Geobacter Sulfurreducens Biofilms Research Articles

Influence of support materials on the electroactive behavior, structure and gene expression of wild type and GSU1771-deficient mutant of Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens DL1 is a metal-reducing dissimilatory bacterium frequently used to produce electricity in bioelectrochemical systems (BES). The biofilm formed on electrodes is one of the most important factors for efficient electron transfer; this is possible due to the production of type IV pili and c-type cytochromes that allow it to carry out extracellular electron transfer (EET) to final acceptors. In this study, we analyzed the biofilm formed on different support materials (glass, hematite (Fe2O3) on glass, fluorine-doped tin oxide (FTO) semiconductor glass, Fe2O3 on FTO, graphite, and stainless steel) by G. sulfurreducens DL1 (WT) and GSU1771-deficient strain mutant (Δgsu1771). GSU1771 is a transcriptional regulator that controls the expression of several genes involved in electron transfer. Different approaches and experimental tests were carried out with the biofilms grown on the different support materials including structure analysis by confocal laser scanning microscopy (CLSM), characterization of electrochemical activity, and quantification of relative gene expression by RT-qPCR. The gene expression of selected genes involved in EET was analyzed, observing an overexpression of pgcA, omcS, omcM, and omcF from Δgsu1771 biofilms compared to those from WT, also the overexpression of the epsH gene, which is involved in exopolysaccharide synthesis. Although we observed that for the Δgsu1771 mutant strain, the associated redox processes are similar to the WT strain, and more current is produced, we think that this could be associated with a higher relative expression of certain genes involved in EET and in the production of exopolysaccharides despite the chemical environment where the biofilm develops. This study supports that G. sulfurreducens is capable of adapting to the electrochemical environment where it grows.

C-di-GMP and AHL signals-triggered chemical communication under electrical signaling disruption restores Geobacter sulfurreducens biofilm formation.

Electrogenic biofilms, which have attracted considerable attention in simultaneous wastewater treatment and energy recovery in bioelectrochemical systems, are regulated by chemical communication and potassium channel-mediated electrical signaling. However, how these two communication pathways interact with each other has not been thoroughly investigated. This study first explored the roles of chemical communication, including intracellular bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and extracellular N-acyl-homoserine lactone (AHL)-mediated quorum sensing, in electrogenic biofilm formation through an integrated analysis of transcriptomics and metabolomics. Electrical signaling disruption inhibited the formation and electroactivity of Geobacter sulfurreducens biofilm, which was mainly ascribed to the reduction in biofilm viability and extracellular protein/polysaccharide ratio. The upregulation of expression levels of genes encoding c-di-GMP and AHL synthesis by transcriptomic analysis, and the increased secretion of N-butanoyl-L-homoserine lactone by metabolomic analysis confirmed the enhancement of chemical communication under electrical signaling disruption, thus indicating a compensatory mechanism among different signaling pathways. Furthermore, protein-protein interaction network showed the convergence of different signaling pathways, with c-di-GMP-related genes acting as central bridges. This study highlights the interaction of different signaling pathways, especially the resilience of c-di-GMP signaling to adverse external stresses, thereby laying the foundation for facilitating electrogenic biofilm formation under adverse conditions in practical applications.

Study of the influence of nanoscale porosity on the microbial electroactivity between expanded graphite electrodes and Geobacter sulfurreducens biofilms.

Expanded graphite (EG) electrodes gather several advantages for their utilization in microbial electrochemical technologies (MET). Unfortunately, the low microbial electroactivity makes them non-practical for implementing them as electrodes. The objective of this work is to explore the enhancement of microbial electroactivity of expanded graphite (commercial PV15) through the generation of nanopores by CO2 treatment. The changes in properties were thoroughly analysed by TG, XRD, Raman, XPS, gas adsorption, SEM and AFM, as well as microbial electroactivity in the presence of Geobacter sulfurreducens. Nanopores remarkably enhance the microbially derived electrical current (60-fold increase). Given the inaccessibility of micron-sized bacteria to these nanopores, it is suggested that the electric charge exchanged by electroactive microorganisms might be greatly affected by the capability of the electrode to compensate these charges through ion adsorption. The increased microbial current density produced on activated PV15 opens the possibility of using such materials as promising electrodes in MET.

