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- Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerolPublication . González-Pajuelo, M.; Andrade, J. C.; Vasconcelos, I.Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l 1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.
- Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisationPublication . Andrade, José Carlos; Vasconcelos, IsabelContinuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/ glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l−1 h−1 and a 84% (w/w) glycerol consumptionwere attained when cultures were grown at pH 6 and D = 0.05 h−1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.
- Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivityPublication . González-Pajuelo, M.; Andrade, J. C.; Vasconcelos, I.The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l-1. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l-1, at D=0.05–0.3 h-1, and from 60–70 g l-1, at D=0.05 and 0.1 h-1. 30 g l-1 of 1,3-propanediol and the highest reported value of productivity, 10.3 g l-1 h-1, was achieved at D=0.30 h-1 and 60 g l-1 of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l-1 of feed glycerol and a dilution rate higher than 0.10 h-1; moreover, at D=0.30 h-1 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.
- 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.
- A GAC biofilm reactor for the continuous degradation of 4-chlorophenol: treatment efficiency and microbial analysisPublication . Carvalho, M.; Vasconcelos, I.; Bull, A.; Castro, P.Using a continuous enrichment technique, a bacterial consortium capable of degrading 4-chlorophenol (4-CP) was obtained from the rhizosphere of Phragmites australis. A granular activated carbon (GAC) biofilm reactor was established using this consortium, and the degradation of 4-CP was investigated under continuous flow operation using a feed of 20-50 mg l-1 with a hydraulic residence time of 17 min over a 6-month period. Chloride liberation occurred throughout the operation, and the reactor had 4-CP removal efficiencies of 69-100%. Periods of lower performance were attributed to clogging of the column with biomass and the formation of channels. Subsequently, the immobilized biofilm was subjected to a starvation period of 5 months, after which its degradative capacity was still maintained. The microbial consortium was characterized during the continuous flow experiment and dynamic population changes were observed throughout. One isolate recovered from the biofilm was shown to be capable of degrading 4-CP as a sole carbon and energy source.
- 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).
- Method for analysis of heavy sulphur compounds using gas chromatography with flame photometric detectionPublication . Moreira, N.; Pinho, P. Guedes de; Vasconcelos, I.A method for analysis of heavy sulphur compounds in wines, based on gas chromatography (GC) with flame photometric detection, is reported. Wine samples preparation includes a dichloromethane liquid–liquid extraction followed by concentration under a nitrogen atmosphere. The extracted fraction was also analysed by GC–mass spectrometry. The method enables high recovery of sulphur compounds in wine and satisfies the requirements of repeatability and sensitivity. Applications of the method to red, white and Port wines are reported.
- Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape mustPublication . Moreira, N.; Mendes, F.; Pinho, P. Guedes de; Hogg, T.; Vasconcelos, I.Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h⁻¹ for H. uvarum, 0.23 h⁻¹ for H. guilliermondii and 0.18 h⁻¹ for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3- mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.
- 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.