Isolation and Characterization of Human Gut Bacteria Capable of Extracellular Electron Transport by Electrochemical Techniques

Microorganisms are known to exhibit extracellular electron transfer (EET) in a wide variety of habitats. However, as for the human microbiome which significantly impacts our health, the role and importance of EET has not been widely investigated. In this study, we enriched and isolated the EET-capable bacteria from human gut microbes using an electrochemical enrichment method and examined whether the isolates couple EET with anaerobic respiration or fermentation. Upon the use of energy-rich or minimum media (with acetate or lactate) for electrochemical enrichment with the human gut sample at an electrode potential of +0.4 V [vs. the standard hydrogen electrode (SHE)], both culture conditions showed significant current production. However, EET-capable pure strains were enriched specifically with minimum media, and subsequent incubation using the δ-MnO2-agar plate with lactate or acetate led to the isolation of two EET-capable microbial strains, Gut-S1 and Gut-S2, having 99% of 16S rRNA gene sequence identity with Enterococcus avium (E. avium) and Klebsiella pneumoniae (K. pneumoniae), respectively. While the enrichment involved anaerobic respiration with acetate and lactate, further electrochemistry with E. avium and K. pneumoniae revealed that the glucose fermentation was also coupled with EET. These results indicate that EET couples not only with anaerobic respiration as found in environmental bacteria, but also with fermentation in the human gut

Electroactivity across the cell wall of Gram-positive bacteria

Funding Information: This work was supported by national funds through FCT? Funda??o para a Ci?ncia e a Tecnologia, I.P. Project UIDB/04612/2020, UIDP/04612/2020 and PTDC/BIA-BQM/30176/2017, and by the European Union's Horizon 2020 research and innovation programme under grant agreement No 810856. Funding Information: This work was supported by national funds through FCT– Fundação para a Ciência e a Tecnologia, I.P., Project UIDB/04612/2020, UIDP/04612/2020 and PTDC/BIA-BQM/30176/2017, and by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 810856. Publisher Copyright: © 2020 The Author(s) Copyright: Copyright 2021 Elsevier B.V., All rights reserved.The growing interest on sustainable biotechnological processes for the production of energy and industrial relevant organic compounds have increased the discovery of electroactive organisms (i.e. organisms that are able to exchange electrons with an electrode) and the characterization of their extracellular electron transfer mechanisms. While most of the knowledge on extracellular electron transfer processes came from studies on Gram-negative bacteria, less is known about the processes performed by Gram-positive bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria lack an outer-membrane and contain a thick cell wall, which were thought to prevent extracellular electron transfer. However, in the last decade, an increased number of Gram-positive bacteria have been found to perform extracellular electron transfer, and exchange electrons with an electrode. In this mini-review the current knowledge on the extracellular electron transfer processes performed by Gram-positive bacteria is introduced, emphasising their electroactive role in bioelectrochemical systems. Also, the existent information of the molecular processes by which these bacteria exchange electrons with an electrode is highlighted. This understanding is fundamental to advance the implementation of these organisms in sustainable biotechnological processes, either through modification of the systems or through genetic engineering, where the organisms can be optimized to become better catalysts.publishersversionpublishe

Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis Pathogenesis

In this study, we explored the extracellular electron transfer (EET) capabilities of two bacterial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gut and the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significant electrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode surface, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence of cell-surface redox substrate potentially mediating EET was revealed by the redox-based staining method and electrochemical voltammetry assay. However, medium swapping analyses and the addition of flavins, a model redox mediator, suggest that the current production is dominated by soluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cell surface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrast to OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redox molecules. Further, the mixture of the two strains did not increase the current production from OTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The present work revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm

Metabolic Current Production by an Oral Biofilm PathogenCorynebacterium matruchotii