Periodic step polarization accelerates electron recovery by electroactive biofilms (EABs).

Relatively low rate of electron recovery is one of the factors that limit the advancement of bioelectrochemical systems. Here, new periodic polarizations were investigated with electroactive biofilms(EABs) enriched from activated sludge and Geobacter sulfurreducens biofilms. When representative anode potentials (Ea ) were applied, redox centers with midpoint potentials (Emid ) higher than Ea were identified by localized cyclic voltammetry. The electrons held by these redox centers were accessible when Ea was raised to 0.4 V (vs. Ag/AgCl). New periodic polarizations that discharge at 0.4 V recovered electrons faster than normal periodic and fixed-potential polarizations. The best-performing periodic step polarization accelerated electron recovery by 23%-24% and 12%-76% with EABs and G. sulfurreducens biofilms, respectively, compared to the fixed-potential polarization. Quantitative reverse transcription polymerase chain reactionshowed an increased abundance of omcZ mRNA transcripts from G. sulfurreducens after periodic step polarization. Therefore, both the rate of energy recovery by EABs and the performance of bioelectrochemical systems can be enhanced by improving the polarization schemes.

Methylmercury formation in biofilms of Geobacter sulfurreducens.

Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens. Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy. Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10-14 L ∙ cell-1 ∙ h-1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms. These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

Microbial biofilms as living photoconductors due to ultrafast electron transfer in cytochrome OmcS nanowires

Light-induced microbial electron transfer has potential for efficient production of value-added chemicals, biofuels and biodegradable materials owing to diversified metabolic pathways. However, most microbes lack photoactive proteins and require synthetic photosensitizers that suffer from photocorrosion, photodegradation, cytotoxicity, and generation of photoexcited radicals that are harmful to cells, thus severely limiting the catalytic performance. Therefore, there is a pressing need for biocompatible photoconductive materials for efficient electronic interface between microbes and electrodes. Here we show that living biofilms of Geobacter sulfurreducens use nanowires of cytochrome OmcS as intrinsic photoconductors. Photoconductive atomic force microscopy shows up to 100-fold increase in photocurrent in purified individual nanowires. Photocurrents respond rapidly (<100 ms) to the excitation and persist reversibly for hours. Femtosecond transient absorption spectroscopy and quantum dynamics simulations reveal ultrafast (~200 fs) electron transfer between nanowire hemes upon photoexcitation, enhancing carrier density and mobility. Our work reveals a new class of natural photoconductors for whole-cell catalysis.

Enhancing electrical outputs of the fuel cells with Geobacter sulferreducens by overexpressing nanowire proteins.

Protein nanowires are critical electroactive components for electron transfer of Geobacter sulfurreducens biofilm. To determine the applicability of the nanowire proteins in improving bioelectricity production, their genes including pilA, omcZ, omcS and omcT were overexpressed in G.sulfurreducens. The voltage outputs of the constructed strains were higher than that of the control strain with the empty vector (0.470-0.578 vs. 0.355 V) in microbial fuel cells (MFCs). As a result, the power density of the constructed strains (i.e. 1.39-1.58 W m-2 ) also increased by 2.62- to 2.97-fold as compared to that of the control strain. Overexpression of nanowire proteins also improved biofilm formation on electrodes with increased protein amount and thickness of biofilms. The normalized power outputs of the constructed strains were 0.18-0.20 W g-1 that increased by 74% to 93% from that of the control strain. Bioelectrochemical analyses further revealed that the biofilms and MFCs with the constructed strains had stronger electroactivity and smaller internal resistance, respectively. Collectively, these results demonstrate for the first time that overexpression of nanowire proteins increases the biomass and electroactivity of anode-attached microbial biofilms. Moreover, this study provides a new way for enhancing the electrical outputs of MFCs.

Water evaporation-induced electricity with Geobacter sulfurreducens biofilms.

