DEKKERA BRUXELLENSIS: STUDIES ON CARBON AND NITROGEN SOURCES METABOLISM AND ITS RESPONSE TO ACETIC ACID STRESS.

The lineages of Dekkera bruxellensis and Saccharomyces cerevisiae separated approximately 200 million years ago, but they share several industrially relevant traits, such as the ability to produce ethanol under aerobic conditions (Crabtree effect), high tolerance towards ethanol and acid, and ability to grow without oxygen. Beside a huge adaptability, D. bruxellensis exhibits a broader spectrum of consumable carbon and nitrogen sources in comparison to S. cerevisiae. This yeast is famous as a spoilage yeast in food and beverage industries and contaminates ethanol production process. Despite its economic importance and physiological interest, D. bruxellensis has not been well studied yet in detail. To characterize its carbon metabolism and regulation, we investigated how galactose is used as carbon source by this yeast. Here we show that in D. bruxellensis under aerobic conditions and on ammonium-based media galactose is a not-fermentable carbon source, in contrast to S. cerevisiae which can ferment also this sugar. The expression of genes involved in different metabolic pathways was also analysed. We report that genes involved in galactose utilization, respiratory metabolism, TCA cycle, glyoxylate cycle and gluconeogenesis are repressed in glucose-based media. These results indicate that in D. bruxellensis glucose repression operates similarly to what occurs in S. cerevisiae. In contrast to Saccharomyces cerevisiae, D. bruxellensis can use nitrate as sole nitrogen source. 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 \u2018\u2018Custers effect\u2019\u2019, in this way improving its fermentative metabolism. Acetic acid, due to its toxic effects, is used in food industry as a preservative against microbial spoilage. We investigated how this yeast responds when exposed to acetic acid. A detailed analysis of acetic acid metabolism was performed on three strains which exhibited a different resistance. Our studies show that D. bruxellensis behaves, from a metabolic point of view, more similarly to S. cerevisiae, being unable to metabolize acetic acid in presence of glucose. The presence of acetic acid affected the growth, causing a reduction of growth rate, glucose consumption rate, ethanol production rate as well as biomass and ethanol yield. Interestingly, the cells continued to produce acetic acid

Development of Biorefinery Schemes for the Valorization of Agro-Industrial Wastes: Production of Polyhydroxyalkanoates

Aiming to reduce the cost of production of polyhydroxyalkanoates (PHAs), it was verified the use of two agro-industrial wastes as alternatives carbon sources for the production of: A- poly(hydroxybutyrate-co-hydroxyvalerate), from olive mill wastewater (2012 campaign; 60% dephenolised). It was possible to used up to 25% v/v in the culture media due to the presence of polyphenolic inhibitors in the matrix. B- polyhydroxybutyrate (PHB) with OMW belonging to 2013 (>70% dephenolised): the culture medium could contain 100% v/v without causing inhibition. An integrated biorefinery scheme was defined and tested sequentially: polyphenols recovery, organic acids PHAs and biogas production. C- PHB from dephenolised and fermented grape pomace. An integrated biorefinery scheme was proposed for the first time for the valorisation of grape pomace. D- medium chain length polyhydroxyalkanoates by performing the anaerobic acidogenic digestion of GP before dephenolisation. From the research work on OMW, a study about total polyphenols determination by colorimetric method was carried out. Indeed, solid phase extraction break-through curves obtained during polyphenols recovery experiment were employed as a tool for the analysis. To increase the volatile fatty acids (VFAs) concentration, for obtaining a feeding solution which allow fed-batch fermentation, it was proposed to concentrate VFAs produced in acidogenic digestion using nanofiltration (NF). A preliminary feasibility study has been carried out, getting rejections in the range 30-90%, using a plant-counter and prepared in the laboratory of VFAS solution (C2 to C6) salts and buffers in distilled water. The optimization of the PHAs downstream process was also studied. It is been developed a procedure for the extraction of PHAs by cell pretreatment with, heat, acids, digestion with NaOH and ethanol-water washing. In this study it was verified that the implementation of a pre-treatment with H2SO4 allows to recover 85% of PHAs products reaching a purity of >95%

Production of bioethanol from the effluents of dairies by Kluyveromyces marxianus

Il siero di latte e la scotta sono effluenti provenienti rispettivamente dal processo di trasformazione del latte in formaggio e ricotta. Il siero di latte contiene minerali, lipidi, lattosio e proteine; la scotta contiene principalmente lattosio. Il siero può essere riutilizzato in diversi modi, come l'estrazione di proteine o per l’alimentazione animale, mentre la scotta è considerata solamente un rifiuto. Inoltre, a causa degli ingenti volumi di siero prodotti nel mondo, vengono a crearsi seri problemi ambientali e di smaltimento. Destinazioni alternative di questi effluenti, come le trasformazioni biotecnologiche, possono essere un modo per raggiungere il duplice obiettivo di migliorare il valore aggiunto dei processi agroindustriali e di ridurre il loro impatto ambientale. In questo lavoro sono state studiate le condizioni migliori per produrre bioetanolo dal lattosio del siero e della scotta. Kluyveromyces marxianus è stato scelto come lievito lattosio-fermentante. Sono state effettuate fermentazioni su scala di laboratorio aerobiche e anaerobiche in batch, fermentazioni semicontinue in fase dispersa e con cellule immobilizzate in alginato di calcio,. Diverse temperature sono state testate per migliorare la produzione di etanolo. Le migliori prestazioni, per entrambe le matrici, sono state raggiunte a basse temperature (28°C). Anche le alte temperature sono compatibili con buone rese di etanolo nelle fermentazioni con siero. Ottimi risultati si sono ottenuti anche con la scotta a 37°C e a 28°C. Le fermentazioni semicontinue in fase dispersa danno le migliori produzioni di etanolo, in particolare con la scotta. Invece, l'uso di cellule di lievito intrappolate in alginato di calcio non ha migliorato i risultati di processo. In conclusione, entrambi gli effluenti possono essere considerati adatti per la produzione di etanolo. Le buone rese ottenute dalla scotta permettono di trasformare questo rifiuto in una risorsa.Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused by several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental problems for disposal. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of agroindustrial processes and reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch aerobic and anaerobic fermentations and semicontinuous fermentations in dispersed phase and in immobilized phase were carried out of whey, scotta at a laboratory scale. Different temperatures were also tested in order to try to improve the ethanol production. The best performances for both matrices were reached at low temperatures (28°C). High temperatures are also compatible with good ethanol yields in whey fermentations. Very good results are also obtained with scotta at 37°C and at 28°C. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, in particular with scotta. Instead, the use of yeast cells entrapped in calcium alginate did not improve the process results. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow to transform this waste in a source