Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

Performance of a combined electrotrophic and electrogenic biofilm operated under long-term, continuous cycling.

OBJECTIVES: Evaluate electrochemically active biofilms as high energy density rechargeable microbial batteries toward providing persistent power in applications where traditional battery technology is limiting (, remote monitoring applications). RESULTS: Here we demonstrated that an electrochemically active biofilm was able to store and release electrical charge for alternating charge/discharge cycles of up to 24 h periodicity (50% duty cycle) with no significant decrease in average current density (0.16 ± 0.04 A/m2) for over 600 days. However, operation at 24 h periodicity for > 50 days resulted in a sharp decrease in the current to nearly zero. This current crash was recoverable by decreasing the periodicity. Overall, the coulombic efficiency remained near unity within experimental error (102 ± 3%) for all of the tested cycling periods. Electrochemical characterization here suggests that electron transfer occurs through multiple routes, likely a mixture of direct and mediated mechanisms. CONCLUSIONS: These results indicate that bidirectional electrogenic/electrotrophic biofilms are capable of efficient charge storage/release over a wide range of cycling frequency and may eventually enable development of sustainable, high energy density rechargeable batteries.

Metagenomic and Metatranscriptomic Characterization of a Microbial Community That Catalyzes Both Energy-Generating and Energy-Storing Electrode Reactions.

Electroactive bacteria are living catalysts, mediating energy-generating reactions at anodes or energy storage reactions at cathodes via extracellular electron transfer (EET). The Cathode-ANode (CANode) biofilm community was recently shown to facilitate both reactions; however, the identities of the primary constituents and underlying molecular mechanisms remain unknown. Here, we used metagenomics and metatranscriptomics to characterize the CANode biofilm. We show that a previously uncharacterized member of the family Desulfobulbaceae, Desulfobulbaceae-2, which had <1% relative abundance, had the highest relative gene expression and accounted for over 60% of all differentially expressed genes. At the anode potential, differential expression of genes for a conserved flavin oxidoreductase (Flx) and heterodisulfide reductase (Hdr) known to be involved in ethanol oxidation suggests a source of electrons for the energy-generating reaction. Genes for sulfate and carbon dioxide reduction pathways were expressed by Desulfobulbaceae-2 at both potentials and are the proposed energy storage reactions. Reduction reactions may be mediated by direct electron uptake from the electrode or from hydrogen generated at the cathode potential. The Desulfobulbaceae-2 genome is predicted to encode at least 85 multiheme (≥3 hemes) c-type cytochromes, some with as many as 26 heme-binding domains, that could facilitate reversible electron transfer with the electrode. Gene expression in other CANode biofilm species was also affected by the electrode potential, although to a lesser extent, and we cannot rule out their contribution to observed current. Results provide evidence of gene expression linked to energy storage and energy-generating reactions and will enable development of the CANode biofilm as a microbially driven rechargeable battery. IMPORTANCE Microbial electrochemical technologies (METs) rely on electroactive bacteria to catalyze energy-generating and energy storage reactions at electrodes. Known electroactive bacteria are not equally capable of both reactions, and METs are typically configured to be unidirectional. Here, we report on genomic and transcriptomic characterization of a recently described microbial electrode community called the Cathode-ANode (CANode). The CANode community is able to generate or store electrical current based on the electrode potential. During periods where energy is not needed, electrons generated from a renewable source, such as solar power, could be converted into energy storage compounds to later be reversibly oxidized by the same microbial catalyst. Thus, the CANode system can be thought of as a living "rechargeable battery." Results show that a single organism may be responsible for both reactions demonstrating a new paradigm for electroactive bacteria.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Biofilm structure, dynamics, and ecology of an upscaled biocathode wastewater microbial fuel cell.

A microbial fuel cell (MFC) system containing modular half-submerged biocathode was operated for 6 months in an 800 L flow-through system with domestic wastewater. For the first time, spatial and temporal differences in biofilm communities were examined on large three-dimensional electrodes in a wastewater MFC. Biocathode microbial community analysis showed a specialized biofilm community with electrogenic and electrotrophic taxa forming during operation, suggesting potentially opposing electrode reactions. The anodic community structure shifted during operation, but no spatial differences were observed along the length of the electrode. Power output from the system was most strongly influenced by pH. Higher power densities were associated with the use of solids-dewatering filtrate with increased organic matter, conductivity, and pH. The results show that the biocathode was the rate-limiting step and that future MFC design should consider the effect of size, shape, and orientation of biocathodes on their community assembly and electrotrophic ability.

