generation in microbial fuel cells

Modification of exoelectrogens with conjugated polymers is an efficient strategy to improve extracellular electron transfer (EET) from individual cells to electrode. However, due to the hydrophobic nature of conductive polymers, improving cell-electrode adhesion needs to be addressed. In this research, we report the modification of Shewanella oneidensis MR-1 by successively in situ coating individual cells with polypyrrole (PPy) and adhesive polydopamine (PDA). The PPy-PDA modified cells display improved conductivity and adhesion. We employ PPy-PDA modified cells as anode in microbial fuel cells (MFCs) and find that electro-activity of anode is greatly improved as direct EET is improved, and riboflavin secretion which is conducive to indirect EET is enhanced. The maximum power density of MFCs employing PPy-PDA modified cells is 11.8 and 4.8 higher than that of PPy modified and unmodified cells, respectively. Our results indicate this in situ modification of exoelectrogens with PPY-PDA offers a facile and promising strategy for performance improvement of MFCs.

Electricity from anaerobic methane oxidation by a single methanogenic archaeon Methanosarcina barkeri

Anaerobic oxidation of methane (AOM) coupled with electron transfer to electrodes has recently showed great promise for energy harvesting in microbial electrochemical reactors. To date this process has been reported limited to the syntrophic cooperation of archaea and bacteria in mixed cultures. It remains unknown whether pure archaea alone can catalyze electricity production from methane. Here we report the performance of AOM-driven electricity generation by a single methanogen Methanosarcina barkeri. At a poised electrode potential of +300 mV vs. SHE, a maximum current of 649.7 mA m−2 was achieved from methane with an appreciable Coulombic efficiency up to 86.9%. The electrogenic mechanism analysis demonstrated that M. barkeri donated most of electrons to electrodes directly and secreted soluble redox-active compounds to facilitate indirect extracellular electron transfer (EET). Electrochemical in situ Fourier transform infrared spectra and metabolic inhibitions suggested the methyl-coenzyme M reductase α-subunit (McrA), NiFe hydrogenases and cytochrome proteins were involved in the AOM-EET process. This work provides the first proof of concept for AOM-powered electricity production by a single archaeon via reversed methanogenesis and suggests methanogens may play an important role in coupling AOM with the reduction of solid electron acceptors in natural environments.

Extracellular electron transfer in fermentative bacterium Anoxybacter fermentans DY22613T isolated from deep-sea hydrothermal sulfides

Dissimilatory Fe(III)-reducing bacteria (DIRBs) could reduce extracellular Fe(III) to Fe(II) via extracellular electron transfer (EET), playing an important role in biogeochemical cycling of Fe(III). Previous studies have noted the key role of multi-heme c-type cytochromes (MHCs) involved in EET by respiratory-type DIRBs, and proposed indirect electron transfer through the use of redox electron shuttles (e.g., flavins) or Fe(III)-chelation. However, knowledge about the EET of fermentative DIRBs was vitally scarce. Here, Anoxybacter fermentans DY22613T is a typical fermentative DIRB isolated from deep-sea hydrothermal sulfides, and it could utilize soluble Fe(III)-citrate and solid Fe(III)-bearing minerals as extracellular electron acceptors. Unlike respiratory-type DIRBs that utilize MHCs, this strain lacked MHCs to mediate EET. Besides, it did not adopt Fe(III)-chelation to mediate indirect EET. Nonetheless, genes encoding biosynthesis pathway of redox molecules (e.g., flavins) were found in its genome and their gene expression was up-regulated with Fe(III) reduction, suggesting redox molecules may mediate indirect EET by this strain. Subsequent physiological and biochemical tests further demonstrated endogenous riboflavin acted as main electron shuttles to mediate indirect EET by this strain, and menaquinone, indole played an assistant role in this process. Besides, this strain could employ exogenous humic acids to facilitate indirect EET. The mode of exogenous and endogenous redox molecules to co-mediate indirect EET by fermentative A. fermentans DY22613T, expands our knowledge about EET of fermentative DIRBs, and would contribute to better understand its ecological role in the biogeochemistry cycle of iron.

Rediverting Electron Flux with an Engineered CRISPR-ddAsCpf1 System to Enhance the Pollutant Degradation Capacity of Shewanella oneidensis.

Pursuing efficient approaches to promote the extracellular electron transfer (EET) of extracellular respiratory bacteria is essential to their application in environmental remediation and waste treatment. Here, we report a new strategy of tuning electron flux by clustered regularly interspaced short palindromic repeat (CRISPR)-ddAsCpf1-based rediverting (namely STAR) to enhance the EET capacity of Shewanella oneidensis MR-1, a model extracellular respiratory bacterium widely present in the environment. The developed CRISPR-ddAsCpf1 system enabled approximately 100% gene repression with the green fluorescent protein (GFP) as a reporter. Using a WO3 probe, 10 representative genes encoding for putative competitive electron transfer proteins were screened, among which 7 genes were identified as valid targets for EET enhancement. Repressing the valid genes not only increased the transcription level of the l-lactate metabolism genes but also affected the genes involved in direct and indirect EET. Increased riboflavin production was also observed. The feasibility of this strategy to enhance the bioreduction of methyl orange, an organic pollutant, and chromium, a typical heavy metal, was demonstrated. This work implies a great potential of the STAR strategy with the CIRPSR-ddAsCpf1 system for enhancing bacterial EET to favor more efficient environmental remediation applications.

High performance of microbial fuel cell afforded by metallic tungsten carbide decorated carbon cloth anode

Developing high efficient anode is of great significances for the application of microbial fuel cells. Tungsten carbide attracts our attention due to its platinum-like metallic conductivity, corrosion resistance and low cost. In this work, tungsten carbide nanoparticles are pasted on carbon cloth as the anode for mediator-less microbial fuel cell. The surface of obtained anode is quickly attached with highly dense biofilm that accounts for a fast extracellular electron transfer rate. Microbial community structure analysis at the genus level shows that the main exoelectrogens are Geobacter, Geothrix and Pseudomonas, all of which can completely decompose acetate and transfer electrons through both direct and indirect extracellular electron transfer pathways. Consequently, it contributes to higher power density (3.26 W m−2), better chemical oxygen demand removal rate (95.5%) and greater coulombic efficiency (83.2%), which are 2.14-fold, 1.22-fold, 1.71-fold of that obtained by the naked carbon cloth anode (1.52 W m−2, 78.1%, 48.6%), respectively. Especially for the coulombic efficiency, it is obviously higher than others’ reported works in the similar microbial fuel cells system, being attributable to the synergistic effect of exoelectrogen. In virtue of the above advantages, tungsten carbide is a potential candidate for the further application of microbial fuel cells.

Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm

Desulfovibrio alaskensis G20 biofilms were cultivated on 316 steel, 1018 steel, or borosilicate glass under steady-state conditions in electron-acceptor limiting (EAL) and electron-donor limiting (EDL) conditions with lactate and sulfate in a defined medium. Increased corrosion was observed on 1018 steel under EDL conditions compared to 316 steel, and biofilms on 1018 carbon steel under the EDL condition had at least twofold higher corrosion rates compared to the EAL condition. Protecting the 1018 metal coupon from biofilm colonization significantly reduced corrosion, suggesting that the corrosion mechanism was enhanced through attachment between the material and the biofilm. Metabolomic mass spectrometry analyses demonstrated an increase in a flavin-like molecule under the 1018 EDL condition and sulfonates under the 1018 EAL condition. These data indicate the importance of S-cycling under the EAL condition, and that the EDL is associated with increased biocorrosion via indirect extracellular electron transfer mediated by endogenously produced flavin-like molecules.

