Dissertations / Theses on the topic 'Settore CHIM/11 - Chimica e Biotecnologia delle Fermentazioni'

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.

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.

This PhD research project was aimed at the development of new biocatalytic processes to produce natural sugars by selection and characterisation of new enzymes able to produce fructooligosaccharides. Biochemical studies were performed to obtain information on the mechanism of action and understand the structural elements that define the activity. After the development of the biotransformation conditions, a continuous production of FOS was studied and a cheap separation method of the transformation products was also assessed, in order to obtain FOS in purified form. Mass spectrometry studies were performed on the purified enzymes after purification from wild-type strains. After a screening for FOS production from sucrose two microorganisms were chosen for their activity up to 30 % (w/w) conversion and differences in FOS mixture production: CF215 = Cladosporium cladosporioides CK1 = Penicilium sizovae CF215 produce a mixture similar to the commercial product Actilight®, while CK1 produce for almost kestose (GF3). Under the optimised biotransformation conditions the maximum accumulation of FOS was 56 % (w/w) and 31 % (w/w) for CF215 and CK1 respectively. We were able to isolate and characterise seven different carbohydrates such as 1-kestose, 1-nystose, 1-fructofuranosylnystose, 6-kestose, neo-kestose and neo-nystose for CF215 while CK1 produce only 1-kestose, 1-nystose, 1-fructofuranosylnystose and 6-kestose. Another oligosaccharides was isolated and fully characterised from CF215 mixture, named blastose (Fru-β(26)-Glc). An immobilization study was carried using the DALGEEs (Dried Alginate Entrapped Enzymes) method on the mycelium of CF215 strain. The maximum accumulation of FOS using DALGEEs mycelium was 51 % (w/w), reached in common buffer and seawater. With this cheap technique we develop a continuous FOS production using the facilities of Flow chemistry. The reactor, filled with DALGEEs and celite, was stable for months and the maximum accumulation of FOS was 52% (w/w). At this flow stream of FOS mixture we added a batch step to purify the FOS from glucose that represent the 26 % (w/w) of entire mixture. Glucose Oxidase from Novozymes® named Glyzyme® MONO 10.000 BG was employed and the result was the reduction of glucose from 26 % (w/w) to 3% (w/w). This purification step was added for two reasons: to obtain a cheap and fast method for FOS purification from glucose and to simplify the blastose HPLC purification. 56 mg of purified blastose were obtained and utilised to perform a pioneer study of blastose prebiotic action. The growth of 5 different lactobacillus strains (Lactobacillus paracasei DG, Lactobacillus rhamnosus GG, Lactobacillus paracasei SHIROTA, Lactobacillus johnsonii LC1 and Lactobacillus reuteri ATCC55730) were followed with the addiction of different carbohydrates as only carbon source (glucose, Actilight®, inulin, blastose). The best results were reached with Lactobacillus johnsonii LC1 where the Vmax using 0.5 % (w/v) of blastose was higher than glucose 0.5 % (w/v) (1.125 ± 0.023 1/h and 0.521 ± 0.054 1/h respectively). In the second part of this PhD project the purification of the enzymes involved in FOS formation was achieved after several chromatographic steps. The molecular weight (MW) of the two proteins was ≈50 kDA for the enzyme from Cladosporium cladosporioides (monomeric) and ≈75 kDa for the one from Penicilium sizovae (monomeric). The enzyme from C. cladosporioides was biochemically characterised and shown a Km of 129 ± 6 mM, Vmax of 2.83 ± 0.04 U/mL, Kcat of 2.88 ± 0.04 1/s and a Kcat/Km of 22.3 ± 1.4 1/M*s with sucrose and a Km of 268 ± 6 mM, Vmax of 0.0328 ± 0.003 U/mL, Kcat of 0.0334 ± 0,003 1/s and Kcat/Km of 0.124 ± 0.014 1/M*s with 1-kestose. A mass spectrometry MALDI-TOF analysis study was performed on the protein, showing a MW of 61178 Da. A trypsin digestion was performed and the fragments analysed but we didn’t found match in databases. The molecular study of the protein was stopped until protein sequence elucidation.

This PhD thesis is divided into four chapters that report the development of different microbial biocatalysts aimed at the valorization of food and biotechnological industrial by-products. The experimental parts are preceded by an introduction aimed to give a background on the impact of biocatalysis on green chemistry and, more generally on circular economy, together with a systematic review of the main molecular biology approaches employed to genetically engineer acetic acid bacteria. The second chapter reports the optimization of a biocatalytic system for the regioselective hydroxylation of different terpenes employing a Mycobacterium sp. CYP153A6 monoxygenase. In chapter 3 and in chapter 4 is described the development of different recombinant acetic acid bacteria strains, aimed at the production of highly added value products, such as perillic acid, starting from limonene, a cheap and highly available substrate derived from the agro-food industry. Limonene was employed as a pure compound (chapter 3) or via fermentation of orange peel wastes (chapter 4). The last chapter deals with the isolation and characterization of a bacterial cellulose (BC) producer strain, namely Komagataeibacter rhaeticus ENS9b, able to produce BC from acetate and crude glycerol, a by-product from the biodiesel production process.

Questa tesi descrive le biotrasformazioni di acidi biliari con ceppi derivanti da campionamenti in Italia e in Ecuador di microrganismi appartenenti al phylum degli Attinobatteri. Dal campionamento fatto in Italia lo screening di 86 ceppi (Capitolo 2) ha permesso di individuarne uno particolarmente interessante, che dopo caratterizzazione, è risultato essere Pseudomonas alcaliphila, Questo ceppo ha prodotto in rese quantitative il 12-idrossi-androstane-1,4-diene-3,17-dione (12-HADD) 2a (95%) partendo dall’acido desossicolico 1a. Risultati analoghi si sono ottenuti con l’acido colico 1b, l’acido chenodesossicolico 1c e l’acido iodesossicolico 1d che hanno fornito rispettivamente 7,12-HADD 2b (23%), 7-HADD 2c (52%) e 6-HADD 2d (83%) (Capitolo 3) . Vengono riportate inoltre, le biotrasformazioni di acido colico, acido desossicolico, acido iodesossicolico con batteri isolati dal macello rurale di Cayambe (provincia di Pichincha, Ecuador) (Capitolo 4) che hanno portato alla sintesi di bendigoli e altri metaboliti. I ceppi più attivi sono stati caratterizzati e appartengono al genere Pseudomonas e Rhodococcus. L’acido colico 1a ha fornito il 3-cheto derivato 2a (45%) e il 3-cheto-4-ene derivato 3a (45%) con P. mendocina ECS10, il 3,12-dicheto-4-ene derivato 4a (60%) con Rh. erythropolis ECS25 e il 9,10-secosteroide 6 (15%) con Rh. erythropolis ECS12. Il bendigolo F 5a (20%) è stato ottenuto con P. fragi ECS22. Dalla biotrasformazione di acido desossicolico 1b si è ottenuto con P. prosekii ECS1 e Rh. erythropolis ECS25 il 3-cheto derivato 2b (20% e 61% rispettivamente), mentre il 3-cheto-4-ene derivato 3b è stato ottenuto con P. prosekii ECS1 e P. mendocina ECS10 (22 e 95% rispettivamente). P. fragi ECS9 ha dato inoltre il bendigolo A 8b (80%). Infine, dalla biotrasformazione di acido iodesossicolico (1c) con P.mendocina ECS10 si è ottentuto il 3-cheto derivato 2c (50%) e con Rh. erythropolis ESC12 il 6-idrossi-3-cheto-23,24-dinor-5-colan-22-oico 9c (66%). Il bendigolo G 5c (13%) è stato ottenuto dalla biotrasformazione con P. prosekii ECS1 e il bendigolo H 8c con P. prosekii ECS1 e Rh. erythropolis ESC12 (20 e 16% rispettivamente).
This research describes the biotransformation of bile acids with microbial strains belonging to the phylum Actinobacter, sampled in Italy and Ecuador. From the 86 Italian samples (Chapter 2) a promising isolate was characterized and identified as Pseudomonas alcaliphila. This strain produced high yields of 12-hydroxy-androstane-1,4-diene-3,17-dione (12-HADD) 2a (95%) from deoxycholic acid 1a. Similar results were obtained with cholic 1b, chenodeoxycholic 1c and hyodeoxycholic acid 1d; which yielded 7,12-HADD 2b (23%), 7-HADD 2c (52%) e 6-HADD 2d (83%) (Chapter 3). In addition, information concerning biotransformations of cholic, deoxycholic, and hyodeoxycholic acids, performed with Ecuadorian strains isolated from the rural slaughterhouse at Cayambe (Pichincha province, Ecuador) is presented in Chapter 4. In this case, biotransformations produced bendigoles and other metabolites. The most promising samples belong to genus Pseudomonas and Rhodococcus. Cholic acid 1a produced 3-keto derivative 2a (45%) 3-keto-4-ene derivative 3a (45%) with P. mendocina ECS10, 3,12-diketo-4-ene derivative 4a (60%) with Rh. erythropolis ECS25 and 9,10-secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F 5a (20%) was obtained with P. fragi ECS22. Biotransformation of deoxycholic acid 1b with P. prosekii ECS1 and Rh. erythropolis ECS25 produced 3-keto derivative 2b (20% e 61% respectively), whereas 3-keto-4-ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22 e 95% respectively). P. fragi ECS9 also produced bendigole A 8b (80%). Finally, biotransformation of hyodeoxycholic (1c) with P.mendocina ECS10 produced 3-keto derivative 2c (50%) and 6-hydroxy-3-keto-23,24-dinor-5-cholan-22-oico 9c (66%) with Rh. erythropolis ESC12. Bendigole G 5c (13%) was obtained from biotransformation with P. prosekii ECS1 and bendigole H 8c with P. prosekii ECS1 and Rh. erythropolis ESC12 (20 e 16% respectively).

The present thesis has the aim of develop and find new and more environmental friendly synthetic routes for the synthesis of active pharmaceutical ingredients (APIs) and pharmaceutically interesting intermediates, exploiting the advantages of the combination between flow chemistry and biocatalysis. Indeed, biocatalytic processes in continuous flow reactors have attracted attention in recent years for carrying out continuous manufacturing systems with high level of intensification. Flow processing has the potential to accelerate heterogeneous biotransformations due to biocatalyst high local concentration and enhanced mass transfer, making large-scale production more economically feasible in significantly smaller equipment with a substantial decrease in reaction time, from hours to a few minutes, and improvement in space–time yield, with increases of up to 650-fold as compared to batch processes. Moreover, biocatalyst stability is enhanced by working in an environment where harsh mixing is avoided. Overall, these features result in reduced inventory, waste and energy requirements of the flow biocatalytic process, as compared to the conventional batch mode. In particular, I focused my attention on redox reactions, since for these the traditional chemical procedures and reagents are far from being sustainable and environmental friendly. For example, for oxidative reactions the most used chemical reactives are Chromium VI (a well known cancerogenic agent), Dess-Martin periodinane (a potential explosive reagent) and the Swern reagent, a thiol based compound that produces dimethyl sulphide as co-product. Moreover, the traditional chemical methods are not able to reach the selectivity and specificity that is possible to achieve with biocatalytical systems. Briefly, the projects I was involved in during my PhD and that are present in the thesis are: 1. Development of a new synthetic route to obtain Captopril, using both chemical and biocatalyzed reactions and exploiting the advantages of flow chemistry, that allows to perform continuous synthesis; 2. Development of a flow based biocatalyzed oxidation with immobilized whole cells of Acetobacter aceti in order to obtain enantiomerically pure mono-carboxylic acids, starting from the corresponding diols; 3. Stereoselective reduction of ketones and di-ketones, in order to obtain enantiomerically pure mono-alcohol products, using together two enzymes (ketoreductase from Pichia glucozyma and a glucodehydrogenase from Bacillus megaterium) in a Flow Chemistry pcked bed reactor; 4. Stereoselective reduction of 2,2-disubstituted 1,3-cyclopenta- and 1,3 cyclohexanediones using both whole cells and a purified ketoreductase from Pichia glucozyma, to obtain enantiomerically pure mono-alcohols products, that can be important intermediates in the synthesis of various steroids. 5. Use of an immobilized transaminase from Halomonas elongata able to perform transaminations in both directions (from amine to aldehydes, and from aldehydes to amine) with the Flow Reactor technology; To reach the goal, I both used whole cells and purified enzymes as biocatalysts, either in a free or immobilized form, and I exploited many advantages of continuous flow technology, as for example downstream processes (i.e., in-line acidifications, extractions and purifications) that allowed me to in-line purify the products, thus avoiding the traditional work-up procedures, reducing the operational times and the amount of organic solvents used. In almost all cases, important results were achieved, as faster kinetics, cleaner procedures that required less purification steps, complete stereo- and regioselectivity, higher conversions and productivities compared to batch procedures, increased stability of the biocatalyst, that could be used for several cycles, thus reducing the waste.

Two new biocatalyst-tool have been studied: starting from interesting whole-cell application in the agro, food and zootechnical field, the hydrolytic activity was related to two carboxylesterases that have been purified until electrophoretic omogeneity. The enzyme from Kluyveromyces marxianus CBS 1553 was also studied from catalytic and biochemical point of view: the overall activity and stability over a wide range of condition indicate that this enzyme has the potential to be exploited in biotechnological applications.

In this PhD thesis the first application of acetylacetoin synthase (AAS), by B. licheniformis DSM 13, as a biocatalyst for the stereoselective formation of C-C bonds is described. AAS, a thiamine diphosphate (ThDP) dependent enzyme, catalyzes the condensation of dialkyl or alkyl-aryl-1,2-diketones into the corresponding α-hydroxy-β-diketones with the elimination of a carboxylic acid. The reactions were carried out using a single α-diketone as donor and acceptor (homo-coupling) or two different α-diketones (cross-coupling). The AAS enzymatic reaction of a new C-C bond formation is highly chemo-, regio- and enantioselective. The α-hydroxy-β-diketones obtained from the reactions of homo- and cross-coupling were reduced with acetylacetoin reductase (AAR), a dehydrogenase obtained from the same bacterium. The combined use of AAS and AAR allowed the preparation of a new range of optically pure α-alkyl-α,β-dihydroxyketones starting from commercial α-diketones. The stereochemistry of the enantiopure syn-α-alkyl-α,β-dihydroxyketones was assigned on the basis of NOE experiments, while their absolute configuration was determined transforming one of these compounds in the natural product (+)-citreodiol. The absolute configuration of α-alkyl-α, β-dihydroxyketones confirmed the S-stereospecificity of the AAR-reduction and R-stereospecificity of AAS homo and cross-coupling reactions. On the basis of the AAS activity, an alternative synthetic biomimetic route, reminiscent the ThDP-dependent enzymes activity, was studied. Both thiamine hydrochloride and its simplified analogue, thiazolium salt, act as pre-catalysts coupled with an appropriate basis and are able to activate α-diketones such as acyl-anion equivalents that can be transferred to enable ketonic acceptors as α-diketones and α-ketoesters. These carboligation reactions have been optimized in catalytic conditions using polyethylene glycol (PEG400), an eco-friendly reaction medium, that made easier the reaction workup allowing, in addition, the catalyst recycling. A further synthetic application of AAS was the chemo-enzymatic synthesis of the natural aroma of green tea. The chirality of this compound, closely related to its organoleptic properties, is actually studied in our laboratories. The versatility of AAS as biocatalyst for C-C bond forming reactions and the raised interest by its particular applications in organic synthesis promped us to purify the enzyme, with the ultimate goal of identifying the gene encoding for AAS in the genome of B. licheniformis DSM 13.

