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In this study, the effect of modulating fulvic acid (FA) concentrations (0, 25 and 50 mg/L) on nitrogen removal in a bioelectrochemical hydrogen autotrophic denitrification system (BHDS) was investigated. Results showed that FA increased the nitrate (NO3--N) removal rate of the BHDSs from 37.8 to 46.2 and 45.2 mg N/(L·d) with a current intensity of 40 mA. The metagenomic analysis revealed that R2 (25 mg/L) was predominantly populated by autotrophic denitrifying microorganisms, which enhanced denitrification performance by facilitating electron transfer. Conversely, R3 (50 mg/L) exhibited an increase in genes related to the heterotrophic process, which improved the denitrification performance through the collaborative action of both autotrophic and heterotrophic denitrification pathways. Besides, the study also identified a potential for nitrogen removal in Serpentinimonas, which have been rarely studied. The interesting set of findings provide valuable reference for optimizing BHDS for nitrogen removal and promoting specific denitrifying genera within the system.

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The extracellular degradation of antibiotics facilitated by bio-nanoparticles is significant in the field of waste valorization. Among different bio-nanoparticles, bio-FeS nanoparticles stand out for their convenient and cost-effective synthesis. Nevertheless, there is a lack of understanding regarding the extracellular degradation of pollutants driven by bio-FeS nanoparticles. Hence, this study aimed to investigate the role of bio-FeS nanoparticles in the extracellular degradation of tetracycline under aerobic and anaerobic conditions. The findings demonstrated that bio-FeS nanoparticles generated hydroxyl radical (·OH), which significantly contributes to the degradation of tetracycline in both aerobic and anaerobic environments. The production of ·OH in anaerobic conditions was primarily attributed to the limited formation of FeS2 during the biosynthesis of nanoparticles, which was very different from aerobic conditions. The bio-FeS nanoparticles facilitated extracellular electron transport by promoting electron shuttles and Fe(II)/Fe(III) cycling, resulting in the continuous production of ·OH. The degradation pathways showed differences under aerobic and anaerobic conditions, with intermediates exhibiting higher toxicity and greater cellular damage under aerobic conditions. However, in anaerobic conditions, bio-FeS nanoparticles enabled the successful integration of intracellular and extracellular degradation of tetracycline. This research proposed a new avenue for biocatalysis and environmental remediation.

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In this study, we explored the extracellular electron transfer (EET) capabilities of two bacterial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gut and the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significant electrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode surface, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence of cell-surface redox substrate potentially mediating EET was revealed by the redox-based staining method and electrochemical voltammetry assay. However, medium swapping analyses and the addition of flavins, a model redox mediator, suggest that the current production is dominated by soluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cell surface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrast to OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redox molecules. Further, the mixture of the two strains did not increase the current production from OTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The present work revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm.

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During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

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Biological nitrogen removal in low C/N environment is challenging in wastewater treatment for a long time. Autotrophic ammonium oxidation is promising due to the no need of carbon source addition, but alternative electron acceptors other than oxygen has to be widely investigated. Recently, microbial electrolysis cell (MEC), which applies a polarized inert electrode as the electron harvester, has been proved effective to oxidize ammonium with electroactive biofilm. That is, anodic microbes stimulated by exogenous low power can extract electron from ammonium and transfer electron to electrodes. This review aims to consolidate the recent advances in anodic ammonium oxidation in MEC. Various technologies based on different functional microbes and mechanisms of these processes are reviewed. Thereafter, the crucial factors influencing the ammonium oxidation technology are discussed. Challenges and prospects of anodic ammonium oxidation in ammonium-containing wastewater treatment are also proposed to provide valuable insights on the technologic reference and potential value of MEC in ammonium-containing wastewater treatment.

