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

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

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Colony-Based Electrochemistry Reveals Electron Conduction Mechanisms Mediated by Cytochromes and Flavins in Shewanella oneidensis.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

The varied roles of pilA-N, omcE, omcS, omcT, and omcZ in extracellular electron transfer by Geobacter sulfurreducens.

Geobacter sulfurreducens mediates extracellular electron transfer (EET) reactions with different substrates, such as solid-phase Fe(III)-containing minerals, anodes and the cells of Geobacter metallireducens. To compare their roles in EET, the pilA-N, omcE, omcS, omcT and omcZ genes of G. sulfurreducens were systematically deleted. All mutants showed impaired and varied ability to form biofilms on nonconductive surface. Deletion of omcE also impaired bacterial ability to reduce ferrihydrite, but its impacts on the ability for anode reduction and the co-culture of G. metallireducens-G. sulfurreducens were minimal. The mutant without omcS showed diminished ability to reduce ferrihydrite and to form the co-culture, but was able to regain its ability to reduce anodes. Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture. Deletion of all tested genes abolished bacterial ability to reduce ferrihydrite and anodes. Triple-deletion of all omcS, omcT and omcZ abolished the ability of G. sulfurreducens to co-culture with G. metallireducens. However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens, which show their indispensable roles in direct electron transfer from G. metallireducens to G. sulfurreducens. Thus, the roles of pilA-N, omcE, omcS, omcT and omcZ for G. sulfurreducens in EET vary substantially, which also suggest that possession of PilA-N and multiple cytochromes of different structures enables G. sulfurreducens to mediate EET reactions efficiently with substrates of different properties.

Engineering Outer Membrane Vesicles to Increase Extracellular Electron Transfer of Shewanella oneidensis.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

Multiple roles of released c-type cytochromes in tuning electron transport and physiological status of Geobacter sulfurreducens.

Dissimilatory metal-reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c-type cytochromes (c-Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free-moving electron carriers in growth to promote electron transport. However, the type of these released c-Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c-Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c-Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c-Cyts accelerated the process of microbial extracellular electron transfer and mediated long-distance electron transfer. In particular, the presence of c-Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c-Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c-Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments.

A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1.

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 µA·cm-2 (ΔmtrA/omcA/mtrC), nearly equivalent to that of WT cells (194 µA·cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in ΔcymA and ΔmtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 µA·cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

Enhancing the extracellular electron transfer ability via Polydopamine@S. oneidensis MR-1 for Cr(VI) reduction.

Microbial reduction of hexavalent chromium (Cr (VI)) shows better efficiency and cost-effectiveness. However, immobilization of Cr (III) remains a challenge as there is a limited supply of electron donors. A greener and cleaner option for donating external electrons was using bioelectrochemical systems to perform the microbial reduction of Cr(VI). In this system, we constructed a polydopamine (PDA) decorated Shewanella oneidensis MR-1 (S. oneidensis MR-1) bioelectrode with bidirectional electron transport, abbreviated as PDA@S. oneidensis MR-1. The conjugated PDA distributed on the intracellular and extracellular of individual S. oneidensis MR-1 has been shown to accelerate electron transfer by outer membrane C-type cytochromes and flavin-bound MtrC/OmcA pathway by various electrochemical analyses. As expected, the PDA@S. oneidensis MR-1 biofilm achieved 88.1% Cr (VI) removal efficiency (RE) and 58.1% Cr (III) immobilization efficiency (IE) within 24 h under the autotrophic conditions at the optimal voltage (-150 mV) compared with the control potential (0 mV). The PDA@S. oneidensis MR-1 biofilm showed increased RE activity was attributed to the shortening of the distance between individual bacteria by PDA. This research provides a viable strategy for in situ bioremediation of Cr(VI) polluted aquatic environment.

Boosting bioelectricity generation in microbial fuel cells via biomimetic Fe-N-S-C nanozymes.

