Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termica

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

Biofilms on exposed monumental stones: mechanism of formation and development of new control methods

Within the stone monumental artefacts artistic fountains are extremely favorable to formation of biofilms, giving rise to biodegradation processes related with physical-chemical and visual aspect alterations, because of their particular exposure conditions. Microbial diversity of five fountains (two from Spain and three from Italy) was investigated. It was observed an ample similarity between the biodiversity of monumental stones reported in literature and that one found in studied fountains. Mechanical procedures and toxic chemical products are usually employed to remove such phototrophic patinas. Alternative methods based on natural antifouling substances are recently experimented in the marine sector, due to their very low environmental impact and for the bio settlement prevention on partially immersed structures of ships. In the present work groups of antibiofouling agents (ABAs) were selected from literature for their ability to interfere, at molecular level, with the microbial communication system “quorum sensing”, inhibiting the initial phase of biofilm formation. The efficacy of some natural antibiofoulants agents (ABAs) with terrestrial (Capsaicine - CS, Cinnamaldehyde - CI) and marine origin (Zosteric Acid - ZA, poly-Alkyl Pyridinium Salts – pAPS and Ceramium botryocarpum extract - CBE), incorporated into two commercial coatings (Silres BS OH 100 - S and Wacker Silres BS 290 - W) commonly used in stone conservation procedures were evaluated. The formation of phototrophic biofilms in laboratory conditions (on Carrara marble specimens and Sierra Elvira stone) and on two monumental fountains (Tacca’s Fountain 2 - Florence, Italy and Fountain from Patio de la Lindaraja - Alhambra Palace, Granada, Spain) has been investigated in the presence or absence of these natural antifouling agents. The natural antibiofouling agents, at tested concentrations, demonstrated a certain inhibitory effect. The silane-siloxane based silicone coating (W) mixing with ABAs was more suitable with respect to ethyl silicate coating (S) and proved efficacy against biofilm formation only when incompletely cured. The laboratory results indicated a positive action in inhibiting the patina formation, especially for poly-alkyl pyridinium salts, zosteric acid and cinnamaldehyde, while on site tests revealed a good effect for zosteric acid.

Methoden und Modelle zur Phytoplanktonanalyse am Beispiel einer Restaurierungsmaßnahme an der Bleiloch Talsperre

Methoden und Modelle zur Phytoplanktonanalyse am Beispiel einer Restaurierungsmaßnahme an der Bleiloch Talsperre Zusammenfassung An der hypertrophen Bleiloch Talsperre (Thüringen, BRD) werden die Auswirkungen der 1997 in Betrieb genommenen partiellen Destratifikation (0- 20m) untersucht. Ziel der Maßnahme war die Hemmung des Phytoplanktonwachstums durch Lichtlimitation.Da es nicht gelang, auch die Oberflächenlammelle in die Durchmischung einzubeziehen, wurde auch keine Reduktion des Phytoplanktons beobachtet. Seine zeitliche Dynamik nahm deutlich zu und es traten neben typischen Längsgradienten auch extreme, kleinräumige Konzentrationsunterschiede auf. Die vorher häufigen Cyanobakterien spielten jedoch keine quantitativ bedeutsame Rolle mehr. Es wird diskutiert, wie die Belüftung die Saisonalität des Planktons stört und zu Verschiebungen der Zusammensetzung führt. Der Zusammenhang zwischen der Chlorophyll a Menge und der Stabilität der Wassersäule sowie der Einfluß wachstumsbestimmender Größen werden modelliert. Methodische Schwerpunkte umfassen die Auswertung von Phytoplankton-Pigmentdaten (HPLC-Analyse) und die Entwicklung einer Durchflußzytometer-Methode. Bei letzerer werden an lebenden Phytoplanktern die Vorwärtsstreuung sowie vier Fluoreszenzen (2 Phycoerythrine, Phycocyanin und Chlorophyll a, 514nm Anregungswellenlänge) gemessen. Zur Klassifizierung von Algen anhand von Referenzen werden zwei Methoden entwickelt, Möglichkeiten des Sortierens aufgezeigt und es wird ein Vergleich mit Mikroskopie und HPLC- Pigmentanalyse durchgeführt. Anhand von Modellrechnungen wird demonstriert, wie sich systematische bzw. zufällige Abweichungen der Pigmentstöchiometrien auf die Berechnung von Algengruppenanteilen auswirken. Strategien zur Auswertung von Pigmentdaten werden empfohlen.

