Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens, offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c-type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH\'s potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction.

The bacterial anode in microbial fuel cells was modified by increasing the biofilm\'s adhesion to the anode material using kaolin and graphite nanoparticles. The MFCs were inoculated with G. sulfurreducens, kaolin (12.5 g·L-1), and three different concentrations of graphite (0.25, 1.25, and 2.5 g·L-1). The modified anode with the graphite nanoparticles (1.25 g·L-1) showed the highest electroactivity and biofilm viability. A potential of 0.59, 0.45, and 0.23 V and a power density of 0.54 W·m-2, 0.3 W·m-2, and 0.2 W·m-2 were obtained by the MFCs based on kaolin-graphite nanoparticles, kaolin, and bare anodes, respectively. The kaolin-graphite anode exhibited the highest Coulombic efficiency (21%) compared with the kaolin (17%) and the bare (14%) anodes. Scanning electron microscopy and confocal laser scanning microscopy revealed a large amount of biofilm on the kaolin-graphite anode. We assume that the graphite nanoparticles increased the charge transfer between the bacteria that are in the biofilm and are far from the anode material. The addition of kaolin and graphite nanoparticles increased the attachment of several bacteria. Thus, for MFCs that are fed with wastewater, the modified anode should be prepared with a pure culture of G. sulfurreducens before adding wastewater that includes non-exoelectrogenic bacteria.

Graphene\'s exceptional electronic and mechanical properties make it a promising material for bioelectronic applications; however, understanding its interaction with electrogenic bacteria is crucial to harness its full potential. This study investigates the interface between electrogenic bacteria and graphene with Raman spectroscopy by analyzing the distinctive spectral fingerprints to understand electron energy and distribution via this non-destructive and label-free method. We find that the presence of bacteria induces a distinct red-shift in the G peak positions of graphene, indicating electron doping. Correspondingly, the bacteria demonstrate a predilection for attachment on hole-rich sites on the graphene sheet, evidenced by the comparative analysis of pre- and post-spatial Raman mapping, revealing their consistent presence within the hole-doped 2D peak position range of 2673.89-2675.43 cm-1. This affinity of bacteria is due to the overall higher Fermi level (∼4.9 ± 0.2 eV) of these regions, which favors electron transfer. These findings demonstrate the potential of leveraging the graphene\'s electronic properties in engineering graphene-based biosensors. Tuning graphene\'s charge carrier concentration would enable the promotion or prevention of bacterial attachment, facilitating capture of specific bacteria or development of antimicrobial surfaces. This approach enables clean, efficient, and accurate study of graphene-based bacterial systems, driving significant advancements and enhancing their performance.

Ferrous sulfide nanoparticles (nFeS) have proven to be effective in removing heavy metals (HMs) from wastewater. One such approach, which has garnered much attention as a sustainable technology, is via the in situ microbial synthesis of nFeS. Here, a sulfate-reducing bacteria (SRB) strain, Geobacter sulfurreducens, was used to initially biosynthesize ferrous sulfide nanoparticles (SRB-nFeS) and thereafter remove HMs from acid mine drainage (AMD). SRB-nFeS was characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM) coupled to an energy dispersive spectrometer (EDS), three-dimensional excitation-emission matrix (3D-EEM) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Such characterization showed that SRB mediated the reduction of SO42- to S2- to form nFeS, where the metabolized substances functioned as complexing agents which coordinated with nFeS to form biofunctional SRB-nFeS with improved stability. One advantage of this synthetic route was that the attachment of nFeS to the bacterial surface protected SRB cells from HM toxicity. Furthermore, due to a synergistic effect between nFeS and SRB, HM removal from both solution and AMD by SRB-nFeS was enhanced relative to the constituent components. Thus, after 5 consecutive cycles of HM removal, SRB-nFeS removed, Pb(Ⅱ) (92.6%), Cd(Ⅱ) (78.7%), Cu(Ⅱ) (76.0%), Ni(Ⅱ) (62.5%), Mn(Ⅱ) (62.2%), and Zn(Ⅱ) (88.5%) from AMD This study thus provides new insights into the biosynthesis of SRB-nFeS and its subsequent practical application in the removal of HMs from AMD.

Bioelectrochemical systems (BESs) are unique devices that harness the metabolic activity of electroactive microorganisms (EAMs) to convert chemical energy stored in organic substrates into electrical energy. Enhancing electron transfer efficiency between EAMs and electrodes is the key to practical implementation of BESs. Considering the role of outer membrane vesicles (OMVs) in mediating electron transfer of EAMs, a genetic engineering strategy to achieve OMVs overproduction was explored to enhance electron transfer efficiency and the underlying mechanisms were investigated. This study constructed a mutant strain of Geobacter sulfurreducens that lacked the ompA gene encoding an outer membrane protein. Experimental results showed that the mutant strain produced more OMVs and possessed higher electron transfer efficiency in Fe(III) reduction, dye degradation and current generation in BESs than the wild-type strain. More cargoes such as c-type cytochromes, functional proteins, eDNA, polysaccharides and signaling molecules that might be favorable for electron transfer and biofilm formation were found in OMVs produced by ompA-deficient anodic biofilm, which possibly contributed to the improved electron transfer efficiency of ompA-deficient biofilm. The results indicate that overproduction of OMVs in EAMs might be a potential strategy to enhance BESs performance.

