Microbial Electrochemical Technologies Research Articles

Hierarchical modulation of extracellular electron transfer processes in microbial fuel cell anodes for enhanced power output through improved Geobacter adhesion

Although microbial fuel cell (MFC) technology is nearing practical realization, the challenge of attaining higher power output continues to impede its widespread implementation. In this study, we successfully developed free-standing three-dimensional high-performance anode materials for MFCs using a straightforward "one-step" process based on garlic. The resulting anode not only promotes the accumulation of biomass and the enrichment of exoelectrogen Geobacter but also facilitates interfacial extracellular electron transfer. This led to rapid start-up within just three days, a robust power density of 12.37 W/m3, and an impressive 24.04 % coulombic efficiency in mixed-culture MFC reactors. Our investigation into the underlying mechanisms of exocellular electron transfer revealed improvements in both direct electron transfer and mediated electron transfer processes. Notably, the fabrication of carbonized garlic MFC anodes proves to be technically competitive and economically relevant. It not only paves the way for the development of high-performance MFCs but also provides valuable insights for the rational design of materials related to microbial electrochemical technologies.

Effect of model methanogens on the electrochemical activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes

Combining anaerobic digestion (AD) and microbial electrochemical technologies (MET) in AD-MET holds great potential. Methanogens have been identified as one cause of decreased electrochemical activity and deterioration of Geobacter spp. biofilm anodes. A better understanding of the different interactions between methanogenic genera/species and Geobacter spp. biofilms is needed to shed light on the observed reduction in electrochemical activity and stability of Geobacter spp. dominated biofilms as well as observed changes in microbial communities of AD-MET. Here, we have analyzed electrochemical parameters and changes in the microbial community of Geobacter spp. biofilm anodes when exposed to three representative methanogens with different metabolic pathways, i.e., Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii. M. barkeri negatively affected the performance and stability of Geobacter spp. biofilm anodes only in the initial batches. In contrast, M. formicicum did not affect the stability of Geobacter spp. biofilm anodes but caused a decrease in maximum current density of ~37%. M. soehngenii induced a coloration change of Geobacter spp. biofilm anodes and a decrease in the total transferred charge by ~40%. Characterization of biofilm samples after each experiment by 16S rRNA metabarcoding, whole metagenome nanopore sequencing, and shotgun sequencing showed a higher relative abundance of Geobacter spp. after exposure to M. barkeri as opposed to M. formicicum or M. soehngenii, despite the massive biofilm dispersal observed during initial exposure to M. barkeri.

Chemical-physical parameters and microbial community changes induced by electrodes polarization inhibit PCB dechlorination in a marine sediment

Microbial reductive dechlorination of organohalogenated pollutants is often limited by the scarcity of electron donors, that can be overcome with microbial electrochemical technologies (METs). In this study, polarized electrodes buried in marine sediment microcosms were investigated to stimulate PCB reductive dechlorination under potentiostatic (−0.7 V vs Ag/AgCl) and galvanostatic conditions (0.025 mA·cm−2-0.05 mA·cm−2), using graphite rod as cathode and iron plate as sacrificial anode. A single circuit and a novel two antiparallel circuits configuration (2AP) were investigated. Single circuit polarization impacted the sediment pH and redox potential (ORP) proportionally to the intensity of the electrical input and inhibited PCB reductive dechlorination. The effects on the sediment’s pH and ORP, along with the inhibition of PCB reductive dechlorination, were mitigated in the 2AP system. Electrodes polarization stimulated sulfate-reduction and promoted the enrichment of bacterial clades potentially involved in sulfate-reduction as well as in sulfur oxidation. This suggested the electrons provided were consumed by competitors of organohalide respiring bacteria and specifically sequestered by sulfur cycling, which may represent the main factor limiting the applicability of METs for stimulating PCB reductive dechlorination in marine sediments.

