Fabrication of bio-abiotic hybrid living hydrogel for bifunctional electrochemical conversion.

Design and intelligent use renewable natural bioenergy is an important challenge. Electric microorganism-based materials are being serve as an important part of bioenergy devices for energy release and collection, calling for suitable skeleton materials to anchor live microbes. Herein we verified the feasibility of constructing bio-abiotic hybrid living materials based on the combination of gelatin, Li-ions and exoelectrogenic bacteria Shewanella oneidensis manganese-reducing-1 (MR-1). The gelatin-based mesh contains abundant pores, allowing microbes to dock and small molecules to diffuse. The hybrid materials hold plentiful electronegative groups, which effectively anchor Li-ions and facilitate their transition. Moreover, the electrochemical characteristics of the materials can be modulated through changing the ratios of gelatin, bacteria and Li-ions. Based on the gelatin-Li-ion-microorganism hybrid materials, a bifunctional device was fabricated, which could play dual roles alternatively, generation of electricity as a microbial fuel cell and energy storage as a pseudocapacitor. The capacitance and the maximum voltage output of the device reaches 68 F g-1 and 0.67 V, respectively. This system is a new platform and fresh start to fabricate bio-abiotic living materials for microbial electron storage and transfer. We expect the setup will extend to other living systems and devices for synthetic biological energy conversion.

Structural study of a polysaccharide component of nfnB mutant of Shewanella vesiculosa HM13.

Shewanella vesiculosa HM13 is a Gram-negative bacterium able to produce a large amount of extracellular membrane vesicles. These nanoparticles carry a major protein P49, the loading of which seems to be influenced by the glycans decorating the membrane. Here we report the structural characterization, using chemical analyses and NMR spectroscopy, of the capsular polysaccharides isolated from the nfnB-mutant strain of S. vesiculosa HM13, which is unable to load P49 on the membrane vesicles. In addition to the polysaccharide corona isolated and characterized from the parental strain, the nfnB-mutant strain released another polysaccharide composed of disaccharide repeating units having the following structure. →4)-ß-D-Glc-(1 â†’ 3)-ß-D-GlcNAc-(1→.

Total Synthesis of the Conjugation-Ready Tetrasaccharide Repeating Unit of Shewanella japonica Type Strain KMM 3299T.

Here we report the first total synthesis of the conjugation-ready tetrasaccharide repeating unit of Shewanella japonica type strain KMM 3299T. The presence of rare deoxyamino sugars and installation of three consecutive 1,2-cis glycosidic linkages makes the synthesis formidable. The challenging late-stage oxidation was overcome by using a galacturonate donor. The total synthesis was completed via a longest linear sequence of 22 steps in an overall yield of 3.5% starting from d-mannose.

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.

Interface Visualization of Bio-material Interaction Via Cryo-AEM Using the Biosynthesis of Iron-Based Nanoparticles as a Model.

Although interaction between organisms and nonorganisms is vital in environmental processes, it is difficult to characterize at nanoscale resolution. Biosynthesis incorporates intracellular and extracellular processes involving crucial interfacial functions and electron and substance transfer processes, especially on the inorganic-organic interface. This work chooses the biosynthesis of iron-based nanoparticles (nFe) as a model for biomaterial interaction and employs Cryo-AEM (i.e., S/TEM, EELS, and EDS analysis based on sample preparation with cryo-transfer holder system), combined with CV, Raman, XPS, and FTIR to reveal the inorganic-organic interface process. The inorganic-organic interactions in the biosynthesis of iron-based nanoparticles by Shewanella oneidensis MR-1 (M-nFe) were characterized by changes in electron cloud density, and the corresponding chemical shifts of Fe and C EELS edges confirm that M-nFe acquires electrons from MR-1 on the interface. Capturing intact filamentous-like, slightly curved, and bundled structure provides solid evidence of a "circuit channel" for electron transfer between organic and inorganic interface. CV results also confirm that adding M-nFe can enhance electron transfer from MR-1 to ferric ions. A mechanism for the synthesis of M-nFe with MR-1 based on intracellular and extracellular conditions under facultative anaerobic was visualized, providing a protocol for investigating the organic-inorganic interface.

A biocompatible surface display approach in Shewanella promotes current output efficiency.

The biology-material hybrid method for chemical-electricity conversion via microbial fuel cells (MFCs) has garnered significant attention in addressing global energy and environmental challenges. However, the efficiency of these systems remains unsatisfactory due to the complex manufacturing process and limited biocompatibility. To overcome these challenges, here, we developed a simple bio-inorganic hybrid system for bioelectricity generation in Shewanella oneidensis (S. oneidensis) MR-1. A biocompatible surface display approach was designed, and silver-binding peptide AgBP2 was expressed on the cell surface. Notably, the engineered Shewanella showed a higher electrochemical sensitivity to Ag+, and a 60 % increase in power density was achieved even at a low concentration of 10 µM Ag+. Further analysis revealed significant upregulations of cell surface negative charge intensity, ATP metabolism, and reducing equivalent (NADH/NAD+) ratio in the engineered S. oneidensis-Ag nanoparticles biohybrid. This work not only provides a novel insight for electrochemical biosensors to detect metal ions, but also offers an alternative biocompatible surface display approach by combining compatible biomaterials with electricity-converting bacteria for advancements in biohybrid MFCs.

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.

Mechanism of Reduction of Aqueous U(V)-dpaea and Solid-Phase U(VI)-dpaea Complexes: The Role of Multiheme c-Type Cytochromes.

The biological reduction of soluble U(VI) complexes to form immobile U(IV) species has been proposed to remediate contaminated sites. It is well established that multiheme c-type cytochromes (MHCs) are key mediators of electron transfer to aqueous phase U(VI) complexes for bacteria such as Shewanella oneidensis MR-1. Recent studies have confirmed that the reduction proceeds via a first electron transfer forming pentavalent U(V) species that readily disproportionate. However, in the presence of the stabilizing aminocarboxylate ligand, dpaea2- (dpaeaH2═bis(pyridyl-6-methyl-2-carboxylate)-ethylamine), biologically produced U(V) persisted in aqueous solution at pH 7. We aim to pinpoint the role of MHC in the reduction of U(V)-dpaea and to establish the mechanism of solid-phase U(VI)-dpaea reduction. To that end, we investigated U-dpaea reduction by two deletion mutants of S. oneidensis MR-1-one lacking outer membrane MHCs and the other lacking all outer membrane MHCs and a transmembrane MHC-and by the purified outer membrane MHC, MtrC. Our results suggest that solid-phase U(VI)-dpaea is reduced primarily by outer membrane MHCs. Additionally, MtrC can directly transfer electrons to U(V)-dpaea to form U(IV) species but is not strictly necessary, underscoring the primary involvement of outer membrane MHCs in the reduction of this pentavalent U species but not excluding that of periplasmic MHCs.

[Advances in electrochemically active biofilm of Shewanella oneidensis MR-1].

Facing the increasingly severe energy shortage and environmental pollution, electrocatalytic processes using electroactive microorganisms provide a new alternative for achieving environmental-friendly production. Because of its unique respiratory mode and electron transfer ability, Shewanella oneidensis MR-1 has been widely used in the fields of microbial fuel cell, bioelectrosynthesis of value-added chemicals, metal waste treatment and environmental remediation system. The electrochemically active biofilm of S. oneidensis MR-1 is an excellent carrier for transferring the electrons of the electroactive microorganisms. The formation of electrochemically active biofilm is a dynamic and complex process, which is affected by many factors, such as electrode materials, culture conditions, strains and their metabolism. The electrochemically active biofilm plays a very important role in enhancing bacterial environmental stress resistance, improving nutrient uptake and electron transfer efficiency. This paper reviewed the formation process, influencing factors and applications of S. oneidensis MR-1 biofilm in bio-energy, bioremediation and biosensing, with the aim to facilitate and expand its further application.

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.

An electrochemical system for the rapid and accurate quantitation of microbial exoelectrogenic ability.

