Silvia Galafassi

Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis

The increasing cost of vegetable oils is turning the use of microbial lipids into a competitive alternative for the production of biodiesel fuel. The oleaginous yeast Rhodotorula graminis is able to use a broad range of carbon sources for lipid production, and is able to resist some of the inhibitors commonly released during hydrolysis of lignocellulosic materials. Using undetoxified corn stover hydrolysate as substrate, the yeast achieved a lipid productivity and lipid content of 0.21 g/L/h and 34%w/w, respectively. The corresponding results with crude glycerol as carbon source were 0.15 g/L/h and 54%w/w, respectively. Therefore, R. graminis appears to be a suitable candidate for fermentation processes involving renewable resources.

Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.

The oxygen level determines the fermentation pattern in Kluyveromyces lactis

Yeasts belonging to the lineage that underwent whole-genome duplication (WGD) possess a good fermentative potential and can proliferate in the absence of oxygen. In this study, we analyzed the pre-WGD yeast Kluyveromyces lactis and its ability to grow under oxygen-limited conditions. Under these conditions, K. lactis starts to increase the glucose metabolism and accumulates ethanol and glycerol. However, under more limited conditions, the fermentative metabolism decreases, causing a slow growth rate. In contrast, Saccharomyces cerevisiae and Saccharomyces kluyveri in anaerobiosis exhibit almost the same growth rate as in aerobiosis. In this work, we showed that in K. lactis, under oxygen-limited conditions, a decreased expression of RAG1 occurred. The activity of glucose-6-phosphate dehydrogenase also decreased, likely causing a reduced flux in the pentose phosphate pathway. Comparison of related and characterized yeasts suggests that the behavior observed in K. lactis could reflect the lack of an efficient mechanism to maintain a high glycolytic flux and to balance the redox homeostasis under hypoxic conditions. This could be a consequence of a recent specialization of K. lactis toward living in a niche where the ethanol accumulation at high oxygen concentrations and the ability to survive at a low oxygen concentration do not represent an advantage.

Generation of an evolved Saccharomyces cerevisiae strain with a high freeze tolerance and an improved ability to grow on glycerol

Glycerol is a residue generated during biodiesel production and represents around 10% of the total product output. Biodiesel production is currently having a significant impact on glycerol price, leading to an increased interest in the use of glycerol as a cheap substrate for fermentation processes. We have analysed the growth kinetics of two wild-type strains of Saccharomyces cerevisiae grown on synthetic media containing glycerol as the sole carbon and energy source. Both strains were initially unable to grow when cultivated under these conditions, and an unusually long lag phase was necessary prior to the appearance of slow-growing cells. Following the application of an “evolutionary engineering” approach, we obtained S. cerevisiae strains with an improved ability to grow on glycerol. We report here the isolation of an evolved strain that exhibits a reduction of the lag phase, a threefold increase of the specific growth rate and a higher glycerol consumption rate compared to wild-type strains. The evolved strain has retained its fermentative activity, producing ethanol at the same rate and yield as the wild type. Interestingly, the yeast biomass obtained by cultivating the evolved strain on synthetic glycerol-based media also showed a high viability after prolonged storage at −20°C. The strategy adopted in our study could be easily applied to obtain S. cerevisiae strains with new industrially relevant traits, such as an improved ability to use cheap substrates and high resistance to freeze and thaw procedures.

Coevolution with bacteria drives the evolution of aerobic fermentation in Lachancea kluyveri

The Crabtree positive yeasts, such as Saccharomyces cerevisiae, prefer fermentation to respiration, even under fully aerobic conditions. The selective pressures that drove the evolution of this trait remain controversial because of the low ATP yield of fermentation compared to respiration. Here we propagate experimental populations of the weak-Crabtree yeast Lachancea kluyveri, in competitive co-culture with bacteria. We find that L. kluyveri adapts by producing quantities of ethanol lethal to bacteria and evolves several of the defining characteristics of Crabtree positive yeasts. We use precise quantitative analysis to show that the rate advantage of fermentation over aerobic respiration is insufficient to provide an overall growth advantage. Thus, the rapid consumption of glucose and the utilization of ethanol are essential for the success of the aerobic fermentation strategy. These results corroborate that selection derived from competition with bacteria could have provided the impetus for the evolution of the Crabtree positive trait.

Characterization of lipid accumulation and lipidome analysis in the oleaginous yeasts Rhodosporidium azoricum and Trichosporon oleaginosus

The influence of cultural conditions on lipid production was investigated in two species, Trichosporon oleaginosus and Rhodosporidium azoricum. We showed that nitrogen limitation is not the main factor triggering the mechanism of lipid accumulation in T. oleaginosus. Moreover, a scarce availability of oxygen negatively affected lipid synthesis to a lesser extent in T. oleaginosus than in R. azoricum. This highlights how the importance of controlling fermentation parameters is strictly linked to the yeast species employed. We showed that these parameters affect the activity of important enzymes, influencing the metabolic fluxes into different pathways, in particular pentose phosphate pathway and cytoplasmic pyruvate bypass. Furthermore, T. oleaginosus exhibited wider substrate flexibility, faster growth and higher lipid accumulation in fed-batch cultivation. Microbial oils obtained from both yeasts proved a valuable feedstock, alternative to vegetable oils, for advanced diesel biofuel production.

Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499.

Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for Δ9-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn’t show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response.