Internal Redox Polarity of an Individual G. sulfurreducens Bacterial Cell Attached to an Inorganic Substrate.

Bacterial cell polarity is an internal asymmetric distribution of subcellular components, including proteins, lipids, and other molecules that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Mechanisms and factors controlling formation of this cell internal asymmetry are not completely understood. As a step in this direction, in the present work, we develop an approach for analyzing how information about inorganic substrate can be non-genetically coded inside an individual bacterial cell. As a model system, we use G. sulfurreducens cells attached to an inorganic mineral, mica. The approach utilizes confocal Raman microscopy, Gaussian deconvolution, and Principal Component Analysis (PCA) and allows for quick label-free identification of the molecular signature of cytochrome intracellular location and the cell to substrate binding down to the level of individual bacterial cells. Our results describe a spectroscopic signature of cell adhesion and how the information about cell adhesion can be coded inside individual bacterial cells.

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 (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.

Biofilm as a redox conductor: a systematic study of the moisture and temperature dependence of its electrical properties.

Some microbial biofilms are electrically conductive. However, the mechanism of electron transport remains unclear. Here, we show that µm-scale long-distance electron transport through electrode-grown Geobacter sulfurreducens biofilms occurs via redox conduction, as determined by electrical measurements performed under varied hydration states and temperatures.

Alamethicin suppresses methanogenesis and promotes acetogenesis in bioelectrochemical systems.

Microbial electrosynthesis (MES) systems with mixed cultures often generate a variety of gaseous and soluble chemicals. Methane is the primary end product in mixed-culture MES because it is the thermodynamically most favorable reduction product of CO2. Here, we show that the peptaibol alamethicin selectively suppressed the growth of methanogens in mixed-culture MES systems, resulting in a shift of the solution and cathode communities to an acetate-producing system dominated by Sporomusa, a known acetogenic genus in MES systems. Archaea in the methane-producing control were dominated by Methanobrevibacter species, but no Archaea were detected in the alamethicin-treated reactors. No methane was detected in the mixed-culture reactors treated with alamethicin over 10 cycles (∼ 3 days each). Instead, acetate was produced at an average rate of 115 nmol ml(-1) day(-1), similar to the rate reported previously for pure cultures of Sporomusa ovata on biocathodes. Mixed-culture control reactors without alamethicin generated methane at nearly 100% coulombic recovery, and no acetate was detected. These results show that alamethicin is effective for the suppression of methanogen growth in MES systems and that its use enables the production of industrially relevant organic compounds by the inhibition of methanogenesis.

Thermally activated long range electron transport in living biofilms.

Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.

Effects of constant or dynamic low anode potentials on microbial community development in bioelectrochemical systems.

In bioelectrochemical systems, exoelectrogenic bacteria respire with anode electrodes as their extracellular electron acceptor; therefore, lower anode potentials can reduce the energy gain to each microbe and select against ones that are not able to respire at a lower potential range. Often fully developed anode communities are compared across bioelectrochemical systems with set anode potentials or fixed external resistances as different operational conditions. However, the comparative effect of the resulting constantly low versus dynamically low anode potentials on the development of anode microbial communities as well as the final cathode microbial communities has not been directly demonstrated. In this study, we used a low fixed anode potential of -250 mV and a higher-current control potential of -119 mV vs. Standard Hydrogen Electrode to approximately correspond with the negative peak anode potential values obtained from microbial fuel cells operated with fixed external resistances of 1 kΩ and 47 Ω, respectively. Pyrosequencing data from a 2-month time series show that a lower set anode potential resulted in a more diverse community than the higher- and variable-potential systems, likely due to the hindered enrichment of a Geobacter-dominated community with limited energy gain at this set potential. In this case, it appears that the selective pressure caused by the low set potential was counteracted by the low energy gain over a 2-month time scale. The air cathode microbial community with constant low anode potentials showed delayed enrichment of denitrifiers or perchlorate-reducing bacteria compared to the fixed external resistance condition.

Exoelectrogenic biofilm as a template for sustainable formation of a catalytic mesoporous structure.

Mesoporous structures can increase catalytic activity by maximizing the ratio of surface area to volume, but current synthesis techniques utilize expensive polymers and toxic chemicals. A Geobacter sulfurreducens biofilm was used as a sustainable template to form mesoporous Pd structures while eliminating the need for synthetic chemicals. The bulk of the biofilm material was removed by thermal treatments after nanoparticle formation, producing a catalytic Pd mesoporous (pore size 9.7 ± 0.1 nm) structure attached to the graphite electrode with a 1.5-2 µm thick backbone composed of nanoparticles (∼200 nm). A control electrode electrochemically plated with Pd in the absence of a biofilm exhibited a variable planar Pd base (∼0.5-3 µm thick) with sporadic Pd extrusions (∼2 µm across, 1-5 µm tall) from the surface. The biotemplated mesoporous structure produced 15-20% higher stable current densities during H2 oxidation tests than the electrochemically plated control electrode, even though 30% less Pd was present in the biotemplated catalyst. These results indicate that electroactive biofilms can be used as a sustainable base material to produce nanoporous structures without the need for synthetic polymers. Biotechnol. Bioeng. 2014;111: 2349-2354. © 2014 Wiley Periodicals, Inc.

Microbial community composition is unaffected by anode potential.

There is great controversy on how different set anode potentials affect the performance of a bioelectrochemical system (BES). It is often reported that more positive potentials improve acclimation and performance of exoelectrogenic biofilms, and alter microbial community structure, while in other studies relatively more negative potentials were needed to achieve higher current densities. To address this issue, the biomass, electroactivity, and community structure of anodic biofilms were examined over a wide range of set anode potentials (-0.25, -0.09, 0.21, 0.51, and 0.81 V vs a standard hydrogen electrode, SHE) in single-chamber microbial electrolysis cells. Maximum currents produced using a wastewater inoculum increased with anode potentials in the range of -0.25 to 0.21 V, but decreased at 0.51 and 0.81 V. The maximum currents were positively correlated with increasing biofilm biomass. Pyrosequencing indicated biofilm communities were all similar and dominated by bacteria most similar to Geobacter sulfurreducens. Differences in anode performance with various set potentials suggest that the exoelectrogenic communities self-regulate their exocellular electron transfer pathways to adapt to different anode potentials.

Marinobacter: A case study in bioelectrochemical chassis evaluation.

The junction of bioelectrochemical systems and synthetic biology opens the door to many potentially groundbreaking technologies. When developing these possibilities, choosing the correct chassis organism can save a great deal of engineering effort and, indeed, can mean the difference between success and failure. Choosing the correct chassis for a specific application requires a knowledge of the metabolic potential of the candidate organisms, as well as a clear delineation of the traits, required in the application. In this review, we will explore the metabolic and electrochemical potential of a single genus, Marinobacter. We will cover its strengths, (salt tolerance, biofilm formation and electrochemical potential) and weaknesses (insufficient characterization of many strains and a less developed toolbox for genetic manipulation) in potential synthetic electromicrobiology applications. In doing so, we will provide a roadmap for choosing a chassis organism for bioelectrochemical systems.

Nanoliter scale electrochemistry of natural and engineered electroactive bacteria.

Bacterial extracellular electron transfer (EET) is envisioned for use in applied biotechnologies, necessitating electrochemical characterization of natural and engineered electroactive biofilms under conditions similar to the target application, including small-scale biosensing or biosynthesis platforms, which is often distinct from standard 100 mL-scale stirred-batch bioelectrochemical test platforms used in the laboratory. Here, we adapted an eight chamber, nanoliter volume (500 nL) electrochemical flow cell to grow biofilms of both natural (Biocathode MCL community, Marinobacter atlanticus, and Shewanella oneidensis MR1) or genetically modified (S. oneidensis ΔMtr and S. oneidensis ΔMtr + pLB2) electroactive bacteria on electrodes held at a constant potential. Maximum current density achieved by unmodified strains was similar between the nano- and milliliter-scale reactors. However, S. oneidensis biofilms engineered to activate EET upon exposure to 2,4-diacetylphloroglucinol (DAPG) produced current at wild-type levels in the stirred-batch reactor, but not in the nanoliter flow cell. We hypothesize this was due to differences in mass transport of DAPG, naturally-produced soluble redox mediators, and oxygen between the two reactor types. Results presented here demonstrate, for the first time, nanoliter scale chronoamperometry and cyclic voltammetry of a range of electroactive bacteria in a three-electrode reactor system towards development of miniaturized, and potentially high throughput, bioelectrochemical platforms.

Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera.