Critical perspective on the elimination of emerging contaminants from industrial wastewater via microbial electrochemical technologies

AbstractIndustrial water pollution originating from various industries like textile, dairy, oil, and petrochemical industries, etc. is a huge concern globally and has led to devastating effects on the environment due to the release of refractory emerging contaminants (ECs). These ECs of concern have attracted wide devotion from the scientific community due to their recalcitrant nature and disastrous effects on plants, aquatic life forms, and humans. In this regard, conventional wastewater treatment technologies such as coagulation, flocculation, membrane technologies, electrocoagulation, and other biological technologies like sequencing batch reactor, anaerobic up‐flow sludge blanket reactor, etc., are inefficient in removing ECs from the industrial effluent, while conventional advanced oxidation processes incur high cost due to the extensive requirement of energy for the degradation of ECs. To overcome this issue, microbial electrochemical technologies (METs) can be employed. For instance, METs have shown promising results in the degradation of various ECs, such as microbial fuel cells, which have shown nearly 92% to 98% removal of sulfamethoxazole with simultaneous power recovery. Alizarin yellow R, nitrobenzene, and Congo red were degraded by microbial electrolysis cells with removal efficiency in the range of 88% to 98%, demonstrating their superiority in the elimination of trace contaminants. Similarly, almost 100% mineralization of pyraclostrobin was noticed for the bio‐electro‐Fenton process, showing the elevated potential of these neoteric technologies for the remediation of recalcitrant pollutants. Thus, the current review article aims to critically analyze the intervention of METs for the elimination of ECs from industrial wastewater.

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.

Electrical current disrupts the electron transfer in defined consortia.

Improving methane production through electrical current application to anaerobic digesters has garnered interest in optimizing such microbial electrochemical technologies, with claims suggesting direct interspecies electron transfer (DIET) at the cathode enhances methane yield. However, previous studies with mixed microbial communities only reported interspecies interactions based on species co-occurrence at the cathode, lacking insight into how a poised cathode influences well-defined DIET-based partnerships. To address this, we investigated the impact of continuous and discontinuous exposure to a poised cathode (-0.7 V vs. standard hydrogen electrode) on a defined consortium of Geobacter metallireducens and Methanosarcina barkeri, known for their DIET capabilities. The physiology of DIET consortia exposed to electrical current was compared to that of unexposed consortia. In current-exposed incubations, overall metabolic activity and cell numbers for both partners declined. The consortium, receiving electrons from the poised cathode, accumulated acetate and hydrogen, with only 32% of the recovered electrons allocated to methane production. Discontinuous exposure intensified these detrimental effects. Conversely, unexposed control reactors efficiently converted ethanol to methane, transiently accumulating acetate and recovering 88% of electrons in methane. Our results demonstrate the overall detrimental effect of electrochemical stimulation on a DIET consortium. Besides, the data indicate that the presence of an alternative electron donor (cathode) hinders efficient electron retrieval by the methanogen from Geobacter, and induces catabolic repression of oxidative metabolism in Geobacter. This study emphasizes understanding specific DIET-based interactions to enhance methane production during electrical stimulation, providing insights for optimizing tailored interspecies partnerships in microbial electrochemical technologies.

Burning questions: Exploring the limits of microbial electrochemical technology for industrial biotechnological applications.

Microbial electrochemical technology (MET) has proven to be a promising solution to overcome the redox and energy metabolic constraints, enabling high yields of biosynthesis beyond stoichiometric limits. While there is room for improvement in extracellular electron transfer rates and productivity of the target compounds, it is crucial to think in advance about which bioprocess could be electrified and what would face major challenges. In this opinion paper, I presented and addressed interfacial electron transfer capacity of MET, whether built on biofilm or planktonic cells, and also discussed the upper limits of the MET system for biosynthesis of chemicals accordingly. Potential future application scenarios of different MET were also briefly addressed. This opinion paper aims to encourage the community to rethink the design and development of microbial electrochemical technologies for potential future applications in industrial biotechnology.