The development of a simple and direct assay for quantifying microbial metabolic activity is important for identifying antibiotic drugs. Current production capabilities of environmental bacteria via the process called extracellular electron transport (EET) from the cell interior to the exterior is well investigated in mineral-reducing bacteria and have been used for various energy and environmental applications. Recently, the capability of human pathogens for producing current has been identified in different human niches, which was suggested to be applicable for drug assessment, because the current production of a few strains correlated with metabolic activity. Herein, we report another strain, a highly abundant pathogen in human oral polymicrobial biofilm,Corynebacterium matruchotii, to have the current production capability associated with its metabolic activity. It showed the current production of 50 nA/cm(2)at OD(600)of 0.1 with the working electrode poised at +0.4 V vs. a standard hydrogen electrode in a three-electrode system. The addition of antibiotics that suppress the microbial metabolic activity showed a significant current decrease (>90%), establishing that current production reflected the cellular activity in this pathogen. Further, the metabolic fixation of atomically labeled(13)C (31.68% +/- 2.26%) and(15)N (19.69% +/- 1.41%) confirmed by high-resolution mass spectrometry indicated thatC. matruchotiicells were metabolically active on the electrode surface. The identified electrochemical activity ofC. matruchotiishows that this can be a simple and effective test for evaluating the impact of antibacterial compounds, and such a method might be applicable to the polymicrobial oral biofilm on electrode surfaces, given four other oral pathogens have already been shown the current production capability

A Human Pathogen Capnocytophaga Ochracea Exhibits Current Producing Capability

Microbial extracellular electron transfer (EET) in diverse environments has gained increasing attention. However, the EET capability of oral pathogens and associated mechanisms has been scarcely studied. Here, our results suggest that the Capnocytophaga ochracea, an etiological human pathogen showed current production and demonstrated a rate enhancement of electron transport at a high cell-density. C. ochracea produced ∼10-fold more current at an OD600 of 0.5 associated with twice a higher glucose consumption rate per cell, compared to 0.1, measured in a three-electrode electrochemical system by single-potential amperometry at +0.2 V (vs Ag/AgCl [sat. KCl]). During current production, the accumulation of the redox molecules on the electrode was observed at high OD600 compared to low OD600. Apart from cell released redox active product, externally added redox active additives enhanced the electron transport, suggesting the EET capability of C. ochracea via electron mediator. A higher metabolic activity via single-cell assay (based on anabolic incorporation of 15NH4+) in cells that did not attach to the electrode strongly suggests the EET rate enhancement through an electron mediator. As bacterial populations play a role in the pathogenesis of human infections such as periodontitis, our results suggest that population-induced EET mechanisms may facilitate in-vivo colonization of C. ochracea

Thermodynamic analysis of heat recovery steam generator in combined cycle power plant

Combined cycle power plants play an important role in the present energy sector. The main challenge in designing a combined cycle power plant is proper utilization of gas turbine exhaust heat in the steam cycle in order to achieve optimum steam turbine output. Most of the combined cycle developers focused on the gas turbine output and neglected the role of the heat recovery steam generator which strongly affects the overall performance of the combined cycle power plant. The present paper is aimed at optimal utilization of the flue gas recovery heat with different heat recovery steam generator configurations of single pressure and dual pressure. The combined cycle efficiency with different heat recovery steam generator configurations have been analyzed parametrically by using first law and second law of thermodynamics. It is observed that in the dual cycle high pressure steam turbine pressure must be high and low pressure steam turbine pressure must be low for better heat recovery from heat recovery steam generator

Biogenesis of Outer Membrane Vesicles Concentrates the Unsaturated Fatty Acid of Phosphatidylinositol in Capnocytophaga ochracea

Bacterial outer membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by bacteria that facilitate oral biofilm formation via cellular aggregation and intercellular communication. Recent studies have revealed that Capnocytophaga ochracea is one of the dominant members of oral biofilms; however, their potential for OMV production has yet to be investigated. This study demonstrated the biogenesis of OMVs in C. ochracea associated with the concentration of unsaturated fatty acids of phosphatidylinositol (PI) and characterized the size and protein profile of OMVs produced at growth phases. Transmission electron microscopy showed isolated spherical structures from cells stained with heavy metals, indicating the production of OMVs with a size ranging from 25 to 100 nm. Lipidome analysis revealed the presence of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, and PI as the main lipids. Some unsaturated fatty acids of PI were present specifically in OMV and little in the outer membrane, suggesting that OMVs are generated from a specific region of the membrane through blebbing rather than a random process such as cell lysis. Furthermore, the lack of similar PI accumulation in the OMV of Porphyromonas gingivalis suggests that C. ochracea has a different biogenesis mechanism. The blebbing mechanism was further supported by higher OMV production occurring at the exponential phase in comparison to the stationary phase, where cell lysis is more likely to occur. Further, comparative protein profile of OMVs isolated under different growth phases may indicate that the OMV cargo does not largely vary with growth phases. The present study provides a basis for further understanding the roles of C. ochracea OMVs in oral biofilms as well as systemic diseases that C. ochracea involves