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Shewanella oneidensis MR-1 is a promising electroactive microorganism in environmental bioremediation, bioenergy generation, and bioproduct synthesis. Accelerating the extracellular electron transfer (EET) pathway that enables efficient electron exchange between microbes and extracellular substances is critical for improving its electrochemical properties. However, the potential genomic engineering strategies for enhancing EET capabilities are still limited. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated dual-deaminase base editing system, named in situ protospacer-adjacent motif (PAM)-flexible dual base editing regulatory system (iSpider), for precise and high-throughput genomic manipulation. The iSpider enabled simultaneous C-to-T and A-to-G conversions with high diversity and efficiency in S. oneidensis. By weakening DNA glycosylase-based repair pathway and tethering two copies of adenosine deaminase, the A-to-G editing efficiency was obviously improved. As a proof-of-concept study, the iSpider was adapted to achieve multiplexed base editing for the regulation of the riboflavin biosynthesis pathway, and the optimized strain showed an approximately three-fold increase in riboflavin production. Moreover, the iSpider was also applied to evolve the performance of an inner membrane component CymA implicated in EET, and one beneficial mutant facilitating electron transfer could be rapidly identified. Taken together, our study demonstrates that the iSpider allows efficient base editing in a PAM-flexible manner, providing insights into the design of novel genomic tools for Shewanella engineering.

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To explore the application of biosensor in real-time monitoring of composite heavy metal polluted wastewater in view of the low performance of MFC sensor, this study used sodium alginate to immobilize biochar to the anode of MFC biosensor, and conducted a study on the sensor performance and related biological processes. The results showed that under the optimal HRT conditions, the output power of the MFC-sensor (BC-300) was 0.432 W/m3 after biochar modification, which was much higher than the highest power density of CG and BC-0 of 0.117 and 0.088 W/m3. The correlation coefficient was greater than that of the control group at the plating wastewater concentration of 0.1-1.0 M and had a wider detection range, and the time to recover the output voltage was 1/3 of that of the control group. The biochar significantly promoted the sensitivity, interference resistance, recovery and anti-interference performance of the MFC-sensor. The intrinsic mechanism was that the composition and structure of biochar lead to a 1.53 fold increase in the abundance of electrogenic microorganisms and the abundance of functional genes such as cytochrome c (MtrABC, CymA, Cox, etc.) and flavin (riba, Rib B, gdh, ushA, IDH, etc.) increased by about 1.03-3.20 times, which promoted the shift of electrons from intracellular to extracellular receptors and significantly improved the electron transfer and the energy metabolism efficiency. The results of this study can provide a reference for the application of MFCsensor to the detection of complex heavy metal effluents.

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Fe(II) clays are common across many environments, making them a potentially significant microbial substrate, yet clays are not well established as an electron donor. Therefore, we explored whether Fe(II)-smectite supports the growth of Sideroxydans lithotrophicus ES-1, a microaerophilic Fe(II)-oxidizing bacterium (FeOB), using synthesized trioctahedral Fe(II)-smectite and 2% oxygen. S. lithotrophicus grew substantially and can oxidize Fe(II)-smectite to a higher extent than abiotic oxidation, based on X-ray near-edge spectroscopy (XANES). Sequential extraction showed that edge-Fe(II) is oxidized before interior-Fe(II) in both biotic and abiotic experiments. The resulting Fe(III) remains in smectite, as secondary minerals were not detected in biotic and abiotic oxidation products by XANES and Mössbauer spectroscopy. To determine the genes involved, we compared S. lithotrophicus grown on smectite versus Fe(II)-citrate using reverse-transcription quantitative PCR and found that cyc2 genes were highly expressed on both substrates, while mtoA was upregulated on smectite. Proteomics confirmed that Mto proteins were only expressed on smectite, indicating that ES-1 uses the Mto pathway to access solid Fe(II). We integrate our results into a biochemical and mineralogical model of microbial smectite oxidation. This work increases the known substrates for FeOB growth and expands the mechanisms of Fe(II)-smectite alteration in the environment.

