Shewanella Oneidensis Research Articles

Low-frequency seismic responses during microbial biofilm formation in sands

This study investigates changes in low-frequency attenuation responses of sands during microbial formation of soft viscous biofilms, or extracellular polymeric substances (EPS). The resonant column experiments were conducted with two model bacteria Shewanella oneidensis MR1 and Leuconostoc mesenteroides, while monitoring changes in the wave velocities and damping ratios associated with EPS formation in sands. The results show that the accumulation of soft, viscous EPS hardly changes the wave velocities, both the shear and flexural modes. By contrast, the low-frequency attenuations, both torsional and flexural damping ratios, show significant increases with the accumulation of highly viscous EPS. It is found that the contribution of EPS to seismic responses of water-saturated sands is mainly limited to the pore fluid component, causing additional energy dissipation during wave propagation, but with no or minimal impact on skeletal stiffness or no involvement in seismic stress transfer. With these unique and unprecedented low-frequency seismic data of biofilm-associated sands, the results suggest that the formation and accumulation of soft, viscous EPS or biofilms by bacterial activities can be detected by monitoring seismic attenuation and can also alter the seismic attenuation responses of sands, such as the case under earthquake loading or blast-induced compaction.

Engineered cytochrome fused extracellular matrix enabled efficient extracellular electron transfer and improved performance of microbial fuel cell

Microbial fuel cell (MFC) was a promising technology for energy harvesting from wastewater. However, inefficient bacterial extracellular electron transfer (EET) limited the performance as well as the applications of MFC. Here, a new strategy to reinforce the EET by engineering synthetic extracellular matrix (ECM) with cytochrome fused curli was developed. By genetically fusing a minimal cytochrome domain (MCD) with the curli protein CsgA and heterogeneously expressing in model exoelectrogen of Shewanella oneidensis MR-1, the cytochrome fused electroactive curli network was successfully constructed and assembled. Interestingly, the strain with the MCD fused synthetic ECM delivered about 2.4 times and 2.0 times higher voltage and power density output than these of wild type MR-1 in MFC. More impressively, electrochemical analysis suggested that this synthetic ECM not only introduced cytochrome of MCD, but also attracted more self-secreted electrochemically active substances, which might facilitate the EET and improve the MFC performance. This work demonstrated the possibility to manipulation the EET with ECM engineering, which opened up new path for exoelectrogen design and engineering.

Microbial iron reduction does not release microplastics from organo‐metallic aggregates

AbstractIron flocculants play a major role in the remediation of water bodies, removing particulate pollutants such as microplastics through floc formation. Such flocs are prone to microbial iron reduction while lying on top of anoxic sediments, which possibly leads the release of bound microplastics. In this study, Shewanella oneidensis was employed to simulate the impact of microbial iron reduction on the release of polyethylene spheres from sunken flocs in 120 d batch experiments. Most of the flocs iron (oxyhydr)oxides were reduced (70–90%), but this did not affect their integrity. Only a negligible proportion (0.2–2.7%) of polyethylene spheres was released, while the majority remained bound inside the floc matrix. This study exemplifies that flocs are quite stable, even when experiencing microbial iron reduction under anoxic conditions. Thereby incorporation into such aggregates may display a potential mode of long‐term microplastics storage in freshwater sediments.

Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.

Diverse Protein Architectures and α-N-Methylation Patterns Define Split Borosin RiPP Biosynthetic Gene Clusters.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.

Biochar-Mediated Degradation of Roxarsone by Shewanella oneidensis MR-1.

It is widely believed that biochar plays an essential role in sequestrating pollutants. The impacts of biochar on microbial growth, and consequently on the environmental fate of pollutants, however, remains poorly understood. In this study, wheat-straw-derived biochar was used to investigate how biochar amendment affected Shewanella oneidensis MR-1 growth and roxarsone transformation in water under anaerobic conditions. Three biochar with different physicochemical properties were used to mediate the roxarsone degradation. The results showed that the degradation rate of roxarsone could be accelerated by the increase of biochar pyrolysis temperature. From the characterization of biochar, the total specific surface area, micropore surface area and micropore volume of biochar increase, but the average pore diameter decreases as the pyrolysis temperature increases. Through infrared spectroscopy analysis, it was found that as the pyrolysis temperature increases, the degree of condensation of biochar increases, thereby increasing the pollutant removal rate. From the changes of the relative concentration of MR-1 and its secreted extracellular polymer content, the growth promotion ability of biochar also increases as the pyrolysis temperature increases. These results suggest that wheat-straw-derived biochar may be an important agent for activating microbial growth and can be used to accelerate the transformation of roxarsone, which could be a novel strategy for roxarsone remediation.