Microbial extracellular electron transfer (EET) plays a vital role in globally important environmental phenomena, including bioremediation, bioenergy generation, and biofuel production. The quantitation of microbial exoelectrogenic ability is fundamental to studying the process of EET. However, there is no accurate and time-saving protocol to directly evaluate EET ability, hindering our understanding and application of EET. In this work, we proposed an accurate and rapid quantitation system for measuring EET ability using a gold-coated membrane filter as a working electrode. The quantitation signals could be recorded within 1 h and accurately normalized by the number of cells with outstanding repeatability and reproducibility. Further, this method could be distinguished microbial direct EET performances of different growth stages, and the results showed the middle logarithmic growth stage of Shewanella onedensis MR-1 had the best electrochemical activity. This method can be widely used for different types of electroactive microorganisms, including gram-negative bacteria, gram-positive bacteria, and fungi. Due to its time savings, accurate quantification and easy operation, this method provides a standard way to assess the role of EET ability.

Electrochemical Characteristics of Shewanella loihica PV-4 on Reticulated Vitreous Carbon (RVC) with Different Potentials Applied.

The current output of an anodic bioelectrochemical system (BES) depends upon the extracellular electron transfer (EET) rate from electricigens to the electrodes. Thus, investigation of EET mechanisms between electricigens and solid electrodes is essential. Here, reticulated vitreous carbon (RVC) electrodes are used to increase the surface available for biofilm formation of the known electricigen Shewanella loihica PV-4, which is limited in conventional flat electrodes. S. loihica PV-4 utilizes flavin-mediated EET at potential lower than the outer membrane cytochromes (OMC), while at higher potential, both direct electron transfer (DET) and mediated electron transfer (MET) contribute to the current output. Results show that high electrode potential favors cell attachment on RVC, which enhances the current output. DET is the prevailing mechanism in early biofilm, while the contribution of MET to current output increased as the biofilm matured. Electrochemical analysis under starvation shows that the mediators could be confined in the biofilm. The morphology of biofilm shows bacteria distributed on the top layer of honeycomb structures, preferentially on the flat areas. This study provides insights into the EET pathways of S. loihica PV-4 on porous RVC electrodes at different biofilm ages and different set potential, which is important for the design of real-world BES.

Accumulation and Pulse Electron Paramagnetic Resonance Spectroscopic Investigation of the 4-Oxidobenzyl Radical Generated in the Radical S-Adenosyl-l-methionine Enzyme HydG.

The radical S-adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN- ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB•), which is the precursor to the HydG p-cresol byproduct. Native HydG only generates a small amount of 4-OB•, limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB• is significantly increased in reactions using HydG variants, in which the "dangler Fe" to which CO and CN- bind is missing or substituted by a redox-inert Zn2+ ion. This allows for the detailed characterization of 4-OB• using high-field EPR and electron nuclear double resonance spectroscopy to extract its g-values and 1H/13C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB• models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB•. Overall, our results depict a clearer electronic structure of the transient 4-OB• radical and provide new insights into the radical SAM chemistry of HydG.

Identification dehydratase domains from Schizochytrium sp. and Shewanella sp. and distinct functions in biosynthesis of fatty acids.

Polyunsaturated fatty acid (PUFA) synthase is a special and effective enzyme for PUFA synthesis, and dehydratase (DH) domain played a crucial role in it. In this work, we compared four different DH domains from different strains (Schizochytrium sp. HX-308 and Shewanella sp. BR-2) and different gene clusters. First bioinformatics analysis showed that DH1, 2 and DH3 were similar to FabA and PKS-DH, respectively, and all of them got a hot-dog structure. Second, four DH domains were expressed in Escherichia coli that increased biomass. Especially, Schi-DH1,2 presented the highest dry cell weight of 2.3 g/L which was 1.62 times of that of control. Fatty acids profile analysis showed that DH1,2 could enhance the percentage of unsaturated fatty acids, especially DH1,2 from Schizochytrium sp., while DH3 benefited for the saturated fatty acid biosynthesis. Furthermore, five kinds of fatty acids were added to the medium to study the substrate preferences. Results revealed that DH1,2 domain preferred to acting on C16:0, while DH3 domain trended acting on C14:0 and C15:0, which illustrated DH from different clusters do have specific substrate preference. Besides, DH expression could save the cell growth inhibition by mid-chain fatty acids. This study provided more information about the catalysis mechanism of polyunsaturated fatty acid synthase and might promote the modification study based on this enzyme.