Water evaporation–induced electricity generators (WEGs) have recently attracted extensive research attention as an emerging renewable energy–harvesting technology that harvests electricity directly from water evaporation. However, the low power output, limited available material, complicated fabrication process, and extremely high cost have restricted wide applications of this technology. Here, a facile and efficient WEG prototype based on Geobacter sulfurreducens biofilm was demonstrated. The device can generate continuous electric power with a maximum output power density of ~685.12 μW/cm2, which is two orders of magnitude higher than that of previously reported analogous devices. The superior performance of the device is attributed to the intrinsic properties of the G. sulfurreducens biofilm, including its hydrophilicity, porous structure, conductivity, etc. This study not only presents the unprecedented evaporating potential effect of G. sulfurreducens biofilms but also paves the way for developing hydrovoltaic technology with biomaterials.

What is the Role of Individual Species within Bidirectional Electroactive Microbial Biofilms: A Case Study on Desulfarculus baarsii and Desulfurivibrio alkaliphilus

AbstractDesulfarculus baarsii and Desulfurivibrio alkaliphilus are strictly anaerobic bacteria existing in marine sediments. D. baarsii gains energy through reducing sulphate and D. alkaliphilus is able to reduce elemental sulphur, thiosulphate and polysulphide in seawater. Both organisms were previously identified as key organisms in sediment derived, bidirectional electroactive biofilms. Here, we investigated the electrochemical performance of these two bacteria in bio‐electrochemical systems and their possible involvement in anodic and cathodic reactions. The results show that D. baarsii was unable to donate or accept electrons to/from an electrode, while D. alkaliphilus was able to catalyse both anodic and cathodic reactions and interact with the electrode through direct or potentially indirect electron transfer. Raman spectra of D. alkaliphilus electrode biofilms showed a high similarity to Geobacter sulfurreducens biofilms, including the specific bands of cytochromes b and c, explaining the electroactivity of D. alkaliphilus in bioelectrochemical reactions.

Competition of two highly specialized and efficient acetoclastic electroactive bacteria for acetate in biofilm anode of microbial electrolysis cell

Maintaining functional stability of microbial electrolysis cell (MEC) treating wastewater depends on maintaining functional redundancy of efficient electroactive bacteria (EAB) on the anode biofilm. Therefore, investigating whether efficient EAB competing for the same resources (electron donor and acceptor) co-exist at the anode biofilm is key for the successful application of MEC for wastewater treatment. Here, we compare the electrochemical and kinetic properties of two efficient acetoclastic EAB, Geobacter sulfurreducens (GS) and Desulfuromonas acetexigens (DA), grown as monoculture in MECs fed with acetate. Additionally, we monitor the evolution of DA and GS in co-culture MECs fed with acetate or domestic wastewater using fluorescent in situ hybridization. The apparent Monod kinetic parameters reveal that DA possesses higher jmax (10.7 ± 0.4 A/m2) and lower KS, app (2 ± 0.15 mM) compared to GS biofilms (jmax: 9.6 ± 0.2 A/m2 and KS, app: 2.9 ± 0.2 mM). Further, more donor electrons are diverted to the anode for respiration in DA compared to GS. In acetate-fed co-culture MECs, DA (98% abundance) outcompete GS for anode-dependent growth. In contrast, both EAB co-exist (DA: 55 ± 2%; GS: 24 ± 1.1%) in wastewater-fed co-culture MECs despite the advantage of DA over GS based on kinetic parameters alone. The co-existence of efficient acetoclastic EAB with high current density in MECs fed with wastewater is significant in the context of functional redundancy to maintain stable performance. Our findings also provide insight to future studies on bioaugmentation of wastewater-fed MECs with efficient EAB to enhance performance.

Electric field stimulates production of highly conductive microbial OmcZ nanowires.

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of previously unknown cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S/cm), and 3-fold higher stiffness (1.5 GPa), than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function, and observe pH-induced conformational switching to β-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modelling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically-produced, highly-conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.

Physiological potential of extracellular polysaccharide in promoting Geobacter biofilm formation and extracellular electron transfer

Geobacter sulfurreducens biofilms have promising applications in renewable energy, pollutant bioremediation, and bioelectronic applications. Genetically manipulating G. sulfurreducens biofilms is an effective strategy to improve the capacity of extracellular electron transfer (EET). Extracellular polysaccharide, a sticky component surrounding microbes, plays an important role in EET. Herein, we constructed a mutant of G. sulfurreducens strain PCA overexpressing the gene GSU1501 (part of the ATP-dependent exporter of the polysaccharide biosynthesis gene operon), designated strain PCA-1501, to increase EET capacity. Experimental results showed that the overexpression of GSU1501 increased extracellular polysaccharide secretion by 25.5%, which promoted the formation of biofilm with higher thickness and viability, as well as the content of extracellular c-type cytochromes. Compared with the control strain, the mutant showed a higher capacity of Fe(III) oxide reduction and current generation (increased by 20.4% and 22.2%, respectively). Interestingly, the overexpression of GSU1501 hindered the pili formation by reducing the transcription level of pilA; a compensatory relationship between extracellular polysaccharide and pili in promoting biofilm formation deserves further investigation. This study provides a feasible method to promote the EET capacity of G. sulfurreducens biofilms, which benefit their bioelectrochemical applications.

Overestimation of biofilm conductance determined by using the split electrode as the microbial respiration

Advancing the application of bioelectrochemical systems necessitates the comprehensive understanding of electron transfer process in electrode-associated biofilm. While in situ electrical measurement by using the split electrode can illustrate the electron conduction in biofilm, the accuracy and interpretation of the obtained data is still in debate. Here, conductance of Geobacter sulfurreducens biofilm is measured by using the split electrode as substrate concentration is changed. Biofilm conductance decreases with the current production as the consumption of substrate. In addition, the measured conductance increases with the area of drain electrode (13.8 ± 1.8 μS for 0.5 cm2 and 29.4 ± 4.2 μS for 1 cm2). These results suggest the measured conductance of biofilm is influenced by microbial metabolism. This effect will diminish when the measurement is performed by using electrochemical impedance spectroscopy or under non-turnover condition. Our results provide the insight on the electrochemical origin of biofilm conductance determined by the in situ electrical measurement.

A High‐Performance Membraneless Microfluidic Microbial Fuel Cell for Stable, Long‐Term Benchtop Operation Under Strong Flow

AbstractStrong control over experimental conditions in microfluidic channels provides a unique opportunity to study and optimize membraneless microbial fuel cells (MFCs), particularly with respect to the role of flow. However, improved performance and transferability of results to the wider MFC community require improvements to device stability under all applied operational conditions. To address these challenges, we present an easy‐to‐fabricate membraneless MFC that combines i) O2 protection via a gas diffusion barrier, ii) integrated graphite electrodes, and iii) optimized electrode placement to avoid cross‐contamination under all applied flow rates. Attention to all of these design features in the same platform resulted in the operation of a MFC with a pure‐culture anaerobic Geobacter sulfurreducens biofilm for half a year, that is, six times longer than previously reported, without the use of an oxygen scavenger. As a result of higher device stability under high flow rates, power densities were four times higher than reported previously for microfluidic MFCs with the same biofilm.

Potassium channel blocker inhibits the formation and electroactivity of Geobacter biofilm

Bacteria in biofilms are able to utilize potassium ion channel-mediated electrical signaling to achieve cell–cell communication. However, it remains unclear whether these signals play a role in Geobacter sp. when surrounded by an intense electric field. This study used a potassium channel blocker (tetraethylammonium, TEA) that interfered with the release of K+ but not bacterial growth to demonstrate that potassium ion channel-mediated electrical signaling affected the formation and electroactivity of Geobacter sulfurreducens. The results showed that 5 mM TEA slowed the formation of Geobacter sulfurreducens biofilm, and the current density was ~50% lower than in the control. The electrochemical analyses showed that the electroactivity of the biofilms with TEA addition was inferior. In particular, the micrometer- scale biofilm with TEA exhibited fewer high current peaks, and the species of outermost groups that participated in the electron transfer in Geobacter sulfurreducens biofilms was different from the control. This work provides initial evidence to reveal the role of potassium channels in Geobacter sulfurreducens electroactive biofilms.

Effect of electrode spacing on electron transfer and conductivity of Geobacter sulfurreducens biofilms

To understand electron transport in electrochemically active biofilms, it is necessary to elucidate the heterogeneous electron transport across the biofilm/electrode interface and in the interior of G. sulfurreducens biofilms bridging gaps of varying widths. The conductivity of Geobacter sulfurreducens biofilm bridging nonconductive gaps with widths of 5µm, 10µm, 20µm and 50µm is investigated. Results of electrochemical gating measurement show that biofilm conductivity peaks at the potential of −0.35V vs. Ag/AgCl. The biofilm conductivity increases with gap width (10.4±0.2µScm−1 in 5µm gap, 13.3±0.2µScm−1 in 10µm gap, 16.7±1.4µScm−1 in 20µm gap and 41.8±2.02µScm−1 in 50µm gap). These results revealed that electron transfer in G. sulfurreducens biofilm is a redox-driven. In addition, higher biofilm conductivities and lower charge transfer resistances are observed in all gaps under a turnover condition than in those under a non-turnover condition. Our results offer insights into the spatial heterogeneity of biofilm structure and extracellular electron transfer in electrochemically active biofilms.

Tuning Geobacter sulfurreducens biofilm with conjugated polyelectrolyte for increased performance in bioelectrochemical system

Bioelectrochemical systems (BESs) are emerging as a platform technology with great application potentials such as wastewater remediation and power generation. Materials for electrode/microorganism modification are being examined in order to improve the current production in BESs. Herein, we report that the current production increased almost one fold in single-chamber BES reactors, by adding a conjugated polyelectrolyte (CPE-K) in the growth medium to co-form the anodic biofilm with Geobacter sulfurreducens cells. The CPE-K treated BESs had a maximum current density as high as 12.3 ± 0.5 A/m2, with that of the controls being 6.2 ± 0.7 A/m2. Improved current production was sustained even after CPE-K was no longer added to the medium. It was demonstrated that increased current resulted from improvement of certain biofilm properties. Analysis using electrochemical impedance spectroscopy (EIS) showed that CPE-K addition decreased the charge transfer resistance at the cell/electrode interface and the diffusion resistance through the biofilm. Protein quantification showed increased biomass growth on the electrode surface, and confocal scanning microscopy images revealed enhanced biofilm permeability. These results demonstrated for the first time that conjugated polyelectrolytes could be used for G. sulfurreducens biofilm augmentation to achieve high electricity production through tuning the anodic biofilm in BESs.

(Keynote) Application of Electrochemical Surface Plasmon Resonance (ESPR) to the Study of Electroactive Microbial Biofilms

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode interface (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.

Backbone assignment of cytochrome PccH, a crucial protein for microbial electrosynthesis in Geobacter sulfurreducens.

Microbial electrosynthesis is an emerging green technology that explores the capability of a particular group of microorganisms to drive their metabolism toward the production of hydrogen or value-added chemicals from electrons supplied by electrode surfaces. The cytochrome PccH showed the largest increase in transcription when electrons are supplied to Geobacter sulfurreducens biofilms. Gene knock-out experiments have shown that the electron transfer toward G. sulfurreducens cells was completely inhibited by the deletion of the gene encoding for cytochrome PccH. This identifies a crucial role for this protein in G. sulfurreducens microbial electrosynthesis mechanisms, which are currently unknown. In this work, we present the backbone (1H, 13C and 15N) and heme assignment for PccH in the oxidized state. The data obtained paves the way to identify and structurally map the molecular interaction regions between the cytochrome PccH and its physiological redox partners.

A Generalized Kinetic Framework Applied to Whole‐Cell Bioelectrocatalysis in Bioflow Reactors Clarifies Performance Enhancements for Geobacter Sulfurreducens Biofilms

AbstractA common kinetic framework for studies of whole‐cell catalysis is vital for understanding and optimizing bioflow reactors. In this work, we demonstrate the applicability of a flow‐adapted version of Michaelis‐Menten kinetics to an electrocatalytic bacterial biofilm. A three‐electrode microfluidic biofilm flow reactor measured increased turnover rates by as much as 50 % from a Geobacter sulfurreducens biofilm as flow rate was varied. Based on parameters from the applied kinetic framework, flow‐induced increases to turnover rate, catalytic efficiency and device reaction capacity could be linked to an increase in catalytic biomass. This study demonstrates that a standardized kinetic framework is critical for quantitative measurements of new living catalytic systems in flow reactors and for benchmarking against well‐studied catalytic systems such as enzymes.