Anode performance has been regarded as a crucial factor determining long-term stability and electricity generation of microbial fuel cells (MFCs), which restricts by the difficult extracellular electron transfer (EET) on the microbe/anode interface. Herein, inspired by biological enzyme systems, this study synthesized the biomimetic nanozymes with Fe-N-S-C active sites as the anode materials of MFCs, which was similar to the hemes of c-type cytochromes (c-Cyts) for boosting EET process. As excepted, an obviously faster start-up and a much higher power density were achieved by the MFCs equipped with Fe-N-S-C nanozymes (startup time, 3.5 d; power density, 2366 ± 34 mW m-2) than that based on traditional carbon cloth (startup time, 5.6 d; power density, 1009 ± 26 mW m-2). Such unique features of Fe-N-S-C nanozymes anode not only greatly favored the bacterial adhesion and the electroactive bacteria enrichment on the anode surface, but also efficiently facilitated the EET process between the electroactive bacteria and anode surface. This study provided a feasible strategy for designing the novel MFC anode materials from the perspective of bionic enzyme.

Genetic Manipulation of Desulfovibrio ferrophilus and Evaluation of Fe(III) Oxide Reduction Mechanisms.

The sulfate-reducing microbe Desulfovibrio ferrophilus is of interest due to its relatively rare ability to also grow with Fe(III) oxide as an electron acceptor and its rapid corrosion of metallic iron. Previous studies have suggested multiple agents for D. ferrophilus extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme c-type cytochromes. However, the previous lack of a strategy for genetic manipulation of D. ferrophilus limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of D. ferrophilus genes of interest with an antibiotic resistance gene via double-crossover homologous recombination. Genes were identified that are essential for flagellum-based motility and the expression of the two types of D. ferrophilus pili. Disrupting flagellum-based motility or expression of either of the two pili did not inhibit Fe(III) oxide reduction, nor did deleting genes for multiheme c-type cytochromes predicted to be associated with the outer membrane. Although redundancies in cytochrome or pilus function might explain some of these phenotypes, overall, the results are consistent with D. ferrophilus primarily reducing Fe(III) oxide via an electron shuttle. The finding that D. ferrophilus is genetically tractable not only will aid in elucidating further details of its mechanisms for Fe(III) oxide reduction but also provides a new experimental approach for developing a better understanding of some of its other unique features, such as the ability to corrode metallic iron at high rates and accept electrons from negatively poised electrodes. IMPORTANCE Desulfovibrio ferrophilus is an important pure culture model for Fe(III) oxide reduction and the corrosion of iron-containing metals in anaerobic marine environments. This study demonstrates that D. ferrophilus is genetically tractable, an important advance for elucidating the mechanisms by which it interacts with extracellular electron acceptors and donors. The results demonstrate that there is not one specific outer surface multiheme D. ferrophilus c-type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the D. ferrophilus genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that D. ferrophilus has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like Shewanella and Geobacter species. Thus, the ability to genetically manipulate D. ferrophilus is likely to lead to new mechanistic concepts in electromicrobiology.

Electron transfer of extremophiles in bioelectrochemical systems.

The interaction of bacteria and archaea with electrodes is a relatively new research field which spans from fundamental to applied research and influences interdisciplinary research in the fields of microbiology, biochemistry, biotechnology as well as process engineering. Although a substantial understanding of electron transfer processes between microbes and anodes and between microbes and cathodes has been achieved in mesophilic organisms, the mechanisms used by microbes under extremophilic conditions are still in the early stages of discovery. Here, we review our current knowledge on the biochemical solutions that evolved for the interaction of extremophilic organisms with electrodes. To this end, the available knowledge on pure cultures of extremophilic microorganisms has been compiled and the study has been extended with the help of bioinformatic analyses on the potential distribution of different electron transfer mechanisms in extremophilic microorganisms.

Biomolecular Insights into Extracellular Pollutant Reduction Pathways of Geobacter sulfurreducens Using a Base Editor System.