Extracellular electron transfer of Enterobacter cloacae SgZ-5T via bi-mediators for the biorecovery of palladium as nanorods

In nature, microbes use extracellular electron transfer (EET) to recover noble metals. Most attention has been paid to the biorecovery process occurring intracellularly and on the cell surface. In this work, we report that Pd nanorods could be biosynthesized by Enterobacter cloacae SgZ-5T in the extracellular space. This bacterium possesses both a direct EET pathway through membrane redox systems and an indirect EET pathway via the self-secreted electron carrier hydroquinone (HQ). When exposed to Pd(II), the bacteria adjusted their metabolic pathway and membrane-bound proteins to secrete riboflavin (RF). However, no HQ was detected in the supernatant in presence of Pd(II). No significant change was observed through metabolomic analysis regarding the abundance of HQ in presence of Pd(II) compared to Pd(II)-free supernatant. Similar results were also obtained through transcriptomic analysis of YqjG gene encoding glutathionyl-HQ reductase synthase. X-ray photoelectron spectroscopic evidence indicated that HQ may adsorb to the surface of Pd nanorods. Moreover, the gene encoding RF synthase (ribE) was up-regulated in the present of Pd(II), suggesting that this bioreduction process induced RF synthase, which had been shown in previous results. The UV–vis spectroscopy data demonstrated that the Pd(II) reduction rate was enhanced by 5%, 5.5% and 30% by the addition of 3.33 μM HQ, 3.33 μM RF and the both, respectively. All these results revealed that the bi-mediators secreted by bacteria were beneficial for biorecovery of Pd. This work is of significance for understanding metal biorecovery processes and natural biogeochemical processes.

Shewanella putrefaciens CN32 outer membrane cytochromes MtrC and UndA reduce electron shuttles to produce electricity in microbial fuel cells

The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c-type cytochromes (OM c-Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c-Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact. Strikingly, the depression of EET by the deletion of mtrC could be partially relieved by acclimation, which might be due to an increase in the transmembrane transport of electron shuttles and/or the activation of other redox proteins. These results suggested that MtrC may be the primary reductase of flavins to ensure fast indirect EET, which plays a crucial role in MFC electricity generation.

Bioelectrochemical systems for simultaneously production of methane and acetate from carbon dioxide at relatively high rate

This study describes the performance of bioelectrochemical systems, based on electrochemically active mixed culture, capable of reducing CO2 to CH4 and CH3COOH via direct and/or indirect extracellular electron transfer. The metabolic pathway and end products of this mixed culture were highly dependent on the set cathode potentials. Only CH4 and H2 were produced when the cathode potentials were set in the range from −850 to −950 mV (vs. Ag/AgCl). At potentials more negative than −950 mV, CH4, H2 and CH3COOH were simultaneously produced. With a relatively large cathode surface area of 49 cm−2, CH4 and CH3COOH were produced at high rates of 129.32 mL d−1 and 94.73 mg d−1, respectively (at potential of −1150 mV). The highest current capture efficiency reached to 97% in batch potentiostatic experiments. These results presented here suggest that mixed culture show the ability to directly accept electrons from the electrode and abiotically produce H2 to convert CO2 into various organic compounds.

Microbial generation of H2 or CH4 coupled to wastewater treatment in bioelectrochemical systems

Microbial biocathodes can be employed to produce reduced value-added compounds, such as H2 or CH4 in bioelectrochemical systems (BES). Here we show that selected anaerobic microbial cultures are capable of catalyzing H2 or CH4 production by using a carbon electrode as electron donor, via direct or indirect extracellular electron transfer mechanisms. The potential of biocathodes for gaseous biofuels production coupled to wastewater treatment in BES is discussed.

Bioelectrochemical reduction of CO 2 to CH 4 via direct and indirect extracellular electron transfer by a hydrogenophilic methanogenic culture

This study describes the performance of a microbial biocathode, based on a hydrogenophilic methanogenic culture, capable of reducing carbon dioxide to methane, at high rates (up to 0.055 ± 0.002 mmol d −1 mgVSS −1) and electron capture efficiencies (over 80%). Methane was produced, at potentials more negative than −650 mV vs. SHE, both via abiotically produced hydrogen gas (i.e., via hydrogenophilic methanogenesis) and via direct extracellular electron transfer. The relative contribution of these two mechanisms was highly dependent on the set cathode potential. Both cyclic voltammetry tests and batch potentiostatic experiments indicated that the capacity for extracellular electron transfer was a constitutive trait of the hydrogenophilic methanogenic culture. In principle, both electrons and carbon dioxide required for methane production could be obtained from a bioanode carrying out the oxidation of waste organic substrates.