Electron transport bifurcation in bioanode with the metabolic shift to nitrate reduction

Electron transport bifurcation in bioanode determines the performance of microbial electrochemical technologies with the presence of an alternative electron acceptor. Here, the bioanode responses including electron transfer efficiency, microbial community, and microbial structure are investigated with the metabolic shift from current production to denitrification. Electrochemical measurements including cyclic voltammetry and electrochemical impedance spectra are performed to identify the change of electron transfer pathways in bioanode. Electron transfer efficiency for electrode reduction decreases ∼17 % with nitrate reduction. Biofilm resistance and charge transfer resistance increase from 23.3 Ω and 22.5 Ω to 36.6 Ω and 61.4 Ω with the metabolic shift, respectively. These results are mainly due to the loss of exoelectrogens inhabited in bioanode. Confocal imaging results indicate the elevated proportion of inactive cells in bioanode as the denitrification. Our results propose a possible mechanism for electron transfer bifurcation in bioanode with the metabolic shift from electrode reduction to soluble electron acceptor reduction.

Advances in microbial electrochemical technology mediated microbial metabolism enhanced remediation of petroleum hydrocarbon contaminated soil

Advances in microbial electrochemical technology mediated microbial metabolism enhanced remediation of petroleum hydrocarbon contaminated soil

(Invited) Using the Model Bacterium Geobacter Sulfurreducens to Understand Microbial Electrochemical Conversion of Nitrogen Gas into Ammonium

There is a growing need to decrease reliance on the Haber-Bosch process. To convert nitrogen gas (N2) into ammonia (NH3) for fertilizers, this temperature- and pressure-intensive process consumes roughly 2–3% of global energy and releases over 740 million tons of CO2 each year. Many microorganisms naturally convert or “fix” N2 into ammonium (NH4 +) at room temperature and pressure using an enzyme called the nitrogenase. Attempts to increase NH4 + yields from microorganisms through genetic engineering are seeing signs of success, but we lack methods to increase NH4 + generation rates and overcome one of the largest challenges: O2 gas. Increasing O2 gas concentrations typically increases metabolic rates of aerobic microorganisms; however, in the case of microorganisms that fix N2 (called diazotrophs), O2 can shut down the nitrogenase and stop cell growth. To overcome these challenges, we are using electricity to drive N2 fixation in exoelectrogenic bacteria. These bacteria have the unique ability to naturally transfer electrons to anode electrodes in microbial electrochemical technologies (METs). Increasing the voltage applied to METs increases the metabolic rates of exoelectrogens. The anode chamber is kept anaerobic, which eliminates O2-driven inhibition of the nitrogenase. We previously showed that the N2 fixation rates of a mixed microbial community in a MET increased more than three times when the applied whole-cell potential increased from 0.7 V to 1.0 V. By adding a chemical inhibitor that prevented the incorporation of NH4 + into larger biomolecules, NH4 + was excreted by the cells and into the medium. Based on the acetylene reduction assay (a proxy for N2 fixation), we estimated that we recovered about 10% of the theoretical NH4 +generated by the cells. If close to 100% of the theoretical NH4 + could be excreted from the cells and recovered, we predicted an energy demand of around 3 MJ/mol-NH4 +, which is close to the range of value reported for the Haber-Bosch process. To develop METs that generate NH4 + at competitive production rates and energy demands, we are now focusing on the model exoelectrogenic diazotroph Geobacter sulfurreducens. Using a single organism with a fully sequenced genome allows us to understand how the N2 fixation process responds to electrochemical variables. Towards these efforts, we conducted a transcriptomic analysis of G. sulfurreducens at two fixed anode potentials: +0.15 V and −0.15 V. This approach revealed which genes are turned on and off in response to these potentials. The presence of NH4 + decreased the expression of genes associated with N2 fixation which was expected due to the known sensitivity of nitrogenases to NH4 +. On the other hand, the two anode potentials had a dramatic and unexpected impact on the expression levels of N2 fixation genes. At −0.15 V, nitrogenase genes were significantly up-regulated, as were genes associated with NH4 + uptake and transport, such as glutamine and glutamate synthetases. Considering that −0.15 V provides less energy to the cells relative to +0.15 V (based on thermodynamic predictions), the results suggest that the cells responded to this highly energy-constrained environment by increasing expression of N2 fixation pathways. Based on our new knowledge of how G. sulfurredcuens regulates N2 fixation and NH4 + production, we have developed new strains of G. sulfurreducens that can excrete NH4 + during N2 fixation. The new engineering toolkit that we have created in this bacterium can now be leveraged to maximize NH4 + production in METs.

