Insoluble Electron Acceptors Research Articles

The bidirectional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition.

Bidirectional extracellular electron transfer (EET) is crucial to upholding microbial metabolism with insoluble electron acceptors or donors in anoxic environments. Investigating bidirectional EET-capable microorganisms is desired to understand the cell-cell and microbe-mineral interactions and their role in mineral cycling besides leveraging their energy generation and conversion, biosensing, and bio-battery applications. Here, we report on iron cycling by haloalkaliphilic Geoalkalibacter halelectricus via bidirectional EET under haloalkaline conditions. It efficiently reduces Fe3+ oxide (Fe2O3) to Fe0 at a 0.75 ± 0.08 mM/mgprotein/d rate linked to acetate oxidation via outward EET and oxidizes Fe0 to Fe3+ at a 0.24 ± 0.03 mM/mgprotein/d rate via inward EET to reduce fumarate. Bioelectrochemical cultivation confirmed its outward and inward EET capabilities. It produced 895 ± 23 µA/cm2 current by linking acetate oxidation to anode reduction via outward EET and reduced fumarate by drawing electrons from the cathode (‒2.5 ± 0.3 µA/cm2) via inward EET. The cyclic voltammograms of G. halelectricus biofilms revealed redox moieties with different formal potentials, suggesting the involvement of different membrane components in bidirectional EET. The cyclic voltammetry and GC-MS analysis of the cell-free spent medium revealed the lack of soluble redox mediators, suggesting direct electron transfer by G. halelecctricus in achieving bidirectional EET. By reporting on the first haloalkaliphilic bacterium capable of oxidizing and reducing insoluble Fe0 and Fe3+ oxide, respectively, this study advances the limited understanding of the metabolic capabilities of extremophiles to respire on insoluble electron acceptors or donors via bidirectional EET and invokes the possible role of G. halelectricus in iron cycling in barely studied haloalkaline environments. IMPORTANCE Bidirectional extracellular electron transfer (EET) appears to be a key microbial metabolic process in anoxic environments that are depleted in soluble electron donor and acceptor molecules. Though it is an ecologically important and applied microbial phenomenon, it has been reported with a few microorganisms, mostly from nonextreme environments. Moreover, direct electron transfer-based bidirectional EET is studied for very few microorganisms with electrodes in engineered systems and barely with the natural insoluble electron acceptor and donor molecules in anoxic conditions. This study advances the understanding of extremophilic microbial taxa capable of bidirectional EET and its role in barely investigated Fe cycling in highly saline-alkaline environments. It also offers research opportunities for understanding the membrane components involved in the bidirectional EET of G. halelectricus. The high rate of Fe3+ oxide reduction activity by G. halelectricus suggests its possible use as a biocatalyst in the anaerobic iron bioleaching process under neutral-alkaline pH conditions.

Anammox with alternative electron acceptors: perspectives for nitrogen removal from wastewaters.

In the context of the anaerobic ammonium oxidation process (anammox), great scientific advances have been made over the past two decades, making anammox a consolidated technology widely used worldwide for nitrogen removal from wastewaters. This review provides a detailed and comprehensive description of the anammox process, the microorganisms involved and their metabolism. In addition, recent research on the application of the anammox process with alternative electron acceptors is described, highlighting the biochemical reactions involved, its advantages and potential applications for specific wastewaters. An updated description is also given of studies reporting the ability of microorganisms to couple the anammox process to extracellular electron transfer to insoluble electron acceptors; particularly iron, carbon-based materials and electrodes in bioelectrochemical systems (BES). The latter, also referred to as anodic anammox, is a promising strategy to combine the ammonium removal from wastewater with bioelectricity production, which is discussed here in terms of its efficiency, economic feasibility, and energetic aspects. Therefore, the information provided in this review is relevant for future applications.

Extracellular Electron Transfer of Weak Electricigens in the Presence of a Competing Electron Acceptor

To maximise microbial electroactivity in bioelectrochemical systems, soluble electron acceptors are typically omitted as they compete with the electrode. While practical, this approach provides engineered conditions that do not reflect the natural environment of electroactive microorganisms, which may contain both soluble and insoluble electron acceptors. This study investigates the behaviour of weak electricigens, a relatively understudied category of microorganisms whose members switch between non-electroactive and electroactive states. Enrichments were performed in microbial fuel cells containing both an electrode and the soluble alternative fumarate to probe extracellular electron transfer of weak electricigens. Using fluorescence spectroscopy, chromatography and voltammetry, the electron shuttle riboflavin was not found in these conditions but was found in controls in which only the electrode was available to reduce. Despite this dichotomy in ability to perform riboflavin-based mediated electron transfer, communities of weak electricigens were similarly electroactive in each condition (19.36 ± 0.9 mW m−2 vs 20.25 ± 2.0 mW m−2). 16S rRNA gene sequencing revealed similar communities enriched in each condition, but with differing abundance. Understanding extracellular electron transfer in natural environments is of both fundamental and applied interest, as it can inform the design of real-world bioelectrochemical systems whose influents are likely to contain competing electron acceptors.

Direct extracellular electron transfer to an indium tin doped oxide electrode via heme redox reactions in Desulfovibrio ferrophilus IS5

Soluble redox mediators have been hypothesized to play a major role in cellular electron transportation to extracellular insoluble electron acceptors in sulfate-reducing bacteria (SRB) through a diffusion-based electron shuttle mechanism. Herein, in vivo electrochemistry shows that outer-membrane cytochromes (OMCs) directly mediated extracellular electron transfer (EET) in Desulfovibrio ferrophilus IS5. Anodic current production by IS5, coupled with lactate oxidation, was detected on indium-tin-doped oxide electrodes poised at +400 mV (vs. standard hydrogen electrode), which was the sole electron acceptor. Subsequent electrolyte replacement experiments with a fresh one showed that cells attached to the electrode contributed to 73% of current production by direct EET, while the electron shuttle mechanism may have contributed to the rest. Differential pulse voltammetry detected two redox species at E = −180 and 20 mV, with the former being most likely assignable to cell-surface cytochromes because nitric oxide decreased the peak current at −180 mV, as previously demonstrated with OMCs in iron-reducing bacteria. The EET onset potential at −200 mV further supports the notion that OMCs mediated EET in IS5, and that EET is thermodynamically favorable for reducing Fe(III) oxide. These results suggest that the direct EET mechanism regulates the reduction rate of insoluble substrates in SRB.

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

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

Soluble Iron Enhances Extracellular Electron Uptake by Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) is a process that microorganisms use to reduce or oxidize external insoluble electron acceptors or donors. Much of our mechanistic understanding of this process is derived from studies of transmembrane cytochrome complexes and extracellular redox shuttles that mediate outward EET to anodes and external electron acceptors. In contrast, there are knowledge gaps concerning the reverse process of inward EET from external electron donors to cells. Here, we describe a role for soluble iron (exogenous FeCl2) in enhancing EET from cathodes to the model EET bacterium Shewanella oneidensis MR-1, with fumarate serving as the intracellular electron acceptor. This iron concentration-dependent electron uptake was eradicated upon addition of an iron chelator and occurred only in the presence of fumarate reductase, confirming an electron pathway from cathodes to this periplasmic enzyme. Moreover, S. oneidensis mutants lacking specific outer membrane and periplasmic cytochromes exhibited significantly decreased current levels relative to wild-type. These results indicate that soluble iron can function as an electron carrier to the EET machinery of S. oneidensis.

Geobacter sulfurreducens' Unique Metabolism Results in Cells with a High Iron and Lipid Content.

Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.

Geoalkalibacter halelectricus SAP-1 sp. nov. possessing extracellular electron transfer and mineral-reducing capabilities from a haloalkaline environment.

The extracellular electron transfer (EET)-capable electroactive microorganisms (EAMs) play crucial roles in mineral cycling and interspecies electron transfer in different environments and are used as biocatalysts in microbial electrochemical technologies. Studying EAMs from extreme environments is desired to advance the electromicrobiology discipline, understanding their unique metabolic traits with implications to extreme microbiology, and develop specific bioelectrochemical applications. Here, we present a novel haloalkaliphilic bacterium named Geoalkalibacter halelectricus SAP-1, isolated from a microbial electroactive biofilm enriched from the haloalkaline lake sediments. It is a rod-shaped Gram-negative heterotrophic anaerobe that uses various carbon and energy sources and respires on soluble and insoluble terminal electron acceptors. Besides 16S-rRNA and whole-genome sequence-based phylogeny, the GGDC values of 21.7%, ANI of 78.5%, and 2.77% genomic DNA GC content difference with the closest validly named species Geoalkalibacter ferrihydriticus (DSM 17813T ) confirmed its novelty. When grown with the solid-state electrode as the only electron acceptor, it produced 460 ± 23 μA/cm2 bioelectrocatalytic current, thereby confirming its electroactivity. Further electrochemical analysis revealed the presence of membrane redox components with a high formal potential, putatively involved in the direct mode of EET. These are distinct from EET components reported for any known electroactive microorganisms, including well-studied Geobacter spp., Shewanella spp., and Desulfuromonas acetexigens. The capabilities of G. halelectricus SAP-1 to respire on soluble and insoluble electron acceptors including fumarate, SO4 2- , Fe3+ , and Mn4+ suggests its role in cycling these elements in haloalkaline environments.

Cytochrome OmcS Is Not Essential for Extracellular Electron Transport via Conductive Pili in Geobacter sulfurreducens Strain KN400.

The multi-heme c-type cytochrome OmcS is one of the central components used for extracellular electron transport in the Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens, is currently a matter of debate. Therefore, we investigated the function of OmcS in the G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than the DL-1 strain. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and that the cells expressed conductive filaments. Replacing the wild-type pilin gene with the aro-5 pilin gene eliminated the OmcS-deficient strain's ability to transport electrons to insoluble electron acceptors and diminished filament conductivity. These results are consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer. IMPORTANCE OmcS has been widely studied and noted to be one of the key components for extracellular electron exchange by the Geobacter sulfurreducens strain DL-1. However, the true importance of OmcS warrants further investigation because it is well known that few bacteria, even within the Geobacteraceae family, contain OmcS homologs, and many bacteria that are capable of extracellular electron transfer lack an abundance of any type of outer surface c-type cytochrome. In addition, there is debate about the importance of OmcS filaments in the mechanism of extracellular electron transport to insoluble electron acceptors by G. sulfurreducens. It has been suggested that filaments comprised of OmcS rather than e-pili are the predominant conductive filaments expressed by G. sulfurreducens. However, the results presented here, along with multiple other sources of evidence, indicate that OmcS filaments cannot be the primary, conductive, protein nanowires expressed by G. sulfurreducens.

Anaerobic ammonium oxidation (anammox) promoted by pyrogenic biochar: Deciphering the interaction with extracellular polymeric substances (EPS)

Efficient biological nitrogen removal (BNR) by anaerobic ammonium oxidation (anammox) can be achieved with presence of redox-active pyrogenic biochar that potentially acting as an insoluble electron acceptor. Anammox bacteria and other symbiotic consortia are surrounded by extracellular polymeric substances (EPS) forming aggregate architecture, which also contains electrochemical-active biomolecules such as aromatic proteins and humic substances. Therefore, understanding the role of EPS is necessary in biochar-promoting anammox process. Herein, we investigated the influence of biochar with granular-sized (GP) and micrometer-sized (MP) particle sizes on microbiology and characteristics of EPS in anammox sludge. Addition of GP and MP biochar not only improved the BNR efficiency by 17.5% and 34.6%, respectively, but also increased the relative abundance of Candidatus Brocadia. The bulk and bound EPS contents substantially decreased in biochar-amended groups, while more slime EPS was produced. Spectroscopic (FTIR, Raman, and circular dichroism) and electrochemical (voltammetry and impedance spectrum) analyses revealed that biochar addition enhanced the structural integrity and electron-transfer capability of anammox sludge. EPS depletion led to a steep decrease in BNR efficiency (21.5% vs 83.0% with EPS-retained sludge), whereas it resumed to 42.1% in the presence of MP biochar. Electron transport system activity data showed that biochar replenished the loss of anaerobic respiration metabolism due to EPS depletion. In summary, these results suggested that EPS possibly work as transient mediator for shuttling electrons from ammonium oxidation to soluble (nitrite) and insoluble electron acceptors (redox-active biochar).

Electrolocation? The evidence for redox-mediated taxis in Shewanella oneidensis.

Shewanella oneidensis is a dissimilatory metal reducing bacterium and model for extracellular electron transfer (EET), a respiratory mechanism in which electrons are transferred out of the cell. In the last 10years, migration to insoluble electron acceptors for EET has been shown to be nonrandom and tactic, seemingly in the absence of molecular or energy gradients that typically allow for taxis. As the ability to sense, locate, and respire electrodes has applications in bioelectrochemical technology, a better understanding of taxis in S. oneidensis is needed. While the EET conduits of S. oneidensis have been studied extensively, its taxis pathways and their interplay with EET are not yet understood, making investigation into taxis phenomena nontrivial. Since S. oneidensis is a member of an EET-encoding clade, the genetic circuitry of taxis to insoluble acceptors may be conserved. We performed a bioinformatic analysis of Shewanella genomes to identify S. oneidensis chemotaxis orthologs conserved in the genus. In addition to the previously reported core chemotaxis gene cluster, we identify several other conserved proteins in the taxis signaling pathway. We present the current evidence for the two proposed models of EET taxis, "electrokinesis" and flavin-mediated taxis, and highlight key areas in need of further investigation.

Relating Translation Efficiency to Protein Networks Provides Evolutionary Insights in Shewanella and Its Implications for Extracellular Electron Transfer.

Shewanella species are well-known for their extracellular electron transfer (EET) capacity, by which these microorganisms can transfer the electrons from intracellular environment to extracellular space for the reduction of the extracellular insoluble electron acceptors. Using a time-stamped data for the paired protein-mRNA, we investigate the impact of differential translation on the EET process of Shewanella oneidensis MR-1. Firstly, differentially translated proteins when O2 levels are switched from high-O2 to low-O2 are identified by using a soft clustering method, 629 up-regulated translated proteins and 767 down-regulated translated proteins are considered to reflect the changes from inactivated to activated EET process. Then, we showed that the degrees of connectivity of differentially translated proteins were significantly larger than those of non-differentially translated proteins, and thereby these differentially translated proteins will be more important in the protein networks. After that, we networked these differentially translated proteins to construct the differentially translated sub-networks, and discussed the most important proteins that are involved in the EET process with the help of centralization analysis of these differentially translated networks. Furthermore, we also studied the differentially translated operonic genes. Taking together, this work searches the key proteins that potentially activated the EET process from a translational efficiency viewpoint.

Bioelectrochemical Microfluidic Platform for Cultivation and Integrated Characterization of Exoelectrogenic Biofilms

Microbial cultivation with the aid of microfluidic flow chambers has a great potential to form biofilms on an easy to handle laboratory scale. Our microfluidic cultivation platform with a modular design offers versatility and precision in terms of simulating different environments to study multiple growth conditions1. Thus, a long-term cultivation of biofilms can easily be obtained with the possibility of integrated on-line optical analysis. In this paper we will present our recent development of a bioelectrochemical microfluidic platform that allows cultivation and characterization of exoelectrogenic biofilms under anoxic conditions.Electroactive biofilms will be cultivated and characterized in the microfluidic bioelectrochemical system focusing on electroactive microorganisms such as Shewanella oneidensis. The conductive intracellular protein chain of these microbes enables extracellular electron transfer (EET) from the cytoplasm through the cell membranes to an insoluble electron acceptor, such as an anode 2. Different deletion strains were cultivated and could be examined and compared with each other by chronoamperometry, electrochemical impedance spectroscopy, cyclic voltammetry and confocal laser scanning microscopy (CLSM). Genetically engineered strains focus on probing key genes that affect the biofilm forming properties of Shewanella oneidensis 3. In-situ fluorescence microscopy studies of biofilm formation and interaction will also be presented based on our modular microfluidic bioelectrochemical platform. We will also present our ongoing research on artificial electroactive biofilms that integrate electrodeposited nanostructures with a biocompatible hydrogel to form an artificial biofilm matrix.In summary, electroactive biofilm growth under anoxic conditions using an anode as electron sink can be studied using our microfluidic flow technology. Examples of the versatility of the system as well as future outlooks on further biofilm growth conditions will be presented.

Synergistic improvement of Shewanella loihica PV-4 extracellular electron transfer using a TiO2@TiN nanocomposite

Extracellular electron transfer (EET) allows microorganisms to perform anaerobic respiration using insoluble electron acceptors, including minerals and electrodes. EET-based applications require efficient electron transfer between living and non-living systems. To improve EET efficiency, the TiO2@TiN nanocomposite was used to form hybrid biofilms with Shewanella loihica PV-4 (PV-4). Chronoamperometry showed that peak current was increased 4.6-fold via the addition of the TiO2@TiN nanocomposite. Different biofilms were further tested in a dual-chamber microbial fuel cell. The PV-4 biofilm resulted a maximum power density of 33.4 mW/m2, while the hybrid biofilm of the TiO2@TiN nanocomposite with PV-4 yielded a 92.8% increase of power density. Electrochemical impedance spectroscopy analyses showed a lower electron-transfer resistance in the hybrid biofilm. Biological measurements revealed that both flavin secretion and cytochrome c expression were increased when the TiO2@TiN nanocomposite presented. These results demonstrated that the TiO2@TiN nanocomposite could synergistically enhance the EET of PV-4 through altering its metabolism. Our findings provide a new strategy for optimizing biotic-abiotic interactions in bioelectrochemical systems.

Cultivating electroactive microbes—from field to bench

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

Preventing Hydrogen Disposal Increases Electrode Utilization Efficiency by Shewanella oneidensis

Many bacteria use hydrogen anaerobically as both a source and sink for electrons; consuming hydrogen when it is plentiful and producing it when concentrations are low enough to allow proton reduction. While this can increase an organism’s competitiveness, hydrogen uptake or excretion can also make it difficult to control electron flux to a specific product. For example, when Shewanella oneidensis strain MR-1 is used to oxidize organic molecules and recover electrons in microbial electrochemical devices, small changes in ambient hydrogen concentrations could dramatically alter the efficiency of electron capture at the anode due to this organism’s respiratory flexibility. When new three-electrode reactor designs created to minimize oxygen intrusion during anodic growth were tested with lactate-oxidizing S. oneidensis, current production decreased significantly in reactors vented to remove hydrogen produced at the counter electrode, suggesting a role for hydrogen uptake or disposal when cells used electrodes as electron acceptors. A ∆hydA∆hyaB mutant lacking both hydrogenases reversed this trend, and nearly doubled current production rates. This increase was shown to be due to the efficiency of lactate oxidation, as 90% of electrons were recovered as electricity in the ∆hydA∆hyaB mutant compared to only 50% for wild type. Experiments with Fe(III) oxide provided additional evidence that S. oneidensis generates hydrogen reducing equivalents during reduction of insoluble electron acceptors, while experiments with cells incubated with Fe(III) citrate showed increased survival of wild-type compared to ∆hydA∆hyaB in stationary phase. Together these data show how the multiple routes of electron disposal of S. oneidensis, while beneficial under changing conditions, limits the efficiency of electron recovery in electrochemical systems, and demonstrates a simple approach to increasing current production rates in systems where hydrogen is being captured as a product.

Spectro-Electrochemical Investigation of the Effects of Flavin Electron Transporters in Artificial and Bacterial Biofilms

Bacterial biofilms have the capacity to develop and thrive in virtually all circumstances and surfaces, even in the most challenging environmental conditions. The pervasive and recalcitrant existence of biofilms renders them to be a significant safety risk and economical encumbrance in a wide array of industries and technologies, and therefore is a priority area of research. It is essential to develop an improved understanding of the mechanisms implicated during biofilm formation, such as in the case of the diffusion of bacterial-secreted extracellular electron transporters, which are purported to play an important role during biocorrosion by exoelectrogenic bacteria. Hence, a fundamental understanding of electron transfer mechanisms between bacteria and extracellular electron acceptors will contribute insight to our understanding of charge transport and chemistry at the biofilm – external insoluble electron acceptor interface. In the present work, Attenuated Total Reflection - Fourier transform-infrared (ATR-FTIR) spectroscopy has been coupled to electrochemical techniques for the nondestructive, in situ spectro-electrochemical monitoring of biofilms in real-time. Shewanella sp. have been selected for this investigation due to their adaptable exoelectrogenic respiratory capacities and their notable ability to reduce metals via several different mechanisms of extracellular electron transfer mechanisms, including self-secreted flavin shuttles. Gold-thin film model substrates have been used due to their inert nature and for their ability to permit precise manipulation of the substrate surface polarization. Additionally, hydrogels comprised of calcium cross-linked alginate have been used to mimic the architectural features of extracellular polymeric substances which are integral to a bacterial biofilm, to allow the study of electron-transporting flavin molecules in an artificial biofilm. The results will demonstrate patterns of diffusion, akin to how flavins would diffuse in a naturally occurring biofilm, and how polarization affects this process. Furthermore, insight will be gained on how the redox behavior of flavins can influence the development and evolution of a biofilm. This interdisciplinary approach should shed light on bacterial electron transfer mechanisms which could contribute towards emerging technologies which seek to better understand such mechanisms for novel antifouling strategies, renewable energies, and bioremediation.

Electron shuttles enhance anaerobic oxidation of methane coupled to iron(III) reduction

Anaerobic oxidation of methane (AOM) has recently been coupled with the reduction of insoluble electron acceptors such as iron minerals. However, effects of electron shuttles (ESs) on this process and the underlying coupling mechanisms remain not well understood. Here, we evaluated AOM-coupled ferrihydrite reduction by a mixed culture in the absence and presence of ESs. The results showed that ESs (AQS, flavin, HA and AQDS) significantly enhanced the rate (up to 7.4 times) of AOM-dependent ferrihydrite reduction compared with the control. The enhancements were linearly related with the electron transfer capacity of ESs. Illumina high-throughput sequencing and DNA-based stable isotope probing revealed that the AOM-coupled iron reduction depended on the syntrophic interaction of Methanobacterium and the partner bacteria. Methanobacterium as the dominant microorganism, did not assimilate methane into its biomasses. However, it played a crucial role in the partial oxidation of methane into an intermediate (i.e. propionate), which was then assimilated by the partner bacteria (e.g. Cellulomonas, Desulfovibrio, Actinotalea, etc.) for ferrihydrite reduction. This work suggests that ESs in natural environments can mitigate the methane emissions by facilitating the AOM process and biogeochemical cycles of iron.

Preparation of Naphthalene Dianhydride Bithiophene Copolymers by Direct Arylation Polycondensation and the Latent Pigment Approach.

An alternating naphthalene dianhydride bithiophene copolymer (PNDAT2) is prepared by a combined direct arylation polycondensation and the latent pigment approach. PNDAT2 is the first reported example of an alternating conjugated polymer containing naphthalene dianhydride, the oxo-analogue of naphthalene diimide often used in electron-acceptor conjugated polymers. PNDAT2 is resistant to organic solvents and can be generated directly as film by thermal treatment of the soluble tetraester precursor PNTET2. PNDAT2 is characterized by a LUMO level of -3.9 eV, similar to that of established naphthalene diimide containing soluble copolymers. This route to insoluble electron acceptor copolymers by thermal cleavage of soluble precursors is an alternative to classical cross-linking or orthogonal processing strategies.

Thermophiles; or, the Modern Prometheus: The Importance of Extreme Microorganisms for Understanding and Applying Extracellular Electron Transfer.

Approximately four billion years ago, the first microorganisms to thrive on earth were anaerobic chemoautotrophic thermophiles, a specific group of extremophiles that survive and operate at temperatures ∼50 – 125°C and do not use molecular oxygen (O2) for respiration. Instead, these microorganisms performed respiration via dissimilatory metal reduction by transferring their electrons extracellularly to insoluble electron acceptors. Genetic evidence suggests that Gram-positive thermophilic bacteria capable of extracellular electron transfer (EET) are positioned close to the root of the Bacteria kingdom on the tree of life. On the contrary, EET in Gram-negative mesophilic bacteria is a relatively new phenomenon that is evolutionarily distinct from Gram-positive bacteria. This suggests that EET evolved separately in Gram-positive thermophiles and Gram-negative mesophiles, and that EET in these bacterial types is a result of a convergent evolutionary process leading to homoplasy. Thus, the study of dissimilatory metal reducing thermophiles provides a glimpse into some of Earth’s earliest forms of respiration. This will provide new insights for understanding biogeochemistry and the development of early Earth in addition to providing unique avenues for exploration and discovery in astrobiology. Lastly, the physiological composition of Gram-positive thermophiles, coupled with the kinetic and thermodynamic consequences of surviving at elevated temperatures, makes them ideal candidates for developing new mathematical models and designing innovative next-generation biotechnologies.KEY CONCEPTSAnaerobe: organism that does not require oxygen for growth.Chemoautotroph: organism that obtains energy by oxidizing inorganic electron donors.Convergent Evolution: process in which organisms which are not closely related independently evolve similar traits due to adapting to similar ecological niches and/or environments.Dissimilatory Metal Reduction: reduction of a metal or metalloid that uses electrons from oxidized organic or inorganic electron donors.Exoelectrogen: microorganism that performs dissimilatory metal reduction via extracellular electron transfer.Extremophiles: organisms that thrive in physical or geochemical conditions that are considered detrimental to most life on Earth.Homoplasy: a character shared by a set of species that is not shared by a common ancestorNon-synonymous Substitutions (Ka): a substitution of a nucleotide that changes a codon sequence resulting in a change in the amino acid sequence of a protein.Synonymous Substitutions (Ks): a substitution of a nucleotide that may change a codon sequence, but results in no change in the amino acid sequence of a protein.Thermophiles: a specific group of extremophiles that survive and operate at temperatures ∼50–125°C.