Surfactants and their aerobic degradation products

Die Ziele der vorliegenden Arbeit waren 1) die Entwicklung und Validierung von sensitiven und substanz-spezifischen Methoden für die quantitative Bestimmung von anionischen, nichtionischen und amphoteren Tensiden und deren Metaboliten in wässrigen Umweltproben unter Einsatz leistungsfähiger, massenspektrometrischer Analysengeräte,2) die Gewinnung von aeroben, polaren Abbauprodukten aus Tensiden in einem die realen Umweltbedingungen simulierenden Labor-Festbettbioreaktor (FBBR), dessen Biozönose oberflächenwasserbürtig war,3) zur Aufklärung des Abbaumechanismus von Tensiden neue, in 2) gewonnene Metabolite zu identifizieren und massenspektrometrisch zu charakterisieren ebenso wie den Primärabbau und den weiteren Abbau zu verfolgen,4) durch quantitative Untersuchungen von Tensiden und deren Abbauprodukten in Abwasser und Oberflächenwasser Informationen zu ihrem Eintrag und Verhalten bei unterschiedlichen hydrologischen und klimatischen Bedingungen zu erhalten,5) das Verhalten von persistenten Tensidmetaboliten in Wasserwerken, die belastetes Oberflächenwasser aufbereiten, zu untersuchen und deren Vorkommen im Trinkwasser zu bestimmen,6) mögliche Schadwirkungen von neu entdeckten Metabolite mittels ökotoxikologischer Biotests abzuschätzen,7) durch Vergleich der Felddaten mit den Ergebnissen der Laborversuche die Umweltrelevanz der Abbaustudien zu belegen. Die Auswahl der untersuchten Verbindungen erfolgte unter Berücksichtigung ihres Produktionsvolumens und der Neuheit auf dem Tensidmarkt. Sie umfasste die Waschmittelinhaltsstoffe lineare Alkylbenzol-sulfonate (LAS), welches das Tensid mit der höchsten Produktionsmenge darstellte, die beiden nichtionischen Tenside Alkylglucamide (AG) und Alkylpolyglucoside (APG), ebenso wie das amphotere Tensid Cocamidopropylbetain (CAPB). Außerdem wurde der polymere Farbübertragungsinhibitor Polyvinylpyrrolidon (PVP) untersucht.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

Bioremediation implies the use of living organisms, primarily microorganisms, to convert environmental contaminants into less toxic forms. The impact of the consequences of hydrocarbon release in the environment maintain a high research interest in the study of microbial metabolisms associated with the biodegradation of aromatic and aliphatic hydrocarbons but also in the analysis of microbial enzymes that can convert petroleum substrates to value-added products. The studies described in this Thesis fall within the research field that directs the efforts into identifying gene/proteins involved in the catabolism of n-alkanes and into studying the regulatory mechanisms leading to their oxidation. In particular the studies were aimed at investigating the molecular aspects of the ability of Rhodococcus sp. BCP1 to grow on aliphatic hydrocarbons as sole carbon and energy sources. We studied the ability of Rhodococcus sp. BCP1 to grow on gaseous (C2-C4), liquid (C5-C16) and solid (C17-C28) n-alkanes that resulted to be biochemically correlated with the activity of one or more monooxygenases. In order to identify the alkane monooxygenase that is involved in the n-alkanes degradation pathway in Rhodococcus sp. BCP1, PCR-based methodology was applied by using degenerate primers targeting AlkB monooxygenase family members. As result, a chromosomal region, including the alkB gene cluster, was cloned from Rhodococcus sp. BCP1 genome. We characterized the products of this alkB gene cluster and the products of the orfs included in the flanking regions by comparative analysis with the homologues in the database. alkB gene expression studies were carried out by RT-PCR and by the construction of a promoter probe vector containing the lacZ gene downstream of the alkB promoter. B-galactosidase assays revealed the alkB promoter activity induced by n-alkanes and by n-alkanes metabolic products. Furthermore, the transcriptional start of alkB gene was determined by primer extension procedure. A proteomic approach was subsequently applied to compare the protein patterns expressed by BCP1 growing on n-butane, n-hexane, n-hexadecane or n-eicosane with the protein pattern expressed by BCP1 growing on succinate. The accumulation of enzymes specifically induced on n-alkanes was determined. These enzymes were identified by tandem mass spectrometry (LC/MS/MS). Finally, a prm gene, homologue to the gene family coding for soluble di-iron monooxygenases (SDIMOs), has been isolated from Rhodococcus sp. BCP1 genome. This gene product could be involved in the degradation of gaseous n-alkanes in this Rhodococcus strain. The versatility in utilizing hydrocarbons and the discovery of new remarkable metabolic activities outline the potential applications of this microorganism in environmental and industrial biotechnologies.

Biodegradazione aerobica cometabolica di cloroformio: studio di fattibilità in reattori a biomassa adesa

Chlorinated Aliphatic Hydrocarbons (CAHs) are widespread wastewater and groundwater contaminants and represent a real danger for human health and environment. This research is related to the biodegradation technologies to treat chlorinated hydrocarbons. In particular the study of this thesis is focused on chloroform cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow biofilm reactors, which are used for in-situ and on-site treatments. The work was divided in two parts: in the first one an experimental study has been conducted in two packed-bed reactors (PBRs) for a period of 370 days; in the second one a fluid dynamics and kinetic model has been developed in order to simulate the experimental data concerning a previous study made in a 2-m continuous-flow sand-filled reactor. The goals of the first study were to obtain preliminary information on the feasibility of chloroform biodegradation by BCP1 under attached-cell conditions and to evaluate the applicability of the pulsed injection of growth substrate and oxygen to biofilm reactors. The pulsed feeding represents a tool to control the clogging and to ensure a long bioreactive zone. The operational conditions implemented in the PBRs allowed the attainment of a 4-fold increase of the ratio of chloroform degraded to substrate consumed, in comparison with the phase of continuous substrate supply. The second study was aimed at identifying guidelines for optimizing the oxygen/substrate supply schedule, developing a reliable model of chloroform cometabolism in porous media. The tested model led to a suitable interpretation of the experimental data as long as the ratio of CF degraded to butane consumed was ≤ 0.27 mgchloroform /mgbutane. A long-term simulation of the best-performing schedule of pulsed oxygen/substrate supply indicated the attainment of a steady state condition characterized by unsatisfactory bioremediation performances, evidencing the need for a further optimization of the pulsed injection technique.

Effetto di un packaging innovativo sulla qualità di nettarine

L’imballaggio alimentare si può definire come un sistema coordinato per disporre i beni per il trasporto, la distribuzione, la conservazione, la vendita e l’utilizzo. Uno dei materiali maggiormente impiegati, nell’industria alimentare, per la produzione di imballaggi sono le materie plastiche. Esse sono sostanze organiche derivanti da petrolio greggio, sono composti solidi allo stato finito, ma possono essere modellate allo stato fluido. Un imballaggio alimentare deve svolgere determinate funzioni tra cui: - contenimento del prodotto - protezione del prodotto da agenti esterni - logistica - comunicativa - funzionale - ecologica L'ultimo punto sopracitato è il principale problema delle materie plastiche derivanti dal petrolio greggio. Questi materiali sono difficilmente riciclabili perché spesso un imballaggio è composto da più materiali stratificati o perché si trova a diretto contatto con gli alimenti. Inoltre questi materiali hanno un lungo tempo di degradazione (da 100 a 1000 anni) che ne rendono difficile e costoso lo smaltimento. Per questo nell’ultimo decennio è cominciata la ricerca di un materiale plastico, flessibile alle esigenze industriali e nel contempo biodegradabile. Una prima idea è stata quella di “imitare la natura” cercando di replicare macromolecole già esistenti (derivate da amido e zuccheri) per ottenere una sostanza plastico-simile utilizzabile per gli stessi scopi, ma biodegradabile in circa sei mesi. Queste bioplastiche non hanno preso piede per l’alto costo di produzione e perché risulta impossibile riconvertire impianti di produzione in tutto il mondo in tempi brevi. Una seconda corrente di pensiero ha indirizzato i propri sforzi verso l’utilizzo di speciali additivi aggiunti in minima misura (1%) ai classici materiali plastici e che ne permettono la biodegradazione in un tempo inferiore ai tre anni. Un esempio di questo tipo di additivi è l’ECM Masterbatch Pellets che è un copolimero di EVA (etilene vinil acetato) che aggiunto alle plastiche tradizionali rende il prodotto finale completamente biodegradabile pur mantenendo le proprie caratteristiche. Scopo di questo lavoro di tesi è stato determinare le modificazioni di alcuni parametri qualitativi di nettarine di Romagna(cv.-Alexa®) confezionate-con-film-plastici-tradizionali-e-innovativi. I campioni di nettarine sono stati confezionati in cestini in plastica da 1 kg (sigillati con un film flow-pack macroforato) di tipo tradizionale in polipropilene (campione denominato TRA) o vaschette in polipropilene additivato (campione denominato BIO) e conservati a 4°C e UR 90-95% per 7 giorni per simulare un trasporto refrigerato successivamente i campioni sono stati posti in una camera a 20°C e U.R. 50% per 4 giorni al fine di simulare una conservazione al punto vendita. Al tempo 0 e dopo 4, 7, 9 e 11 giorni sono state effettuate le seguenti analisi: - coefficiente di respirazione è stato misurata la quantità di CO2 prodotta - indice di maturazione espresso come rapporto tra contenuto in solidi solubili e l’acidità titolabile - analisi di immagine computerizzata - consistenza della polpa del frutto è stata misurata attraverso un dinamometro Texture Analyser - contenuto in solidi totali ottenuto mediante gravimetria essiccando i campioni in stufa sottovuoto - caratteristiche sensoriali (Test Accettabilità) Conclusioni In base ai risultati ottenuti i due campioni non hanno fatto registrare dei punteggi significativamente differenti durante tutta la conservazione, specialmente per quanto riguarda i punteggi sensoriali, quindi si conclude che le vaschette biodegradabili additivate non influenzano la conservazione delle nettarine durante la commercializzazione del prodotto limitatamente ai parametri analizzati. Si ritiene opportuno verificare se il processo di degradazione del polimero additivato si inneschi già durante la commercializzazione della frutta e soprattutto verificare se durante tale processo vengano rilasciati dei gas che possono accelerare la maturazione dei frutti (p.e. etilene), in quanto questo spiegherebbe il maggiore tasso di respirazione e la più elevata velocità di maturazione dei frutti conservati in tali vaschette. Alimentary packaging may be defined as a coordinate system to dispose goods for transport, distribution, storage, sale and use. Among materials most used in the alimentary industry, for the production of packaging there are plastics materials. They are organic substances deriving from crude oil, solid compounds in the ended state, but can be moulded in the fluid state. Alimentary packaging has to develop determinated functions such as: - Product conteniment - Product protection from fieleders agents - logistic - communicative - functional - ecologic This last term is the main problem of plastic materials deriving from crude oil. These materials are hardly recyclable because a packaging is often composed by more stratified materials or because it is in direct contact with aliments. Beside these materials have a long degradation time(from 100 to 1000 years) that make disposal difficult and expensive. For this reason in the last decade the research for a new plastic material is begin, to make industrial demands more flexible and, at the same time, to make this material biodegradable: At first, the idea to “imitate the nature” has been thought, trying to reply macromolecules already existents (derived from amid and sugars) to obtain a similar-plastic substance that can be used for the same purposes, but it has to be biodegradable in about six months. These bioplastics haven’t more success bacause of the high production cost and because reconvert production facilities of all over the wolrd results impossible in short times. At second, the idea to use specials addictives has been thought. These addictives has been added in minim measure (1%) to classics plastics materials and that allow the biodegradation in a period of time under three years. An example of this kind of addictives is ECM Masterbatch Pellets which is a coplymer of EVA (Ethylene vinyl acetate) that, once it is added to tradizional plastics, make final product completely biodegradable however maintaining their own attributes. The objective of this thesis work has been to determinate modifications of some Romagna’s Nectarines’ (cv. Alexa®) qualitatives parameters which have been packaged-with traditional and innovative-plastic film. Nectarines’ samples have been packaged in plastic cages of 1 kg (sealed with a macro-drilled flow-pack film) of traditional type in polypropylene (sample named TRA) or trays in polypropylene with addictives (sample named BIO) and conservated at 4°C and UR 90-95% for 7 days to simulate a refrigerated transport. After that, samples have been put in a camera at 20°C and U.R. 50% for 4 days to simulate the conservation in the market point. At the time 0 and after 4, 7, 9 and 11 days have been done the following analaysis: - Respiration coefficient wherewith the amount CO2 producted has been misurated - Maturation index which is expressed as the ratio between solid soluble content and the titratable acidity - Analysis of computing images - Consistence of pulp of the fruit that has been measured through Texture Analyser Dynanometer - Content in total solids gotten throught gravimetry by the drying of samples in vacuum incubator - Sensorial characteristic (Panel Test) Consequences From the gotten results, the two samples have registrated no significative different scores during all the conservation, expecially about the sensorial scores, so it’s possible to conclude that addictived biodegradable trays don’t influence the Nectarines’ conservation during the commercialization of the product qualifiedly to analized parameters. It’s advised to verify if the degradation process of the addicted polymer may begin already during the commercialization of the fruit and in particular to verify if during this process some gases could be released which can accelerate the maturation of fruits (p.e. etylene), because all this will explain the great respiration rate and the high speed of the maturation of fruits conservated in these trays.

Intensificazione di processi biologici per la Bioremediation aerobica di suoli contaminati

Enzyveba, a partially characterized complex consortium of not-adapted microorganisms developed through prolonged stabilization of organic wastes, was found to markedly intensify the aerobic remediation of aged PAH- and PCB-contaminated soil by acting as a source of exogenous specialized microorganisms and nutrients. Thus, Enzyveba was tested in the bioremediation of Diesel (G1) and HiQ Diesel (G2) contaminated soils under aerobic slurry-phase conditions by means of a chemical, microbiological, ecotoxicological integrated analytical procedure. The addition of Enzyveba resulted in a higher availability of cultivable specialized bacteria and fungi but this resulted in a slight intensification of soil remediation, probably because of the high content of nutrients and specialized microorganisms of the soil. In many cases, the biotreatability of soils impacted by diesel fuel is limited by their poor content of autochthonous pollutant-degrading microorganisms. Thus, bioaugmentation with stable and reproducible cultures with the required broad substrate specificity might be the solution for a successful remediation. Two microbial consortia, ENZ-G1 and ENZ-G2, were enriched from Enzyveba on G1 and G2. Both consortia consist of a similar composition of bacterial and fungal species. They exhibited a comparable and significant biodegradation capability by removing about 90% of 1 g/l of diesel fuel under liquid culture conditions. Given their remarkable biodegradation potential, richness of quite diverse microbes, stability and resistance after cryopreservation at -20 °C for several months, both consortia appear very interesting candidates for bioaugmentation on site. The mycoflora of a soil historically contaminated by high concentration of PCBs was characterised before, at the beginning and at the end of the biotreatment mentioned above. Several mitosporic fungi isolated from soil grew in presence of a mixture of three PCBs congeners when also glucose was provided. This is the first study in which 5 strains of mitosporic species able to biodegrade PCB are reported in the literature.