Biological Anolyte Regeneration System for Redox Flow Batteries

Redox flow batteries (RFBs) are valuable energy storage devices with applications in grid storage, and redox-active small organic molecules (ROMs) have shown promise as charge storage species for RFB applications. However, electrolyte degradation is a common mechanism in RFBs, and the chemical instability of ROMs limits their practical application in commercial RFB devices. Recently, microbial electrochemical technologies have been studied, taking advantage of the natural oxidation microbes undergo when exposed to a carbon source. Here, an aqueous redox flow battery with anolyte phenazine-1-carboxylic acid (PCA) and catholyte (Ferrocenylmethyl)trimethylammonium (FcN) is explored with a genetically engineered, phenazine-producing E. coli phzAG-SM strain. E. coli phzAG-SM is a genetically engineered bacterial strain that has phenazine gene clusters responsible for producing phenazine species, specifically PCA and pyocyanin (PYO). Standard E. coli strains like E. coli BL21 do not have genes responsible for the phenazine biosynthetic pathways that result in the production of phenazine species. This genetically engineered strain has the ability to not only excrete its own phenazine metabolites, but also to intake the phenazine species present in solution and increase phenazine production rates. In a redox flow battery, the electrolyte species are subjugated to changes in potential during charging and discharging cycles. Over time, the phenazine anolyte species can degrade or decompose. The role of the genetically engineered E. coli in the anolyte chamber is to repair the degraded/decomposed phenazine species, producing a longer lasting battery with more stable phenazine-based electrolyte species. Herein, we demonstrate the design of an RFB system where ROMs are continually regenerated by genetically engineered microorganisms to maintain RFB cycling stability. This work represents a new strategy for improving the stability of RFB systems because, under the influence of genetically engineered microbes, the anolyte species does not display degradation after battery cycling.

(Invited) Enhancing the Performance of Microbial Fuel Cells Via Metabolic Engineering of Escherichia coli for Phenazine Production

Establishing an efficient and successful extracellular electron transfer (EET) between microorganisms and electrode surfaces plays a critical role in the design, development, and application of mediated microbial electrochemical technologies, such as microbial fuel cells (MFCs). Most microbial-based systems require the use of artificial, redox-active mediator systems in order to facilitate and/or increase electron transfer. Our previous work established an exogenous phenazine-based library as a mediator system to enable electron transfer from the model microorganism Escherichia coli. However, the addition of exogenous mediators to a microbial electrochemical system has certain limiting downsides, specifically with regard to mediator toxicity to cells and increased operational expenses. Herein, we demonstrate the metabolic and genetic engineering of E. coli to self-generate phenazine metabolites endogenously by introducing the phenazine biosynthetic pathway from Pseudomonas aeruginosa into E. coli. This pathway consists of a seven-gene phenazine biosynthetic cluster phzA-G responsible for the synthesis of phenazine-1-carboxylic acid (PCA) and two phenazine accessory genes phzM and phzS to produce pyocyanin (PYO). We present the characterization of the engineered E. coli cells via electrochemical measurements, RNA sequencing, and microscopy imaging. Finally, the engineered E. coli cells were used for the design of a microbial fuel cell with enhanced performances, demonstrating a maximum power density increase from 305 mW/m2 with non-engineered E. coli cells to 1555 mW/m2 with the genetically engineered, phenazine-producing E. coli. Our results indicate that introducing a heterologous electron shuttle into E. coli can be an efficient approach to establishing efficacious mediation in living bioelectrochemical systems and improving the performance of MFCs.

Responses of syntrophic microbial communities and their interactions with polystyrene nanoplastics in a microbial electrolysis cell

