Caparica, Portugal
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Miguel C.,REQUIMTE CQFB | Pinto J.V.,New University of Lisbon | Melo M.J.,REQUIMTE CQFB
Dyes and Pigments | Year: 2014

Vermilion red, mercury sulphide (α-HgS), was one of the most important reds in art and its use as a pigment dates back to Antiquity. In medieval Europe, it could be mined as cinnabar, or produced as vermilion by heating mercury with sulphur. This work aims to study the production of synthetic vermilion as a medieval pigment and to confirm which was the source (mineral or artificial) of the reds used in Portuguese medieval illuminations. The production of synthetic vermilion was based on the process described in the Judaeo-Portuguese medieval treatise "The book on how to make colours", using materials and technologies as close as possible to the medieval ones. The reaction mechanism was studied by following the heating process by X-ray diffraction, and it was possible to conclude that the transformation from black cubic β-HgS into red hexagonal α-HgS is a solid-state phase transition, occurring at 235 C. This result is contrary to what published in technical art literature, in which this process is described as a sublimation. Moreover, Scanning Electron Microscopy evidenced a sinterization effect on the artificial vermilion, not found in medieval original samples nor in paints prepared with mineral cinnabar from Almadén (Spain). Red mercury sulphide, natural and synthetic, was then prepared as a parchment-glue paint and compared to proteinaceous red paints from 12th-13th century miniatures produced in important medieval monasteries, previously fully characterized by a multi-analytical approach (μ-Energy dispersive X-ray fluorescence, μ-Fourier Transform Infrared Spectroscopy, Raman microscopy). A comparative Electron probe microanalysis of the red paints point to a mineral provenance for medieval vermilion found in Portuguese illuminations. © 2013 Elsevier Ltd. All rights reserved.

Lanham A.B.,REQUIMTE CQFB | Oehmen A.,REQUIMTE CQFB | Saunders A.M.,University of Aalborg | Carvalho G.,REQUIMTE CQFB | And 3 more authors.
Water Research | Year: 2013

This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic-aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A2O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems. © 2013 Elsevier Ltd.

Lanham A.B.,REQUIMTE CQFB | Oehmen A.,REQUIMTE CQFB | Saunders A.M.,University of Aalborg | Carvalho G.,REQUIMTE CQFB | And 3 more authors.
Water Research | Year: 2014

This study investigates, for the first time, the application of metabolic models incorporating polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) towards describing the biochemical transformations of full-scale enhanced biological phosphorus removal (EBPR) activated sludge from wastewater treatment plants (WWTPs). For this purpose, it was required to modify previous metabolic models applied to lab-scale systems by incorporating the anaerobic utilisation of the TCA cycle and the aerobic maintenance processes based on sequential utilisation of polyhydroxyalkanoates, followed by glycogen and polyphosphate. The abundance of the PAO and GAO populations quantified by fluorescence in situ hybridisation served as the initial conditions of each biomass fraction, whereby the models were able to describe accurately the experimental data. The kinetic rates were found to change among the four different WWTPs studied or even in the same plant during different seasons, either suggesting the presence of additional PAO or GAO organisms, or varying microbial activities for the same organisms. Nevertheless, these variations in kinetic rates were largely found to be proportional to the difference in acetate uptake rate, suggesting a viable means of calibrating the metabolic model. The application of the metabolic model to full-scale sludge also revealed that different Accumulibacter clades likely possess different acetate uptake mechanisms, as a correlation was observed between the energetic requirement for acetate transport across the cell membrane with the diversity of Accumulibacter present. Using the model as a predictive tool, it was shown that lower acetate concentrations in the feed as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis, which could explain why the anaerobic TCA pathway seems to be more relevant in full-scale WWTPs than in lab-scale systems. © 2014 Elsevier Ltd.

Dantas J.M.,Requimte CQFB | Morgado L.,Requimte CQFB | Marques A.C.,Requimte CQFB | Salgueiro C.A.,Requimte CQFB
Journal of Physical Chemistry B | Year: 2014

The increase of conductivity of electrolytes favors the current production in microbial fuel cells (MFCs). Adaptation of cell cultures to higher ionic strength is a promising strategy to increase electricity production. The bacterium Geobacter sulfurreducens is considered a leading candidate for MFCs. Therefore, it is important to evaluate the impact of the ionic strength on the functional properties of key periplasmic proteins that warrants electron transfer to cell exterior. The effect of the ionic strength on the functional properties of triheme cytochrome PpcA, the most abundant periplasmic cytochrome in G. sulfurreducens, was investigated by NMR and potentiometric methods. The redox properties of heme IV are the most affected ones. Chemical shift perturbation measurements on the backbone NMR signals, at increasing ionic strength, also showed that the region close to heme IV is the most affected due to the large number of positively charged residues, which confer a highly positive electrostatic surface around this heme. The shielding of these positive charges at high ionic strength explain the observed decrease in the reduction potential of heme IV and shows that PpcA was designed to maintain its functional mechanistic features even at high ionic strength. © 2014 American Chemical Society.

Corvo M.C.,REQUIMTE CQFB | Sardinha J.,REQUIMTE CQFB | Menezes S.C.,Petrobras | Einloft S.,Pontifical Catholic University of Rio Grande do Sul | And 4 more authors.
Angewandte Chemie - International Edition | Year: 2013

Where is CO2? The intermolecular interactions of [C 4mim]BF4 and [C4mim]PF6 ionic liquids and CO2 have been determined by high-pressure NMR spectroscopy in combination with molecular dynamic simulations. The anion and the cation are both engaged in interactions with CO2. A detailed picture of CO2 solvation in these ILs is provided. CO2 solubility is essentially determined by the microscopic structure of the IL. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Silva M.A.,Requimte CQFB | Valente R.C.,Requimte CQFB | Pokkuluri P.R.,Argonne National Laboratory | Turner D.L.,New University of Lisbon | And 2 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2014

The periplasmic sensor domains GSU582 and GSU935 are part of methyl-accepting chemotaxis proteins of the bacterium Geobacter sulfurreducens containing one c-type heme and a PAS-like fold. Their spectroscopic properties were shown previously to share similar spectral features. In both sensors, the heme group is in the high-spin form in the oxidized state and low-spin after reduction and binding of a methionine residue. Therefore, it was proposed that this redox-linked ligand switch might be related to the signal transduction mechanism. We now report the thermodynamic and kinetic characterization of the sensors GSU582 and GSU935 by visible spectroscopy and stopped-flow techniques, at several pH and ionic strength values. Despite their similar spectroscopic features, the midpoint reduction potentials and the rate constants for reduction by dithionite are considerably different in the two sensors. The reduction potentials of both sensors are negative and well framed within the typical anoxic subsurface environments in which Geobacter species predominate. The midpoint reduction potentials of sensor GSU935 are lower than those of GSU582 at all pH and ionic strength values and the same was observed for the reduction rate constants. The origin of the different functional properties of these closely related sensors is rationalized in the terms of the structures. The results suggest that the sensors are designed to function in different working potential ranges, allowing the bacteria to trigger an adequate cellular response in different anoxic subsurface environments. These findings provide an explanation for the co-existence of two similar methyl-accepting chemotaxis proteins in G. sulfurreducens. © 2014 Elsevier B.V.

Dantas J.M.,Requimte CQFB | Morgado L.,Requimte CQFB | Pokkuluri P.R.,Argonne National Laboratory | Turner D.L.,New University of Lisbon | Salgueiro C.A.,Requimte CQFB
Biochimica et Biophysica Acta - Bioenergetics | Year: 2013

Extracellular electron transfer is one of the physiological hallmarks of Geobacteraceae. Most of the Geobacter species encode for more than 100 c-type cytochromes which are, in general, poorly conserved between individual species. An exception to this is the PpcA family of periplasmic triheme c-type cytochromes, which are the most abundant proteins in these bacteria. The functional characterization of PpcA showed that it has the necessary properties to couple electron/proton transfer, a fundamental step for ATP synthesis. The detailed thermodynamic characterization of a PpcA mutant, in which the strictly conserved residue phenylalanine 15 was replaced by leucine, showed that the global redox network of cooperativities among heme groups is altered, preventing the mutant from performing a concerted electron/proton transfer. In this work, we determined the solution structure of PpcA F15L mutant in the fully reduced state using NMR spectroscopy by producing 15N-labeled protein. In addition, pH-dependent conformational changes were mapped onto the structure. The mutant structure obtained is well defined, with an average pairwise root-mean-square deviation of 0.36 Å for the backbone atoms and 1.14 Å for all heavy atoms. Comparison between the mutant and wild-type structures elucidated the contribution of phenylalanine 15 in the modulation of the functional properties of PpcA. © 2013 Elsevier B.V.

Morgado L.,Requimte CQFB | Fernandes A.P.,Requimte CQFB | Londer Y.Y.,Argonne National Laboratory | Bruix M.,CSIC - Institute of Physical Chemistry "Rocasolano" | Salgueiro C.A.,Requimte CQFB
Biochemical and Biophysical Research Communications | Year: 2010

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural-functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (13C/15N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of 1H-13C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature. © 2010 Elsevier Inc. All rights reserved.

Cruz F.J.A.L.,Requimte CQFB | Esteves I.A.A.C.,Requimte CQFB | Mota J.P.B.,Requimte CQFB
Colloids and Surfaces A: Physicochemical and Engineering Aspects | Year: 2010

Grand canonical Monte Carlo (GCMC) simulations are employed to study the adsorption equilibrium properties of methane, ethane, ethylene, propane, and propylene onto homogeneous bundles of single-walled carbon nanotubes, at room temperature, from 10-4 bar up to 90% vapor pressure. Individual adsorption isotherms for the internal volume of a bundle and for its external adsorption sites are separately calculated for individual nanotube diameters in the range 11.0 Å ≤ D ≤ 18.1 Å. External adsorption is further decomposed into the contributions from its two main adsorption sites - external grooves and exposed surfaces of the peripheral tubes - based on a geometrical model for the average groove volume that takes into account the molecular nature of the adsorbate. Both intrabundle confinement and adsorption onto the grooves lead to type I isotherms, which are modeled with Langmuirian-type equations. Adsorption on the exposed surfaces of the peripheral tubes in a bundle gives rise to a type II isotherm, which is described by the BET model with a finite number of adsorbed layers. The linear combination of the Langmuir isotherm model for adsorption onto groove sites and the BET isotherm model produces a composite isotherm that is in good agreement with the GCMC isotherm for overall adsorption onto the external sites of a bundle. The influence of adsorbate molecular length and existence of an unsaturated chemical bond in its molecular skeleton are studied by monitoring the dependence of the Henry constant and zero-coverage isosteric heat of adsorption with the dispersive energy for the solid-fluid pair potential of each adsorbate. Our results show that the adsorptive properties are especially influenced by the presence of a double bond in the case of small molecules, such as the ethane/ethylene pair. © 2009 Elsevier B.V. All rights reserved.

Morgado L.,Requimte CQFB | Dantas J.M.,Requimte CQFB | Simoes T.,Requimte CQFB | Londer Y.Y.,Argonne National Laboratory | And 3 more authors.
Bioscience Reports | Year: 2013

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e- /H+ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met58, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e- /H+ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways. © 2013 The Author(s).

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