Unveiling the chemotactic response and mechanism of Shewanella oneidensis MR-1 to nitrobenzene

Bioreduction by electroactive bacteria (EAB) is considered as a potential and cost-effective approach for the removal of nitroaromatic compounds (NACs). However, little is known about how the widespread EAB sense and respond to slightly soluble NACs in aquatic environments. Here, the chemotactic behaviors of Shewanella oneidensis MR-1, a model EAB, toward several NACs were examined and their underlying molecular mechanism was elucidated. S. oneidensis MR-1 was found to exhibit a strong chemotactic response to nitrobenzene (NB), but not to other selected NACs under aerobic conditions. To sense NB, this bacterium requires both the histidine kinase (CheA-3)-involved chemotactic signal transduction pathway and an inner-membrane c-type cytochrome CymA. Such a chemotactic response is mediated by an energy taxis mechanism. Additionally, external riboflavin was shown to greatly enhance the Shewanella taxis toward NB, implying a feasible way to increase the bioavailability of NACs. The present study deepens our understanding of the role of microbial chemotaxis in the removal of NACs and provides more options for the bioremediation of NAC-contaminated sites.

Changes in Magnetic Properties of Magnetite Nanoparticles Upon Microbial Iron Reduction

AbstractThe magnetic signals of magnetite nanoparticles (NPs) preserved in rocks, soils, and sediments are effective index for paleoenvironmental reconstruction. It has been demonstrated that magnetite NPs can serve as a terminal electron sink for the microbial respiration (i.e., microbial iron reduction). The magnetic properties of magnetite NPs may be altered by microbial iron reduction, which is a critical but often overlooked process in paleomagnetism. In this study, three magnetite NPs with different particle sizes were reduced by a dissimilatory iron‐reducing bacterium (Shewanella oneidensis MR‐1) under a non‐growth condition mimicking that of the early Earth and modern oligotrophic environment. The changes in magnetic, chemical as well as crystallographic properties of the magnetite NPs during the microbial reduction process were examined. Our results showed that the bioreduction rate of magnetite NPs was mainly controlled by their particle size and redox state. In addition, the microbial iron reduction could affect both the crystallographic and magnetic properties of three types of magnetite NPs used herein. After bioreduction, the crystal lattice parameters and magnetic susceptibility of the magnetite NPs increased, while their remanence recording capability and coercivity decreased (i.e., “softer” magnetism). Furthermore, bioreduced magnetite NPs had a larger remanence loss near the Verwey transition region with low‐temperature magnetic analysis. These results indicate that the microbial reduction of magnetite NPs deserves attention when sedimentary magnetites are used in paleoenvironment reconstruction.

Effect of TOC Concentration of Humic Substances as an Electron Shuttle on Redox Functional Groups Stimulating Microbial Cr(VI) Reduction.

Humic substances as an electron shuttle play an essential role in the biogeochemistry processes. However, the influence of total organic carbon (TOC) concentrations of humic substances on microbial Cr(VI) reduction remains unclear. In this study, the rates and extents of Cr(VI) reduction by Shewanella oneidensis MR-1 in the presence of Leonardite humic acids (LHA) and Pahokee peat humic acids (PPHA) with different TOC concentrations were evaluated. We found that the enhanced reduction in Cr(VI) was associated with TOC concentrations of 2.5–50 mg C/L of HA samples. The result shows that HA as an electron shuttle impacted both rates and extents of microbial Cr (VI) reduction, which delivered differently in terms of low TOC concentration range of 2.5 to 15 mg C/L and high concentration range of 15–50 mg C/L. The rates of Cr(VI) reduction significantly enhanced in the low TOC concentration range of HA compared to a high concentration range. The highest acceleration rate of Cr(VI) reduction was achieved at 15 mg C/L of HA. The quinone-like fluorophore was responsible for the main redox-active functional groups of HA by the three-dimensional excitation-emission spectroscopy. The fluorescence intensity of quinone-like fluorophore of HA in the low TOC concentration range was positively correlated with its acceleration coefficient, corresponding to the highest microbial Cr(VI) reduction rate obtained in 15 mg C/L of HA. These findings highlighted the effect of the TOC concentration of HA on microbial Cr(VI) reduction processes. It emphasized that the low TOC concentration of HA contributed to the high rates of Cr(VI) reduction, which is critical for better understanding the fate of Cr(VI) and evaluating the effectiveness of Cr(VI) restoration strategies in the future.

Solution-Deposited and Patternable Conductive Polymer Thin-Film Electrodes for Microbial Bioelectronics.

