Silver nanoparticles boost charge-extraction efficiency in Shewanella microbial fuel cells.

Microbial fuel cells (MFCs) can directly convert the chemical energy stored in organic matter to electricity and are of considerable interest for power generation and wastewater treatment. However, the current MFCs typically exhibit unsatisfactorily low power densities that are largely limited by the sluggish transmembrane and extracellular electron-transfer processes. Here, we report a rational strategy to boost the charge-extraction efficiency in Shewanella MFCs substantially by introducing transmembrane and outer-membrane silver nanoparticles. The resulting Shewanella-silver MFCs deliver a maximum current density of 3.85 milliamperes per square centimeter, power density of 0.66 milliwatts per square centimeter, and single-cell turnover frequency of 8.6 × 105 per second, which are all considerably higher than those of the best MFCs reported to date. Additionally, the hybrid MFCs feature an excellent fuel-utilization efficiency, with a coulombic efficiency of 81%.

Single cell electron collectors for highly efficient wiring-up electronic abiotic/biotic interfaces.

By electronically wiring-up living cells with abiotic conductive surfaces, bioelectrochemical systems (BES) harvest energy and synthesize electric-/solar-chemicals with unmatched thermodynamic efficiency. However, the establishment of an efficient electronic interface between living cells and abiotic surfaces is hindered due to the requirement of extremely close contact and high interfacial area, which is quite challenging for cell and material engineering. Herein, we propose a new concept of a single cell electron collector, which is in-situ built with an interconnected intact conductive layer on and cross the individual cell membrane. The single cell electron collector forms intimate contact with the cellular electron transfer machinery and maximizes the interfacial area, achieving record-high interfacial electron transfer efficiency and BES performance. Thus, this single cell electron collector provides a superior tool to wire living cells with abiotic surfaces at the single-cell level and adds new dimensions for abiotic/biotic interface engineering.

Carbon dots-fed Shewanella oneidensis MR-1 for bioelectricity enhancement.

Bioelectricity generation, by Shewanella oneidensis (S. oneidensis) MR-1, has become particularly alluring, thanks to its extraordinary prospects for energy production, pollution treatment, and biosynthesis. Attempts to improve its technological output by modification of S. oneidensis MR-1 remains complicated, expensive and inefficient. Herein, we report on the augmentation of S. oneidensis MR-1 with carbon dots (CDs). The CDs-fed cells show accelerated extracellular electron transfer and metabolic rate, with increased intracellular charge, higher adenosine triphosphate level, quicker substrate consumption and more abundant extracellular secretion. Meanwhile, the CDs promote cellular adhesion, electronegativity, and biofilm formation. In bioelectrical systems the CDs-fed cells increase the maximum current value, 7.34 fold, and power output, 6.46 fold. The enhancement efficacy is found to be strongly dependent on the surface charge of the CDs. This work demonstrates a simple, cost-effective and efficient route to improve bioelectricity generation of S. oneidensis MR-1, holding promise in all relevant technologies.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes.

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Mechanism study of photo-induced gold nanoparticles formation by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1, a bioelectricity generating bacterium, is broadly used in bioremediation, microbial fuel cell and dissimilatory reduction and recovery of precious metals. Herein, we report for the first time that photo induction as a trigger to stimulate gold nanoparticles (Au@NPs) formation by MR-1, with wavelength and light intensity as two key variables. Results indicated that sigmoidal model is the best fit for Au@NPs formation at various wavelengths (with R2 > 0.97). Light intensity in terms of photosynthetic photon flux density (PPFD) critically influences the rate constant in the low-light intensity region (PPFD < 20), while wavelength controls the maximum rate constant in the high-light region (PPFD > 20). By deletion of Mtr pathway genes in MR-1, we proposed the mechanism for light induced Au@NP formation is the excitation effect of light on certain active groups and extracellular polymeric substances (EPS) on the cell surface. Also, the release of electrons from proteins and co-enzyme complexes enhance electron generation. To the best of our knowledge, this is the first-attempt to explore the effect of photo-induction on Au@NPs production by MR-1, which provides an alternative cost-effective and eco-friendly process in green chemical industry.

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography.

Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that S. oneidensis OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.

Anaerobic Respiration on Self-Doped Conjugated Polyelectrolytes: Impact of Chemical Structure.

We probe anaerobic respiration of bacteria in the presence of conjugated polyelectrolytes (CPEs). Three different CPEs were used to probe how structural variations impact biocurrent generation from Shewanella oneidensis MR-1. For the self-doped anionic CPE only, absorption spectroscopy shows that the addition of S. oneidensis MR-1 leads to the disappearance of the polaron (radical cation) band at >900 nm and an increase in the band at 735 nm due to the neutral species, consistent with electron transfer from microbe to polymer. Microbial three-electrode electrochemical cells (M3Cs) show an increase in the current generated by S. oneidensis MR-1 with addition of the self-doped CPE relative to other CPEs and controls. These experiments combined with in situ cyclic voltammetry suggest that the doped CPE facilitates electron transport to electrodes and reveal structure-function relationships relevant to developing materials for biotic/abiotic interfaces.

Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.

Deciphering the aggregation mechanism of bacteria (Shewanella oneidensis MR1) in the presence of polyethyleneimine: Effects of the exopolymeric superstructure and polymer molecular weight.

Aggregation tests between bacteria and Polyethyleneimine (PEI) of low (600g/mol) and high (750,000g/mol) molecular weight were performed in order to address the physico-chemical mechanisms underlying the interactions between cationic polymer and bacterial membranes. The selected strain, Schewanella oneidensis MR-1, produces a lipopolysaccharide (LPS) of various lengths depending on the growth conditions. Optical density, bioaggregate size, electrophoretic mobility measurements, TEM and AFM observations, and cell lysis tests (crystal violet release), were collected to describe the PEI-mediated aggregation of LPS-O-antigen-free and LPS-O-antigen-decorated bacteria. The results show that PEI of low molecular weight (600g/mol) fails to aggregate bacteria, whereas PEIs of higher molecular weight (60,000 and 750,000g/mol) lead to flocculation at low polymer concentrations. In addition, the LPS-O antigen bacterial superstructure is shown to act as a protective barrier, thus delaying the harmful effects of the cationic polymer. Despite this protection, the interaction of bacterial membranes with increasing concentrations of PEI leads to a series of deleterious processes including biosurface modification (peeling, membrane permeabilization and/or lysis), aggregation of bacterial cells, and complexation of PEI with both released biosurface fragments and cytoplasmic residues issued from lysis.

In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy.

Shewanella oneidensis MR-1 is an electroactive bacterium, capable of reducing extracellular insoluble electron acceptors, making it important for both nutrient cycling in nature and microbial electrochemical technologies, such as microbial fuel cells and microbial electrosynthesis. When allowed to anaerobically colonize an Ag/AgCl solid interface, S. oneidensis has precipitated silver nanoparticles (AgNp), thus providing the means for a surface enhanced confocal Raman microscopy (SECRaM) investigation of its biofilm. The result is the in-situ chemical mapping of the biofilm as it developed over time, where the distribution of cytochromes, reduced and oxidized flavins, polysaccharides and phosphate in the undisturbed biofilm is monitored. Utilizing AgNp bio-produced by the bacteria colonizing the Ag/AgCl interface, we could perform SECRaM while avoiding the use of a patterned or roughened support or the introduction of noble metal salts and reducing agents. This new method will allow a spatially and temporally resolved chemical investigation not only of Shewanella biofilms at an insoluble electron acceptor, but also of other noble metal nanoparticle-precipitating bacteria in laboratory cultures or in complex microbial communities in their natural habitats.

[Biosynthesis of selemium nanobars by Shewallena oneidensis MR-1].

OBJECTIVE: We used Shewallena oneidensis MR-1 to produce selemium (Se) nanobars and studied the influence of Se(IV) concentrations and incubation time on nanobars production. METHODS: We incubated Shewallena oneidensis MR-1 under anaerobic condition with Luria-Bertani (LB) liquid medium containing 0.1, 1.0, 10.0 or 100.0 mmol/L Se (IV) in Na2SeO3, to determine the optimal Se (IV) concentration for bacterial growth. Then, we incubated Shewallena oneidensis MR-1 with the optimal Se (IV) concentration and collected deposits 24 and 72 h after anearobic incubation. We used scanning electron microscopy, energy-dispersive X-ray and X-ray diffraction to analyse the deposits. RESULTS: The cross sectional diameter and length of deposits that were produced by Shewallena oneidensis MR-1 after 24 h incubation with 1 mmol/L Se(IV) was around 80 nm and 2-3 µm, respectively. However, the deposits after 72 h incubation exceeded the size limit of nano material. Furthermore, the energy-dispersive X-ray and the X-ray diffraction spectroscopy confirmed that the deposits were elemental Se. CONCLUSION: This study provides a viable method for the biosynthesis of Se nanoban Shewallena oneidensis MR-1 can produce a large number of Se nanobars at exponential phase under 0.1 mmol/L Se (IV).