The contribute of the Analytical Chemistry in the study of complex matrices, as for instance foodstuff, is crucial. In particular the development of chromatographic methods allows to quantify important technological, microbiological and physiological markers. The ensemble of all these information furthers a deeper and higher knowledge. This Ph.D. project was designed to define the tools and evaluate the contribution of an important class of molecule on wine aroma. Grape products are really important in the cultural and dietary Italian traditions; moreover Italy is both the first producer and the first consumer around the world. Oenological products have been largely studied since the early ’70, even if the lack of biochemical and microbiological knowledge and the poor sensitivity of the analytical techniques did not allow to carry an deep study on sulfur compounds. This project, planned to bid analytical and oenological chemistry, focussed its attention on the study of sulfur compounds. Dealing with complex matrices, the first problem we had to face was the matrix effects. The overcoming of such issue or the attempts in reducing it, costs to chemical laboratory, wasting of time and money to prepare suitable internal standards. In this thesis we proposed a statistical approach based on the variance component model able to handle matrix effects. The benefits obtained by this approach definitely outweigh both the slightly worse sensitivity and uncertainty. Another topic discussed in the thesis is the definition of analytical methods to quantify sulfur compounds. This subject has been studied distinguishing sulfur compounds into two sub-groups: o fermentative sulfur compounds: molecules produced by yeast metabolisms from amino acidic precursors. o varietal sulfur compounds: molecules present in specific variety, as conjugated with cysteine and glutathione. The free forms, which are sensorially active, are released during fermentation by means of a specific enzymatic activity. This further classification was kept in the development of the analytical methods because the two classes required different sensitivity as well as metabolomics study were separated. Concerning fermentative sulfur compounds a HS-SPME/GC-MS method was optimised and validated. With this method a wide range of sulfur compounds were quantified. Such tool was applied to metabolomic studies, concerning the influence of variety, ageing, yeast strain and other technological practice on the level of 13 fermentative sulfur compounds. The results obtained from the development of the analytical method permitted to highlight the potentiality of the HS-SPME technique in sampling volatile compounds in complex matrices. This approach allows an easier and safer lab conditions, avoiding the use of organic solvents. The metabolomic studies furnished important suggestion on the influence of important oenological variables on the level of important sulfur compounds. The last topic discussed in this thesis concerns the study of varietal sulfur compounds. The lack of suitable analytical technique, sufficiently rapid and avoiding the use of mercuro-organic compounds stimulated our research in verifying the performance of headspace technique in extracting these analytes. We optimised and validated HS-SPME and purge and trap methods and finally we defined a non-parametric robust approach to compare performances from different analytical methods. Owing to the recent interest of the scientific research in trying to understand the formation and evolution of varietal sulfur compounds, we focussed our attention on their precursors. The first step was the synthesis of the putative precursors, followed by the optimisation of LC-MS/MS methods to quantify them. It was possible to identify a new precursor by LC-MS/MS experiments. Finally, by the isolation of the enzyme potentially responsible of the biosynthesis of the varietal thiols precursors, we gave a preliminary explanation of the formation of these glutathionylated precursors during grape ripening. By this project it has been possible to study and apply the newest analytical techniques available and it has been possible to define statistical procedures to overcome the most common issues in studying complex matrices. Furthermore it was clarified the contribution of an important class of molecules, such as sullfur compounds, on oenological matrices.
Il contributo della Chimica Analitica nello studio delle matrici complesse, quali ad esempio quelle alimentari, è cruciale. In particolare lo sviluppo di metodi cromatografici avanzati può permettere di dosare importanti marker tecnologici, microbiologici e fisiologici e quindi consentire un approfondimento delle attuali conoscenze. Questo lavoro è stato concepito allo scopo di definire gli strumenti per valutare il contributo di un’importante classe di molecole all’aroma dei vini. Il vino riveste un ruolo di primaria importanza nelle tradizioni culturali ed alimentari Italiane, inoltre l’Italia risulta primo produttore e primo consumatore mondiale di vino. I prodotti enologici sono stati largamente studiati fin dai primi anni ’70, anche se l’assenza di conoscenze biochimiche e microbiologiche e la mancanza di tecniche analitiche sufficientemente sensibili non ha consentito lo studio esaustivo della classe dei composti solforati. Questa tesi, nata da esigenze legate alla chimica analitica e alla chimica enologica, ha avuto come punto centrale lo studio dei composti solforati nei vini. Il primo problema che si è dovuto affrontare nello studio di matrici complesse come quelle enologiche è stato quello relativo agli effetti matrice. Il superamento o il contenimento di questo problema crea ogni giorno costi in termini di tempo e di preparazione di opportuni standard interni. In questa tesi si è proposto un metodo statistico basato sul modello a componente di varianza in grado di gestire questo problema. I vantaggi sono notevoli, a fronte di una incertezza leggermente maggiore e di una sensibilità di poco peggiorata. Altro argomento trattato è stata la definizione dei metodi analitici in grado di quantificare e quindi di studiare i composti solforati. Questa materia è stata affrontata riconoscendo una prima distinzione all’interno del gruppo degli analiti indagati: o composti solforati fermentativi: molecole derivanti dal metabolismo del lievito a partire da precursori amminoacidici o composti solforati varietali: molecole presenti in specifiche varietà come coniugati della cisteina e del glutatione e rilasciati durante la fermentazione alcolica ad opera di specifiche attività enzimatiche. Questa suddivisione è stata mantenuta anche nello sviluppo dei metodi analitici, in quanto sensibilità richieste e conseguenze metabolomiche erano anch’esse distinte. Per quanto riguarda i composti solforati fermentativi, durante il dottorato di ricerca è stato ottimizzato e validato un metodo HS-SPME/GC-MS per l’analisi di un’ampia gamma di molecole tipicamente derivanti dal metabolismo del lievito. Tale approccio è poi stato la base per studi di natura più squisitamente metabolomica in cui è stato verificato come il cultivar (i.e. la varietà), l’invecchiamento, il ceppo di lievito e altre pratiche tecnologiche potessero influenze il livello e l’evoluzione degli analiti indagati. I risultati ottenuti per quanto attiene lo sviluppo del metodo in spettrometria di massa hanno potuto confermare con la tecnica SPME con campionamento in spazio di testa sia una tecnica di elezione per l’analisi di molecole volatili. Inoltre questo approccio consente una gestione semplice ed enviromental friendly in confronto con i più obsoleti metodi di estrazione con solvente. Gli studi metabolomici hanno consentito di trarre importanti considerazioni su aspetti molto importanti nella definizione della qualità del prodotto finale. Tali risultati consentono anche una trasferibilità nei settori della ricerca applicata e dell’enologia. Come ultima classe di analiti indagati si sono studiati i composti solforati varietali. L’assenza di metodi sensibili e sufficientemente rapidi da essere applicati per studi su un numero significativo di campioni ha spinto la nostra attenzione alla verifica delle potenzialità delle tecniche in spazio di testa nel dosare queste molecole. Si sono ottimizzati e validati metodi HS-SPME e purge and trap ed è stata definita una procedura non parametrica robusta per il confronto delle performance delle diverse procedure. Visto il recente interesse della ricerca scientifica operante nel settore al controllo della formazione ed evoluzione di queste molecole, abbiamo focalizzato la nostra attenzione sui precursori dei composti solforati varietali. Il primo stadio è stata la sintesi di queste molecole, seguito poi dall’ottimizzazione di metodi LC-MS/MS per il dosaggio di queste molecole. In questo contesto è stato possibile identificare per la prima volta un nuovo potenziale precursore di aroma, tramite esperimenti LC-MS/MS. Da ultimo, attraverso l’isolamento dell’enzima responsabile della biosintesi dei precursori d’aroma è stato possibile proporre una preliminare spiegazione alla formazione di tali molecole durante la maturazione della materia prima. In conclusione, grazie a questa tesi di dottorato, è stato possibile approfondire ed applicare alcune delle più moderne tecniche analitiche a disposizione, è stato possibile definire procedure statistiche per superare i più comuni ostacoli nello studio di matrici complesse, ed è stato possibile chiarire il contributo di una classe importante di molecole come i composti solforati nelle matrici enologiche.

Questa Tesi di Dottorato è stata strutturata in due parti differenti precedute da un’introduzione riguardante i principi fondamentali della biocatalisi con particolare attenzione sull’applicazione di cellule intere di lieviti non convenzionali e una nuova chetoreduttasi proveniente dal lievito Pichia glucozyma. Tutte queste tematiche rappresentano il “background” per poter meglio comprendere le parti sperimentali descritte nei successivi paragrafi. Parte 1 Il potenziale biocatalitico rappresentato da cellule intere di P. glucozyma è stato studiato usando substrati di differente natura chimica interessanti sia dal punto di vista farmaceutico che sintetico. Dopo l’ottimizzazione delle condizioni di reazione, effettuata sulla trasformazione dell’acetofenone in (S)-1-feniletanolo, l’attenzione è stata focalizzata sulla riduzione di differenti chetoni aromatici. Tutte le reazioni generalmente avvengono con elevate rese ed elevata selettività per il corrispondente (S)-alcol. Parte 2 Riduzioni catalizzate da una benzil-reduttasi (KRED1-Pglu) NADPH-dipendente proveniente da P. glucozyma, rappresentano un’ interessante strategia sintetica per la riduzione stereoselettiva di chetoni aromatici. Dopo identificazione, la caratterizzazione e l’over-espressione della proteina in E. coli, un metodo di purificazione e conservazione dell’enzima è stato messo a punto. Dopo ottimizzazione dei parametri di reazione, è stato effettuato un accurato studio riguardante i substrati accettati da KRED1-Pglu. Questa proteina preferisce substrati stericamente ingombrati, spesso convertiti con elevata stereoelettività. La differente enantioselettività sperimentalmente osservata, è stata spiegata mediante studi di docking molecolare. In conclusione, due differenti metodi efficienti ed eco-sostenibili per la riduzione stereoselettiva di diversi chetoni aromatici, utilizzando sia cellule intere di lieviti non convenzionali tra cui P. glucozyma CBS 5766 e l’enzima isolato KRED1-Pglu sono stati presentati in questo lavoro di tesi.
The Doctoral Thesis has been structured into two parts preceded by a general introduction that covers basic concepts on biocatalysis with special focus on application of whole cells of unconventional yeasts and a new ketoreducatese from Pichia glucozyma. All these thematic issues give a background to the following two parts, where the experimental results have been deeply discussed. Part 1 The biocatalytic potential of whole cells of P. glucozyma have been studied through biotransformations using substrates of different chemical nature having interesting pharmaceutical and synthetic aspects. After optimization of the reaction conditions through the transformation of acetophenone in (S)-1-phenylethanol, the attention was focused on the reduction of different aromatic ketones. All the reactions occurred with high yields and high enantiomeric excess for the corresponding (S)-alcohols. Part 2 Bioreduction catalysed by NADPH-dependent benzil-reductase (KRED1-Pglu) from Pichia glucozyma provides an attractive approach for selectively reducing a broad range of aromatic ketones. After protein identification, characterization and over-expression in E. coli, we have established a procedure for the purification and storage of KRED1-Pglu. After optimization of the reaction parameters, a thorough study of the substrate range of KRED1-Pglu was conducted. KRED1-Pglu prefers space demanding substrates which are often converted with high stereoselectivity. The observed activities and enantioselectivities were explained with a molecular modelling study for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrate. In conclusion, different efficient and sustainable methods for the stereoselective reduction of prochiral ketones using both whole cells of unconventional yeasts such as P. glucozyma CBS 5766 and the isolated enzyme KRED1-Pglu have been proposed in this PhD work.

Currently, the idea of reducing dairy components as vehicles for probiotic agents has been promoted because of the high proportion of individuals who present lactose intolerance and allergy to milk protein components. Researchers suggest that lactose intolerance occurs in the sensitized intestinal mucosa, causing abdominal cramps, vomiting, intestinal constipation, fecal occult blood loss, and in more severe cases, intestinal obstruction and protein-losing enteropathy. Considering the pathologies related to food intolerance, new alternatives such as soy-based probiotic fermented beverages instead of dairy products have been emphasized in the diet, as they provide a beneficial modulation of the intestinal microbiota. The acidification kinetics was performed with the CINAC system of the different formulations at 42°C. The ternary co-culture composed of Lactococcus lactis (LL), Lactobacillus bulgaricus (LB), Streptococcus thermophilus (ST) was the highest acidification rate (Vmax 11.40 x 10-3 units pH / min) 10.12 hours to reach pH 4.5 (P <0.05), post acidification the Lactococcus lactis (LL) strain had the lowest significant acidification rate when compared to the others at 37ºC p <0.05. And the ternary co-culture Lactobacillus plantarum Lactobacillus bulgaricus (LB) and Streptococcus thermophilus (ST) showed the highest acidification rate when compared to the other p <0.05, at 42ºC, and the highest acidification rate was Lactococcus lactis (LL) with Lactobacillus plantarum (LP). In the microbiological count, all microorganisms at different temperatures and formulations maintained viability above 106 after 28 days of storage at 4ºC. The thermodynamic parameters estimated for nisin irreversible thermal inactivation were energy, enthalpy and Gibbs free energy of activation of 155.1, 152.1-152.3 and 86.2-90.4 kJ/mol, respectively, and an activation entropy of 177.9-186.2 J mol-1 K-1.

Il microbiota intestinale è un insieme di batteri, archea ed eucarioti che risiedono nel tratto intestinale dei mammiferi. La composizione di questa varia comunità microbica è specifica per ogni individuo e crea una complessa e mutualistica relazione con esso evolvendosi durante l’arco della vita (Backhed, 2005; Neish, 2009). Ad oggi, la ricerca su Escherichia coli e sulle altre specie di Enterobatteriacee si è principalmente focalizzata sullo studio della acquisizione di antibiotico-resistenze in ceppi coinvolti in infezioni cliniche conclamate. Al contrario, i ceppi isolati da soggetti sani sono poco caratterizzati, se non in studi comparativi (Johnson et al., 2000; Kudinha et al., 2013; Lee et al., 2019). Il primo progetto ha riguardato la caratterizzazione di ceppi di E. coli e di altre specie di Enterobatteriacee isolati dai campioni fecali di 20 soggetti sani, con lo scopo di comprendere se esistono delle correlazioni tra il genotipo attribuito dalla PFGE, il filogruppo, i determinanti genici di virulenza e le strutture funzionali potenzialmente coinvolte nella colonizzazione e nella virulenza. Dai 20 volontari sani sono stati isolati 51 ceppi di E. coli, dei quali il 27% appartiene al filogruppo B2, il 24% al B1, il 14% all’F, il 14% all’A, il 10% al D, l’8% all’E e il 4% al C. Un totale di12 ceppi appartenenti ai filogruppi B2 e F presentano determinanti genici di virulenza caratteristici di ceppi di E. coli uropatogeni. Dei 51 ceppi testati, solo 2 sono resistenti ai comuni antibiotici usati per il trattamento delle infezioni da Gram-negativi. Dagli stessi campioni fecali dei 20 soggetti sani è stato possibile isolare 34 diversi biotipi di Enterobatteriacee appartenenti a 12 specie: Klebsiella pneumoniae, K. oxytoca, Enterobacter hormaechei, E. ludwigii, E. cloacae, Citrobacter freudnii, C. amalonaticus, Hafnia alvei, Cronobacter sakazakii, Morganella morganii, Serratia liquefaciens e Raoultella planticola. Dei 10 ceppi di K. pneumoniae testati per specifici geni di virulenza, 6 presentano mrkD, ycfM, kpn, e entB, che codificano per diverse strutture delle fimbrie e per un sideroforo. L’88% delle Enterobatteriacee isolate sono capaci di produrre biofilm su terreno minimo M9glu, il 62% di loro producono cellulosa e il 58% sono in grado di sintetizzare curli. Sulla base di queste caratteristiche gli isolati sono simili a ceppi patogeni di interesse clinico. Lo studio ha messo in evidenza come tra i ceppi di E. coli e delle altre specie di Enterobatteriacee isolati dalle feci di soggetti sani, e che come tali risiedono in modo innocuo nel tratto gastrointestinale, siano stati identificati ceppi che potenzialmente potrebbero comportarsi da agenti eziologici di infezioni extra-intestinali. Infatti, presentano numerosi geni di virulenza, sono capaci di produrre biofilm e di trasferire materiale genetico tramite coniugazione. La maggior parte dei ceppi di E. coli isolati però, è risultata suscettibile a tutti gli antibiotici testati. Il secondo progetto ha riguardato lo studio di Clostridium ramosum, un batterio che sembra partecipare all’insorgenza dell’obesità a causa del suo ruolo di modulatore della disponibilità di serotonina nell’epitelio intestinale, che come conseguenza aumenta i livelli di assorbimento dei nutrienti (Mandić et al., 2018). La presenza di C. ramosum è stata analizzata nelle feci di topi soggetti ad una dieta ricca di grassi o obesi, al fine di individuarne i ceppi endogeni. Da un totale di 85 topi, è stato possibile isolare solo 3 ceppi endogeni di C. ramsoum. Nonostante l’obesità dei topi oggetto di studio non è stato possibile isolarne così frequentemente quanto ipotizzato. Probabilmente, in questi topi C. ramosum non ha un ruolo determinante nell’insorgenza dell’obesità.
Gut microbiota is a collection of bacteria, archaea, and eukarya that inhabits the mammalian gastrointestinal tract. The composition of this microbial community is host specific, it evolves throughout lifetime, and can create an intricate and mutually beneficial relationship with the host (Backhed, 2005; Neish, 2009). Through the years, research interest has been manly focused on the characterization of antibiotic resistance acquisition of virulent clinical Escherichia coli and other Enterobacteriaceae species, whereas strains isolated from healthy subjects have been less studied and mostly investigated only in comparative studies (Johnson et al., 2000; Kudinha et al., 2013; Lee et al., 2019). The first project focused on the deep characterization of a collection of E. coli and other Enterobacteriaceae isolated from fecal samples of 20 healthy volunteers, in order to determine whether the relationship between PFGE genotyping, phylogroups, genetic determinants, and functional features can be established among the isolates. A total of 51 different strains of E. coli were collected. According to Clermont phylogrouping, the 27% of E. coli isolates belonged to B2 phylogroup, the 24% to B1, 14% to F, 14% to A, 10% to D, 8% to E and 4% to C. Within the B2 and F strains, 12 shared pattern of virulence genes associated with clinical uropathogenic strains. A total of 49 out of 51 strains were sensitive to all the tested antibiotics. From the same fecal samples it has been possible to collect 34 different Enterobacteriaceae strains belonging to 12 different species: Klebsiella pneumoniae, K. oxytoca, Enterobacter hormaechei, E. ludwigii, E. cloacae, Citrobacter freudnii, C. amalonaticus, Hafnia alvei, Cronobacter sakazakii, Morganella morganii, Serratia liquefaciens, and Raoultella planticola. A total of 6 out of 10 K. pneumoniae strains were positive for mrkD, ycfM, kpn, and entB genes that encode for different fimbrial structures and siderophores. The 88% of Enterobacteriaceae strains isolated were able to produce biofilm in M9Glu minimal medium; the 62% was able to produce cellulose and the 58% to synthetize curli fibers. These features are consistent with the profile of clinical isolates. Strains of E. coli and of other Enterobacteriaceae potentially act like pathogens based on the presence of virulence genes, capacity of biofilm production, and transfer of genetic material through conjugation, albeit they innocuously inhabit the gut of healthy subjects. It is possible that they occasionally act as etiologic agents of extra-intestinal infections. It is relevant that most E. coli strains are susceptible to a wide range of antibiotics. The second project involved the study of Clostridum ramosum, an intestinal bacterium that seems to be involved in obesity development for its role in the modulation of serotonin availability in the intestinal epithelium that is responsible of an increased nutrient absorption (Mandić et al., 2018). The presence of C. ramosum was investigated in mice strains prone to obesity and in mice subjected to a high-fat diet, in order to identify endogenous strains. From a total of 85 mice, it was possible to isolate only 3 strains of endogenous C. ramosum. In spite of the obesity of the mice analysed, the isolation of C. ramosum was not so frequent as hypothesized. Probably the bacterium itself was not involved in the exacerbation of obesity in those mice.

Sustainability is one of the most pressing challenge of our century, this term is becoming a main keyword of political agendas and more in general of mass media. To increase the “greenness of bioprocesses”, academia and industry, especially in the biotechnological and chemical fields, are focusing their studies with the scope to shift from traditional organic synthesis to new processes with reduced ecological foot-print. A good way to increase sustainability could be set up bioprocesses exploiting microorganisms. Nowadays, companies are searching new organisms that, differently from the well characterized Saccharomyces cerevisiae, show to be more resistant to the harsh conditions commonly occurring in industrial fermentations (high salt concentration, temperature and pressure). Due to their peculiar features, non-conventional yeasts (NCYs) seem to be a promising solution. On the other hand, the disadvantage to use these new organisms is related to the few studies and literature data available, especially compared to S. cervisiae. To fill this gap researchers have started to characterize these new species. My PhD work had dual aim: • First to identify good candidates, with specific physiological properties, that could be exploited in bioprocesses. • Second to characterize new promising enzymatic activities useful for industrial applications. In the first studies, I focused my attention on marine yeasts. I chose yeasts isolated from this environment, because their use gives the possibility to perform a seawater-based bioprocess saving large amount of fresh waters, reducing both cost and environmental impact. From our laboratory yeasts collection, I selected, for their halotolerance, two different Debaryomyces hansenii strains. Hence mechanisms involved in osmotic stress response have been investigated employing flow cytometry. I showed that hyper-osmotic stress elicits membrane depolarization and decreases membrane permeability to cationic compounds. This phenomenon reduces ions permeability and can negatively affect the uptake of charged substrate during bioprocesses. My research proceeded with the set up of new fermentation protocols in seawater-based media composed by a mixture of hexose and pentose sugar and cheap nitrogen sources. In these conditions we obtained high biomass yield (0.627) in 40 h of bioprocess. In the second part of my PhD project, I studied NCYs as sources of enzymes. With this aim I identified a nitrilase of marine strain of Meyerozyma guilliermondii, that displayed high activities on aromatic substrate, but also on arylaliphatic and aliphatic ones. These activities were maintained also in presence of high salts concentration. In particular M. guilliermondii nitrilase was able to perform complete dynamic resolution of mandelonitrile in seawaters within in 8 h. In the last part of my PhD, I identified a novel extracellular and cell-bound phytase activity in Cyberlindnera jadinii. This enzyme is suitable as feed additive, indeed activities at pH 4.5 and 37°C (animals gastric pH and temperature) were 26.25 mU/mgd.w. and 58.36 mU/mgd.w., detected as extracellular and cell-bound respectively. Phytase activities had their optimum at 50°C, reaching 37.2 mU/mgd.w. (extracellular) and 146 mU/mgd.w. (cell-bound). Data reported in my PhD work suggest that could be interest to proceed with further characterization on NCYs. New “green” bioprocesses characterized by high productivity could be a key for reach sustainability reducing the ecological impact of industrial production.

The subject of this thesis is the protein kinase CK2 which is a family of enzymes that in humans consists of two catalytic subunits, termed CK2α and CK2α’, and one regulatory subunit, CK2β. CK2 is a highly conserved acidophilic Ser/Thr protein kinase, ubiquitously distributed in different cell compartments. CK2 is a member of the superfamily of eukaryotic protein kinases (EPKs), meaning the catalytic subunit is related by sequence homology and structural features to the other 478 kinases of the superfamily. CK2 shows some singular features like its high constitutive activity and the lack of an defined mode of regulation, which make CK2 unique with respect to the other kinases. With hundreds of substrates, this kinase is involved in several cellular events, resulting essential for the cell viability: CK2β gene knockout in mouse model is lethal even at single cell level, CK2α gene knockout are embryonic lethal at day 10.5 and CK2α' (expressed only in brain and testis) mouse knockout are viable with some defects in spermatogenesis. It participates in cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. Down-regulation of CK2 leads to apoptosis while abnormal over-activation has been found coupled to several diseases: the clinical relevance of CK2 is that high levels of the protein activity have been detected in a number of cancers, such as head and neck, renal, breast, prostate, lung, and kidney. A wide spectrum of cell permeable, fairly specific ATP site directed CK2 inhibitors are currently available which are proving useful to dissect its biological functions and which share the property of inducing apoptosis of cancer cells with no comparable effect on their “normal” counterparts. One of these, CX-4945, has recently entered clinical trials for the treatment of advanced solid tumors, Castelman’s disease and multiple myeloma. CK2 is considered constitutively active enzyme and, unlike many other protein kinases, it does not require phosphorylation for activation. The mechanism of regulation of CK2 is not firmly established yet, however it is clear that it differs from those commonly utilized by other protein kinases. Dozens of crystal structures of CK2 have been solved and highlighted the structural features of the main CK2 entities, the catalytic subunit CK2α, the regulatory subunit CK2β and the tetrameric α2β2 CK2 holoenzyme. Even if the structural knowledge of CK2 is very extended, no high resolution 3D-structure are available for the C-terminal part of CK2α, which has been deleted for the crystallization purpose. Moreover the structure of the CK2α’ has been solved but no structural information are present for the tetrameric holoenzyme with this catalytic subunit. To address this issue, one part of my PhD project focused on the production and the structural characterization of the full-length wild type CK2α and a phosphomimetic mutant in the tetrameric holoenzyme, in order to study the possible structural role of the C-terminus. Starting from the three holoenzyme structures solved we were able to determine some new holoenzyme structural features, in particular the new interface of interaction between the subunits within the tetramer and the so far unknown symmetry of the complex. Moreover we dealt with the development of a purification protocol of the CK2α’2β2 holoenzyme (and a chimeric CK2αα’β2) in quantities appropriate for structural approaches. The second part of the PhD focused on the structural characterization of a new potent dual inhibitor K164 which is specific for CK2 and Pim1; the crystal structure of the inhibitor in complex with the CK2α336 has been solved at 1.25 Å, which is the highest resolution ever reached for CK2.
Il soggetto di questa tesi è la protein chinasi CK2, una famiglia di enzimi che negli uomini è composta da due subunità catalitiche, CK2α e CK2α’, e da una subunità regolatoria, CK2β. CK2 è una Ser/Thr protein chinasi acidofila altamente conservata nel mondo eucariote, presente in differenti compartimenti cellulari. CK2 è un membro della superfamiglia delle protein chinasi eucariotiche (EPKs), con la subunità catalitica correlata mediante omologia di sequenza e caratteristiche strutturali alle altre 478 chinasi della superfamiglia. CK2 mostra alcune caratteristiche singolari, come la sua elevata attività costitutiva e la mancanza di un importante meccanismo di regolazione, il quale rende CK2 unica rispetto alle altre chinasi. Con centinaia di substrati, CK2 è coinvolta in numerosi processi biologici, risultando essenziale per la vitalità cellulare: il knockout del gene CK2β nel modello murino è letale anche a livello di singola cellula, il knockout del gene CK2α è letale al giorno 10.5 dello sviluppo embrionale e il knockout CK2α' (espresso solo nel cervello e testicoli) in topo è vitale con alcuni difetti di spermatogenesi. CK2 partecipa alla progressione del ciclo cellulare, all'espressione genica, alla crescita cellulare e alla differenziazione e all’embriogenesi. Down-regulation di CK2 porta all'apoptosi cellulare mentre una sovra-attivazione anomala è stata trovata accoppiata a diverse malattie: la rilevanza clinica di CK2 risiede nel fatto che alti livelli di attività della proteina sono stati trovati in diversi tipi di tumori, come alla testa e al collo, ai reni, al seno, alla prostata e al polmone. Un ampio spettro di inibitori di CK2, permeabili alle cellule e specifici per il sito dell’ATP, sono attualmente disponibili e si stanno rivelando utili per analizzare le funzioni biologiche della proteina; queste piccole molecole sono in grado di indurre l'apoptosi delle cellule tumorali senza alcun effetto analogo sulle loro controparti "normali". Uno di questi inibitori, CX-4945, è recentemente entrato in studi clinici per il trattamento di tumori solidi avanzati, malattia di Castelman e mieloma multiplo. CK2 è considerato un enzima costitutivamente attivo e, a differenza di molte altre protein chinasi, non richiede fosforilazione per l'attivazione. Il meccanismo di regolazione di CK2 non è stato ancora stabilito, tuttavia è chiaro che si differenzia da quelli comunemente utilizzati dalle altre protein chinasi. Decine di strutture cristallografiche di CK2 sono state risolte e hanno evidenziato le caratteristiche strutturali delle principali entità di CK2: la subunità catalitica CK2α, la subunità regolatoria CK2β e l’oloenzima tetramerico CK2α2β2. Anche se la conoscenza strutturale di CK2 è molto estesa, non è disponibile alcuna struttura 3D a elevata risoluzione per la parte C-terminale di CK2α, che è sempre stata deleta per scopi di cristallizzazione. Inoltre, malgrado la struttura di CK2α' sia stata risolta, non sono presenti alcune informazioni strutturali per l’oloenzima tetramerico con questa subunità catalitica. Per raggiungere questo obiettivo, una parte del mio progetto di dottorato si è focalizzata sulla produzione e sulla caratterizzazione strutturale di CK2α wild type (completa della parte C-terminale) e di un mutante fosfomimetico nell’oloenzima tetramerico, al fine di studiare il possibile ruolo strutturale del C-terminale. Partendo da tre strutture dell’oloenzima risolte abbiamo potuto determinare alcune nuove caratteristiche strutturali dell’oloenzima, in particolare la nuova interfaccia di interazione tra le subunità all'interno del tetramero e la simmetria del complesso, finora sconosciuta. Inoltre ci siamo occupati dello sviluppo di un protocollo di purificazione dell’oloenzima CK2α’2β2 (e di una forma chimerica CK2αα’β2) in quantità appropriate per approcci strutturali. La seconda parte del dottorato si è focalizzata sulla caratterizzazione strutturale del complesso con un nuovo potente inibitore duale (K164) il quale è specifico per CK2 e Pim1; la struttura cristallina di CK2α336 in complesso con l’inibitore è stata risolta a 1.25 Å, che è la più alta risoluzione mai raggiunta per CK2.

This PhD project focuses on the identification, isolation and characterization of new biocatalysts able to generate biologically active molecules with significant enantioselectivity. Through screening, we identified marine strains, from MaCuMBA (Marine Culturable Microorganism for Biotechnological Applications) and BIODEEP (Biotechnologies form the deep) European project collections, which show a marked enantioselectivity on intermediates of molecules of biological interest. Biotransformation substrate range included pramipexole, as main target, but it also embraces other common building blocks for synthetic industrial preparation. The stereoselective reduction of structurally different ketones using halotolerant marine yeasts (Meyerozyma guilliermondii and Rhodotorula mucilaginosa) was studied using cells grown and bio-converted in seawater. The preparation of valuable chemicals through water-saving (bio)processes based on the direct exploitation of seawater is a significant step towards sustainable biocatalysis. By choosing a suitable strain, high yields and stereoselectivity could be achieved in most cases. Notably, high chemoselectivity and enantioselectivity were observed using R. mucilaginosa in the reduction of aromatic β-ketonitriles, which allowed the recovery of the optically pure corresponding alcohols; notably, reduction with whole cells of yeasts generally give a mixture of undesired products, as observed with M. guilliermondii. Keto-reduction potential of thirty-three marine bacterium species was checked and afterwards the possibility to convert this substrate directly into the optically pure amine was investigated: marine bacteria were screened to identify transaminase activity. Based on the previous results in terms of halotolerance and transaminase activity, the marine bacterium strain Virgibacillus pantothenticus 21D was selected for the genome sequencing in order to clone and express an ω-transaminase enzyme. A recombinant non-marine ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for the enantioselective reduction of various cyclic ketones including pramipexole ketone intermediate. Thanks to co-factor recycling system, the purified enzyme showed very promising results. The soluble expression of a novel omega transaminase from a newly isolated halotolerant marine bacterium Virgibacillus pantothenticus was attained. Despite of several standard methodologies applied, the marine wild-type enzyme was total insoluble in E. coli host and it was satisfactorily solubilized by one single-point mutation, allowing the characterization of the new omega transaminase. The enzyme shows an interesting salt and solvent tolerance, in accordance to its origin and it results particularly active on some interesting building blocks molecules.

The subject of this thesis is a family of anion transporters known as SulP/SLC26 (Sulfate Permease/Solute Carrier 26) family, a large and ubiquitous family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions, whose members were found in eubacteria, plants, fungi, and mammals. The clinical relevance of the SulP/SLC26 gene family has been highlighted with the identification of pathogenic mutations related to hereditary genetic human diseases with diverse symptoms that arise as a result of the different substrate specificities and tissue localizations of the different transporters, such as dystrophic dysplasia (SLC26A2), congenital chloride diarrhoea (SLC26A3) and Pendred syndrome (SLC26A4). The SulP/SLC26 family belongs to the APC (Amino Acid-Polyamine-Organocation) superfamily, one of the largest superfamily of secondary carriers. While some members of other families of the APC superfamily have been structurally characterized, very little is known about the molecular organization of the SulP/SLC26 proteins and no high-resolution three-dimensional structure of full-length sequences is available. The SulP/SLC26 anion transporters share a common structural organization: a highly conserved transmembrane domain and a less conserved cytoplasmic C-terminal portion mainly composed of a STAS domain. The name STAS (Sulfate Transporter and Anti-Sigma factor antagonist) is due to a remote but statistically significant sequence similarity with bacterial ASA (Anti-Sigma factor Antagonist) proteins (Aravind and Koonin, 2000). The bacterial ASA proteins are functionally and structurally well characterized in their 3D structure both by NMR spectroscopy and X-ray crystallography. Unlike these proteins, the STAS domains present in anion transporters are poorly characterized in terms of both their function and structure. Despite the fact that their precise role is unclear, the STAS domains play a fundamental role in the function/regulation of SulP/SLC26 anion transporters. In particular, it has been proposed that the STAS domain, like ASA proteins, could have a role in protein/protein interaction; for instance the STAS domains of SCL26A3, -A4, -A6 and -A9 interact with the R domain of CFTR (Cystic Fibrosis Transmembrane conductance Regulator), the transmembrane protein involved in cystic fibrosis disease. So far three 3D structures of STAS domains from different species are available in literature, two from bacteria and one from mammalian, the latter solved during my Master Degree Thesis in the same laboratory where I've attended the PhD. The structural characterization of the full-length SulP/SLC26 transporters and of their STAS domains is fundamental for the comprehension of their mode of action and it is an essential step for the understanding of the functional consequences of the mutations responsible for related pathologies. To address this issue, one part of my PhD project focused on the production and the structural characterization of STAS domains from different species, and mutants of the STAS domain whose 3D structure have been solved, in order to study the anion-binding site and the possible role of the STAS domain in the transport. We identified a fundamental residue for the proper function of the transporter, probably implicated in the anion translocation within the transmembrane domain. The other part of the project dealt with the production of a selection of full-length SulP/SLC26 transporters from different orthologs, both Prokaryotes and Eukaryotes. To this aim, in collaboration with Prof. Frank Bernhard at the Johann Wolfgang Goethe University of Frankfurt (Germany), I used the cell-free (CF) expression method, an emerging technique for the large-scale production of membrane proteins for structural studies. Sample properties after post-translational solubilization have been analyzed by evaluation of homogeneity and protein stability. This is the first quality evaluation of the SulP/SLC26 transporters produced by CF expression mode in quantities appropriate for structural approaches.
Il soggetto di questa tesi è una famiglia di trasportatori di anioni nota come famiglia SulP/SLC26 (Sulfate Permease/Solute Carrier 26), una grande ed ubiquitaria famiglia di proteine di membrana in grado di trasportare un'ampia varietà di anioni monovalenti e divalenti, i cui membri sono stati trovati in eubatteri, piante, funghi, e mammiferi. La rilevanza clinica della famiglia genica SulP/SLC26 è stata evidenziata dall'identificazione di mutazioni patogene connesse a malattie umane genetiche ed ereditarie, con diversi sintomi che sorgono come risultato delle differenti specificità di substrato e localizzazioni tissutali dei differenti trasportatori, come la displasia distrofica (SLC26A2), la diarrea cloridrica congenita (SLC26A3) e la sindrome di Pendred (SLC26A4). La famiglia SulP/SLC26 appartiene alla superfamiglia APC (Amino Acid-Polyamine-Organocation), una delle più grandi superfamiglie di trasportatori secondari. Mentre alcuni membri di altre famiglie della superfamiglia APC sono stati caratterizzati strutturalmente, si sa molto poco riguardo l'organizzazione molecolare delle proteine SulP/SLC26 e non è disponibile nessuna struttura tridimensionale ad elevata risoluzione delle intere sequenze. I trasportatori di anioni SulP/SLC26 condividono un'organizzazione strutturale simile: un dominio transmembrana altamente conservato ed una porzione C-terminale meno conservata principalmente composta da un dominio STAS. Il nome STAS (Sulfate Transporter and Anti-Sigma factor antagonist) è dovuto ad una similarità di sequenza remota ma statisticamente significativa con le proteine batteriche ASA (Anti-Sigma factor Antagonist) (Aravind and Koonin, 2000). Le proteine batteriche ASA sono state ben caratterizzate funzionalmente e strutturalmente nella loro struttura 3D sia mediante la spettroscopia NMR sia mediante cristallografia a raggi X. A differenza di queste proteine, i domini STAS presenti nei trasportatori di anioni sono stati poco caratterizzati sia in termini della loro funzione, sia della loro struttura. Nonostante il fatto che il loro preciso ruolo non sia chiaro, i domini STAS svolgono un ruolo fondamentale nella funzione/regolazione dei trasportatori di anioni SulP/SLC26. In particolare, è stato proposto che il dominio STAS, come le proteine ASA, potesse svolgere un ruolo nell'interazione proteina/proteina; per esempio, domini STAS di SCL26A3, -A4, -A6 e -A9 interagiscono con il dominio R di CFTR (Cystic Fibrosis Transmembrane conductance Regulator), la proteina transmembrana coinvolta nella fibrosi cistica. Finora tre strutture 3D di domini STAS provenienti da specie diverse sono disponibili in letteratura, due da batteri ed una da mammifero, quest'ultima risolta durante la mia tesi di laurea specialistica nello stesso laboratorio dove ho frequentato il dottorato. La caratterizzazione strutturale degli interi trasportatori SulP/SLC26 e dei loro domini STAS è fondamentale per la comprensione del loro modo di azione ed è una fase essenziale per comprendere le conseguenze funzionali delle mutazioni responsabili delle patologie collegate. Per raggiungere questo obiettivo, una parte del mio progetto di dottorato si è focalizzata sulla produzione e caratterizzazione strutturale dei domini STAS provenienti da diverse specie, e mutanti del dominio STAS la cui struttura 3D è stata risolta per studiare il sito di legame dell'anione ed il possibile ruolo del dominio STAS nel trasporto. È stato identificato un residuo fondamentale per il corretto funzionamento del trasportatore, probabilmente implicato nella traslocazione dell'anione all'interno del dominio transmembrana. L'altra parte del progetto riguarda la produzione di una selezione di trasportatori SulP/SLC26 interi provenienti da diversi ortologhi, sia Procarioti che Eucarioti. Per questo scopo, in collaborazione con il Prof. Frank Bernhard presso l'università Johann Wolfgang Goethe di Francoforte (Germania), utilizzai il metodo di espressione cell-free (CF), una tecnica emergente per la produzione a larga scala di proteine di membrana per studi strutturali. Le proprietà dei campioni dopo la solubilizzazione post-traduzionale sono state analizzate mediante la valutazione di omogeneità e della stabilità della proteina. Questa è la prima valutazione della qualità dei trasportatori SulP/SLC26 prodotti mediante il modo di espressione CF in quantità appropriate per approcci strutturali.

Questa tesi di Dottorato è stata strutturata in tre parti, una per ogni tipo di attività enzimatica trattata, preceduta da una breve introduzione riguardante i principi fondamentali della biocatalisi. Parte 1: Sono state studiate le caratteristiche strutturali e biochimiche dell’esterasi (BCE) prodotta per via ricombinante da Bacillus coagulans NCBI 9365. Questo enzima mostra un elevato grado di selettività nei confronti degli esteri (acetato, butirrato e benzoato) dell’1,2-O-isopropilidenglicerolo (IPG) un acetale equivalente chirale del glicerolo, che in entrambe le configurazioni, è un utile prodotto di partenza per la sintesi di molecole naturali biologicamente attive di interesse alimentare e farmaceutico come gliceridi, glicerofosfolipidi, -bloccanti ed antiipertensivi Parte 2: Sono state studiate le potenzialità biocatalitiche di batteri acetici, microrganismi noti per la loro capacità di accumulare prodotti di ossidazione (aldeidi, chetoni e acidi carbossilici) dati dalla trasformazione di alcoli primari e secondari. Questa parte risulta ulteriormente suddivisa in tre capitoli: • La desimmetrizzazione stereoselettiva di dioli achirali diversamente sostituiti, è stata eseguita ottenendo i corrispettivi acidi chirali utilizzando, cellule intere di Acetobacter aceti MIM 2000/28 • È stata studiata la sintesi chemo-enzimatica della forma enantiopura del captopril, noto farmaco utilizzato per il trattamento dell’ipertensione. Particolare attenzione è stata dedicata allo step enzimatico catalizzato da cellule immobilizzate di Acetobacter aceti MIM 2000/28 utilizzate in un sistema in continuo di “flow chemistry”. • La produttività e la stabilità del sistema di ossidazione “flow chemistry” è stato studiato e ottimizzata attraverso l’utilizzo del 2-metil 1,3 propandiolo. Per testare la reale efficienza del sistema in continuo messo a punto, sono in corso di studio le prove effettuate con ulteriori substrati. Parte 3: L'idrossilazione del (R) -limonene in (R)-alcool-perililico è stata studiata attraverso l’utilizzo di cellule intere ricombinanti di E. coli trasformate con l’operone sintetico ottimizzato che codifica per una citocromo P450 monossigenasi (CYP153A6), una ferredossina e una ferredossina reduttasi isolate dal ceppo Mycobacterium sp. HXN-1500
This PhD project has been structured into three parts preceded by a general introduction on basic biocatalysis concepts. Part 1: The recombinant preparation coupled with biochemical and structure characterization a thermostable carboxylesterase (BCE) from Bacillus coagulans NCBI 9365. This enzyme showed high enantioselectivity towards different 1,2-O-isopropylideneglycerol esters (acetate, butyrate and benzoate). Both enantiopure of 1,2-O-isopropylideneglycerol (IPG or solketal), a chiral equivalent of glycerol, are building blocks for the synthesis of -blockers, glycerophospholipids and prostaglandins. Part 2: An insight on the biocatalytic potential of Acetic Acid Bacteria (AAB), a group of bacteria well-known for the ability to oxidize primary and secondary alcohols into partially oxidized organic compounds (aldehydes, carboxylic acids and ketones) as end-products. This part collect three chapters: • the stereoselective desymmetrisation of achiral 2-alkyl-1,3-diols performed by oxidation of one of the two enantiotopic primary alcohol moieties by means of Acetobacter aceti MIM 2000/28 free whole cells to afford the corresponding chiral 2-hydroxymethyl alkanoic acids in batch system • Chemoenzymatic flow synthesis of enantiomerically pure captopril, a widely used antihypertensive drug, with the particular focus on the enzymatic step carried on with immobilized cells of Acetobacter aceti MIM 2000/28 used in a continuous flow reactor. • Productivity and stability of the air-liquid flow system setted up in the previous work, has been studied and optimized, through the use of immobilized form Acetobacter aceti MIM 2000/28 in the oxidation of 2-metyl-1,3-propandiols. To verify the real efficiency of the system, the experiment with other substrate will be carried out. Part 3: Hydroxylation of (R)-limonene into (R)-perillyl alcohol studied using recombinant whole cells harbouring an optimized redox gene operon (CYP153A6) which encodes a cytochrome P450, a ferredoxin, and a ferredoxin reductase from Mycobacterium sp. HXN-1500-

The use of natural sources in economic activities can aid in the resource saving and recycling and reuse of wastes, contributing for a more sustainable world by providing clean technologies in the industrial and agricultural sector in both developed and developing countries. In general, increased and improved global strategies for energy safety, security and mitigation of CO2 emissions from energy production processes are required, especially those aimed at maximizing the energy efficiency by expanding the use of clean energy. This means using fuels that are able to implement the carbon cycle without changing the atmospheric balance (renewable fuels), by developing energy resources in CO2 reduced/neutral systems (Brennan and Owende, 2011; Moraes et al., 2017). The expansion of biofuels production and use is an important issue since it plays primary role in reducing global the climate change. But, in order to insert a new source/technology in the market, several factors are involved such as industrial aspects and economic feasibility, legal restrictions and incentives, international trade, land use, raw material availability and management techniques. At present, ethanol is the main biofuel produced worldwide. Between 2007 and 2015 bioethanol throughput practically doubled, reaching 25 billion gallons per year, even though after 2010 the production was stagnant (AFDC, 2016). This is the result of a number of reasons, to cite: - high dependency on the first-generation crops which need a lot of arable land and compete directly with food/feed production; - need for a complete validation of the lignocellulosic ethanol industry due to unsuitability of the large-scale process because of corrosion problems (mainly in the pretreatment), cost of enzymes, difficult/inhibition of the fermentation step; - difficulty to utilize all lignocellulosic fractions, according to a biorefinery approach, because each biomass has its biological complexity and the related lignocellulosic content/arrangement/recalcitrance changes significantly; - Lacking of investments/incentives (mainly, governmental) after the decrease of petroleum prices occurred at the end of 2014. In fact, based on the type of biomass, bioethanol production is classified as first (raw material saccharine or starch-based – sugarcane and corn); second (lignocellulosic materials); third (microalgal/macroalgal biomass) and fourth (genetically modified cyanobacteria) generation. Sugar cane ensures the lowest bioethanol production costs. In spite of its significant advantage, it is not a viable option for all the regions of the planet owing to climatic and soil limitations (Belincanta et al., 2016). Consequently, countries of the northern hemisphere have been incessantly looking for new technological routes that permit the efficient production of biofuels while respecting environmental and economic sustainability issues, and ‘new’ generations of biomass-to-ethanol processes are proposed. In addition, countries as Brazil have their sugarcane cultivation saturated, i.e., there is no new extensions of arable land to expand significantly the Brazilian ethanol industry. Low production costs are the advantage of first generation bioethanol, with the exception of corn-based one, which has a well-established and economically sustainable technology, while second generation still requires more investigations to become economically competitive, with pretreatment and hydrolysis processes needing to be more effective and largely scalable (Gupta and Verna, 2015). On the other hand, micro and macroalgae have not reached a maturity for designing and operating industrial scale plants yet. Therefore, in the case of third and fourth generation bioethanol, further studies are required to develop a competitive and consolidated technology, taking into account also issues other than technological ones. In third generation bioethanol, microalgae and/or macroalgae biomass are used, which do not have lignin in their cellular structure, and are cultivated with higher growth rates when compared to higher plants. As for this biomass, a suitable process is not available yet, and the related costs cannot be properly estimated. Researchers are currently trying for microalgae: to optimize microalgal productivity and cultivation conditions, as this represents the highest production costs, considering that hydrolysis and fermentation are instead easier compared with lignocellulosics and macroalgae (Jonh et al., 2011; Wei et al., 2013; Hong et al., 2014). Thanks to their high growth rate, and relatively simple biochemical composition (partitioned among carbohydrates, lipids and proteins), microalgae are acknowledged as very promising feedstock for bioethanol production (Chen et al., 2013). Main aspects needing to be developed in this respect are: carbohydrate cultivation (productivity), hydrolysis and ethanolic fermentation and nutrient recycling/recovery from residual medium/biomass. With regard to the open issues recalled above, the aim of this research project has been to address and study how to improve the knowledge and discuss the real potentiality of microalgal biomass as a feedstock for an effective bioethanol production, from a perspective of biomass/carbohydrate productivity (microalgal cultivation) and bioconversion process (hydrolysis and fermentation) in a context of a biorefinery concept. In fact, experimental values about fermentation applications from microalgae are not expanded yet in literature. The topics addressed by this thesis are organized and subdivided in twelve chapters as follows. In Chapter 1, a literature survey to collect and discuss the available information about bioethanol from photosynthetic microorganisms, and to delimit the main lacks to be developed, is done. Chapter 2 shows a basic analysis of an ethanol biorefinery scheme aimed to include microalgal biomass, discussing the main bottlenecks and the processes which must be developed to adequately evaluate the potentiality of this type of biomass for industrial fermentation proposes. Chapter 3 treats specifically of the carbohydrate-rich biomass cultivation from microalgae utilizing nutritional and environmental techniques. Operation mode of microalgae cultivation is discussed as well, and the importance to consider semi-continuous and continuous processes is shown, because batch mode is extensively used but less efficient. Chapter 4 develops a design procedure of a two-unit system composed by a reactor and settler, discussing the influence of operating variables and their limiting values. Specifically, recycle ratio and purge flow rate concepts and effects are extensively studied. In Chapter 5, the carbohydrate cultivation with Synechococcus PCC 7002 is optimized with respect to the carbon source and pH, because a stable pH (greatly influenced by the carbon source) is necessary for this strain and organic buffers exhibit toxicity. An inorganic buffer study (CO2-bicarbonate) is developed and detailed. Chapter 6 shows S. PCC 7002 treating urban wastewater to remove chemical oxygen demand, nitrogen and phosphorous content, thus ensuring a double gain: environmental enhancement and valorization of cyanobacterial biomass. In Chapter 7, continuous cultivation of Chlorella vulgaris in flat-plate photobioreactors to improve carbohydrate productivity is assessed and evaluated using nitrogen limitation as a combination between nitrogen concentration inlet, light intensity and residence time under constant light intensity. Chapter 8 demonstrates that a similar approach used for the continuous cultivation of C. vulgaris is applicable also to Scenedesmus obliquus. Additionally, it is proved that under outdoor conditions (seasonal regime of illumination – summer and winter), a high carbohydrate content can be produced as well. In Chapter 9, the kinetics regarding acidic hydrolysis to biomass solubilization and sugars depolymerization is studied with Chlorella vulgaris biomass. An n order kinetics for biomass solubilization and m order for acid concentration is applied for biomass solubilization, providing values of reaction order and activation energy for microalgae. In addition, a saccharification model based on the Michaelis-Menten model is proposed and validated. Chapter 10 demonstrates how the kinetics considerations determined in the previous chapter can be efficiently applied with the concept of severity factor – CSF (combination between time, temperature and acid concentration). A literature discussion about some assumptions so far considered and the importance to know the biomass nature to determine a coherent range of CSF is provided. Chapter 11 reports ultrasonication as an effective pretreatment method to improve enzyme accessibility and promote a high rate of hydrolysis from Scenedesmus obliquus biomass. Pretreatment time, ultrasonication intensity and biomass concentration are specifically studied in order to minimize the energy consumption since the bottleneck of the pretreatment method is a high energy dissipation. In Chapter 12, ethanolic fermentation is addressed with acidic and enzymatic hydrolysates. A systematic optimization of inoculum concentration and consortium between Saccharomyces cerevisiae and Pichia stipitis is determined. Then, the influence of salinity/matrix characteristics was evaluated to understand possible interferences during fermentation process and exhibited lower biochemical yields than the control conditions. Thus, further fermentations experiments are necessary.
L’obiettivo generale di questo progetto di ricerca è stato di verificare la potenzialità delle microalghe come fonte alternativa di biomassa per la produzione di etanolo. In particolare, sono state discusse teoricamente, sperimentalmente e tramite simulazione di processo la coltivazione, l’idrolisi e la fermentazione della biomassa microalgale. Inizialmente, grazie ad un’ampia ricerca bibliografica ed a prove preliminari effettuate nel Laboratorio Microalghe del Dipartimento di Ingegneria Industriale della Università di Padova si è dimostrato che le specie più promettenti da studiare erano Synechococcus PCC 7002, Chlorella vulgaris e Scenedesmus obliquus, grazie alle loro elevate velocità di crescita e capacità di accumulo di carboidrati, che costituiscono le materia-prima per la produzione di etanolo (fino al 50-60% del peso secco). In particolare, l’attenta analisi della letteratura riguardo a queste specie ha consentito di verificare che: - per la produzione di carboidrati è preferibile sviluppare un processo continuo, perché richiede un solo step, mentre il processo batch ne richiede due, e perciò consente di ottenere produttività significativamente inferiori; - sono disponibili pochi lavori sulla possibilità di usare le microalghe in un processo continuo di questo tipo, mentre sono parecchi i riferimenti al processo batch; - mancano informazioni sulla capacità di produrre carboidrati da parte di S. PCC 7002. In una prima parte del lavoro sono stati quindi pianificati e condotti esperimenti in modalità batch con S. PCC 7002, per studiare come mantenere la stabilità e vitalità della coltura durante tutto il periodo di coltivazione. Si sono rilevati problemi con il controllo del pH, ed é stato approfondito l’uso di bicarbonato come fonte di carbonio assieme ad un tampone inorganico, dimostrando in un primo lavoro che il suo impiego è efficiente per la produzione di biomassa ma insufficiente per accumulare un alto contenuto di carboidrati, a causa di una significativa inibizione osmotica causata dall’alta concentrazione di sodio in soluzione. D’altro canto, l’applicazione di un tampone con sostanze organiche, generalmente usato nella coltivazione di microalghe e cianobatteri, ha evidenziato notevoli fenomeni di tossicità per questa specie. Al contrario, il tampone inorganico CO2-bicarbonato messo a punto successivamente è stato capace di garantire la stabilità del pH durante 12 giorni di coltivazione, ed ha consentito di ottenere 6 g L-1 di biomassa (peso secco) con circa il 60% di contenuto di carboidrati. La coltivazione in continuo di C. vulgaris in un fotobioreattore piatto e sottile è stata studiata per verificare la produzione di carboidrati secondo questa modalità operativa. Il lavoro ha evidenziato l’importanza della riduzione della concentrazione di azoto in entrata al reattore, che va rapportata ai valori di intensità di luce e tempo di residenza per massimizzare la produzione di carboidrati. Si sono misurati valori massimi per la produttività di biomassa e di carboidrati pari a 0.7 e 0.37 g L-1 giorno-1. La stessa procedura é stata usata nello studio del comportamento di S. obliquus, per vedere se l’approccio era valido anche durante la coltivazione all’aperto, simulando la fornitura della luce in modo stagionale. S. obliquus ha mostrato una produttività quasi tre volte maggiore che Chlorella, raggiungendo valori di 0.8 g L-1 giorno-1 (con luce costante) e di 0.71 g L-1 giorno-1 (nell’estate). Questa produttività di carboidrati, se estrapolata a dimensioni industriali, consentirebbe di ottenere tra 45–100 tonbiomass ha-1 anno-1, ben di più di quanto prodotto con le fonti tradizionali di carboidrati. Un sistema reattore-sedimentatore con riciclo parziale di biomassa è generalmente usato a livello industriale in processi di coltivazione e/o fermentazione. Questo sistema fornisce semplicità e diversi vantaggi per la produzione su larga scala. É stato quindi messo a punto un modello per la simulazione di tale processo, nel caso specifico delle microalghe, per verificare l’influenza dei gradi di libertà (tempo di residenza, rapporto di riciclo della biomassa, età della biomassa e sua velocità di sedimentazione) sulle prestazioni. I principali risultati sono: - la definizione di un rapporto di riciclo minimo Rmin, di un intervallo operativo per la stessa variabile, e di un valore massimo per la portata di spurgo di biomassa Fwmax; - la dimostrazione che la perdita di biomassa dalle sommità del sedimentatore abbassa significativamente le prestazioni del sistema; - la costruzione di grafici adimensionali che legano R a θc/θ e FI/FW (età della biomassa/tempo di residenza, e rapporto tra le portate di ingresso e di spurgo); - il confronto fra il modello rigoroso messo a punto ed il modello semplificato generalmente considerato in letteratura. Synechococcus è stata coltivata in acque reflue urbane (sintetiche e reali, con valori di COD pari a 340.0 ± 14.1 mg L-1, di azoto totale pari a 31.0 ± 1.4 mg L-1, e di fosforo totale a 8.20 ± 0.99 mg L-1), con l’obbiettivo di ottenere la depurazione da questi inquinanti. Questa specie è stata molto efficiente nella rimozione di COD, azoto e fosforo totale, raggiungendo valori sotto i limiti di legge in due giorni di coltivazione. L’acqua reflua sintetica ha evidenziato una limitazione dei micronutrienti quando la concentrazione di COD era elevata, differentemente dell’acqua reflua reale, in cui Synechococcus è cresciuta più velocemente. Successivamente, l’idrolisi e la fermentazione di biomassa microalgale sono state studiate con riferimento ai processi di saccarificazione acida ed enzimatica, e con riferimento ai microorganismi Saccharomyces cerevisiae e Pichia stipitis, rispettivamente. L’idrolisi acida, con acido solforico 0-5% v/v, è stata condotta a diverse temperature (110-130 °C) e tempi di reazione (0-60 min) partendo da 100 g L-1 di concentrazione di biomassa (Chlorella vulgaris). Gli zuccheri idrolizzati sono stati recuperati con un valore massimo pari al 92%, ottenuto con il 3% di acido e 20 min di reazione a 120 °C. La solubilizzazione di biomassa ha esibito un ordine di reazione n = 3.63 ± 0.18 ed un’energia di attivazione pari a 41.19 ± 0.18 kJ/mol. Questi valori sono significativamente diversi di quelli trovati per l’idrolisi di matrici lignocellulosiche, generalmente considerata di primo ordine con Ea = 100-200 kJ/mol, e dimostrano che la biomassa microalgale è più suscettibile al trattamento termico catalizzato all’acido in confronto ai lignocellulosici. Un’equazione basata sulla cinetica di Michaelis-Menten modificata per tenere conto della concentrazione di acido è riuscita a modellare tutti i risultati sperimentali, con un valore della costante di semi-saturazione per la biomassa PolKM pari al 42% della concentrazione iniziale, e con una resa di fermentazione di circa il 60%. Prima di realizzare l’idrolisi enzimatica, si é reso necessario procedere ad un’ottimizzazione del pretrattamento della biomassa. È stata studiata l’ultrasonicazione applicando un piano statistico di sperimentazione su tre livelli con 3 esperimenti centrali (in tutto si sono condotte 11 prove). Le variabili ottimizzate sono state l’intensità, il tempo di pretrattamento e la concentrazione di biomassa. I risultati hanno dimostrato che l’intensità e il tempo di trattamento sono più importanti e consentono di ottenere un recupero degli zuccheri superiore al 90%, in 4-8 ore. Si é visto che l’energia spesa nel processo di ultrasonicazione non è direttamente collegata con l’efficienza dell’idrolisi, per cui questa può essere condotta efficientemente anche riducendo il consumo di energia nel pretrattamento. Infine, si sono eseguiti esperimenti di fermentazione dell’idrolizzato ad etanolo con le due specie menzionate (S. cerevisiae e P. stipitis). Si sono ottimizzati la concentrazione di inoculo (7.5 g L-1) ed il consorzio (25% Pichia + 75% Saccharomyces) per avere una produttività tra 5 e 10 g L-1 ora-1 (prossimo al valore industriale). Si è però visto che le velocità di fermentazione sono però più basse a causa di una inibizione dovuta alla accresciuta salinità dell’idrolizzato, un fattore. Per questo motivo, la parte di fermentazione necessita di essere più approfondita al fine di validare l’impiego di questo tipo di biomassa a livello industriale.

Concerns about energy security, fossil fuel prices, and climate change issues, are leading to increasing renewable energy demand. Hydrogen is considered as one of the main possible energy carriers in future, due to its environmental (it can be converted to energy with the solely emission of water) and energetic (energy content of 120 MJ/kg, three times higher of the gasoline content of 44 MJ/kg) unique properties. If hydrogen is currently being produced mainly by fossil sources, its production from renewable sources answers to the demand of more environment-friendly exploiting alternatives, possibly leading to a renewable-based hydrogen economy. Biomasses are an important renewable source ranging from energy-dedicated crops to livestock waste effluents, agro-industrial wastewaters, food-processing industry residues and organic fractions of the municipal solid waste (OFMSW). Thus the agricultural sector may acquire a renewed importance in the mid-term as a producer of energy sources for renewable biohydrogen production. Among the biological ways to exploit biomasses for hydrogen production, this thesis focused its interest on anaerobic dark fermentation, which can simultaneously guarantee the production of an high-value product (H2) at high evolution rates and the treatment of wastes, thus transformed from an environmental pollution and greenhouse gases emissions source into a valuable resource. If on the one hand this process has lots in common with anaerobic digestion, which already is a well-established technology for treating different biomass types in real-scale plants, on the other hand it is a relatively new approach, which needs to be further studied for improving its performances and being concretely applicable. As a matter of fact, the main disadvantage of dark fermentation is its relatively low yield, compared to other bio-hydrogen production methods, which typically are between 2.4 and 3 mol H2/mol glucose. This represents just the 20-25% of the 12 mol of H2 theoretically obtainable by glucose fermentation. Therefore, generally two different (but not mutually exclusive) options could be chosen for improving the process and making it ready for full-scale applications: the optimization of the biological, biochemical, chemical-physical operative parameters that regulate process; or the coupling of this bioprocess with other technologies capable of exploiting the organic matter not fully used by the dark fermentative approach. For example, Microbial Electrolysis Cells (MECs) are able to biologically oxidize the organic matter (from simple substrates like volatile fatty acids, lactic acid, glucose, cellulose, to actual wastewaters) releasing electrons from an anode to a cathode where potentially pure hydrogen can be formed from protons in the water. Papers I and II basically belong to the first strategy. In Paper I indeed, two waste biomasses were co-digested: in consideration that in the Po Valley area (Italy) swine manures (SM) are yearly produced at high waste density levels and could be a cause of environmental problems, this waste was used as a co-substrate for biohydrogen production by the thermophilic fermentation of easily degradable and carbohydrate-rich materials, such as fruit and vegetable market waste (FVMW). Biohydrogen production rates and process stability were thus simultaneously maximized, thanks to the endogenous buffer capacity of manure, through the combination of a suitable composition (as FVMW/SM) of the feeding material and the hydraulic retention time (HRT) of the process. Thus, livestock manure represented not only a renewable source for supplying the production of biohydrogen, but also a source of alkali to be used for avoiding the addition of exogenous chemicals (alkali) to maintain the pH, and so the metabolic pathways and bacterial communities, into an optimal domain for biohydrogen production. To further study and optimize the bio-H2 production in laboratory-scale processes, but also to find applicable tools for favoring dark fermentation application in full-scale biogas plants, Paper II succeeded in obtaining mixed microbial cultures from natural sources (soil-inocula and anaerobically digested materials) which reached high hydrogen yields with glucose and were used to explore the potential of bio-hydrogen production from four organic substrates of possible interest for full-scale plants (market bio-wastes, maize silage, swine manure, OFMSW). In direct prosecution of the positive co-digestion results shown in Paper I and looking for future transfer of this bioprocess technical solutions to full-scale systems, Paper II used the enriched mixed microflora for evaluating the co-fermentation of a mixture of OFMSW and swine manure in a lab-scale continuously-fed CSTR (continuously stirred tank reactor) digester. Despite the good results obtained, our study suggested that further efforts are needed for future applications of effective biohydrogen fermentation in full-scale plants. Paper III and IV are more focused on the second scientific strategy. Paper III joins the interest toward implementation of bio-H2 in full-scale plants and the strategy of improving the overall recovery of the energy contained in the biomass associating hydrogen production to other bioprocesses. Many authors report that the two-stage anaerobic digestion (AD) process, if compared to traditional and extensively real-scale applied single-stage AD, has also other advantages, such as differentiating the biofuel production (bio-hydrogen and bio-methane), potentially reducing the plant dimensions and costs, improving the overall biogas production yields and allowing higher CH4 concentrations in the biogas produced in the second stage, thus decreasing the biogas purification costs. Therefore, a two-stage laboratory-scale CSTR anaerobic digester, fed with a mixture of agricultural and livestock residues, was monitored for a long run (approximately 700 hours) and compared to a similar one-stage reactor. This study obtained a good hydrogen yield per kg of biomass treated and partially confirmed the advantages previously illustrated, even if it reached almost the same overall energy recovery of the single stage process. Aiming at other possible biological strategies to improve the energy and hydrogen recovery efficiency with the use of effluents from a first dark fermentative stage, a relatively new electrohydrogenesis device (MEC) was studied. Paper IV explores the rate and the yield of biogas (a mixture of H2, CH4 and CO2) produced by MEC exploiting an actual industrial wastewater with high methanol content, a compound never before reported to be used in a MEC device. The energetic recovery and treatment performance of the process was evaluated and also compared with a simulation of anaerobic digestion of the same wastewater, revealing the economical competitiveness of the MEC technology with the AD process. This leads to future research perspectives aiming to realize a laboratory-scale two-stage reactor with a MEC using the volatile-rich effluent of a first dark fermentative stage.

Il muco è un colloide viscoso che costituisce una barriera fisica in tutte le superfici epiteliali del corpo umano, inclusi il tratto gastrointestinale, respiratorio, riproduttivo e urinario (Alemao et al., 2020; Bansil & Turner, 2018). É secreto dalle cellule mucipare che producono uno strato protettivo completo già dopo pochi giorni dalla nascita (Bunesova et al., 2018). La barriera mucosa è coinvolta in più funzioni come ad esempio l’assorbimento di cofattori e nutrienti, la lubrificazione ed inoltre svolge un ruolo fondamentale in correlazione con il sistema immunitario (Anthony P. Corfield, 2015). Le mucine sono glicoproteine e sono le maggiori costituenti del muco (Bansil & Turner, 2018). Queste ultime sono caratterizzate da un alto livello di glicosilazione, sono composte da oligosaccaridi quali N-acetilglucosammina (GlcNAc), N-acetilgalattosamina (GalNAc), fucosio, galattosio e acido sialico, i quali rappresentano fino all’80% della massa molecolare (Bansil & Turner, 2006). Lo scopo di questo lavoro è stato quello di identificare, attraverso un approccio cultura dipendente e indipendente, la popolazione batterica intestinale isolata in soggetti sani che fosse in grado di degradare la mucina. Le feci di cinque adulti sani sono state sottoposte a tre step successivi di arricchimento in un terreno di crescita contenente la mucina come unica fonte di carbonio e azoto. La popolazione batterica è stata confrontata prima e dopo l’arricchimento mediante la metodica del 16S rRNA gene profiling. Sono state isolate solamente tre specie di anaerobi capaci di sviluppare su mucina: Clostridium celatum, Clostridium tertium e Paraclostridium bifermentans. Di queste tre specie è stata effettuata l’analisi genomica e in parallelo un’analisi comparativa con altri genomi presenti nella banca dati NCBI GenBank, in modo da approfondire gli aspetti funzionali e metabolici, oltre che determinare la loro capacità di utilizzo della mucina. In tutti i genomi analizzati, sono stati identificati geni codificanti glicosidasi (GH) coinvolte nella degradazione della mucina, con un massimo di 25 GH riscontrate nel genoma di C. celatum. I geni coinvolti nei pathway di utilizzo dei singoli monosaccaridi quali Gal, GlcNAc and GalNAc, sono stati riscontrati in tutti i tre genomi, mentre solo C. celatum è in grado di utilizzare il fucosio. I tre ceppi oggetto di studio sono stati inoltre caratterizzati dal punto di vista fisiologico. In tutti i ceppi è stata registrata crescita batterica in un intervallo di pH fra 5.5 e 8, con un optimum di crescita di pH 6.5-7, producendo etanolo, acido acetico, propionico, formico e idrogeno. È stata osservata una propensione a formare biofilm solamente in C. celatum e P. bifermentans. Come altri membri della classe Clostridia, i tre clostridi sono in grado di produrre spore, attraverso le quali resistere a condizioni di stress. Per testare se la formazione di spore aumentasse la sopravvivenza, sono stati effettuati dei trattamenti di esposizione all’ossigeno e alle alte temperature. In seguito all’esposizione all’ossigeno tutti i ceppi sono stati in grado di ripristinare lo sviluppo delle cellule vegetative, sebbene in C. celatum sia stata osservata una maggiore sensibilità allo stress indotto da ossigeno. Diversamente, il trattamento con temperature fino a 80°C ha causato una riduzione massima di circa un ordine logaritmico in tutte le specie analizzate. Infine, sono in corso test in vivo su modelli murini Balb/c, per testare la capacità dei tre isolati di colonizzare la mucosa intestinale e di produrre effetti sul sistema immunitario. Per determinare il loro impatto sul modello murino a seguito del trattamento, verrà valutata l’espressione di interleuchine come IL-1β e IL-10.
Mucus is a viscoelastic gel barrier that covers wet epithelial surfaces throughout the body including gastrointestinal (GI), respiratory, reproductive, and urinary tracts (Alemao et al., 2020; Bansil & Turner, 2018). It is secreted by goblet and mucous cells and achieves a complete layer already several days after birth (Bunesova et al., 2018). Different roles and functions are carried out by mucus barrier such as gaseous exchange, nutrient and cofactor adsorption, lubrification, chemical sensing and an important relationship with the immune system (Anthony P. Corfield, 2015). The major building block of mucus are represented by mucins glycoproteins (Bansil & Turner, 2018). Mucins are characterised by an high level of glycosylation, oligosaccharides such as N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, galactose and sialic acidic, constitute up to 80% of their molecular mass (Bansil & Turner, 2006). The aim of this study was to investigate gut mucin degraders of healthy subjects through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of enrichment in a medium with only mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Only three species of strict anaerobes able to grow on mucin were isolated: Clostridium celatum, Clostridium tertium, and Paraclostridium bifermentans. Genome analysis of the strains was carried out and compared with other available genome present on NCBI GenBank database to better understand their metabolic and functional potential, and to determine their ability to use mucin. Genes coding for glycoside hydrolases (GHs) involved in mucin degradation were found in all the genomes, with a higher abundance in C. celatum that possess at least 25 GHs. The distribution of genes required for utilization of Gal, GlcNAc and GalNAc were widespread among all the strains, while only C. celatum degrades fucose. The three strains were investigated also in terms of physiological characterization and functional properties. They all were able to grow in a pH range between 5.5 and 8, with an optimum of pH 6.5-7, producing ethanol, acetic, propionic, formic acids, and hydrogen. Biofilm-forming ability was observed in C. celatum and P. bifermentans. Like other members of Clostridia class, the three clostridia produced spore that resisted to stress conditions. To evaluate whether spore formation improve survival, tolerance to oxygen and high temperature exposure were checked. The three strains were able to resume vegetative growth after exposure to oxygen, albeit C. celatum resulted more susceptible to oxygen stress, whereas heat treatment up to 80°C caused a decrease of maximum one Log. In vivo experiment on Balb/c mice are in progress, to test the ability of the three strains to colonize intestinal mucus and to exert some effect on immune system. To assess their impact, the expression of IL-1β and IL-10 will be measured after the treatment.

Le moderne tecnologie di Next Generation Sequencing sono un componente cruciale nello studio di microrganismi e comunità microbiche grazie all’enorme quantità di dati fornita in breve tempo. Con queste tecnologie è possibile identificare e caratterizzare i microrganismi utilizzando un ampio numero di tool bioinformatici che possono sostituire le classiche tecniche di tipizzazione in vitro, portando un risparmio di tempo e risorse. La genomica e la metagenomica possono essere applicate a vari campi e forniscono informazioni sia riguardo un singolo organismo, sia su intere comunità microbiche. I nostri studi sono stati concentrati su due nicchie ecologiche: matrici alimentari e microbiota intestinale umano, entrambi importanti per la salute umana. Un prima parte della tesi descrive la comparazione genomica di 12 ceppi di Leuconostoc carnosum isolati da prodotti a base di carne. Questo batterio è un noto colonizzatore di queste matrici, ricopre un ruolo nel loro deterioramento, ma alcuni ceppi presentano effetti utili alla preservazione. Abbiamo eseguito un sequenziamento whole genome per tutti i ceppi, e dopo l’assembly sono state identificate le caratteristiche genomiche, la presenza di plasmidi, di geni responsabili di antibiotico-resistenza, produzione di batteriocine, sintesi di ammine biogene e abbiamo ricostruito i loro pathway metabolici. La comparazione ha rivelato che i ceppi sono strettamente correlati e condividono la maggior parte delle caratteristiche metaboliche, evidenziando l’adattamento all’ambiente di isolamento grazie alle 23 peptidasi presenti nel genoma core. Questo studio fornisce un approfondimento genomico e metabolico su questo batterio ubiquitario nei prodotti a base di carne. Un secondo progetto della tesi è stato indirizzato a indagare la presenza delle beta-glucuronidasi (GUS) nel microbiota intestinale umano attraverso una strategia metagenomica inedita. Le GUS prodotte dai batteri del microbiota sono capaci di rimuovere le porzioni di acido glucuronico da molti composti e metaboliti, come farmaci e xenobiotici. Queste molecole vengono coniugate con l’acido glucuronico nel fegato per l’escrezione attraverso il tratto gastrointestinale, quindi l’azione enzimatica potrebbe riattivarle permettendone il riassorbimento comportando un’alterazione dell’efficacia del farmaco ed effetti negativi sulla salute. Le GUS sono organizzate in classi in base alle differenze nel loro sito catalitico. Sono stati utilizzati 60 metagenomi ottenuti con shotgun sequencing provenienti da soggetti sani divisi in coorti in base alla provenienza geografica. Da questo set di dati, è stata definita la composizione batterica ed è stata sviluppata ed impiegata una nuova strategia per indagare la distribuzione delle diverse GUS. La beta diversità calcolata con gli indici di Jaccard e Bray-Curtis è stata impiegata per determinare le distanze tra i campioni e determinare le differenze tra essi in termini di distribuzione del tipo di GUS e abbondanza dei batteri che le contengono. Poiché alle differenze strutturali dell’enzima corrisponde una diversa specificità di substrato, e considerando la proporzione delle comunità batteriche contenenti GUS, possiamo valutare che la composizione del microbiota può alterare l’escrezione di alcuni farmaci o xenobioti e determinare un’ampia variabilità interindividuale in termini di risposta al farmaco. Un’ultima parte del progetto di tesi descrive l’applicazione dell’approccio metagenomico in due diversi studi: il primo incentrato a indagare la composizione microbica di campioni fecali arricchiti per identificare specie intestinali capaci di degradare la mucina, e il secondo focalizzato sulla descrizione del microbiota di larve di Hermetia illucens allevate per consumo umano o animale.
The modern Next Generation Sequencing technologies represent a crucial component in the study of microorganisms and microbial communities thanks to the huge quantity of data they can provide in a short period of time. These technologies allow the identification and characterization of microorganisms exploiting a vast number of bioinformatic tools that can replace the standard in vitro typing techniques, resulting in savings of time and resources. Genomics and metagenomics can be applied in different fields and they can provide information on single microorganism or on entire microbial communities. We focused our studies on two ecological niches: food matrices and human gut microbiome, due to their relevance to human health. The first work of this thesis is a comparative genomic study of 12 Leuconostoc carnosum strains isolated from meat-based products. This bacterium is a known colonizer of meat-based food matrices, it plays a role in spoilage, but preservative effects have also been proposed for some strains. In our study we performed whole genome sequencing for all the strains, and after genome assembly we identified their genomic features, the presence of plasmids, and genes related to antibiotic resistance, bacteriocins production, biogenic amines synthesis. We also reconstructed their metabolic pathways. The comparison revealed that the strains are closely related and share most of the metabolic features, confirming the adaptation to the meat environment due to the presence of 23 peptidase genes in their core genome. With this study we provided a deeper insight into the genomic and metabolic features of this bacterium ubiquitous in meat products. The second project of the thesis aimed to investigate through an inedited metagenomic strategy the presence of beta-glucuronidases (GUS) in the human gut microbiome. Beta-glucuronidases (GUS) produced by gut microbiome bacteria can remove glucuronic acid moieties from a vast range of compounds and metabolites, like drugs and xenobiotics. These molecules are conjugated with glucuronic acid in the liver to be excreted in the gastrointestinal tract, so the action of GUS may reactivate them allowing the reabsorption, with unpredictable and different efficacy of drugs and negative effect on health. GUS are classified in classes by the differences in the catalytic site. 60 shotgun sequenced metagenomic samples from healthy subjects, ascribed to five geographically distinct cohorts, have been retrieved. From this dataset, bacterial composition has been defined and a novel pipeline to investigate distribution the different GUS has been developed and utilized. Beta-diversity calculated on Bray-Curtis dissimilarity index has been used to determine the distances among samples and determine the differences among samples in terms of GUS type distribution and abundance of bacteria containing GUS. Since the structural differences in the enzyme involve a different substrate specificity, and taking into account the ratio of bacterial community harbouring GUS genes, we can assess that the microbiota composition can alter the excretion of certain drugs or xenobiotics, and determine a wide interindividual variability in terms of response to drugs. In the third part of the thesis, I present metagenomic analysis carried out in two other different studies, the former aimed to investigate the microbial composition in enriched human faecal samples to identify gut mucin degraders, and the latter focused on the description of the microbiota of Hermetia illucens larvae reared for food or feed consumption.

Le comunità microbiche sono complesse associazioni di microrganismi che coesistono ed interagiscono fra loro, interferendo sulla composizione della matrice che colonizzano. Il loro studio del microbiota può essere condotto con metodi convenzionali e/o con tecniche di high-throughput sequencing. Questo progetto si è incentrato sullo studio del microbiota di matrici diverse (larve di Hermetia illucens, prosciutto cotto confezionato in atmosfera protettiva (MAP) e infiorescenze di Cannabis sativa) con metodi metagenomici e di microbiologia classica. Inoltre, è stata eseguita una caratterizzazione fisiologica e funzionale della specie Leuconostoc carnosum, frequente colonizzatore di prodotti carnei, specie a tutt’oggi poco studiata. È stato analizzato il microbiota di larve H. illucens alimentate con scarti agroalimentari, nella prospettiva di produrre mangimi innovativi per galline ovaiole. Sono state testate 3 diverse temperature di allevamento delle larve per valutare l’influenza sul microbiota dell'insetto, utilizzando tecniche coltura-dipendenti per la valutazione del rischio microbiologico e analisi metagenomiche basate sul sequenziamento del gene 16S rRNA. Le temperature più alte hanno accelerato lo sviluppo larvale e determinato una maggiore biomassa finale e una maggiore mortalità. In base all’analisi metagenomica, il microbiota delle larve è dominato dal genere Providencia e da altri Proteobateria. Nello stadio di prepupe aumentano la complessità della composizione e l’abbondanza relativa di Actinobacteria, Bacteroidetes e Bacilli. Le tecniche coltura dipendenti hanno evidenziato un’elevata carica di mesofili totali (108 ufc/g) e di patogeni opportunisti che possono compromettere la stabilità della biomassa e la safety del prodotto. Un approccio combinato metagenomico e coltura dipendente è stato utilizzato per studiare l’evoluzione del microbiota del prosciutto cotto in MAP, durante la shelf-life secondaria. La qualità del prosciutto cotto dopo l’apertura, durante i 12 giorni di conservazione a 7°C, è stata valutata attraverso analisi microbiologiche, molecolari, sensoriali e chimiche. Lo studio ha evidenziato una grande variabilità nella composizione del microbiota legata soprattutto al produttore, confermando che durante la shelf-life il prodotto è già colonizzato da una comunità complessa. Lo studio del microbiota è stato esteso a inflorescenze di 9 varietà di C. sativa ad uso industriale, raccolte ed essiccate in tre località Italiane, destinate alla produzione di biochemicals di interesse manifatturiero e cosmeceutico. Nel complesso le varietà raccolte a Rovigo presentavano una maggiore contaminazione in aerobi totali. La presenza di Salmonella è stata evidenziata nei campioni ottenuti da Rovigo e Acireale, mentre era assente in quelli raccolti a Caserta. Il progetto di tesi ha riguardato anche lo studio comparativo delle caratteristiche biochimiche, fisiologiche e funzionali della specie L. carnosum, a cui appartengono numerosi ceppi isolati da prosciutto cotto e altre matrici carnee dove possono svolgere una duplice funzione, o preservante o deteriorante. Sono stati analizzati 12 ceppi, che sviluppano a temperature comprese fra 4 e 37° C, in presenza di NaCl fino a 60 g/L. Sei ceppi producono esopolisaccaridi, responsabili della comparsa di essudati mucosi. È emerso un potenziale ruolo protettivo dei ceppi L. carnosum WC0321 e L. carnosum WC0323 nei confronti di Listeria monocytogenes. Tre ceppi selezionati sono stati saggiati per la capacità di colonizzare un modello murino e sono state studiate le loro proprietà immunomodulatorie. I ceppi, nonostante la perdita di vitalità durante il transito gastrointestinale, hanno mostrato diversi effetti immunomodulatori sulla maturazione delle cellule dendritiche in vivo, suggerendo un impatto positivo sulla salute dell’ospite.
Microbial communities are complex associations of microorganisms that coexist and interact with each other, interfering with the composition of the matrix they colonise. The study of microbiota can be conducted with conventional microbiological methods and/or with high-throughput sequencing techniques. In this thesis, the microbiota of different matrices (cooked ham packaged in a protective atmosphere (MAP), larvae of Hermetia illucens, and inflorescences of Cannabis sativa) was characterised by metagenomic analysis and/or classical microbiology techniques. In addition, a physiological and functional characterisation of Leuconostoc carnosum, a frequent colonizer of meat products, species still poorly studied, was performed. Thanks to the disclosure of L. carnosum genome, it was possible to resolve the phylogenetic relationships within the genus Leuconostoc through a phylogenomic approach. A combined metagenomic and culture-dependent approach was used to study the evolution of the microbiota of cooked ham in MAP, in order to determine the secondary shelf-life. The quality of the cooked ham after opening, during 12 days of storage at 4 °C, was assessed through microbiological, molecular, sensory, and chemical analyses. The study highlighted a great variability in the composition of the microbiota, mostly associated to the producer, confirming that during the shelf-life the product is already abundantly colonised by a rather complex community. Biochemical, physiological, and functional features of L. carnosum, frequently isolated from cooked ham and other meat matrices, were determined. This species can perform a dual function in meat products, either preserving or deteriorating them. Twelve strains that developed at temperatures between 4 and 37 °C and in the presence of NaCl up to 60 g/L, were analysed. Six strains produced exopolysaccharides responsible for the appearance of mucous exudates. A potential protective role of L. carnosum WC0321 and L. carnosum WC0323 emerged against Listeria monocytogenes. Three selected strains were tested for the ability to colonize a murine model, and their immunomodulatory properties were investigated. Despite the loss of viability during the passage through the gastrointestinal transit, the strains showed different immunomodulatory effects on dendritic cell maturation in vivo, suggesting a positive impact on the host's health. Phylogenomic analysis of the genus Leuconostoc was carried out. 216 deposited genome sequences were subjected to phylogenomic analysis based on the Average Nucleotide Identity (ANI) and the core genome alignment, resulting in a proposal of phylogenomic reorganization of the genus. Microbiological analysis of two diverse potential ingredients for novel foods/feeds and nutraceutical were carried out on larvae and prepupae of Hermetia illucens, and inflorescences of Cannabis sativa. The microbiota of larvae H. illucens fed with waste from agri-food industries has been determined, in order to develop innovative feed for laying hens. Three rearing temperatures of larvae were assayed to evaluate the influence on the insect microbiota. The higher temperatures favoured faster larval development and resulted in higher final biomass and mortality. The culture-dependent approach showed a high load of total mesophiles (108 cfu/g) and opportunistic pathogens, that can compromise the stability of the biomass and the safety of the product. Inflorescences of 7 varieties of C. sativa for industrial purposes were analysed. They were collected and dried in two Italian locations, with the aim to produce biochemicals for manufacturing and cosmeceutical interest. Overall, the varieties collected in Rovigo showed higher contamination in total aerobes.

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 ‘‘Custers effect’’, 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.

Gli esteri svolgono un ruolo rilevante nell'industria alimentare, sono tra i composti più importanti e versatili di aromi e fragranze naturali in alimenti, bevande e cosmetici. La loro preparazione da substrati naturali e l'utilizzo di bioprocessi (eg. fermentazione o reazioni enzimatiche) è attrattivo, perché il prodotto finale può essere etichettato e commercializzato in UE e USA come naturale. Pertanto, nuovi approcci biotecnologici per ottenere aromi sono desiderati, una volta che siano efficienti e sostenibili. Molti esteri con proprietà aromatiche possono essere ottenuti enzimaticamente usando lipasi che catalizzano reazioni di esterificazione, transesterificazione o interesterificazione. In questa tesi di dottorato abbiamo sviluppato due sistemi per la produzione di esteri con proprietà aromatiche: 1) Un metodo biocatalitico per la preparazione enzimatica di diversi esteri con proprietà aromatiche da alcoli primari (isoamilico, n-esilico, geranilico, cinnamilico, 2-feniletilico e benzilico) ed esteri etilici naturalmente disponibili (formiato, acetato, propionato e butirrato). Le biotrasformazioni sono catalizzate da un'aciltransferasi di Mycobacterium smegmatis (MsAcT) e precedute da eccellenti rese (80- 97%) e brevi tempi di reazione (30-120 minuti), anche quando sono state utilizzate concentrazioni di substrato elevate (fino a 0,5 M). Questa strategia enzimatica rappresenta un'alternativa efficace all'applicazione delle lipasi nei solventi organici e un miglioramento significativo rispetto ai metodi già noti in termini di uso ridotto di solventi organici, aprendo la strada a una preparazione sostenibile ed efficiente degli agenti aromatizzanti naturali. 2) Un metodo biocatalitico con la lipasi legata al micelio liofilizzato di Aspergillus oryzae che catalizza l'esterificazione diretta di alcoli e acido acetico in solvente organico. Ha mostrato un'elevata stabilità verso substrati e prodotti. L'acqua prodotta durante l'esterificazione non ha influenzato in modo significativo l'equilibrio della reazione, consentendo conversioni elevate. Queste caratteristiche sono state sfruttate per preparare esteri con proprietà aromatiche dell'acetato (isoamil e cinnamil acetato) in sistemi in batch e continui. È stato sviluppato un continuous stirred tank membrane reactor (CST-MR) ovvero un reattore continuo a membrana sotto agitazione per garantire una buona produttività e un'elevata stabilità del biocatalizzatore. Entrambi i sistemi di produzione sono promettenti, rappresentano due diverse alternative e possono essere ulteriormente ottimizzati e scalati per gli interessi del settore.
Esters play a significant role in the food industry, they are among the most important and versatile components of natural flavours and fragrances in food, drinks and cosmetics Their preparation starting from natural substrates and using bioprocesses (e.g., fermentation or enzymatic reactions) is appealing, since the final product can be labelled and commercialized in EU and USA as natural. Therefore, new biotechnological approaches for obtaining flavours are highly demanded as long as they are efficient and sustainable. Many flavour/fragrance esters can be enzymatically obtained using lipases that catalyse esterification, transesterification or interesterification reactions. In this PhD thesis we studied two systems for production of flavours esters: 1) A straightforward biocatalytic method for the enzymatic preparation of different flavour esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalysed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and preceded with excellent yields (80-97%) and short reaction times (30-120 minutes), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared to already known methods in terms of reduced use of organic solvents, paving the way to a sustainable and efficient preparation of natural flavouring agents. 2) Mycelium-bound lipase of dry mycelium of Aspergillus oryzae catalysed direct esterification of alcohols and acetic acid in organic solvent, showing high stability towards substrates and products. Water produced during the esterification did not significantly affect the equilibrium of the reaction, allowing for high conversions. These features were exploited for preparing flavour-active acetate esters (e.g., isoamyl and cinnamyl acetate) in batch and continuous systems. A continuous stirred tank membrane reactor (CST-MR) was developed securing good reactor productivity and high biocatalyst stability. Both production systems are promising, represent two different alternatives and can be further optimized and scaled up for the interests of the industry.

Volatile fatty acid (VFAs) represent the final product of anaerobic acidogenic fermentation and can be exploited as precursors for the production of added-value chemicals. The biotechnological conversion of agro-industrial wastes into VFAs can be considered a valuable alternative to waste disposal. However, the productivity of processes, which refer to the carboxylation platform are affected by the purity of produced VFAs. Biosphere SRL (Forli, Italy) had started to valorize wastes to obtain biogas. VFAs were produced using maize silage (MS), vegetable waste (VW) and cheese whey (CW) as feedstock. The co-digestion of VW (70%) MS (30%) (VMW) under continuous operation with hydraulic retention time (HRT) of 6 days and a controlled pH 7 resulted in a VFAs rich solution of 30 g.L-1. The fermentation of CW was performed in a packed bed bioreactor where Vukuopor was the supported material. The HRT, organic loading rate (OLR) and pH were parameters tested under continuous operation. By a HRT of 6 days, ORL of 4.2 g.L-1.d-1, and pH 5.8−6, 16 g·L-1 of total VFAs were produced, with a yield higher than 75%. Electrodialysis (ED) process was studied for the concentration of VFAs from the two acidogenic effluents resulted from the previous mentioned anaerobic acidogenic digestions. Under potentiostat operation the VMW concentration stream was concentrated from 29 g.L-1 to 50 g.L-1. Thought it is expected that a concentration of 100 g.L-1 can be achieved by performing sequential batch. With CW acidified effluent the concentration achieved was similar however the initial solution was poor in VFAs. Experiments with Nanofiltration were performed for the separation of ferulic acid (FA) and sugars from an enzymatic hydrolysate of wheat bran. Eight different membranes were screened at different pressures and solution pH. The best condition was found be membrane NT7450 operated at pH 9 and 16 bar.

Modern society is forced to deal with two main problems: a growing scarcity of resources and a growing production of waste. The unsustainability of the current linear economic model “make, use, dispose” originated the concept of “circular economy” that aims to reduce the environmental impact through measures in favour of a more sustainable development, such as the use of renewable energy and waste recycling. Unlike the traditional linear model, the circular economy keeps resources in use for as long as possible, extracts the maximum value from them whilst in use, then recovers and regenerates products and materials at the end of each service life. The basis of the circular economy could be represented by the biorefinery: a productive system aimed at the sustainable conversion of biomass, including energy crops, food industry by-products and organic wastes, into marketable bio-based products (i.e. chemicals, polymers, food and animal feed) and bioenergy (i.e. liquid and gaseous fuels, such as bioethanol, biodiesel and biogas). Among the wide varieties of bio-based products of biorefineries are the polyhydroxyalkanoates (PHAs), a class of bioplastics with microbial origin, which are completely biodegradable and obtainable from renewable carbon sources. PHAs are now earning great interest since they offer, differently from common bioplastics, a wide range of physico-chemical properties that make them comparable and in several applications possible substitute of traditional plastics, moreover they do not determine environmental damages after their service life as a result of their biodegradability. Nowadays, the main limit that slows down PHAs production is the high production cost that does not make them competitive on the market. In this PhD thesis the production of polyhydroxyalkanoates-PHAs was integrated as a step into simple or more complex biorefinery approaches in which cheese whey, the organic fraction of municipal solid waste (OFMSW), Arundo donax and CO2 were used as feedstocks. Currently PHAs production is accomplished by using pure microbial strains, requiring sterility and a close monitoring of the culture growing conditions, thus increasing the production costs. However, it remains the most widespread method for the highest process yields and volumetric productivities. The use of mixed microbial cultures (MMCs) has started to be investigated as an economically viable option for PHAs production since they do not require sterility, do not need restricted growing conditions, resulting in both energy and economic savings during the fermentation process. Moreover, MMCs are more able than pure cultures to grow on complex substrates, such as low-cost agro-food industry wastes, giving a further possibility to reduce PHAs production costs. For this reason in this PhD thesis MMCs were used as starting inoculum to select PHAs storing bacteria by adopting the four feedstocks above mentioned as carbon sources. Before using these feedstocks for PHAs production, they were submitted to different pre treatments aimed to the production of liquid substrates rich in organic acids, the direct metabolic precursors of PHAs in MMCs. All of these substrates were then used as carbon sources to successfully select MMCs with good PHAs storing ability, and for the massive biopolymer production by using the selected MMCs. The results obtained in terms of PHAs yield and PHAs content on cell dry weight were promising and comparable with the literature related to PHAs production from MMCs and complex feedstocks. In particular, during the accumulation tests, PHAs yield on substrate consumed resulted up to 0.75 ± 0.1 g CODPHA g-1 COD by using fermented cheese whey as carbon source, 0.52 ± 0.1 g CODPHA g-1 COD by using a percolate of the OFMSW, 0.85 ± 0.2 g CODPHA g-1 COD by using fermented hydrolysed Arundo donax, and up to 1.06 ± 0.2 g CODPHA g-1 COD by using a mixture of organic acids produced via microbial electrosynthesis (MES) from CO2. The lowest yield was detected in PHAs production from a percolate of the OFMSW, probably because of the complexity of the carbon source that acted as limiting factor during the selection of a PHAs storing MMC; in facts, in the last work performed, by using the mixture of organic acids produced via MES as substrate, the highest PHAs yield was obtained, due to the purity of the carbon source fed to the MMC that led to a selection of a MMC with high PHAs storing ability. Depending mainly on the composition in organic acids of the substrates fed to the mixed microbial consortia, different biopolymers were produced: in particular, by using substrates rich in butyric, acetic and lactic acids (fermented cheese whey from lactic fermentation, fermented hydrolysed Arundo donax and the mixture of butyric and acetic acids obtained via MES) biopolymers made mainly of hydroxybutyrate (HB) were produced (HB > 96 %); while using substrates containing also propionic and valeric acids (fermented cheese whey from mixed acid fermentation and the percolate of the OFMSW), copolymers made of hydroxybutyrate and of hydroxyvalerate (HV) in different proportion were produced (namely P(HBcoHV)), 60:40 (HB:HV, %) and 55:45 (HB:HV, %), respectively for fermented cheese whey and for the percolate of the OFMSW. Since the traditional downstream processes play a key role in the profitability of the fermentation system, counting for about the 30-50 % of the total production costs, in the last part of this study a novel PHAs extraction method from mixed microbial biomass was developed with encouraging results. In particular, by coupling the use of organic non-ionic surfactants to act on the non-PHA cell mass with the use of the green organic solvent dimethyl carbonate to extract PHAs, it was possible to reach biopolymer recovery yields not so far from the one obtained adopting traditional extraction methods based on the use of chloroform. Excluding food contact, the extracted biopolymers could be used to produce packing materials, shopping bags and biodegradable mulching films to be applied in agriculture.

The bioproduction of materials and energy from renewable sources (industrial biotechnology) is getting more and more interest in order to improve environmental sustainability of chemical industrial processes and to decrease their dependence from oil. Anaerobic digestion of organic waste matrices (agricultural and industrial wastes, organic fraction of municipal wastes, sewage sludges etc.) may play an important role in the implementation of industrial biotechnology being a well developed strategy in the valorization of complex matrices, as it can mineralize them while producing bioenergy in the form of a biogas rich in methane. In this research the potential of anaerobic digestion in the treatment of polluted sewage sludge was studied by developing three set of anaerobic microcosms with sludges differently contaminated by xenobiotic compounds. The effect of different incubating temperatures and of exogenous carbon and vitamine sources was investigated along with the role of the occurring microbial populations in the pollutant degradation activity. So, while confirming the potential of anaerobic digestion for the biomethanization of sewage sludges, this work proved the effectiveness of this technology in the removal of pollutants too. Moreover, since the degradation of lignocellulose appears to be a limiting step in the anaerobic treatment of a wide range of biomass, the possibility of optimizing anaerobic digestion of lignocellulosic substrates was also studied. To this aim a research was carried out at the BOKUUniversity of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, IFA - Tulln, where mixed cellulolytic cultures were isolated from biogas plants while assessing the metabolic pathway leading to cellulose degradation and verifying their capability to grow on lignocellulose too, proving that on the long term such bacterial cultures could be used as inoculum in order to improve the hydrolysis of lignocellulose in anaerobic digestion plants.

The bioproduction of materials and energy from renewable sources (industrial biotechnology) is getting more and more interest in order to improve environmental sustainability of chemical industrial processes and to decrease their dependence from oil. Anaerobic digestion of organic waste matrices (agricultural and industrial wastes, organic fraction of municipal wastes, sewage sludges etc.) may play an important role in the implementation of industrial biotechnology being a well developed strategy in the valorization of complex matrices, as it can mineralize them while producing bioenergy in the form of a biogas rich in methane. In this research the potential of anaerobic digestion in the treatment of polluted sewage sludge was studied by developing three set of anaerobic microcosms with sludges differently contaminated by xenobiotic compounds. The effect of different incubating temperatures and of exogenous carbon and vitamine sources was investigated along with the role of the occurring microbial populations in the pollutant degradation activity. So, while confirming the potential of anaerobic digestion for the biomethanization of sewage sludges, this work proved the effectiveness of this technology in the removal of pollutants too. Moreover, since the degradation of lignocellulose appears to be a limiting step in the anaerobic treatment of a wide range of biomass, the possibility of optimizing anaerobic digestion of lignocellulosic substrates was also studied. To this aim a research was carried out at the BOKUUniversity of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, IFA - Tulln, where mixed cellulolytic cultures were isolated from biogas plants while assessing the metabolic pathway leading to cellulose degradation and verifying their capability to grow on lignocellulose too, proving that on the long term such bacterial cultures could be used as inoculum in order to improve the hydrolysis of lignocellulose in anaerobic digestion plants.

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

Adaptation and acclimation to different temperatures of obligate psychrophilic, facultative psychrophilic and mesophilic yeasts. Production of ω-3 and ω-6 polyunsaturated fatty acids by fermentative way. Obligate psychrophilic, facultative psychrophilic and mesophilic yeasts were cultured in a carbon rich medium at different temperatures to investigate if growth parameters, lipid accumulation and fatty acid composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to lower temperature negatively affected their specific growth rate. Obligate psychrophiles exhibited the highest biomass yield (YX/S), followed by facultative psychrophiles, then by mesophiles. The growth temperature did not influence the YX/S of facultative psychrophiles and mesophiles. Acclimation to lower temperature caused the increase in lipid yield (YL/X) in mesophilic yeasts, but did not affect YL/X in facultative psychrophiles. Similar YL/X were found in both facultative and obligated psychrophiles, suggesting that lipid accumulation is not a distinctive character of adaptation to permanently cold environments. The extent of unsaturation of fatty acids was one major adaptive feature of the yeasts which colonize permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expenses of linoleic acid, whereas it was generally scarce or absent in all the others strains. Increased unsaturation of fatty acids was also an acclimatory response of mesophiles and facultative psychrophiles to lower temperature. It’s well known that omega-3 polyunsaturated fatty acids (PUFAs) display a variety of beneficial effects on various organ systems and diseases, therefore a process for the microbial production of omega-3 PUFAs would be of great interest. This work sought also to investigate if one of the better psychrophilic yeast, Rhodotorula glacialis DBVPG 4785, stimulated by acclamatory responses, produced omega-3 PUFAs. In fact, the adaptation of psychrophilic yeasts to cold niches is related to the production of higher amounts of lipids and to increased unsaturation degree of fatty acids, presumably to maintain membrane fluidity and functionality at low temperatures. Bioreactor fermentations of Rhodotorula glacialis DBVPG 4785 were carried out at 25, 20, 15, 10, 5, 0, and -3°C in a complex medium with high C:N ratio for 15 days. High biomass production was attained at all the temperatures with a similar biomass/glucose yield (YXS), between 0.40 and 0.45, but the specific growth rate of the strain decreased as the temperature diminished. The coefficients YL/X have been measured between a minimum of 0.50 to a maximum of 0.67, but it was not possible to show a clear effect of temperature. Similarly, the coefficient YL/S ranges from a minimum of 0.22 to a maximum of 0.28: again, it does not appear to be any significant changes due to temperature. Among omega-3 PUFAs, only α-linolenic acid (ALA, 18:3n-3) was found at temperatures below to 0°C, while, it’s remarkable, that the worthy arachidonic acid (C20:4,n-6), stearidonic acid (C20:4,n-3) C22:0 and docosahexaenoic acid (C22:6n-3) were produced only at the late exponential phase and the stationary phase of batch fermentations at 0 and -3°C. The docosahexaenoic acid (DHA) is a beneficial omega-3 PUFA that is usually found in fatty fish and fish oils. The results herein reported improve the knowledge about the responses which enable psychrophilic yeasts to cope with cold and may support exploitation of these strains as a new resource for biotechnological applications.

Adaptation and acclimation to different temperatures of obligate psychrophilic, facultative psychrophilic and mesophilic yeasts. Production of ω-3 and ω-6 polyunsaturated fatty acids by fermentative way. Obligate psychrophilic, facultative psychrophilic and mesophilic yeasts were cultured in a carbon rich medium at different temperatures to investigate if growth parameters, lipid accumulation and fatty acid composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to lower temperature negatively affected their specific growth rate. Obligate psychrophiles exhibited the highest biomass yield (YX/S), followed by facultative psychrophiles, then by mesophiles. The growth temperature did not influence the YX/S of facultative psychrophiles and mesophiles. Acclimation to lower temperature caused the increase in lipid yield (YL/X) in mesophilic yeasts, but did not affect YL/X in facultative psychrophiles. Similar YL/X were found in both facultative and obligated psychrophiles, suggesting that lipid accumulation is not a distinctive character of adaptation to permanently cold environments. The extent of unsaturation of fatty acids was one major adaptive feature of the yeasts which colonize permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expenses of linoleic acid, whereas it was generally scarce or absent in all the others strains. Increased unsaturation of fatty acids was also an acclimatory response of mesophiles and facultative psychrophiles to lower temperature. It’s well known that omega-3 polyunsaturated fatty acids (PUFAs) display a variety of beneficial effects on various organ systems and diseases, therefore a process for the microbial production of omega-3 PUFAs would be of great interest. This work sought also to investigate if one of the better psychrophilic yeast, Rhodotorula glacialis DBVPG 4785, stimulated by acclamatory responses, produced omega-3 PUFAs. In fact, the adaptation of psychrophilic yeasts to cold niches is related to the production of higher amounts of lipids and to increased unsaturation degree of fatty acids, presumably to maintain membrane fluidity and functionality at low temperatures. Bioreactor fermentations of Rhodotorula glacialis DBVPG 4785 were carried out at 25, 20, 15, 10, 5, 0, and -3°C in a complex medium with high C:N ratio for 15 days. High biomass production was attained at all the temperatures with a similar biomass/glucose yield (YXS), between 0.40 and 0.45, but the specific growth rate of the strain decreased as the temperature diminished. The coefficients YL/X have been measured between a minimum of 0.50 to a maximum of 0.67, but it was not possible to show a clear effect of temperature. Similarly, the coefficient YL/S ranges from a minimum of 0.22 to a maximum of 0.28: again, it does not appear to be any significant changes due to temperature. Among omega-3 PUFAs, only α-linolenic acid (ALA, 18:3n-3) was found at temperatures below to 0°C, while, it’s remarkable, that the worthy arachidonic acid (C20:4,n-6), stearidonic acid (C20:4,n-3) C22:0 and docosahexaenoic acid (C22:6n-3) were produced only at the late exponential phase and the stationary phase of batch fermentations at 0 and -3°C. The docosahexaenoic acid (DHA) is a beneficial omega-3 PUFA that is usually found in fatty fish and fish oils. The results herein reported improve the knowledge about the responses which enable psychrophilic yeasts to cope with cold and may support exploitation of these strains as a new resource for biotechnological applications.

Faithful replication of DNA from one generation to the next is crucial for long-term species survival. Genomic integrity in prokaryotes, archaea and eukaryotes is dependent on efficient and accurate catalysis by multiple DNA polymerases. Escherichia coli possesses five known DNA polymerases (Pol). DNA polymerase III holoenzyme is the major replicative polymerase of the Escherichia coli chromosome (Kornberg, 1982). This enzyme contains two Pol III cores that are held together by a t dimer (Studwell-Vaughan and O’Donnell, 1991). The core is composed of three different proteins named α-, ε- and θ-subunit. The α-subunit, encoded by dnaE, contains the catalytic site for DNA polymerisation (Maki and Kornberg, 1985), the ε-subunit, encoded by dnaQ, contains the 3′→5′ proofreading exonuclease (Scheuermann, et al., 1983) and the θ-subunit, encoded by hole, that has no catalytic activity (Studwell-Vaughan, and O'Donnell, 1983). The three-subunit α–ε–θ DNA pol III complex is the minimal active polymerase form purified from the DNA pol III holoenzyme complex; these three polypeptides are tightly associated in the core (McHenry and Crow, 1979) Despite a wealth of data concerning the properties of DNA polymerase III in vitro, little information is available on the assembly in vivo of this complex enzyme. In this study it is shown that the C-terminal region of the proofreading subunit is labile and that the ClpP protease and the molecular chaperones GroL and DnaK control the overall concentration in vivo of ε. Two α-helices (comprising the residues E311-M335 and G339-D353, respectively) of the N-terminal region of the polymerase subunit were shown to be essential for the binding to ε. These informations could be utilized to produce a conditional mutator strain in which proofreading activity would be titrated by a a variant that can only bind e and that is polymerase-deficient. In this way the replication of DNA made by DNA Pol-III holoenzyme would accordingly become error-prone.

Faithful replication of DNA from one generation to the next is crucial for long-term species survival. Genomic integrity in prokaryotes, archaea and eukaryotes is dependent on efficient and accurate catalysis by multiple DNA polymerases. Escherichia coli possesses five known DNA polymerases (Pol). DNA polymerase III holoenzyme is the major replicative polymerase of the Escherichia coli chromosome (Kornberg, 1982). This enzyme contains two Pol III cores that are held together by a t dimer (Studwell-Vaughan and O’Donnell, 1991). The core is composed of three different proteins named α-, ε- and θ-subunit. The α-subunit, encoded by dnaE, contains the catalytic site for DNA polymerisation (Maki and Kornberg, 1985), the ε-subunit, encoded by dnaQ, contains the 3′→5′ proofreading exonuclease (Scheuermann, et al., 1983) and the θ-subunit, encoded by hole, that has no catalytic activity (Studwell-Vaughan, and O'Donnell, 1983). The three-subunit α–ε–θ DNA pol III complex is the minimal active polymerase form purified from the DNA pol III holoenzyme complex; these three polypeptides are tightly associated in the core (McHenry and Crow, 1979) Despite a wealth of data concerning the properties of DNA polymerase III in vitro, little information is available on the assembly in vivo of this complex enzyme. In this study it is shown that the C-terminal region of the proofreading subunit is labile and that the ClpP protease and the molecular chaperones GroL and DnaK control the overall concentration in vivo of ε. Two α-helices (comprising the residues E311-M335 and G339-D353, respectively) of the N-terminal region of the polymerase subunit were shown to be essential for the binding to ε. These informations could be utilized to produce a conditional mutator strain in which proofreading activity would be titrated by a a variant that can only bind e and that is polymerase-deficient. In this way the replication of DNA made by DNA Pol-III holoenzyme would accordingly become error-prone.

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.