Microbial fuel cell (MFC) anode communities often reveal just a few genera, but it is not known to what extent less abundant bacteria could be important for improving performance. We examined the microbial community in an MFC fed with formic acid for more than 1 year and determined using 16S rRNA gene cloning and fluorescent in situ hybridization that members of the Paracoccus genus comprised most (approximately 30%) of the anode community. A Paracoccus isolate obtained from this biofilm (Paracoccus denitrificans strain PS-1) produced only 5.6 mW/m(2), whereas the original mixed culture produced up to 10 mW/m(2). Despite the absence of any Shewanella species in the clone library, we isolated a strain of Shewanella putrefaciens (strain PS-2) from the same biofilm capable of producing a higher-power density (17.4 mW/m(2)) than the mixed culture, although voltage generation was variable. Our results suggest that the numerical abundance of microorganisms in biofilms cannot be assumed a priori to correlate to capacities of these predominant species for high-power production. Detailed screening of bacterial biofilms may therefore be needed to identify important strains capable of high-power generation for specific substrates.

Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers.

Bacterial microcompartment (BMC) shells are modular, selectively permeable, nanoscale protein shells that self-assemble from hexagonal and pentagonal building blocks in vivo or in vitro. Natural and engineered BMC shells colocalize and concentrate catalysts and metabolites in their lumen, increasing reaction kinetics. Here, we describe the design and characterization of a shell protein (pseudohexameric/trimeric BMC-T1HO protein) engineered to coordinate a Cu ion in its pore. Several designs, each varying the position of an introduced coordinating histidine residue, were shown to maintain their trimeric oligomerization state upon Cu coordination via chemical denaturation experiments. We measured reversible redox activity from electrode-bound Cu-3His BMC-T1HO variants, with formal potential(s) that were dependent on the Cu coordination site within the discoidal shaped trimer (E°' = +208 to +265 mV vs SHE). These results represent important steps toward expanding the functionality (Cu coordination) and applicability (redox activity on an electrode surface) of engineered BMC reactor architectures.

Activation of Protein Expression in Electroactive Biofilms.

Microbes that form biofilms on electrodes and generate electrical current responses could be integrated into devices to perform sensing, conduct signals, or act as living microprocessors. A challenge in working with these species is the ability to visualize biofilm formation and protein expression in real-time while also measuring current, which is not possible with typical bio-electrochemical reactors. Here, we present a three-dimensional-printed flow cell for simultaneous electrochemistry and fluorescence imaging. Current-producing biofilms of Marinobacter atlanticus constitutively expressing green fluorescent protein were grown on the flow cell working electrode. Increasing current corresponded with increasing surface coverage and was comparable to biofilms grown in typical stirred-batch reactors. An isopropyl ß-d-1-thiogalactopyranoside (IPTG) inducible system driving yellow fluorescent protein was used to assess the spatiotemporal activation of protein expression within the biofilm at different stages of growth and induction dynamics. The response time ranged from 30 min to 5 h, depending on the conditions. These data demonstrate that the electrochemical flow cell can evaluate the performance of an electrically active environmental bacterium under conditions relevant for development as a living electronic sensor.

Spatially Resolved Chemical Analysis of Geobacter sulfurreducens Cell Surface.

Geobacter sulfurreducens is of interest for the highest efficiency of power generation and extremely long extracellular electron transfer (EET) between the bacterium and electrodes. Despite more than 15 years of intensive molecular biological research, there is still no clear answer which molecules are responsible for these processes. In the present work, we look at the problem from another (atomic) perspective and identify the location and shape of the compounds that are known to be conductive, particularly those containing Fe atoms. By using highly sophisticated energy dispersive X-ray spectroscopy combined with high-angle annular dark-field transmission electron microscopy enabling detection, identification, and localization of chemical compounds on the surface at nearly atomic spatial resolution, we analyze Fe spatial distribution within the G. sulfurreducens community. We discover the presence of small Fe-containing particles on the surface of the bacterium cells. The size of the particles (diameter 5.6 nm) is highly reproducible and comparable with the size of a single protein. The particles cover about 2% of the cell surface, which is similar to that expected for molecular conductors responsible for electron transfer through the bacterium cell wall. We find that G. sulfurreducens filaments ("bacterial molecular wires") also contain Fe atoms in their bundles. We observe that the bacterium enable changing the distance between the Fe-containing bundles in the filaments from separated to attached (the latter is needed for the efficient electron transfer between the Fe-containing particles), depending on the bacterium metabolic activity and attachment to extracellular substrates. These results are consistent with the recently published research about the role of Fe atoms in protein molecular conductance ( Phys. Chem. Chem. Phys. , 2018 , 20 , 14072 - 14081 ) and show what type of Fe-containing particles are involved in the bacterial extracellular communication. They can be used for the design and construction of artificial biomolecular wires and bioinorganic interfaces.