Geobacter species are critically involved in elemental biogeochemical cycling and environmental bioremediation processes via extracellular electron transfer (EET), but the underlying biomolecular mechanisms remain elusive due to lack of effective analytical tools to explore into complicated EET networks. Here, a simple and highly efficient cytosine base editor was developed for engineering of the slow-growing Geobacter sulfurreducens (a doubling time of 5 h with acetate as the electron donor and fumarate as the electron acceptor). A single-plasmid cytosine base editor (pYYDT-BE) was constructed in G. sulfurreducens by fusing cytosine deaminase, Cas9 nickase, and a uracil glycosylase inhibitor. This system enabled single-locus editing at 100% efficiency and showed obvious preference at the cytosines in a TC, AC, or CC context than in a GC context. Gene inactivation tests confirmed that it could effectively edit 87.7-93.4% genes of the entire genome in nine model Geobacter species. With the aid of this base editor to construct a series of G. sulfurreducens mutants, we unveiled important roles of both pili and outer membrane c-type cytochromes in long-range EET, thereby providing important evidence to clarify the long-term controversy surrounding their specific roles. Furthermore, we find that pili were also involved in the extracellular reduction of uranium and clarified the key roles of the ExtHIJKL conduit complex and outer membrane c-type cytochromes in the selenite reduction process. This work developed an effective base editor tool for the genetic modification of Geobacter species and provided new insights into the EET network, which lay a basis for a better understanding and engineering of these microbes to favor environmental applications.

Coupling riboflavin de novo biosynthesis and cytochrome expression for improving extracellular electron transfer efficiency in Shewanella oneidensis.

Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c-Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine-tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, and ybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c-Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c-Cyts expression were combined, resulting in 2.34-fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.

Sporomusa ovata as Catalyst for Bioelectrochemical Carbon Dioxide Reduction: A Review Across Disciplines From Microbiology to Process Engineering.

Sporomusa ovata is a bacterium that can accept electrons from cathodes to drive microbial electrosynthesis (MES) of acetate from carbon dioxide. It is the biocatalyst with the highest acetate production rate described. Here we review the research on S. ovata across different disciplines, including microbiology, biochemistry, engineering, and materials science, to summarize and assess the state-of-the-art. The improvement of the biocatalytic capacity of S. ovata in the last 10 years, using different optimization strategies is described and discussed. In addition, we propose possible electron uptake routes derived from genetic and experimental data described in the literature and point out the possibilities to understand and improve the performance of S. ovata through genetic engineering. Finally, we identify current knowledge gaps guiding further research efforts to explore this promising organism for the MES field.

The Role of Exopolysaccharides in Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c-type cytochromes (c-Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c-Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c-Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c-Cyts.

Response of the Anaerobic Methanotrophic Archaeon Candidatus "Methanoperedens nitroreducens" to the Long-Term Ferrihydrite Amendment.

Anaerobic methanotrophic (ANME) archaea can drive anaerobic oxidation of methane (AOM) using solid iron or manganese oxides as the electron acceptors, hypothetically via direct extracellular electron transfer (EET). This study investigated the response of Candidatus "Methanoperedens nitroreducens TS" (type strain), an ANME archaeon previously characterized to perform nitrate-dependent AOM, to an Fe(III)-amended condition over a prolonged period. Simultaneous consumption of methane and production of dissolved Fe(II) were observed for more than 500 days in the presence of Ca. "M. nitroreducens TS," indicating that this archaeon can carry out Fe(III)-dependent AOM for a long period. Ca. "M. nitroreducens TS" possesses multiple multiheme c-type cytochromes (MHCs), suggesting that it may have the capability to reduce Fe(III) via EET. Intriguingly, most of these MHCs are orthologous to those identified in Candidatus "Methanoperedens ferrireducens," an Fe(III)-reducing ANME archaeon. In contrast, the population of Ca. "M. nitroreducens TS" declined and was eventually replaced by Ca. "M. ferrireducens," implying niche differentiation between these two ANME archaea in the environment.

GSU1771 regulates extracellular electron transfer and electroactive biofilm formation in Geobacter sulfurreducens: Genetic and electrochemical characterization.

Type IV pili and the >100c-type cytochromes in Geobacter sulfurreducens are essential for extracellular electron transfer (EET) towards metal oxides and electrodes. A previous report about a mutation in the gsu1771 gene indicated an enhanced reduction of insoluble Fe(III) oxides coupled with increased pilA expression. Herein, a marker-free gsu1771-deficient mutant was constructed and characterized to assess the role of this regulator in EET and the formation of electroactive biofilms. Deleting this gene delayed microbial growth in the acetate/fumarate media (electron donor and acceptor, respectively). However, this mutant reduced soluble and insoluble Fe(III) oxides more efficiently. Heme staining, western blot, and RT-qPCR analyses demonstrated that GSU1771 regulates the transcription of several genes (including pilA) and many c-type cytochromes involved in EET, suggesting the broad regulatory role of this protein. DNA-protein binding assays indicated that GSU1771 directly regulates the transcription of pilA, omcE, omcS, and omcZ. Additionally, gsu1771-deficient mutant biofilms are thicker than wild-type strains. Electrochemical studies revealed that the current produced by this biofilm was markedly higher than the wild-type strains (approximately 100-fold). Thus, demonstrating the role of GSU1771 in the EET pathway and establishing a methodology to develop highly electroactive G. sulfurreducens mutants.

Uranium bioremediation with U(VI)-reducing bacteria.

Uranium (U) pollution is an environmental hazard caused by the development of the nuclear industry. Microbial reduction of hexavalent uranium (U(VI)) to tetravalent uranium (U(IV)) reduces U solubility and mobility and has been proposed as an effective method to remediate uranium contamination. In this review, U(VI) remediation with respect to U(VI)-reducing bacteria, mechanisms, influencing factors, products, and reoxidation are systematically summarized. Reportedly, some metal- and sulfate-reducing bacteria possess excellent U(VI) reduction capability through mechanisms involving c-type cytochromes, extracellular pili, electron shuttle, or thioredoxin reduction. In situ remediation has been demonstrated as an ideal strategy for large-scale degradation of uranium contaminants than ex situ. However, U(VI) reduction efficiency can be affected by various factors, including pH, temperature, bicarbonate, electron donors, and coexisting metal ions. Furthermore, it is noteworthy that the reduction products could be reoxidized when exposed to oxygen and nitrate, inevitably compromising the remediation effects, especially for non-crystalline U(IV) with weak stability.

Tailoring spatial structure of electroactive biofilm for enhanced activity and direct electron transfer on iron phthalocyanine modified anode in microbial fuel cells.

Electroactive biofilm (EAB) has been considered as the core determining electricity generation in microbial fuel cells (MFCs), and its spatial structure regulation for enhanced activity and selectivity is of great concern. In this study, iron phthalocyanine (FePc) was introduced into a carbon cloth (CC) electrode, aiming at improving the affinity between the anode and outer membrane c-type cytochromes (OM c-Cyts) and achieving a highly active EAB. The FePc modified CC anode (FePc-CC) effectively improved the viability of EAB and enriched the Geobacter species up to 44.83% (FePc-CC) from 6.97% (CC). The FePc-CC anode achieved a much higher power density of 2419 mW m-2 than the CC (560 mW m-2) and a remarkable higher biomass loading of 2477.2 ± 84.5 µg cm-2 than the CC (749.3 ± 31.3 µg cm-2). As the charge transfer resistance was decreased by 58.6 times from 395.2 Ω (CC) to 6.74 Ω (FePc-CC), the interfacial reaction rate was accelerated and the direct electron transfer via OM c-Cyts was promoted. This work provides an effective method to improve the EAB activity by regulating its spatial structure, and opens the door toward the development of highly active EAB using metal phthalocyanines in MFCs.