Suchergebnisse

It has been estimated that at least 99 % of the world's microbial biomass exists in form of biofilm, a complex differentiated surface-associated community embedded in a self-produced polymeric matrix enabling microorganisms to develop coordinated and efficient survival strategies. Biofilm formation is a dynamic and cyclical process involving attachment, maturation and a final dispersal phase, and these steps are initiated by a variety of signals. Despite their positive effects in some cases, biofilms can be detrimental in different environmental domains since microorganisms are able to colonize almost all types of surfaces both abiotic and biotic, leading to consequences in terms of social and economic impact. These include human tissues, implantable medical devices, natural aquatic systems, plants, food and industrial lines. Once biofilm is formed, its eradication becomes difficult because its resilience to environmental stresses, disinfectants, and antimicrobial treatments. Plants support a diverse array of microorganisms that exist in form of biofilms. Even if in some cases the association with plants leads to beneficial interactions promoting plant growth, inducing plant defense mechanisms and preventing the deleterious effects of pathogenic microorganisms, in other cases they have a significant negative impact. For instance, in agriculture, plant colonization of fungi and bacteria in form of biofilm is a cause of plant diseases, affecting crop quality and productivity. Indeed, despite the planktonic growth, biofilm lifestyle improves microbial resistance to antimicrobials up to several orders of magnitude, often reducing the possibility of treating biofilm effectively. In addition, due to the worrisome consequences related to the use of these substances on human health and on their persistence in the environment, increasingly regulations are arising to limit antimicrobial application. Furthermore, in addition to the principles of integrated pest management (IPM) embraced by the worldwide legislation aims to recommend alternative approaches to the application of pesticides, an innovative approach could be the use of biocide-free bioactive compounds characterized by novel targets, unique modes of action and properties that are separate from those currently highlighted in the use of antimicrobials. Indeed, the application of non-lethal doses of bio-inspired molecules able to interfere with specific key-steps involved in the biofilm formation process has been suggested as a complementary/alternative strategy to hinder biofilm formation. In addition, this approach also lead to deprive microorganisms of their virulence factors without affecting their viability and decreasing the selection pressure for biocides resistance. In this PhD thesis, the in vitro effects of non-lethal concentrations of several bioactive compounds were evaluated on the biofilm formation of different plant-associated microorganisms. Specifically, the aim of this work was to provide new effective preventive or integrated solutions against bacterial and fungal biofilm formation. In chapter III, the methanol extracts obtained by different plant portions of three seagrass species collected in Vietnam and in India (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) were investigated for their effects in mediating non-lethal interactions on sessile Escherichia coli and Candida albicans cultures taken as models of bacterial and fungal biofilms respectively. The study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Seagrass extracts appeared to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. Results revealed that E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an anti-biofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Methanolic extracts were characterized and major phenolic compounds were identified by MS/MS analysis, showing the unique profile of the E. acoroides leaf extract. In chapter IV, two essential oils (PK and PK-IK) derived from two cultivars of Perilla frutescens, an annual short day plant widely used in therapeutics in the traditional medicine as well as in food preparations in Asian countries. Essential oils were extracted from the leaves and were characterized. Subsequeltly, their ability to affect biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and F. oxysporum have been studied. PK and PK-IK neither inhibited fungal growth nor were they utilized as a carbon energy source. In addition, PK and PK-IK essential oils showed excellent anti-biofilm performances inhibiting conidia germination and reducing conidia adhesion. Furthermore, they revealed a magnificent anti-biofilm effect even during biofilm maturation, affecting biofilm structural development, with a reduction of dried weight, extracellular polysaccharides and proteins. In all cases PK-IK displayed better activity than PK. Thus, the anti-biofilm effects were exploited with a non-lethal mechanism. This research supported the spreading of PK and PK-IK essential oils as biocide-free agents suitable for a preventive or integrative approach for sustainable crop protection. Lastly, in chapter V, a non-lethal concentration of N-Acetylcysteine (NAC) was evaluated on the biofilm formation of Xylella fastidiosa, a phytopathogen bacterium that causes a range of economically important plant diseases worldwide and that has been recently found in Italy in olive plants, where it causes the olive quick decline syndrome (OQSD). NAC is a naturally occurring compound found in several vegetables (including garlic, onion, peppers and asparagus) and it is mostly known in clinical area, in which it is employed at lethal concentrations in the treatment of human diseases due to its ability to reduce bacterial adhesion, inhibit the production of extracellular polysaccharides and promote the dispersion of pre-formed mature biofilms. In this study, N-Acetylcysteine (NAC) was tested for its ability to affect biofilm response of X. fastidiosa CoDiRO strain, mimicking a preventive, a curative and a combination of both approaches. The not-lethal dose 0.08 mg/ml was chosen as representative of plant concentration after its application. NAC did not alter planktonic bacterial growth but promoted biofilm formation in terms of biofilm biomass (above 62 %) and matrix polysaccharides (above 53%) through a ROS-mediated mechanism. Additionally, NAC was not able to destroy X. fastidiosa biofilm when already established on the surface but rather, it was suitable to contain the biofilm infection limiting biofilm dispersal. On the contrary, a combination of both preventive and curative approach has been found promising in biofilm dissolving making it more vulnerable.

Compost is commonly used as an organic fertilizer to recover and preserve the chemical and physical properties of agricultural soils. Effectively, the use of recycled biomasses, residues and by-products, such as compost, is an effective way to improve the environmental and economic sustainability of agroecosystems and agri-food value chains. The use of green compost is increasingly acknowledged in the EU policies as one of the most sustainable approaches to implement the circular economy in agricultural sector due to an economic and environmental re-evaluation of agricultural biomass. Following the raising attention on compost as valuable organic resource for crop productivity, in the recent years the research activity was addressed on the innovative utilization of specific compost fractions such as humic substances and compost teas for crop biostimulation, acting as bio-effectors on biochemical activities, physiological processes and plant microbial interactions The research described in this thesis, is focused on the application of compost as renewable resource for the production of these potential bioactive materials such as humic substances (HS) and compost teas (CTs) as biostimulant to promote the biosynthesis of nutraceutical products in aromatic crop represented by common basil plant. These organic materials were obtained from different composted agricultural biomasses: artichoke, coffee and pepper. Compost teas and humic substances isolated from the different on-farm composts were characterized in details for their chemical properties and molecular composition. Experiments using HS and CTs obtained from artichoke, coffee and pepper composted biomasses highlighted a relationship with the capacity to increase the development of Basil plants with positive effects on phenological parameters such as fresh and dry biomasses and chlorophyll content. Moreover, the plant treatments with compost extracts promoted the synthesis of metabolites with antioxidant proprieties and antibacterial activity against some common human pathogens. In line with the most advanced research approaches, the results indicated the occurrence of structural-activity relationships based on a combination of physical and chemical features of applied organic fractions as triggering factors for biostimulant properties. The data of molecular characterization suggested the prevalent role of hydrophobic conformation and aromatic components, lignin derivatives and peptidic cluster in the modulation of analysed metabolic pathways. The application of metabolomics and lipidomic approaches have shown an increase in the biosynthesis of secondary metabolites with nutraceutical properties such as hydroxycinnamic acids (caffeic, ferulic, trans-cinnamic and p-cumaric) naringenin, resveratrol and coenzyme Q-10, thus confirming the effect of compost derivatives on plant metabolism. The organic materials influenced the production of primary metabolic intermediates such as erythrosis-4-phosphate and phosphoenolpyruvate that activate the production of metabolites involved in the biochemical pathway of schikimic acid. Moreover, the treatment of Basil plants with humic materials such as HS-artichoke improved the yield, antimicrobial and antioxidant properties of Essential oils (EOs) extracted from Basil leaves by steam distillation with a clear dose-response effect on the production of most bioactive chemical components like eucalyptol, methyl-chavicol, eugenol, methyl-Eugenol, trans-α-Bergamotene, geranyl acetate. Finally, medical applications of the HS and CTs extracted from different composted biomasses were carried out using in vitro assays on epithelial adenocarcinoma epithelial colon cancer cells (CaCo-2) and immortalized cells of cervical tumor (HeLa). The results of the determination of cytotoxic activity showed the significant inhibition in the proliferation of these tumorigenic cell lines, thus further supporting the reliable utilization and exploitation of natural compost extracts in pharmaceutical, nutraceutical and cosmetic fields

Dottorato di ricerca in Biotecnologie degli alimenti ; Lo smaltimento delle acque di vegetazione dei frantoi oleari costituisce, attualmente, uno dei principali problemi dal punto di vista ambientale, specialmente nei paesi del Mediterraneo dove si concentra la maggior parte della produzione mondiale di olio di oliva. Le acque di vegetazione sono tra i reflui agro-industriali a più alto tasso inquinante a causa del loro elevato carico organico, caratterizzato soprattutto da composti fenolici e polifenolici ad elevata azione antimicrobica e fitotossica. La purificazione biologica delle acque di vegetazione è particolarmente difficile poiché questo refluo presenta solidi in sospensione e un elevato carico organico, in particolare polifenoli con attività biostatica e/o biocida, che riduce fortemente le prestazione degli impianti di depurazione. Di conseguenza, l'impianto deve prevedere due o più stadi di trattamento che rendono la depurazione complessa e costosa. Attualmente, la normativa vigente consente la pratica dello spandimento delle acque di vegetazione sui terreni agrari; nonostante questa risulti, al momento, essere la soluzione migliore sia dal punto di vista pratico che economico, trova attuazione solo se si ha disponibilità di terreni sufficientemente vicini su cui spargere il refluo e comunque deve essere applicata in maniera controllata dal momento che gli eventuali effetti positivi o negativi sulla composizione, sulla carica microbica e la fertilità del terreno sono ancora oggi oggetto di studio. Inoltre, la migrazione di alcuni composti negli strati più bassi del terreno potrebbe causare la contaminazione di eventuali falde acquifere sottostanti con conseguenze per la salute dell'uomo. Negli ultimi anni sono state proposte soluzioni alternative finalizzate a sfruttare questo refluo, in quanto ricco di composti utili. La valorizzazione delle AV mediante il loro impiego per l'ottenimento di prodotti a medio o alto valore aggiunto attraverso processi fisico-chimici o fermentativi, riveste notevole interesse scientifico. Nelle AV sono presenti una grande varietà di biomolecole come acidi organici, polialcoli, zuccheri semplici e complessi e lipidi che le rendono una possibile base per i processi fermentativi. In virtù del contenuto residuo di lipidi, le AV potrebbero rappresentare un ottimo candidato come terreno liquido di crescita per la produzione di lipasi microbiche. Lo scopo della presente tesi di dottorato è stato quello di mettere a punto un processo fermentativo per la valorizzazione delle AV mediante produzione microbica di enzimi, in particolare enzimi lipolitici, ottenendo al contempo un abbattimento, o quanto meno una riduzione, del loro potere inquinante. Esiste una vasta bibliografia in cui viene presa in esame la produzione di lipasi da numerose specie microbiche tra cui Penicillium e Candida e sia il terreno che il processo fermentativo per la produzione di questo enzima è stato ampiamente ottimizzato. Nella maggior parte dei casi, una buona produzione di lipasi microbica prevede l'utilizzo di terreni sintetici piuttosto complessi che sicuramente incidono in maniera significativa sul prezzo finale del prodotto. Inoltre, negli ultimi anni anche la produzione di preparati enzimatici commerciali contenenti lipasi di origine microbica ha avuto un notevole sviluppo. Sigma, Amano, Roche, Novo Nordisk, etc., forniscono preparati lipolititici con varie composizioni e proprietà catalitiche utilizzati in diversi settori: industria alimentare, farmaceutica, dei detergenti e per la produzione di biodiesel. L'innovazione che dovrebbe introdurre questo lavoro è l'opportunità di produrre lipasi microbiche di possibile interesse industriale utilizzando un substrato costituito da un refluo agro-industriale. Con questa idea, si è cercato di mettere a punto un terreno di produzione a basso costo che permettesse di ottenere buoni livelli di attività e contemporaneamente un abbattimento del carico inquinante del refluo finale. In prima battuta, è stato effettuato uno screening di microrganismi (Geotrichum candidum, NRRL 552, 553; Rhizopus sp, ISRIM 383; Rhizopus arrhizus, NRRL 2286; Rhizopus oryzae, NRRL 6431; Aspergillus oryzae, NRRL 1988, 495; Aspergillus niger, NRRL 334; Candida cylindracea, NRRL Y-17506; Penicillium citrinum, NRRL 1841, 3754, ISRIM 118) in grado di crescere sulle acque di vegetazione producendo lipasi. Le produzioni più elevate di enzima sono state ottenute, in condizioni non-ottimizzate, dopo 168 h con Geotrichum candidum NRRL 553 (0,521 U/ml) e Candida cylindracea (0,460 U/ml). Inoltre, livelli di produzione molto interessanti sono stati raggiunti dopo 72 h con i ceppi di Penicillium citrinum (0,365, 0,320 e 0,375 U/ml per NRRL 1841, NRRL 3754 e ISRIM 118, rispettivamente). Questi ceppi sono stati selezionati per valutare, in via preliminare, l'effetto di alcuni fattori sulla produzione di lipasi quali tipologia di AV, utilizzo di vari oli come induttori di attività e impiego di diverse fonti di azoto. Per quanto riguarda la produzione di lipasi da P. citrinum NRRL 1841 su AV, l'attività è stata influenzata in maniera marcata dal tipo di fonte di azoto ma non era aumentata in maniera significativa dall'aggiunta di oli. Nel caso della produzione di lipasi da C. Cylindracea NRRL Y-17506, il cloruro di ammonio e l'olio di oliva rappresentavano rispettivamente la fonte di azoto e l'induttore più adatto; infatti questo ceppo cresciuto in condizioni parzialmente ottimizzate produceva 9,48 U/ml di attività lipolitica dopo 264 h di fermentazione. Successivamente, la produzione di lipasi da P. citrinum NRRL 1841, utilizzando il terreno a base di AV, è stata ottimizzata in beuta valutando l'effetto del pH iniziale, della concentrazione di azoto e di estratto di lievito secondo un approccio multi-fattoriale. La combinazione ottimizzata dal modello è stata la seguente: pH 6,15, 2,7 g/l NH4Cl e 1,1 g/l YE. La produzione massima raggiunta è stata di 1,242 U/ml. Con il terreno così ottimizzato, al fine di ottenere informazioni sul possibile trasferimento di scala del processo, sono stati condotti altri esperimenti in reattori da banco. Allo scopo, sono stati impiegati due tipi di sistemi, un bioreattore ad agitazione meccanica (STR) e uno ad agitazione pneumatica (Air-lift). In entrambi i casi, l'attività lipolitica extracellulare aveva raggiunto il suo picco massimo dopo 192 h di fermentazione. Tuttavia, il massimo di attività è stato significativamente più alto in STR che in Airlift (0,700 vs 0,420 U/ml, rispettivamente). Sebbene tutti i ceppi studiati sono stati in grado di crescere sulle acque di vegetazione e produrre a livelli significativi attività lipolitica, una particolare attenzione è stata riservata a C. cylindracea (noto anche come C. rugosa) per il notevole interesse applicativo della lipasi prodotta da questo lievito. Inizialmente, si è cercato di ottimizzare in beuta la composizione del terreno di produzione (concentrazione dell'olio di oliva, effetto del glucosio, aggiunta di surfactanti e di vari fattori di crescita) e di valutare in via preliminare l'effetto sulla crescita cellulare e sull'attività di alcune condizioni colturali quali velocità di agitazione e aerazione. La migliore composizione del terreno di produzione si è confermata essere quella contenente 3 g/l di olio di oliva, 2,4 g/l di NH4Cl e 0,5 g/l di estratto di lievito, senza l'aggiunta di glucosio e Tween 80. Inoltre, con lo scopo di valutare la fattibilità tecnica di un trasferimento di scala del bioprocesso e approfondire la messa a punto del processo fermentativo sono stati condotti una serie di esperimenti in bioreattore da banco ad agitazione meccanica (STR). In particolare, utilizzando il terreno a base di AV ottimizzato, si è cercato di ottimizzare alcuni parametri quali pH, velocità di agitazione e aerazione. Per quanto riguarda l'effetto della velocità di agitazione e dell'aerazione sulla produzione enzimatica, sono state prese in esame tre velocità di agitazione (300, 500 e 700 giri/min), mantenute fisse durante tutta la fermentazione, e in più è stato condotto un esperimento in cui si è cercato di mantenere la concentrazione dell'ossigeno disciolto nel mezzo superiore al 20% di saturazione facendo variare la velocità di agitazione tra 300 e 800 giri/min. Mentre per valutare l'effetto del pH, sono stati condotti degli esperimenti a pH 6,5 fisso confrontando la produzione con quella ottenuta a pH libero e a pH mantenuto inferiore a 6,5. La massima produzione di lipasi da C. cylindracea è stata ottenuta in bioreattore lavorando a pH libero e ad una velocità di agitazione costante di 500 giri/min (18,50 U/ml) o ad una velocità di agitazione variabile tra 300 e 800 giri/min in modo da assicurare un valore di ossigeno disciolto nel brodo superiore al 20% di saturazione (18,70 U/ml); in quest'ultimo caso, inoltre, la comparsa del picco massimo è stata anticipata nel tempo favorendo così la produttività oraria del bioprocesso. Per quanto riguarda i reattori a 300 e 700 giri/min, la produzione enzimatica è stata di 2,54 e 11,65 U/ml, rispettivamente. Infine, messo a punto il bioprocesso di produzione della lipasi da C. cylindracea coltivata su un terreno a base di AV, si è cercato di identificare il profilo enzimatico del campione grezzo così ottenuto, dal momento che, come è noto dalla letteratura, questo lievito è in grado di produrre fino a sette isoforme ad attività lipolitica. A tale scopo sono stati condotti degli esperimenti di isoelettrofocalizzazione (IEF) analitica. Nel gel sono stati caricati un campione di lipasi commerciale (Tipo VII, Sigma) e due campioni grezzi ottenuti da C. cylindracea coltivata sul terreno a base di AV, prelevati a due tempi fermentativi diversi e corrispondenti ai due picchi di attività lipolitica raggiunti durante le prove in STR (I° e II° picco di massima attività, 48esima e 192esima ora, rispettivamente). Dai risultati ottenuti, è stato osservato che il campione grezzo era costituito da più isoenzimi con attività lipolitica e che il profilo isoenzimatico aveva una sola banda in comune con quello della lipasi commerciale (Typo VII, Sigma) a cui è stato assegnato pI 4,7. Per quanto riguarda il campione prelevato alla 48esima ora, sono state osservate anche una banda piuttosto intensa a pI 5,1 e una tripletta di bande più deboli a pIs di 5,06, 5,0 e 4,9. Durante la fermentazione il profilo isoenzimatico del campione aveva subito delle modifiche: infatti, alla 192esima ora, le bande a pIs 5,1, 5,0 e 4,9 erano scomparse, mentre era comparsa una banda di attività intensa a cui è stato assegnato un pI di 4,5. Infine, in entrambi i campioni grezzi è stata rilevata una banda tenue a pI 3,8. In conclusione, i buoni livelli di attività enzimatica raggiunti dimostrano la fattibilità tecnica di un processo fermentativo finalizzato alla valorizzazione dei reflui oleari mediante la produzione di lipasi, che può avere promettenti utilizzi in varie applicazioni industriali. Comunque, ulteriori fasi di scale-up del processo sono ancora necessarie al fine di poter effettuare una valutazione sulla fattibilità economica del processo. ; The olive mill wastewater (OMW) disposal is, currently, one of the main environmental problems in all olive-oil producing countries, especially in the Mediterranean area. In fact, for its high organic load, phenolic fraction with phytotoxic effects and antimicrobial activity, the OMW is a highly polluted agro-industrial effluent. The biological treatment can be very difficult since solid residues, high organic load and phenols may strongly reduce the depuration efficiency. Consequently, a possible process should include several technological options, physical, chemical and biological, as well as combinations thereof, thus resulting in increased process costs. At the moment, the Italian legislation allows land spreading of untreated olive mill wastewater that is the best economical solution. Application on agriculture soils is a practice which solves partially the problem of OMW disposal. Positive and negative effects on soil composition and fertility are still under study, so that OMW application must be strictly controlled. Land spreading, in fact, may cause serious negative environmental impact regarding, for instance, groundwater contamination. In the last years, alternative solutions have been proposed in view of the use this waste as a source of valuable compounds. Several recent research studies have reported the possibility of OMW valorization to obtain products of actual or potential industrial interest. The presence in OMW of a wide range of biomolecules such as organic acids, polyalcohols, simple and complex sugars and lipids makes it a potential basis for fermentation processes. In this way, OMW could be a putative candidate as a potentially suitable liquid growth medium for the production of microbial lipases by virtue of its residual lipid content. For these reasons, the objective of the present PhD thesis was to assess the suitability of OMW as growth medium for the production of lipases and to set up a related fermentation process that might lead, at the same time, to a low polluting load final effluent. A large number of microbial strains have been screened for lipase production belonging to several fungal genera, Candida and Penicillium in particular. In literature, numerous methods for lipolytic enzyme production are published and medium composition and cultural conditions have been fully optimised. Neverthless, the most frequently used medium is a chemical defined and complex one, significantly affecting the final product costs. Besides, in the last years, a whole range of microbial lipase preparations has been developed. Sigma, Amano, Roche, Novo Nordisk, etc., provide lipolytic preparations with various compositions and catalytic proprierties employed in areas such as detergent pharmaucetic and food industries and biodiesel production. Our innovative approach consists in the trial of producing microbial lipases using an agroindustrial-waste based medium. Our basic idea, in fact, was that of developing a low cost production medium. Firstly, 12 fungal strains belonging to well-known lypolytic species (Geotrichum candidum, NRRL 552, 553; Rhizopus sp, ISRIM 383; Rhizopus arrhizus, NRRL 2286; Rhizopus oryzae, NRRL 6431; Aspergillus oryzae, NRRL 1988, 495; Aspergillus niger, NRRL 334; Candida cylindracea, NRRL Y-17506; Penicillium citrinum, NRRL 1841, 3754, ISRIM 118) were screened for their ability to grow on undiluited OMW and to produce extracellular lipase activity. The highest lipase productions were obtained under non-optimized conditions after 168 h with Geotrichum candidum NRRL 553 (0.521 U/ml) and Candida cylindracea (0.460 U/ml). Interesting production levels were also achieved after 72 h with strains of Penicillium citrinum (0.365, 0.320 and 0.375 U/ml for NRRL 1841, NRRL 3754 and ISRIM 118, respectively). These strains were then selected to study the effect of culture conditions, such as OMW typology, nitrogen sources and inducers, on the enzyme production. With regard to the lipase production by P. citrinum NRRL 1841, the enzyme activity was significantly influenced by nitrogen addition; on the other hand, the addition of oils resulted in a marked increase in biomass without affecting, however, lipase production. Lipase production by C. cylindracea NRRL Y-17506 was significatly favored by ammonium salts and oil addition. This strain growth in OMW medium containing ammonium chloride and olive oil led to an activity peak of 9.48 U/ml after 264 hours of fermentation. In order to optimise lipase production by P. citrinum in OMW-based medium, the combined effect of three variables (i.e, concentration of NH4Cl, yeast extract and initial pH) was assessed using a multi-factorial design with 'optimizer' function of 'Modde 5.0' program. The optimised combination by the model was as follows: pH 6.15, 2.7 g/l NH4Cl e 1.1 g/l extract yeast. The maximum lipase activity was 1.242 U/ml after 192 hour of fermentation. To gain information on the possible up-scaling of the process, further experiments were performed in 3-l laboratory-scale reactors. Specifically, pneumatically agitated (Airlift) and mechanically agitated (STR) reactors were employed using the optimised OMW-based medium. In both cases, the extracellular lipase peaked 192 h after inoculation. Howewer, the maximum activity was significatly higher in STR with respect to the Airlift (0.700 vs 0.420 U/ml, respectively). Of all strains, C. cylindracea appeared to be particularly interesting and was, therefore, used as the model microorganism to further investigate the feasibility of an OMW substrate. Firstly, the optimisation of medium composition was assessed in shaken cultures. In particular, the effects on the lipase production of olive oil concentration (1, 3, 5 e 10 g/l), glucose (5 g/l), Tween 80 (0,5 g/l) and several growth nutrients (yeast extract, malt extract and peptone) addition were studied. The best medium composition was as follows: diluited OMW (1:2), olive oil 3 g/l, NH4Cl 2.4 g/l and yeast extract 0.5 g/l. The glucose and Tween 80 addition negatively affected the production of lipolytic enzyme. Lipase production by C. cylindracea on OMW-optimized medium was subsequently assessed in mechanically agitated bioreactor (STR). To study the agitation influence on enzyme production, a set of experiments was carried out at three impeller speed, 300, 500 and 700 rpm; moreover, an additional experiment was carried out at dissolved oxygen DO > 20% saturation (agitation speed automatically controlled between 300 and 800 rpm). To evaluate the effect of pH, three conditions were compared: free pH; fixed pH (6.5) maintained constant by addition of HCl 4.0 M and NaOH 4.0 M; pH lower than 6.5 controlled with addition of HCl 4.0 M. The maximum lipase productions were obtained with the pH left free to vary, 500 rpm costant agitation speed (18.5 U/ml) and variable agitation speed between 300 and 800 rpm to ensure a dissolved oxygen value upper to 20% (18.7 U/ml); in the latter thesis the onset of enzyme activity was anticipated thus leading to increased bioprocess productivity. At 300 e 700 rpm agitation speed, the maximum lipase productions were 2.54 and 11.65 U/ml, respectively. Finally, to set up the bioprocess of lipase production by C. Cylindracea grown on OMW-based medium, the isoenzymatic profiles of the raw sample was evaluated. This aspect appears to be very interesting since it is known that commercial C. rugosa lipase is a mixture of 3 isoenzymes namenly Lip 1, Lip2 and Lip 3 but the yeast is able to produce up to seven different isoenzymes (Lip 1-Lip 7). Moreover isoenzymatic profiles can depend on media composition and fermentation conditions. With this aim, a set of analitycal isoelectrofocusing experiments were carried out. In the gels, a sample of commercial lipase (Type VII, Sigma) and two raw samples of lipase by C. cylindracea grown on OMW-optimized medium and corresponding to two lipolytic activity peaks (1st and 2nd peak, 48esime and 192esime hour of fermentation, respectively) obtained in STR, were loaded. The results suggest that the raw samples were constituted of more lipolytic isoenzymes with the isoenzymatic profile having only one band in common with that of the commercial lipase (assigned pI 4.7). The sample corresponding to the 1st activity peak showed a strong band at pI 5.1 and a triplette of weak bands at pIs 5.06, 5.0 e 4.9. Moreover, the isoenzymatic profiles changed during fermentation; in fact, the bands at pIs 5.1, 5.0 and 4.9 disappeared and a new strong band at pI 4.5 formed. Finally, in both raw samples a band at pI 3.8 was observed. OMWs valorisation by its use as growth medium for lipase production by C. cylindracea NRRL Y-17506 and P. citrinum NRRL 1841 appears to be possible and promising. Moreover, the investigation for further up-scaling is need to evaluate the economic fattibility of the bioprocess.