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.

Electroactive bacteria can display notable plasticity in their response to magnetic field (MF), which prompted bioelectrochemical system as promising candidates for magnetic sensor applications. In this study, we explored the sensing and stimulatory effect of MF on current generation by Geobacter sulfurreducens, and elucidated the related molecular mechanism at the transcriptomic level. MF treatment significantly enhanced electricity generation and overall energy efficiency of G. sulfurreducens by 50 % and 22 %, respectively. The response of current to MFs was instantaneous and reversible. Cyclic voltammetry analysis of the anode biofilm revealed that the redox couples changed from -0.31 to -0.39 V (vs. Ag/AgCl), suggesting that MFs could alter electron transfer related components. Differential gene expression analysis further verified this hypothesis, genes associated with electron transfer were upregulated in G. sulfurreducens under MF treatment relative to the control group, specifically, genes encoding periplasmic c-type cytochromes (ppcA and ppcD), outer membrane cytochrome (omcF, omcZ, omcB), pili (pilA-C, pilM, and pilV2), and ribosome. The enhanced bacterial extracellular electron transfer process was also linked to the overexpression of the NADH dehydrogenase I subunit, the ABC transporter, transcriptional regulation, and ATP synthase. Overall, our findings shed light on the molecular mechanism underlying the effects of magnetic field stimuli on EAB and provide a theoretical basis for its further application in magnetic sensors and other biological system.

Geobacter sulfurreducens is part of a specialized group of microbes with the unique ability to exchange electrons with insoluble materials, such as iron oxides and electrodes. Therefore, G. sulfurreducens plays an essential role in the biogeochemical iron cycle and microbial electrochemical systems. In G. sulfurreducens this ability is primarily dependent on electrically conductive nanowires that link internal electron flow from metabolism to solid electron acceptors in the extracellular environment. Here we show that when carrying conjugative plasmids, which are self-transmissible plasmids that are ubiquitous in environmental bacteria, G. sulfurreducens reduces insoluble iron oxides at much slower rates. This was the case for all three conjugative plasmids tested (pKJK5, RP4 and pB10). Growth with electron acceptors that do not require expression of nanowires was, on the other hand, unaffected. Furthermore, iron oxide reduction was also inhibited in Geobacter chapellei, but not in Shewanella oneidensis where electron export is nanowire-independent. As determined by transcriptomics, presence of pKJK5 reduces transcription of several genes that have been shown to be implicated in extracellular electron transfer in G. sulfurreducens, including pilA and omcE. These results suggest that conjugative plasmids can in fact be very disadvantageous for the bacterial host by imposing specific phenotypic changes, and that these plasmids may contribute to shaping the microbial composition in electrode-respiring biofilms in microbial electrochemical reactors.

The reduction of Sb(V)-bearing ferrihydrite by Geobacter sulfurreducens was studied to determine the fate of the metalloid in Fe-rich systems undergoing redox transformations. Sb(V) added at a range of concentrations adsorbed readily to ferrihydrite, and the loadings had a pronounced impact on the rate and extent of Fe(III) reduction and the products formed. Magnetite dominated at low (0.5 and 1 mol%) Sb(V) concentrations, with crystallite sizes decreasing at higher Sb loadings: 37-, 25-, and 17-nm particles for no-Sb, 0.5% Sb, and 1% Sb samples, respectively. In contrast, goethite was the dominant end product for samples with higher antimony loadings (2 and 5 mol%), with increased goethite grain size in the 5% Sb sample. Inductively coupled mass spectrometry (ICP-MS) analysis confirmed that Sb was not released to solution during the bioreduction process, and X-ray photoelectron spectroscopy (XPS) analyses showed that no Sb(III) was formed throughout the experiments, confirming that the Fe(III)-reducing bacterium Geobacter sulfurreducens cannot reduce Sb(V) enzymatically or via biogenic Fe(II). These findings suggest that Fe (bio)minerals have a potential role in limiting antimony pollution in the environment, even when undergoing redox transformations. IMPORTANCE Antimony is an emerging contaminant that shares chemical characteristics with arsenic. Metal-reducing bacteria (such as Geobacter sulfurreducens) can cause the mobilization of arsenic from Fe(III) minerals under anaerobic conditions, causing widespread contamination of aquifers worldwide. This research explores whether metal-reducing bacteria can drive the mobilization of antimony under similar conditions. In this study, we show that G. sulfurreducens cannot reduce Sb(V) directly or cause Sb release during the bioreduction of the Fe(III) mineral ferrihydrite [although the sorbed Sb(V) did alter the Fe(II) mineral end products formed]. Overall, this study highlights the tight associations between Fe and Sb in environmental systems, suggesting that the microbial reduction of Fe(III)/Sb mineral assemblages may not lead to Sb release (in stark contrast to the mobilization of As in iron-rich systems) and offers potential Fe-based remediation options for Sb-contaminated environments.

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.