Microbial bioelectronic devices integrate naturally occurring or synthetically engineered electroactive microbes with microelectronics. These devices have a broad range of potential applications, but engineering the biotic-abiotic interface for biocompatibility, adhesion, electron transfer, and maximum surface area remains a challenge. Prior approaches to interface modification lack simple processability, the ability to pattern the materials, and/or a significant enhancement in currents. Here, a novel conductive polymer coating that significantly enhances current densities relative to unmodified electrodes in microbial bioelectronics is reported. The coating is based on a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) crosslinked with poly(2-hydroxyethylacrylate) (PHEA) along with a thin polydopamine (PDA) layer for adhesion to an underlying indium tin oxide (ITO) electrode. When used as an interface layer with the current-producing bacterium Shewanella oneidensis MR-1, this material produces a 178-fold increase in the current density compared to unmodified electrodes, a current gain that is higher than previously reported thin-film 2D coatings and 3D conductive polymer coatings. The chemistry, morphology, and electronic properties of the coatings are characterized and the implementation of these coated electrodes for use in microbial fuel cells, multiplexed bioelectronic devices, and organic electrochemical transistor based microbial sensors are demonstrated. It is envisioned that this simple coating will advance the development of microbial bioelectronic devices.

Enhancing the sensitivity of water toxicity detection based on suspended Shewanella oneidensis MR-1 by reversing extracellular electron transfer direction.

Water toxicity detection is of great significance to ensure the safety of water supply. With suspended electrochemically active bacteria (EAB) as the sensing element, a novel microbial electrochemical sensor (MES) has recently been reported for the real-time detection of water toxicity, but its practical applications need to further improve the sensitivity. Extracellular electron transfer (EET) is an important factor affecting MES performance. In the study, the EET of suspended EAB-based MES was optimized to further enhance the sensitivity. Firstly, by using a model EAB stain Shewanella oneidensis MR-1, it was revealed that the sensitivity was increased at most 2.7 times with inward EET (i.e., cathodic polarization). Then, a novel conjecture based on electron transfer and energy fluxes was proposed and testified to explain this phenomenon. Finally, three key operating parameters of inward EET were orthogonally optimized. The optimized parameters of inward EET included a potential of - 0.5V, a cell density of 1.8 × 108CFU/mL, and an electron acceptor concentration of 15mM.

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria.

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.

Current Production Capability of Drug-Resistant Pathogen Enables Its Rapid Label-Free Detection Applicable to Wastewater-Based Epidemiology.

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.

Development of Whole Genome-Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.

Electrochemical Microwell Plate to Study Electroactive Microorganisms in Parallel and Real-Time.

Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models Shewanella oneidensis and Geobacter sulfurreducens with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities (j max) and coulombic efficiencies (CE) that were well in line with literature data. The co-cultivation of S. oneidensis and G. sulfurreducens led to an increased current density of j max of 88.57 ± 14.04 µA cm−2 (lactate) and j max of 99.36 ± 19.12 µA cm−2 (lactate and acetate). Further, a decreased time period of reaching j max and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.

Comparative Study of Roxarsone Reduction by Shewanella oneidensis MR-1 and Cellulomonas sp. Strain Cellu-2a

Roxarsone, an organoarsenic compound, has been used extensively as an additive in poultry feed to promote animal growth. Although roxarsone is moderately toxic, it can be converted into more toxic compounds upon exposure to the environment, resulting in human health risks. This article reports on a comparative study of roxarsone reduction by fermentative, anaerobic Cellulomonas sp. Cellu-2a and a widely distributed metal-reducing bacterium, Shewanella oneidensis MR-1. The strain Cellu-2a showed a faster roxarsone reduction capability by fermentative growth than by MR-1 in anaerobic respiration. With an environmentally relevant mineral, hydrous ferric oxide (HFO) included in the incubations, there was a rather rapid abiotic (indirect, heterogeneous) reduction of roxarsone by the solid phase Fe(II) in HFO generated by reduction of Fe(III) of HFO by MR-1, while still direct reduction was major reaction by Cellu-2a even with HFO. This study provides evidence of direct and/or indirect bacterial reduction of roxarsone in fermentative conditions as well as in anaerobic respiration conditions, at exposure to the environment, which would assist understanding of environmental behaviors of roxarsone in biological settings with HFO, a naturally occurring ubiquitous mineral class.

Conjugated Polyelectrolyte/Bacteria Living Composites in Carbon Paper for Biocurrent Generation.

Successful practical implementation of bioelectrochemical systems (BES) requires developing affordable electrode structures that promote efficient electrical communication with microbes. Recent efforts have centered on immobilizing bacteria with organic semiconducting polymers on electrodes via electrochemical methods. This approach creates a fixed biocomposite that takes advantage of the increased electrode's electroactive surface area (EASA). Here, it is demonstrated that a biocomposite comprising the water-soluble conjugated polyelectrolyte CPE-K and electrogenic Shewanella oneidensis MR-1 can self-assemble with carbon paper electrodes, thereby increasing its biocurrent extraction by ≈6-fold over control biofilms. A ≈1.5-fold increment in biocurrent extraction is obtained for the biocomposite on carbon paper relative to the biocurrent extracted from gold-coated counterparts. Electrochemical characterization revealed that the biocomposite stabilized with the carbon paper more quickly than atop flat gold electrodes. Cross-sectional images show that the biocomposite infiltrates inhomogeneously into the porous carbon structure. Despite an incomplete penetration, the biocomposite can take advantage of the large EASA of the electrode via long-range electron transport. These results show that previous success on gold electrode platforms can be improved when using more commercially viable and easily manipulated electrode materials.

The influence of iron-binding ligands in the corrosion of carbon steel driven by iron-reducing bacteria

Iron reducing bacteria (IRB) are thought to accelerate the corrosion of steel by removing the Fe(III)-oxide passivating layer through iron respiration. We investigated the effect of the iron-binding ligands oxalate, malonate, and succinate on the corrosion of carbon steel driven by the IRB Shewanella oneidensis. These dicarboxylates were found to accelerate the corrosion of carbon steel driven by IRB up to 2.6 times more than the abiotic experiment without dicarboxylates. Iron dissolution was enhanced by dicarboxylates, and this influenced the ability of planktonic cells to engage in iron respiration. The strong iron-binding ligands oxalate and malonate supported iron reduction by planktonic cells, whereas in the experiments with succinate or without dicarboxylate, a direct contact mechanism with the solid Fe(III)-oxide was observed. Faster microbial respiration rates were found in experiments with succinate than with oxalate or malonate, suggesting a competition for iron between the microbial cells and the strong iron-binding ligands.

Biotic Process Dominated the Uptake and Transformation of Ag+ by Shewanella oneidensis MR-1.

Silver ions (Ag+) directly emitted from industrial sources or released from manufactured Ag nanoparticles (AgNPs) in biosolid-amended soils have raised concern about the risk to ecosystems. However, our knowledge of Ag+ toxicity, internalization, and transformation mechanisms to bacteria is still insufficient. Here, we combine the advanced technologies of hyperspectral imaging (HSI) and single-particle inductively coupled plasma mass spectrometry to visualize the potential formed AgNPs inside the bacteria and evaluate the contributions of biological and non-biological processes in the uptake and transformation of Ag+ by Shewanella oneidensis MR-1. The results showed a dose-dependent toxicity of Ag+ to S. oneidensis MR-1 in the ferrihydrite bioreduction process, which was primarily induced by the actively internalized Ag. Moreover, both HSI and cross-section high-resolution transmission electron microscopy results confirmed that Ag inside the bacteria existed in the form of particulate. The Ag mass distribution in and around live and inactivated cells demonstrated that the uptake and transformation of Ag+ by S. oneidensis MR-1 were mainly via biological process. The bioaccumulation of Ag+ may be lethal to bacteria. A better understanding of the uptake and transformation of Ag+ in bacteria is central to predict and monitor the key factors that control Ag partitioning dynamics at the biointerface, which is critical to develop practical risk assessment and mitigation strategies.

Evidence for Horizontal and Vertical Transmission of Mtr-Mediated Extracellular Electron Transfer among the Bacteria.

ABSTRACTSome bacteria and archaea have evolved the means to use extracellular electron donors and acceptors for energy metabolism, a phenomenon broadly known as extracellular electron transfer (EET). One such EET mechanism is the transmembrane electron conduit MtrCAB, which has been shown to transfer electrons derived from metabolic substrates to electron acceptors, like Fe(III) and Mn(IV) oxides, outside the cell. Although most studies of MtrCAB-mediated EET have been conducted in Shewanella oneidensis MR-1, recent investigations in Vibrio and Aeromonas species have revealed that the electron-donating proteins that support MtrCAB in Shewanella are not as representative as previously thought. This begs the question of how widespread the capacity for MtrCAB-mediated EET is, the changes it has accrued in different lineages, and where these lineages persist today. Here, we employed a phylogenetic and comparative genomics approach to identify the MtrCAB system across all domains of life. We found mtrCAB in the genomes of numerous diverse Bacteria from a wide range of environments, and the patterns therein strongly suggest that mtrCAB was distributed through both horizontal and subsequent vertical transmission, and with some cases indicating downstream modular diversification of both its core and accessory components. Our data point to an emerging evolutionary story about metal-oxidizing and -reducing metabolism, demonstrates that this capacity for EET has broad relevance to a diversity of taxa and the biogeochemical cycles they drive, and lays the foundation for further studies to shed light on how this mechanism may have coevolved with Earth’s redox landscape.