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BACKGROUND: Electric energy is not collected and utilized in biobutanol fermentation. The reason is that the yields of electron shuttles and nanowires are not enough to gather and transfer all electrons to the electrode in liquid fermentation. However, the solid matrix of the adsorption carrier may be conducive to the collection and transfer of electrons because of its good adsorption and conductivity. Therefore, this first-attempt study coupled microbial fuel cell (MFC) with adsorption carrier solid-state fermentation (ACSF). In addition, the effect and mechanism of adsorption carrier solid-state fermentation on power generation were explored. RESULTS: The power generation performance and fermentation performance were improved by ACSF. The power density by polyurethane and carbon felt carrier solid-state fermentation (PC) was 12 times that by no carrier fermentation (NC). The biobutanol yield of absorbent cotton and carbon felt carrier solid-state fermentation (ACC) was increased by 36.86%. Moreover, the mechanism was explored via metabolic flux analysis, cyclic voltammetry and scanning electron microscopy. The results of metabolic flux analysis showed that more electrons were produced and more carbon flowed to biobutanol production. The cyclic voltammetry results revealed that more riboflavin was produced to enhance extracellular electron transport (EET) by ACSF. The scanning electron microscopy image showed that the adsorption capacity and aggregation degree of bacteria were increased on the electrode and nanowires were observed by ACSF. CONCLUSIONS: A new fermentation mode was established by coupling MFC with ACSF to improve substrate utilization, which will provide crucial insights into the fermentation industry. In addition, the ACSF is an effective method to enhance power generation performance and fermentation performance.

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A rapid and label-free method for the detection of drug-resistant pathogens is in high demand for wastewater-based epidemiology. As recently shown, the extent of electrical current production (Ic) is a useful indicator of a pathogen's metabolic activity. Therefore, if drug-resistant bacteria have extracellular electron transport (EET) capability, a simple electric sensor may be able to detect not only the growth as a conventional plating technique but also metabolic activity specific for drug-resistant bacteria in the presence of antibiotics. Here, one of the multidrug-resistant pathogens in wastewater, Klebsiella pneumoniae, was shown to generate Ic, and the extent of Ic was unaffected by the microbial growth inhibitor, kanamycin, while the current was markedly decreased in environmental EET bacteria Shewanella oneidensis. Kanamycin differentiated Ic in K. pneumonia and S. oneidensis within 3 h. Furthermore, the detection of K. pneumoniae was successful in the presence of S. oneidensis in the electrochemical cell. These results clarify the advantage of detecting drug-resistant bacteria using whole-cell electrochemistry as a simple and rapid method to detect on-site drug-resistant pathogens in wastewater, compared with conventional colony counting, which takes a few days.

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Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

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Many species of bacteria are naturally capable of types of electron transport not observed in eukaryotic cells. Some species live in environments containing heavy metals not typically encountered by cells of multicellular organisms, such as arsenic, cadmium, and mercury, leading to the evolution of enzymes to deal with these environmental toxins. Bacteria also inhabit a variety of extreme environments, and are capable of respiration even in the absence of oxygen as a terminal electron acceptor. Over the years, several of these exotic redox and electron transport pathways have been discovered and characterized in molecular-level detail, and more recently synthetic biology has begun to utilize these pathways to engineer cells capable of detecting and processing a variety of metals and semimetals. One such application is the biologically controlled synthesis of nanoparticles. This review will introduce the basic concepts of bacterial metal reduction, summarize recent work in engineering bacteria for nanoparticle production, and highlight the most cutting-edge work in the characterization and application of bacterial electron transport pathways.

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BACKGROUND: Halotolerant Fe (III) oxide reducers affiliated in the family Desulfuromonadaceae are ubiquitous and drive the carbon, nitrogen, sulfur and metal cycles in marine subsurface sediment. Due to their possible application in bioremediation and bioelectrochemical engineering, some of phylogenetically close Desulfuromonas spp. strains have been isolated through enrichment with crystalline Fe (III) oxide and anode. The strains isolated using electron acceptors with distinct redox potentials may have different abilities, for instance, of extracellular electron transport, surface recognition and colonization. The objective of this study was to identify the different genomic signatures between the crystalline Fe (III) oxide-stimulated strain AOP6 and the anode-stimulated strains WTL and DDH964 by comparative genome analysis. RESULTS: The AOP6 genome possessed the flagellar biosynthesis gene cluster, as well as diverse and abundant genes involved in chemotaxis sensory systems and c-type cytochromes capable of reduction of electron acceptors with low redox potentials. The WTL and DDH964 genomes lacked the flagellar biosynthesis cluster and exhibited a massive expansion of transposable gene elements that might mediate genome rearrangement, while they were deficient in some of the chemotaxis and cytochrome genes and included the genes for oxygen resistance. CONCLUSIONS: Our results revealed the genomic signatures distinctive for the ferric iron oxide- and anode-stimulated Desulfuromonas spp. strains. These findings highlighted the different metabolic abilities, such as extracellular electron transfer and environmental stress resistance, of these phylogenetically close bacterial strains, casting light on genome evolution of the subsurface Fe (III) oxide reducers.