Electronic control of redox reactions inside Escherichia coli using a genetic module.

Microorganisms regulate the redox state of different biomolecules to precisely control biological processes. These processes can be modulated by electrochemically coupling intracellular biomolecules to an external electrode, but current approaches afford only limited control and specificity. Here we describe specific electrochemical control of the reduction of intracellular biomolecules in Escherichia coli through introduction of a heterologous electron transfer pathway. E. coli expressing cymAmtrCAB from Shewanella oneidensis MR-1 consumed electrons directly from a cathode when fumarate or nitrate, both intracellular electron acceptors, were present. The fumarate-triggered current consumption occurred only when fumarate reductase was present, indicating all the electrons passed through this enzyme. Moreover, CymAMtrCAB-expressing E. coli used current to stoichiometrically reduce nitrate. Thus, our work introduces a modular genetic tool to reduce a specific intracellular redox molecule with an electrode, opening the possibility of electronically controlling biological processes such as biosynthesis and growth in any microorganism.

Iron can be microbially extracted from Lunar and Martian regolith simulants and 3D printed into tough structural materials.

In-situ resource utilization (ISRU) is increasingly acknowledged as an essential requirement for the construction of sustainable extra-terrestrial colonies. Even with decreasing launch costs, the ultimate goal of establishing colonies must be the usage of resources found at the destination of interest. Typical approaches towards ISRU are often constrained by the mass and energy requirements of transporting processing machineries, such as rovers and massive reactors, and the vast amount of consumables needed. Application of self-reproducing bacteria for the extraction of resources is a promising approach to reduce these pitfalls. In this work, the bacterium Shewanella oneidensis was used to reduce three different types of Lunar and Martian regolith simulants, allowing for the magnetic extraction of iron-rich materials. The combination of bacterial treatment and magnetic extraction resulted in a 5.8-times higher quantity of iron and 43.6% higher iron concentration compared to solely magnetic extraction. The materials were 3D printed into cylinders and the mechanical properties were tested, resulting in a 400% improvement in compressive strength in the bacterially treated samples. This work demonstrates a proof of concept for the on-demand production of construction and replacement parts in space exploration.

Bacterial extracellular electron transfer: a powerful route to the green biosynthesis of inorganic nanomaterials for multifunctional applications.

Synthesis of inorganic nanomaterials such as metal nanoparticles (MNPs) using various biological entities as smart nanofactories has emerged as one of the foremost scientific endeavors in recent years. The biosynthesis process is environmentally friendly, cost-effective and easy to be scaled up, and can also bring neat features to products such as high dispersity and biocompatibility. However, the biomanufacturing of inorganic nanomaterials is still at the trial-and-error stage due to the lack of understanding for underlying mechanism. Dissimilatory metal reduction bacteria, especially Shewanella and Geobacter species, possess peculiar extracellular electron transfer (EET) features, through which the bacteria can pump electrons out of their cells to drive extracellular reduction reactions, and have thus exhibited distinct advantages in controllable and tailorable fabrication of inorganic nanomaterials including MNPs and graphene. Our aim is to present a critical review of recent state-of-the-art advances in inorganic biosynthesis methodologies based on bacterial EET using Shewanella and Geobacter species as typical strains. We begin with a brief introduction about bacterial EET mechanism, followed by reviewing key examples from literatures that exemplify the powerful activities of EET-enabled biosynthesis routes towards the production of a series of inorganic nanomaterials and place a special emphasis on rationally tailoring the structures and properties of products through the fine control of EET pathways. The application prospects of biogenic nanomaterials are then highlighted in multiple fields of (bio-) energy conversion, remediation of organic pollutants and toxic metals, and biomedicine. A summary and outlook are given with discussion on challenges of bio-manufacturing with well-defined controllability.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

The degradation activities for three seaweed polysaccharides of Shewanella sp. WPAGA9 isolated from deep-sea sediments.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

Combining two optimized and affordable methods to assign chemoreceptors to a specific signal.

Chemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies.