Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium spp

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

Vanillin production from ferulic acid with Pseudomonas fluorescens BF13-1p4

Bioconversion of ferulic acid to vanillin represents an attractive opportunity for replacing synthetic vanillin with a bio-based product, that can be label “natural”, according to current food regulations. Ferulic acid is an abundant phenolic compound in cereals processing by-products, such as wheat bran, where it is linked to the cell wall constituents. In this work, the possibility of producing vanillin from ferulic acid released enzymatically from wheat bran was investigated by using resting cells of Pseudomonas fluorescens strain BF13-1p4 carrying an insertional inactivation of vdh gene and ech and fcs BF13 genes on a low copy number plasmid. Process parameters were optimized both for the biomass production phase and the bioconversion phase using food-grade ferulic acid as substrate and the approach of changing one variable while fixing the others at a certain level followed by the response surface methodology (RSM). Under optimized conditions, vanillin up to 8.46 mM (1.4 g/L) was achieved, whereas highest productivity was 0.53 mmoles vanillin L-1 h-1). Cocktails of a number of commercial enzyme (amylases, xylanases, proteases, feruloyl esterases) combined with bran pre-treatment with steam explosion and instant controlled pressure drop technology were then tested for the release of ferulic acid from wheat bran. The highest ferulic acid release was limited to 15-20 % of the ferulic acid occurring in bran, depending on the treatment conditions. Ferulic acid 1 mM in enzymatic hydrolyzates could be bioconverted into vanillin with molar yield (55.1%) and selectivity (68%) comparable to those obtained with food-grade ferulic acid after purification from reducing sugars with a non polar adsorption resin. Further improvement of ferulic acid recovery from wheat bran is however required to make more attractive the production of natural vanillin from this by-product.

Food safety in wine: optimization of analytical controls and evaluation of production technologies

This PhD thesis has been proposed to validate and then apply innovative analytical methodologies for the determination of compounds with harmful impact on human health, such as biogenic amines and ochratoxin A in wines. Therefore, the influence of production technology (pH, amino acids precursor and use of different malolactic starters) on biogenic amines content in wines was evaluated. An HPLC method for simultaneous determination of amino acids and amines with precolumnderivatization with 9-Fluorenyl-methoxycarbonyl chloride (FMOC-Cl) and UV detection was developed. Initially, the influence of pH, time of derivatization, gradient profile were studied. In order to improve the separation of amino acids and amines and reduce the time of analysis, it was decided to study the influence of different flows and the use of different columns in the chromatographic method. Firstly, a C18 Luna column was used and later two monolithic columns Chromolith in series. It appeared to be suitable for an easy, precise and accurate determination of a relatively large number of amino acids and amines in wines. This method was then applied on different wines produced in the Emilia Romagna region. The investigation permitted to discriminate between red and white wines. Amino acids content is related to the winemaking process. Biogenic amines content in these wines does not represent a possible toxicological problem for human health. The results of the study of influence of technologies and wine composition demonstrated that pH of wines and amino acids content are the most important factors. Particularly wines with pH > 3,5 show higher concentration of biogenic amines than wines with lower pH. The enrichment of wines by nutrients also influences the content of some biogenic amines that are higher in wines added with amino acids precursors. In this study, amino acids and biogenic amines are not statistically affected by strain of lactic acid bacteria inoculated as a starter for malolactic fermentation. An evaluation of different clean-up (SPE-MycoSep; IACs and LLE) and determination methods (HPLC and ELISA) of ochratoxin A was carried out. The results obtained proved that the SPE clean-up are reliable at the same level while the LLE procedures shows lowest recovery. The ELISA method gave a lower determination and a low reproducibility than HPLC method.

Quality and safety of oils and fats obtained as co-products or by-products of the food chain and destined to animal production

The aim of the first part of this thesis was to evaluate the effect of trans fatty acid- (TFA), contaminant, polycyclic aromatic hydrocarbon (PAH)- and oxidation productenriched diets on the content of TFA and conjugated linoleic acid (CLA) isomers in meat and liver of both poultry and rabbit. The enriched feedings were prepared with preselected fatty co-and by-products that contained low and high levels of TFA (low, palm fatty acid distillate; high, hydrogenated palm fatty acid distillate), environmental contaminants (dioxins and PCBs) (two different fish oils), PAH (olive oil acid oils and pomace olive oil from chemical refining, for low and high levels) and oxidation products (sunflower-olive oil blend before and after frying), so as to obtain single feedings with three enrichment degrees (high, medium and low) of the compound of interest. This experimental set-up is a part of a large, collaborative European project (http://www.ub.edu/feedfat/), where other chemical and health parameters are assessed. Lipids were extracted, methylated with diazomethane, then transmethylated with 2N KOH/methanol and analyzed by GC and silver-ion TLC-GC. TFA and CLA were determined in the fats, the feedings, meat and liver of both poultry and rabbit. In general, the level of TFA and CLA in meat and liver mainly varied according to those originally found in the feeding fats. It must be pointed out, though, that TFA and CLA accumulation was different for the two animal species, as well as for the two types of tissues. The TFA composition of meat and liver changes according to the composition of the oils added to the feeds with some differences between species. Chicken meat with skin shows higher TFA content (2.6–5.4 fold) than rabbit meat, except for the “PAH” trial. Chicken liver shows higher TFA content (1.2–2.1 fold) than rabbit liver, except for the “TRANS” and “PAH” trials. In both chicken and rabbit meats, the TFA content was higher for the “TRANS” trial, followed by the “DIOXIN” trial. Slight differences were found on the “OXIDATION” and “PAH” trends in both types of meats. In both chicken and rabbit livers, the TFA content was higher for the “TRANS” trial, followed by those of the “PAH”, “DIOXIN” and “OXIDATION” trials. This trend, however, was not identical to that of feeds, where the TFA content varied as follows: “TRANS” > “DIOXIN” >“PAH” > “OXIDATION”. In chicken and rabbit meat samples, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, except for the “DIOXIN” trial where C18:3 TFA > C18:2 TFA. In chicken and rabbit liver samples of the “TRANS” and “OXIDATION” trials, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, whereas C18:3 TFA > C18:2 in the “DIOXIN” trial. Slight differences were found on the “PAH” trend in livers from both species. The second part of the thesis dealt with the study of lipid oxidation in washed turkey muscle added with different antioxidants. The evaluation on the oxidative stability of muscle foods found that oxidation could be measured by headspace solid phase microestraction (SPME) of hexanal and propanal. To make this method effective, an antioxidant system was added to stored muscle to stop the oxidative processes. An increase in ionic strength of the sample was also implemented to increase the concentration of aldehydes in the headspace. This method was found to be more sensitive than the commonly used thiobarbituric acid reactive substances (TBARs) method. However, after antioxidants were added and oxidation was stopped, the concentration of aldehydes decreased. It was found that the decrease in aldehyde concentration was due to the binding of the aldehydes to muscle proteins, thus decreasing the volatility and making them less detectable.

Use of sub-lethal high pressure homogenization (HPH) treatments to enhance functional properties of lactic acid bacteria probiotic strains

The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

New catalytic processes for the synthesis of adipic acid

The aim of my Ph.D. research was to study the new synthetic ways for the production of adipic acid. Three different pathways were studied: i) oxidation of cyclohexanone with molecular oxygen using Keggin – heteropolycompounds as the catalyst, ii) Baeyer – Villiger oxidation of cyclohexanone with hydrogen peroxide in the presence of two different heterogeneous catalysts, titanium silicalite and silica grafted decatungstate, iii) two step synthesis of adipic acid starting from cyclohexene via 1,2-cyclohexanediol. The first step was catalyzed by H2WO4 in the presence of the phase transfer catalyst, the oxidant was hydrogen peroxide. The second step, oxidation of 1,2 – cyclohexanediol was performed in the presence of oxygen and the heterogeneous catalyst – ruthenium on alumina. The results of my research showed that: i) Oxidation of cyclohexanone with molecular oxygen using Keggin heteropolycompounds is possible, anyway the conversion of ketone is low and the selectivity to adipic acid is lowered by the consecutive reaction to from lower diacids. Moreover it was found out, that there are two mechanisms involved: redox type and radicalic chain-reaction autoxidation. The presence of the different mechanism is influenced by the reaction condition. ii) It is possible to perform thermally activated oxidation of cyclohexanone and obtain non negligible amount of the products (caprolactone and adipic acid). Performing the catalyzed reaction it was demonstrated that the choice of the reaction condition and of the catalyst plays a crucial role in the product selectivity, explaining the discrepancies between the literature and our research. iii) Interesting results can be obtained performing the two step oxidation of cyclohexene via 1,2-cyclohexanediol. In the presence of phase transfer catalyst it is possible to obtain high selectivity to alcohol with stoichiometric amount of oxidant. In the second step of the synthesis, the conversion of alcohol is rather low with modest selectivity to adipic acid

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Study of thiabendazole resistance and volatile organic compounds production of Penicillium expansum strains

The years of excessive use of thiabendazole to control Penicillium expansum has induced the development of resistance. Sensitivity of fourty eight strains collected from orchards and packinghouses in Emilia Romagna to pure and commercial TBZ was determined in vitro on TBZ amended medium (400μg/mL). Out of 48 strains, 35 were thiabendazole-sensitive (S) and 13 were thiabendazole-resistant (R). Microtiter assay adapted to P. expansum, showed EC50 values ranging from 54 to 320 μg/mL for ten TBZ-resistant strains. At the highest dose (50 μg/mL), resistant strains growth was not inhibited and the reported MICs value were >1000 μg/mL. Therefore, preliminary screening combined with microtiter assay, can be a good strategy to test susceptibility to TBZ. Mutations in the β-tubulin gene were studied on amino acid sequences from residue 167 to residue 357 of 10 P. expansum strains. Mutation at codon 198 was associated with TBZ-resistance. However, its absence in 3 resistant strains can be explained by the involvement of other mechanisms. Moreover, a P. expansum strain LB8/99 showed good antifungal effect against some fungal pathogens through double petri dish assay. It inhibited both mycelium growth and conidia germination of B. cinerea, C. acutatum, and M. laxa, and reduced significantly by 53% and 18% respectively P. expansum. Three major VOCS: geosmin, phenethyl alcolhol (PEA) and an unknown substance were identified by GC-MS analysis. Consistent fumigation of fungal pathogens with PEA (1230 mg/mL), inhibited both conidia germination and mycelium growth of all pathogens, except conidia germination of P. expansum that was reduced by 90% with respect to control. While, the concentration of PEA produced naturally by LB8/99 was ineffective in controlling the pathogens and seemed to have a synergic or additive effect with the other VOCS. Investigations to study the biofumigant effect of LB8/99 on other commodities like seeds and seedlings are in progress.

Sustainable processes using heterogeneous acid catalysts. Some examples of industrial interest.

In recent years the need for the design of more sustainable processes and the development of alternative reaction routes to reduce the environmental impact of the chemical industry has gained vital importance. Main objectives especially regard the use of renewable raw materials, the exploitation of alternative energy sources, the design of inherently safe processes and of integrated reaction/separation technologies (e.g. microreactors and membranes), the process intensification, the reduction of waste and the development of new catalytic pathways. The present PhD thesis reports results derived during a three years research period at the School of Chemical Sciences of Alma Mater Studiorum-University of Bologna, Dept. of Industrial Chemistry and Materials (now Dept. of Industrial Chemistry “Toso Montanari”), under the supervision of Prof. Fabrizio Cavani (Catalytic Processes Development Group). Three research projects in the field of heterogeneous acid catalysis focused on potential industrial applications were carried out. The main project, regarding the conversion of lignocellulosic materials to produce monosaccharides (important intermediates for production of biofuels and bioplatform molecules) was financed and carried out in collaboration with the Italian oil company eni S.p.A. (Istituto eni Donegani-Research Center for non-Conventional Energies, Novara, Italy) The second and third academic projects dealt with the development of green chemical processes for fine chemicals manufacturing. In particular, (a) the condensation reaction between acetone and ammonia to give triacetoneamine (TAA), and (b) the Friedel-Crafts acylation of phenol with benzoic acid were investigated.

A study of new and more sustainable catalytic routes for the synthesis of adipic acid

The aim of my PhD research project was to investigate new and more sustainable routes, compared to those currently used, for the production of adipic acid (AA). AA is a very important chemical intermediate. The main use of AA is the production of Nylon-6,6 fibers, resins, polyesters, plasticizers. My project was divided into two parts: 1. The two-step oxidation of cyclohexene, where the latter is first oxidized into trans-1,2-cyclohexanediol (CHD) with aqueous hydrogen peroxide, and then the glycol is transformed into AA by reaction with molecular oxygen. Various catalysts were investigated in this process, both heterogeneous (alumina-supported Ru(OH)x and Au nanoparticles supported on TiO2, MgO and Mg(OH)2) and homogeneous (polyoxometalates). We also studied the mechanism of CHD oxidation with oxygen in the presence of these catalysts. 2. Baeyer-Villiger oxidation of cyclohexanone with aqueous hydrogen peroxide into ɛ-caprolactone, as a first step on the way to produce AA. Study on the mechanism of the uncatalyzed (thermal) oxidation of cyclohexanone were also carried out. Investigation on how the different heterogeneous catalysts affect the formation of the reaction products and their distribution was done.

Methane production through anaerobic digestion of dedicated energy crops

Methane yield of ligno-cellulosic substrates (i.e. dedicated energy crops and agricultural residues) may be limited by their composition and structural features. Hence, biomass pre-treatments are envisaged to overcome this constraint. This thesis aimed at: i) assessing biomass and methane yield of dedicated energy crops; ii) evaluating the effects of hydrothermal pre-treatments on methane yield of Arundo; iii) investigating the effects of NaOH pre-treatments and iv) acid pre-treatments on chemical composition, physical structure and methane yield of two dedicated energy crops and one agricultural residue. Three multi-annual species (Arundo, Switchgrass and Sorghum Silk), three sorghum hybrids (Trudan Headless, B133 and S506) and a maize, as reference for AD, were studied in the frame of point i). Results exhibit the remarkable variation in biomass yield, chemical characteristics and potential methane yield. The six species alternative to maize deserve attention in view of a low need of external inputs but necessitate improvements in biodegradability. In the frame of point ii), Arundo was subjected to hydrothermal pre-treatments at different temperature, time and acid catalyst (with and without H2SO4). Pre-treatments determined a variable effect on methane yield: pre-treatments without acid catalyst achieved up to +23% CH4 output, while pre-treatments with H2SO4 catalyst incurred a methanogenic inhibition. Two biomass crops (Arundo and B133) and an agricultural residue (Barley straw) were subject to NaOH and acid pre-treatments, in the frame of point iii) and iv), respectively. Different pre-treatments determined a change of chemical and physical structure and an increase of methane yield: up to +30% and up to +62% CH4 output in Arundo with NaOH and acid pre-treatments, respectively. It is thereby demonstrated that pre-treatments can actually enhance biodegradability and subsequent CH4 output of ligno-cellulosic substrates, although pre-treatment viability needs to be evaluated at the level of full scale biogas plants in a perspective of profitable implementation.

Glycerol oxidehydration to acrylic acid on complex mixed-metal oxides

The project of this Ph.D. thesis is based on a co-supervised collaboration between Università di Bologna, ALMA MATER STUDIORUM (Italy) and Instituto de Tecnología Química, Universitat Politècnica de València ITQ-UPV (Spain). This Ph.D. thesis is about the synthesis, characterization and catalytic testing of complex mixed-oxide catalysts mainly related to the family of Hexagonal Tungsten Bronzes (HTBs). These materials have been little explored as catalysts, although they have a great potential as multifunctional materials. Their peculiar acid properties can be coupled to other functionalities (e.g. redox sites) by isomorphous substitution of tungsten atoms with other transition metals such as vanadium, niobium and molybdenum. In this PhD thesis, it was demonstrated how it is possible to prepare substituted-HTBs by hydrothermal synthesis; these mixed-oxide were fully characterize by a number of physicochemical techniques such as XPS, HR-TEM, XAS etc. They were also used as catalysts for the one-pot glycerol oxidehydration to acrylic acid; this reaction might represent a viable chemical route to solve the important issue related to the co-production of glycerin along the biodiesel production chain. Acrylic acid yields as high as 51% were obtained and important structure-reactivity correlations were proved to govern the catalytic performance; only fine tuning of acid and redox properties as well as the in-framework presence of vanadium are fundamental to achieve noteworthy yields into the acid monomer. The overall results reported herein might represent an important contribution for future applications of HTBs in catalysis as well as a general guideline for a multifaceted approach for their physicochemical characterization.

Chemopreventive effects of eicosapentaenoic acid free fatty acid in a murine model of colitis-associated colorectal cancer

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyps formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control, resulted in the enrichment of Lactobacillus species in the gut microbiota and led to tumor suppressor miR34-a induction. In conclusion, our data suggest that EPA-FFA is an effective chemopreventive agent in CAC.