ABSTRACT
Yeasts are able to act as biosorbents, as their cell wall includes several components capable of binding organic xenobiotic compounds that can potentially be removed during various fermentation processes. In the present investigation, two novel Saccharomyces cerevisiae strains (LMBF-Y 16 and LMBF-Y-18), previously isolated from grapes, were studied regarding their physiological behavior (dry cell weight-DCW production, substrate uptake, and ethanol and glycerol biosynthesis) during fermentations of grape must, in some cases enriched with commercial glucose and fructose (initial total sugar concentration approximately 150 and 250 g/L, respectively). Myclobutanil (a chiral triazole fungicide broadly used as a protective agent of vine) was also added to the culture media at various concentrations in order to assess the ability of the yeasts to simultaneously perform alcoholic fermentations and detoxify the medium (i.e., to remove the fungicide). In the first set of experiments and for both tested strains, trials were carried out in either 250 mL or 2.0 L agitated shake flasks in either synthetic glucose-based experiments or grape musts. Since the results obtained in the trials where the cultures were placed in 2.0 L flasks with grape musts as substrates were superior in terms of both DCW and ethanol production, these experimental conditions were selected for the subsequent studies. Both strains showed high fermentative efficiency, producing high amounts of DCW (9.5-10.5 g/L) in parallel with high ethanol production, which in some cases achieved values very close to the maximum theoretical ethanol production yield (≈0.49 g of ethanol per g of sugar). When using grape must with initial total sugars at approximately 250 g/L (very high gravity fermentation media, close to winemaking conditions), significantly high ethanol quantities (i.e., ranging between 105 and 123 g/L) were produced. Myclobutanil addition slightly negatively affected sugar conversion into ethanol; however, in all cases, ethanol production was very satisfactory. A non-negligible myclobutanil removal during fermentation, which ranged between 5%-27%, as a result of the adsorptive or degradative capacity of the yeast was also reported. The presence of myclobutanil had no effect on DCW production and resulted in no significant differences in the biosynthesis of glycerol. Therefore, these newly isolated yeast strains could be excellent candidates for simultaneous high ethanol production and parallel pesticide removal in a general biorefinery concept demonstrating many environmental benefits.
ABSTRACT
Oleochemical activities (e.g. biodiesel production, fat saponification) generate annually very high amounts of concentrated glycerol-containing waters (called crude glycerol) as the principal residues of these processes. Crude glycerol is an industrial residue the valorization of which attracts remarkable and constantly increasing interest. In the current investigation, biodiesel-derived glycerol was employed as substrate for yeast and fungal strains cultivated under nitrogen-limited conditions in shake flasks. Glucose was employed as reference substrate. Several yeasts (Candida diddensiae, Candida tropicalis, Pichia ciferrii, Williopsis saturnus, Candida boidinii, and Candida oleophila) rapidly assimilated glucose and converted it into ethanol, despite aerobic conditions imposed, and were Crabtree-positive. None of these yeasts produced ethanol during growth on glycerol or accumulated significant quantities of lipid during growth on glucose or glycerol. Only Rhodosporidium toruloides produced notable lipid quantities from glucose and to lesser extent from glycerol. Yarrowia lipolytica LFMB 20 produced citrate ≈58 g/L growing on high-glucose media, while on high-glycerol media ≈42 g/L citrate and ≈18 g/L mannitol. During growth on glucose/glycerol blends, glycerol was assimilated first and thereafter glucose was consumed. Fungi produced higher lipid quantities compared with yeasts. High lipid quantities were produced by Mortierella ramanniana, Mucor sp., and mainly Mortierella isabellina, with glycerol being more adequate for M. ramanniana and glucose for Mucor sp. and M. isabellina. M. isabellina ATHUM 2935 produced lipids of 8.5 g/L, 83.3% w/w in dry cell weight (DCW) and conversion yield per unit of glucose consumed ≈0.25 g/g. The respective values on glycerol were 5.4 g/L, 66.6% w/w in DCW and ≈0.22 g/g. Lipids of all microorganisms were analyzed with regards to their fatty acid composition, and M. isabellina presented the closest similitude with rapeseed oil. Crude lipids produced by this fungus and extracted with chloroform/methanol blend, were composed mostly of triacylglycerols, thus indicating that these solvents are adequate for triacylglycerol extraction.