Microbial electrochemical technologies are promising for simultaneous energy recovery and wastewater treatment. Although the inhibitory effects of emerging pollutants, particularly micro/nanoplastics (MPs/NPs), on conventional wastewater systems have been extensively studied, the current understanding of their impact on microbial electrochemical systems is still quite limited. Microplastics are plastic particles ranging from 1 μm to 5 mm. However, nanoplastics are smaller plastic particles ranging from 1 to 100 nm. Due to their smaller size and greater surface area, they can penetrate deeper into biofilm structures and cell membranes, potentially disrupting their integrity and leading to changes in biofilm composition and function. This study first reports the impact of polystyrene nanoplastics (PsNPs) on syntrophic anode microbial communities in a microbial electrolysis cell. Low concentrations of PsNPs (50 and 250 μg/L) had a minimal impact on current density and hydrogen production. However, 500 μg/L of PsNPs decreased the maximum current density and specific hydrogen production rate by ∼43 % and ∼48 %, respectively. Exposure to PsNPs increased extracellular polymeric substance (EPS) levels, with a higher ratio of carbohydrates to proteins, suggesting a potential defense mechanism through EPS secretion. The downregulation of genes associated with extracellular electron transfer was observed at 500 μg/L of PsNPs. Furthermore, the detrimental impact of 500 μg/L PsNPs on the microbiome was evident from the decrease in 16S rRNA gene copies, microbial diversity, richness, and relative abundances of key electroactive and fermentative bacteria. For the first time, this study presents the inhibitory threshold of any NPs on syntrophic electroactive biofilms within a microbial electrochemical system.

Microbial electricity-driven anaerobic phenol degradation in bioelectrochemical systems

Microbial electrochemical technologies have been extensively employed for phenol removal. Yet, previous research has yielded inconsistent results, leaving uncertainties regarding the feasibility of phenol degradation under strictly anaerobic conditions using anodes as sole terminal electron acceptors. In this study, we employed high-performance liquid chromatography and gas chromatography-mass spectrometry to investigate the anaerobic phenol degradation pathway. Our findings provide robust evidence for the purely anaerobic degradation of phenol, as we identified benzoic acid, 4-hydroxybenzoic acid, glutaric acid, and other metabolites of this pathway. Notably, no typical intermediates of the aerobic phenol degradation pathway were detected. One-chamber reactors (+0.4 V vs. SHE) exhibited a phenol removal rate of 3.5 ± 0.2 mg L−1 d−1, while two-chamber reactors showed 3.6 ± 0.1 and 2.6 ± 0.9 mg L−1 d−1 at anode potentials of +0.4 and + 0.2 V, respectively. Our results also suggest that the reactor configuration certainly influenced the microbial community, presumably leading to different ratios of phenol consumers and microorganisms feeding on degradation products.

Comparing theoretical and practical biomass yields calls for revisiting thermodynamic growth models for electroactive microorganisms

Research on electroactive microorganisms (EAM) often focuses either on their physiology and the underlying mechanisms of extracellular electron transfer or on their application in microbial electrochemical technologies (MET). Thermodynamic understanding of energy conversions related to growth and activity of EAM has received only a little attention. In this study, we aimed to prove the hypothesized restricted energy harvest of EAM by determining biomass yields by monitoring growth of acetate-fed biofilms presumably enriched in Geobacter, using optical coherence tomography, at three anode potentials and four acetate concentrations. Experiments were concurrently simulated using a refined thermodynamic model for EAM. Neither clear correlations were observed between biomass yield and anode potential nor acetate concentration, albeit the statistical significances are limited, mainly due to the observed experimental variances. The experimental biomass yield based on acetate consumption (YX/ac = 37 ± 9 mgCODbiomass gCODac−1) was higher than estimated by modeling, indicating limitations of existing growth models to predict yields of EAM. In contrast, the modeled biomass yield based on catabolic energy harvest was higher than the biomass yield from experimental data (YX/cat = 25.9 ± 6.8 mgCODbiomass kJ−1), supporting restricted energy harvest of EAM and indicating a role of not considered energy sinks. This calls for an adjusted growth model for EAM, including, e.g., the microbial electrochemical Peltier heat to improve the understanding and modeling of their energy metabolism. Furthermore, the reported biomass yields are important parameters to design strategies for influencing the interactions between EAM and other microorganisms and allowing more realistic feasibility assessments of MET.

Microbial Electrochemical Systems and Membrane Bioreactor Technology for Wastewater Treatment

AbstractMembrane bioreactors (MBRs) and microbial electrochemical systems (MESs) are promising technologies for wastewater treatment and generation of electricity from organic matter. The application of MBRs is limited due to membrane fouling and high energy consumption. The novel design of the coupling system of microbial fuel cell and membrane bioreactor (MFC‐MBR), which efficiently combines microbial degradation in MFCs and uses the microelectric field to reduce and mitigate membrane fouling in MBR. The fundamentals of extracellular electron transport, the performance of MFCs, different types of MBR systems, and the novelty of the MFC‐MBR coupling system for wastewater treatment are reviewed.