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The development of a simple and direct assay for quantifying microbial metabolic activity is important for identifying antibiotic drugs. Current production capabilities of environmental bacteria via the process called extracellular electron transport (EET) from the cell interior to the exterior is well investigated in mineral-reducing bacteria and have been used for various energy and environmental applications. Recently, the capability of human pathogens for producing current has been identified in different human niches, which was suggested to be applicable for drug assessment, because the current production of a few strains correlated with metabolic activity. Herein, we report another strain, a highly abundant pathogen in human oral polymicrobial biofilm, Corynebacterium matruchotii, to have the current production capability associated with its metabolic activity. It showed the current production of 50 nA/cm2 at OD600 of 0.1 with the working electrode poised at +0.4 V vs. a standard hydrogen electrode in a three-electrode system. The addition of antibiotics that suppress the microbial metabolic activity showed a significant current decrease (>90%), establishing that current production reflected the cellular activity in this pathogen. Further, the metabolic fixation of atomically labeled 13C (31.68% ± 2.26%) and 15N (19.69% ± 1.41%) confirmed by high-resolution mass spectrometry indicated that C. matruchotii cells were metabolically active on the electrode surface. The identified electrochemical activity of C. matruchotii shows that this can be a simple and effective test for evaluating the impact of antibacterial compounds, and such a method might be applicable to the polymicrobial oral biofilm on electrode surfaces, given four other oral pathogens have already been shown the current production capability.

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Once pathogens form a biofilm, they become more tolerant to drugs and quicker to recover from physical removal than planktonic cells. Because such robustness of a biofilm is associated with the active metabolism of its constituent microbes, establishment of a direct assay quantifying biofilm's metabolic activity is important for developing antibiofilm substrates and techniques. Current production capability via extracellular electron transport (EET) was recently found in Gram-positive pathogens, which we hypothesized to correlate with the metabolic activity of their biofilm. Here, we identified current production from the biofilm of oral pathogen Streptococcus mutans that enables the electrochemical assessments of their metabolic activity in situ which conventionally require gene insertion for a fluorescent protein expression. Single-potential amperometry (SA) showed that S. mutans produced an anodic current and formed a biofilm within 8 h on a +0.4 V electrode vs a standard hydrogen electrode (SHE) in the presence of the electron donor glucose. Current production was significantly decreased by the addition of a metabolic inhibitor Triclosan. Furthermore, the anabolic activity of a single cell using high-resolution mass spectroscopy revealed that higher current production resulted in a higher metabolic fixation of an atomically labeled nitrogen 15N. These results demonstrate that current production in S. mutans reflects its metabolic activity. Given electrochemical impedance spectroscopy (EIS) helps quantifying the bacterial cell adhesion on the electrode, combination of EIS and SA could be a novel assay for EET capable pathogens for quantifying their time-dependent metabolic activity, cellular electrode coverage and physiological response to antibiofilm compounds.

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Bacterial extracellular electron transport (EET) plays an important role in many natural and engineering processes. Some periplasmic non-heme redox proteins usually coexist with c-type cytochromes (CTCs) during the EET process. However, in contrast to CTCs, little is known about the roles of these non-heme redox proteins in EET. In this study, the transcriptome of Shewanella decolorationis S12 showed that the gene encoding a periplasmic sulfite dehydrogenase molybdenum-binding subunit SorA was significantly up-regulated during electrode respiration in microbial fuel cells (MFCs) compared with that during azo-dye reduction. The maximum current density of MFCs catalyzed by a mutant strain lacking SorA (ΔsorA) was 25% higher than that of wild strain S12 (20 vs. 16 µA/cm2). Both biofilm formation and the current generation of the anodic biofilms were increased by the disruption of sorA, which suggests that the existence of SorA in S. decolorationis S12 inhibits electrode respiration. In contrast, disruption of sorA had no effect on respiration by S. decolorationis S12 with oxygen, fumarate, azo dye, or ferric citrate as electron acceptors. This is the first report of the specific effect of a periplasmic non-heme redox protein on EET to electrode and provides novel information for enhancing bacterial current generation.

