In situ electrochemical characterization of a microbial fuel cell biocathode running on wastewater

Funding Information: FCT?Funda??o para a Ci?ncia e a Tecnologia, Portugal (Grant number grant No. FRH/BP D/33864/2009). This work was supported by the Associate Laboratory for Green Chemistry (LAQV) which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020). Acknowledgments: S.V.R. acknowledges the financial support from FCT (Portuguese Foundation for Science and Technology) for Postdoctoral Research grant No. FRH/BPD/33864/2009. This work was supported by the Associate Laboratory for Green Chemistry?LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).The electrochemical features of microbial fuel cells’ biocathodes, running on wastewater, were evaluated by cyclic voltammetry. Ex situ and in situ electrochemical assays were performed and the redox processes associated with the presence of microorganisms and/or biofilms were attained. Different controls using sterile media (abiotic cathode microbial fuel cell) and membranes covering the electrodes were performed to evaluate the source of the electrochemistry response (surface biofilms vs. biotic electrolyte). The bacteria presence, in particular when biofilms are allowed to develop, was related with the enhanced active redox processes associated with an improved catalytic activity, namely for oxygen reduction, when compared with the results attained for an abiotic microbial fuel cell cathode. The microbial main composition was also attained and is in agreement with other reported studies. The current study aims contributing to the establishment of the advantages of using biocathodes rather than abiotic, whose conditions are frequently harder to control and to contribute to a better understanding of the bioelectrochemical processes occurring on the biotic chambers and the electrode surfaces.publishersversionpublishe

Screening of Potential Stress Biomarkers in Sweat Associated with Sports Training

The authors acknowledge the financial support of Fundacao para a Ciencia e Tecnologia (FCT/MCTES), grant PTDC/SAU-SOC/28390/2017. Also, this work was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020).Background: Intense and continuous physical training in sports is related with psychological and physiological stress, affecting the health and well-being of athletes. The development of non-invasive sampling methodologies is essential to consider sweat as a potential biological fluid for stress biomarker assessment. In the current work, the identification in sweat samples of potential molecules that may be used as stress biomarkers was pursued. Methods: A sweat pool sample from football players after a 90-min intense training game was studied. Results: An analysis method using liquid chromatography with detection by tandem mass spectrometry (LC-MSMS) to attain a screening profile of sweat composition is presented. The major focus was on neurotransmitters (e.g. monoamines and metabolites) and other biological molecules related with physical training, such as precursors of biogenic amines (phenylaniline, tyrosine, etc.). Conclusions: This study allowed the identification of small biomolecules, neurotransmitters and other related molecules in sweat that are potentially associated with stress conditions. The developed methodology intends to contribute to the assessment and study of physical and psychological stress biomarkers related with intense sports using non-invasive methods.publishersversionpublishe

Direct electrochemical reduction of carbon dioxide by a molybdenum-containing formate dehydrogenase

This work was supported by the Associate Laboratory for Green Chemistry -LAQV, with national funds from FCT/MCTES ( UID/QUI/50006/2019 ). LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract.Formate dehydrogenase enzymes catalyse the reversible two-electron oxidation of formate to carbon dioxide. The class of metal-dependent formate dehydrogenases comprises prokaryotic enzymes holding redox-active centres and a catalytic site, containing either molybdenum or tungsten ion, that mediates the formate/carbon dioxide interconversion. The carbon dioxide reduction is of a particular interest, since it may be a route for its atmospheric mitigation with the simultaneous production of added-value products, as formate-derived compounds. Recently, the periplasmic formate dehydrogenase from Desulfovibrio desulfuricans, a molybdenum-containing enzyme, was proven to be an efficient enzyme for the CO 2 reduction to formate. In this work, the immobilized formate dehydrogenase isolated from Desulfovibrio desulfuricans direct electrochemical behaviour was attained in the presence and absence of substrates and the formal potentials associated with the catalytic centre transitions were determined in non-turnover conditions. The enzyme catalytic activity towards carbon dioxide reduction was observed using direct electrochemical methods.authorsversionpublishe

Bioelectricity generation using long-term operated biocathode: RFLP based microbial diversity analysis

FRH/BPD/33864/2009 UIDB/50006/2020 UIDP/50006/2020In the present work, power generation and substrate removal efficiencies of long-term operated microbial fuel cells, containing abiotic cathodes and biocathodes, were evaluated for 220 days. Among the two microbial fuel cell (MFC) types, the one containing biocathode showed higher power density (54 mW/m2), current density (122 mA/m2) coulombic efficiency (33%), and substrate removal efficiency (94%) than the abiotic cathode containing MFC. Voltammetric analysis also witnessed higher and sustainable electron discharge for the MFC with biocathode, when compared with the abiotic cathode MFC. Over the tested period, both MFC have shown a cell voltage drop, after 150 and 165, days, for the MFC with biocathode and abiotic cathodes, respectively. Polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis identified 281 clones. Bacteria belonging to Acinetobacter, Acidovorax, Pseudomonas and Burkholderia were observed in the abiotic cathode MFC. Bacteria belonging to Geobacter, Cupriavidus and Acidobacteria were observed in the biocathode MFC. Almost similar types of archaea (Methanosarcinales, Methanolinea, Nitrososphaera and Methanomicrobiales) were observed in both MFCs.publishersversionpublishe

Modulation of the Redox Potential and Electron/Proton Transfer Mechanisms in the Outer Membrane Cytochrome OmcF From Geobacter sulfurreducens

PD/00193/2012 UID/FIS/00068/2019 PTDC/BBBBQB/3554/2014 PTDC/BIA-BQM/31981/2017 PD/BD/114445/2016 UID/Multi/04378/2019 ROTEIRO/0031/2013 -PINFRA/22161/2016The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His47), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His47 is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His47 with non-protonatable residues (isoleucine – OmcFH47I and phenylalanine – OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were 15N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His47 on the pH modulation of OmcF’s redox potential. However, the pH effect on the heme substituents’ NMR chemical shifts suggested that the heme propionate P13 also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid–base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.publishersversionpublishe

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

Biochem. J. (2011) 438,485–494 doi:10.1042/BJ2011083

Design and Optimization of Microbial Fuel Cells and Evaluation of a New Air-Breathing Cathode Based on Carbon Felt Modified with a Hydrogel—Ion Jelly®

Funding Information: This research was funded by Fundação para a Ciência e a Tecnologia projects DSAIPA/DS/0117/2020, UIDB/04565/2020, and UIDP/04565/2020, by the Associate Laboratory Institute for Health and Bioeconomy—i4HB project LA/P/0140/2020. This work was supported by the Associate Laboratory for Green Chemistry—LAQV financed by national funds from FCT/MCTES (UIDB/50006/2020). We also thank Fundação para a Ciência e Tecnologia (FCT) for funding (SFRH/BD/77568/2011 (R.N.L.C.); SFRH/BPD/80293/2011 (R.M.A.)). C.M.C. acknowledges FCT for the Ciência 2008 Program; S.V.R. acknowledges the financial support from FCT (Portuguese Foundation for Science and Technology) for a postdoctoral research grant (FRH/BPD/33864/2009). This work was supported by the Associate Laboratory for Green Chemistry—LAQV, financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020); the Institute for Bioengineering and Biosciences—iBB, financed by FCT (UID/BIO/04565/2013); and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317). Publisher Copyright: © 2023 by the authors.The increased demand for alternative sustainable energy sources has boosted research in the field of fuel cells (FC). Among these, microbial fuel cells (MFC), based on microbial anodes and different types of cathodes, have been the subject of renewed interest due to their ability to simultaneously perform wastewater treatment and bioelectricity generation. Several different MFCs have been proposed in this work using different conditions and configurations, namely cathode materials, membranes, external resistances, and microbial composition, among other factors. This work reports the design and optimization of MFC performance and evaluates a hydrogel (Ion Jelly®) modified air-breathing cathode, with and without an immobilized laccase enzyme. This MFC configuration was also compared with other MFC configuration performances, namely abiotic and biocathodes, concerning wastewater treatment and electricity generation. Similar efficiencies in COD reduction, voltage (375 mV), PD (48 mW/m2), CD (130 mA/m2), and OCP (534 mV) were obtained. The results point out the important role of Ion Jelly® in improving the MFC air-breathing cathode performance as it has the advantage that its electroconductivity properties can be designed before modifying the cathode electrodes. The biofilm on MFC anodic electrodes presented a lower microbial diversity than the wastewater treatment effluent used as inocula, and inclusively Geobacteracea was also identified due to the high microbial selective niches constituted by MFC systems.publishersversionpublishe

Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide

PB would thank the PTNMRPhD (PD/00065/2013). VLP thanks the NIH for support (GM141086).The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ∼ −408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UV–visible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.publishersversionpublishe

Cytochrome c stimulates the superoxide anion production by cytochrome b5 reductase in neuronal synaptic plasma membrane vesicles

info:eu-repo/grantAgreement/FCT/5876/147258/PT Work financed by Grants FCT/MEC (UID/Multi/04378/2013). POCI-01-0145-FEDER-007728 and BFU2014-53641-P of the Spanish Ministerio de Economia y Competitividad co-financed by FEDER.authorsversionpublishe

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction