Percorrer por autor "Mendes, Filipa"
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- Alcohols, esters and heavy sulphur compounds production by pure and mixed cultures of apiculate wine yeastsPublication . Moreira, Nathalie; Mendes, Filipa; Hogg, Tim; Vasconcelos, IsabelStrains of Hanseniaspora uvarum, Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used as pure or mixed starter cultures in commercial medium, in order to compare their kinetic parameters and fermentation patterns. In pure and mixed cultures, yeasts presented similar ethanol yield and productivity. Pure cultures of H. uvarum and S. cerevisiae showed a specific growth rate of 0.38 h⁻¹; however, this value decreased when these yeasts were grown in mixed cultures with H. guilliermondii. The specific growth rate of pure cultures of H. guilliermondii was 0.41 h⁻⁻¹ and was not affected by growth of other yeasts. H. guilliermondii was found to be the best producer of 2-phenylethyl acetate and 2-phenylethanol in both pure and mixed cultures. In pure cultures, H. uvarum led to the highest contents of heavy sulphur compounds, but H. guilliermondii and S. cerevisiae produced similar levels of methionol and 2-methyltetrahydrothiophen-3-one. Growth of apiculate yeasts in mixed cultures with S. cerevisiae led to amounts of 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2- methyltetrahydrothiophen-3-one similar to those obtained in a pure culture of S. cerevisiae; however, growth of apiculate yeasts increased methionol contents of fermented media.
- Metabolic engineering of clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerolPublication . González-Pajuelo, María; Meynial-Salles, Isabelle; Mendes, Filipa; Andrade, José Carlos; Vasconcelos, Isabel; Soucaille, PhilippeClostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better ‘‘vitamin B12-free’’ biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3- propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L⁻¹ h⁻¹) and titer (788 mM).
- Microbial conversion of glycerol to 1,3-propanediol$ephysiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1 (pSPD5)Publication . González-Pajuelo, María; Meynial-Salles, Isabelle; Mendes, Filipa; Soucaille, Philippe; Vasconcelos, IsabelClostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3- propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH2 flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.
- Sequential injection kinetic flow assay for monitoring glycerol in a sugar fermentation process by saccharomyces cerevisiaePublication . Hueso Domínguez, Karina B.; Tóth, Ildikó V.; Souto, M. Renata S.; Mendes, Filipa; García De María, Cándido; Vasconcelos, Isabel; Rangel, António O. S. S.A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD+ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD+ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in agreement with those obtained with a high-performance liquid chromatography method.
- Utilização de compostos azotados por estirpes de Saccharomyces cerevisiae durante a fermentação de mostos Loureiro e TrajaduraPublication . Moreira, Nathalie; Mendes, Filipa; Vasconcelos, Isabel