Characterization of the enhancement of zero valent iron on microbial azo reduction.

BACKGROUND: The microbial method for the treatment of azo dye is promising, but the reduction of azo dye is the rate-limiting step. Zero valent iron (Fe(0)) can enhance microbial azo reduction, but the interactions between microbes and Fe(0) and the potential mechanisms of enhancement remain unclear. Here, Shewanella decolorationis S12, a typical azo-reducing bacterium, was used to characterize the enhancement of Fe(0) on microbial decolorization. RESULTS: The results indicated that anaerobic iron corrosion was a key inorganic chemical process for the enhancement of Fe(0) on microbial azo reduction, in which OH(-), H2, and Fe(2+) were produced. Once Fe(0) was added to the microbial azo reduction system, the proper pH for microbial azo reduction was maintained by OH(-), and H2 served as the favored electron donor for azo respiration. Subsequently, the bacterial biomass yield and viability significantly increased. Following the corrosion of Fe(0), nanometer-scale Fe precipitates were adsorbed onto cell surfaces and even accumulated inside cells as observed by transmission electron microscope energy dispersive spectroscopy (TEM-EDS). CONCLUSIONS: A conceptual model for Fe(0)-assisted azo dye reduction by strain S12 was established to explain the interactions between microbes and Fe(0) and the potential mechanisms of enhancement. This model indicates that the enhancement of microbial azo reduction in the presence of Fe(0) is mainly due to the stimulation of microbial growth and activity by supplementation with elemental iron and H2 as an additional electron donor. This study has expanded our knowledge of the enhancement of microbial azo reduction by Fe(0) and laid a foundation for the development of Fe(0)-microbial integrated azo dye wastewater treatment technology.

Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels.

Polygold-FISH for signal amplification of metallo-labeled microbial cells.

An improved in situ hybridization approach (Polygold-FISH) using biotinylated probes targeting multiple locations of the 16 S ribosomal subunit, followed by fluoronanogold-streptavidin labeling and autometallographic enhancement of nanogold particles was developed as a means of signal amplification of metallo-labeled cells, without the need for Catalyzed Reporter Deposition (CARD). Bacterial cells were readily detected based on their gold-particle signal using scanning-electron microscopy and energy-dispersive X-ray spectroscopy when contrasted with controls or cells hybridized with a single probe. Polygold-FISH presents an alternative to CARD-FISH, circumventing the need for aggressive oxidants, which is useful when products of microbial respiration such as those relevant at the microbe-mineral interface could be altered during processing for visualization.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Effects of bio-Au nanoparticles on electrochemical activity of Shewanella oneidensis wild type and ΔomcA/mtrC mutant.

Both Shewanella oneidensis MR-1 wild type and its mutant ΔomcA/mtrC are capable of transforming Au(III) into Au nanoparticles (AuNPs). Cyclic voltammetry reveals a decrease in redox current after the wild type is exposed to Au(III) but an increase in oxidation current for the mutant. The peak current of the wild type is much higher than that of the mutant before the exposure of Au(III), but lower than that of the mutant after the formation of AuNPs. This suggests that damage to the electron transfer chain in the mutant could be repaired by AuNPs to a certain extent. Spectroscopy and SDS-PAGE analysis indicate a decrease in cell protein content after the formation of AuNPs, which provides a convenient way to detect intracellular information on cells.

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens, Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces.

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48-72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N21 days of Serratia to be ca 3 Log10(CFU cm(-2)) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.

Microbial-enzymatic-hybrid biological fuel cell with optimized growth conditions for Shewanella oneidensis DSP-10.

In this work we present a biological fuel cell fabricated by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. This concept is devised as an extension to traditional biochemical methods by incorporating diverse biological catalysts with the aim of powering small devices. In preparing the biological fuel cell anode, novel hierarchical-structured architectures and biofilm configurations were investigated. A method for creating an artificial biofilm based on encapsulating microorganisms in a porous, thin film of silica was compared with S. oneidensis biofilms that were allowed to colonize naturally. Results indicate comparable current and power densities for artificial and natural biofilm formations, based on growth characteristics. As a result, this work describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells.