Filipa Mendes

Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)

ABSTRACT Clostridium 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.

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must

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.

Volatile sulphur compounds in wines related to yeast metabolism and nitrogen composition of grape musts

The influence of nitrogen compounds in grape musts on the content of sulphur compounds of wines was studied. Different vinifications were performed with the addition of methionine (20 mg l −1 ) and/or cysteine (40 mg l −1 ) to grape musts before alcoholic fermentation. Six grape musts, with different nitrogen composition, from cultivars of the ‘Vinhos Verdes’ Region, in Portugal, were used. Addition of methionine to grape musts enhanced the content of wines in 3-(methylthio)-1-propanol, acetic acid-3-(methylthio)propyl ester, 3-(methylthio)propionic acid and some unidentified sulphur compounds. Increase of cysteine concentration in musts led to the production of wines with high levels of hydrogen sulphide and cis-2-methyltetrahydrothiophene-3-OL and also unidentified sulphur compounds; however, the content of 3-(methylthio)propionic acid in the wines decreased considerately with the addition of cysteine to grape musts. This work showed that cultivars from the Vinho Verde Region show different sulphur compound contents. Avesso wines, elaborated from grape musts with low amino acids level, presented the highest total sulphur compounds content. Wines from Azal branco and Alvarinho were characterised by high contents of 4-(methylthio)-1-butanol and 3-(methylthio)propionic acid, respectively. A high content of N-(3-(methylthio) propyl)-acetamide and dimethylsulphone characterise the Loureiro wines. In contrast, Trajadura wines, produced from a must rich in amino acids, presented a low total sulphur compounds content; however, these wines were also characterised by high concentrations of 4-(methylthio)-1-butanol, acetic acid-3-(methylthio)propyl ester and hydrogen sulphide.