Advancements in the development of membranes employed in microbial electrochemical technologies

Energy generation by the use of microbes is being widely studied for meeting the demands of ever-growing global energy consumption. In this veneration, microbial electrochemical technologies (METs) are one such technology, which is used to produce energy simultaneously along with wastewater treatment through the microbial degradation of organic matter. Further, the membranes employed in different types of METs play a crucial role in their operation and efficacy, and also their economic feasibility critically affects the scalability of METs. Therefore, the present review, elucidates the different types of membranes used in both lab-scale and field-scale METs, the performance of the METs in terms of wastewater treatment, coulombic efficiency and energy production with different membranes. Further, recent development in the field of membranes and their application in various types of METs and status quo are included along with proposing the future directions in membrane-based research.

Osmium-grafted magnetic nanobeads improve microbial current generation via culture-free and quick enrichment of electrogenic bacteria

The conventional methods of enriching electrogenic bacteria from the natural microbial inoculum rely on time-consuming pre-incubation, substantially limiting the applicability of microbial electrochemical technologies. Therefore, developing a rapid and feasible approach to enrich electrogenic bacteria is beneficial for implementing microbial electrochemical technologies. Here, labeling electrogenic bacteria from a microbiota was successfully achieved by polymerizing the osmiophilic 3,3′-diaminobenzidine (DAB) monomers to form the DAB polymer and then depositing these on the cell surface, which was catalytically triggered by electron transfer enzymes such as outer-membrane cytochromes (OMCs) on the surface of electrogenic bacteria. These osmiophilic DAB-labeled electrogenic bacteria were rapidly enriched through specific conjugation with magnetic nanoparticle surfaces where folic acid and osmium tetroxide (OsO4) were sequentially grafted to form osmium-DAB conjugation. Higher enrichment of OMC-rich Shewanella oneidensis MR-1 than OMC-deficient Escherichia coli K12 under DAB-labeled magnetic separation was confirmed by imaging, protein, and electrochemical analyses. Upon treating environmental microbiota by the DAB-labeled magnetic separation process, 16S rRNA gene analysis showed that the relative abundance of potential electrogenic bacteria increased, resulting in a 16-fold higher current production than the original sample. Furthermore, the enriched electrogenic bacteria showed higher performance for sustainable bioelectricity production than the initial microbiota during a week and more of electrochemical incubation. Our novel enrichment strategy reveals the massive potential in improving the efficiency of microbial electrochemical technologies and facilitating the identification of electrogenic bacteria from unexplored microbial resources.

Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Protection of electroactive biofilms against hypersaline shock by quorum sensing

Quorum sensing (QS) is an ideal strategy for boosting the operating performance of electroactive biofilms (EABs), but its potential effects on the protection of electroactive biofilms against environmental shocks (e.g., hypersaline shock) have been rarely revealed. In this study, a QS signaling molecule, the N-(3-oxo-dodecanoyl)-L-homoserine lactone, was employed to promote the anti-shock property of the EABs against extreme saline shock. The maximum current density of the QS-regulated biofilm recovered to 0.17 mA/cm2 after 10% salinity exposure, which was much higher than those of its counterparts. The laser scanning confocal microscope confirmed a thicker and more compact biofilm with the presence of the QS signaling molecule. The extracellular polymeric substances (EPS) might play a crucial role in the anti-shocking behaviors, as the polysaccharides in EPS of QS-biofilm had doubled compared to the groups with acylase (the QS quencher). The microbial community analysis indicated that the QS molecule enriched the relative abundance of key species including Pseudomonas sp. and Geobacter sp., which were both beneficial to the stability and electroactivity of the biofilms. The functional genes related to the bacterial community were also up-regulated with the presence of the QS molecule. These results highlight the importance of QS effects in protecting electroactive biofilm under extreme environmental shock, which provides effective and feasible strategies for the future development of microbial electrochemical technologies.