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A flavin-based extracellular electron transfer mechanism (EET) has recently been described for the gram-positive Listeria monocytogenes. The gram-positive, solvent producing Clostridium acetobutylicum is a known flavin producer. Since flavin secretion in C. acetobutylicum can be triggered by a low-iron environment, the interaction of iron with an electrochemical system as well as the consequences for flavin production are investigated. It is shown that iron adsorbs onto the electrode's surface in the form of iron phosphorus compounds but that this iron is still bioavailable. Moreover, a shift in the flavin spectrum of the supernatant from high flavin mononucleotide percentages of 59% to high riboflavin (43-45%) and flavin adenine dinucleotide (FAD, 40-48%) content can be seen by limiting or omitting the iron source from the culture medium. When additionally an electric potential of -600 mV vs. Ag/AgCl (saturated KCl) is applied, the same overall trend is obtained but an increase in flavin concentration and especially in the FAD share between 6 and 27% is observed. This study is a first hint that a flavin-based EET might also take place in solventogenic Clostridia and highlights the importance of further investigation of flavin production and their involvement in EET mechanisms in different species.

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In microbial electrochemical systems, transport of electrons from bacteria to an electrode is the key to its functioning. However, the roles of several electron transport proteins, especially the membrane-bound dehydrogenases which link cellular metabolism to EET pathway are yet to be identified. NDH-2 is a non-proton pumping NADH dehydrogenase located in the inner membrane of several bacteria like Bacillus subtilis, Escherichia coli, etc. Unlike NADH dehydrogenase I, NDH-2 is not impeded by a high proton motive force thus helping in the increase of metabolic flux and carbon utilization. In the current study, NADH dehydrogenase II protein (NDH-2) was heterologously expressed from B. subtilis into E. coli BL21 (DE3) for enhancing electron flux through EET pathway and to understand its role in bioelectrogenesis. We found that E. coli expressing NDH-2 has increased the electron flux through EET and has shown a ninefold increase in current (4.7 µA) production when compared to wild strain with empty vector (0.52 µA). Furthermore, expression of NDH-2 also resulted in increased biofilm formation which can be corroborated with the decrease in charge transfer resistance of NDH-2 strain and increased NADH oxidation. It was also found that NDH-2 strain can reduce ferric citrate at a higher rate than wild type strain suggesting increased electron flux through electron transport chain due to NADH dehydrogenase II activity. Purified NDH-2 was found to be ∼42 kDa and has FAD as a cofactor. This work demonstrates that the primary dehydrogenases like NADH dehydrogenases can be overexpressed to increase the electron flux in EET pathway which can further enhance the microbial fuel cells performance.

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Long-range (>10 µm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within ∼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.

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Extracellular polymeric substances (EPS) play crucial roles in promoting biofilm formation and contribute to electrochemical activities of biofilms in bioelectrochemical systems (BES). In this study, three stratified EPS fractions were extracted from Geobacter biofilms using EDTA-, ultrasound- and heating-based protocols and characterized with chemical, spectral and electrochemical analyses. Results suggested that, for Geobacter biofilms, ultrasound-based extraction protocol was more effective in EPS yield (62.1-66.5 mg C/g dry cell) than EDTA method, and had less cell lysis than heating method. The extraction methods greatly affected the proteins composition in the extracted EPS, indicated by the varied ratios of tryptophan/tyrosine protein-like substances. Electrochemical measurements demonstrated a good correlation between protein concentration and extracellular electron transfer function for both tightly-bound EPS and total EPS. This is the first study to extract and characterize stratified EPS fractions from Geobacter biofilms, and helpful for better understanding the function of EPS in BESs predominated by Geobacter.

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