Novel etheroatom containing aliphatic polyesters for biomedical and environmental applications

Biodegradable polymers for short time applications have attracted much interest all over the world. The reason behind this growing interest is the incompatibility of the polymeric wastes with the environment where they are disposed after usage. Synthetic aliphatic polyesters represent one of the most economically competitive biodegradable polymers. In addition, they gained considerable attention as they combine biodegradability and biocompatibility with interesting physical and chemical properties. In this framework, the present research work focused on the modification by reactive blending and polycondensation of two different aliphatic polyesters, namely poly(butylene succinate) (PBS) and poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE). Both are characterized by good thermal properties, but their mechanical characteristics do not fit the requirements for applications in which high flexibility is requested and, moreover, both show slow biodegradation rate. With the aim of developing new materials with improved characteristics with respect to the parent homopolymers, novel etheroatom containing PBS and PBCE-based fully aliphatic polyesters and copolyesters have been therefore synthesized and carefully characterized. The introduction of oxygen or sulphur atoms along the polymer chains, by acting on chemical composition or molecular architecture, tailored solid-state properties and biodegradation rate: type and amount of comonomeric units and sequence distribution deeply affected the material final properties owing, among all, to the hydrophobic/hydrophilic ratio and to the different ability of the polymer to crystallize. The versatility of the synthesized copolymers has been well proved: as a matter of fact these polymers can be exploited both for biomedical and ecological applications. Feasibility of 3D electrospun scaffolds has been investigated, biocompatibility studies and controlled release of a model molecule showed good responses. As regards ecological applications, barrier properties and eco-toxicological assessments have been conducted with outstanding results. Finally, the ability of the novel polyesters to undergo both hydrolytic and enzymatic degradation has been demonstrated under physiological and environmental conditions.

Response of Microcystis aeruginosa PCC 7806 to different CO2 concentrations

Future climatic change scenarios predict rising of the atmospheric CO2 levels which could favor the proliferation of some harmful bloom-forming cyanobacteria as Microcystis aeruginosa. In the present study, the response of M. aeruginosa strain PCC 7806 to two different partial pressure of CO2 was tested. Sandrini et al. (2013) recently found that several, but not all, M. aeruginosa strains lack the SbtA or BicA HCO3- uptake system genes; the contribution of different Ci transporters to photosynthesis and the difference between low and high affinity activated Ci uptake state were investigated. M. aeruginosa PCC 7806 was cultured in four chemostats containing modified BG11 medium with 10 mM NaNO3 and no presence of NaCl, NaHCO3, Na2CO3 and additional buffers. A wide variety of analysis on samples collected from continuous cultures – such as A750, medium composition, cellular composition, cell counting, mini-PAM, measurements with the O2 optode, Aminco, 77K fluorescence emission spectra – was carried out. Data analysis results showed that the increased CO2 concentration has a big effect on M. aeruginosa PCC 7806. Experiments were performed using the Oxy-4 O2 optode apparatus in order to measure the photosynthetic O2 evolution of samples taken from both batch and chemostat cultures. At low bicarbonate concentration, an evident inhibition of Na+-dependent HCO3- transporter BicA by LiCl at 25 mM was observed. The consequent addition of 25 mM NaCl was able to counteract the Li+ effect at pH 8.0 but not at pH 10.0. In the latter case, only the addition of a higher amount of HCO3- led to photosynthetic O2 evolution suggesting the important role of the BicA transporter. However, further studies are needed to better explain the results obtained as high pH levels might have an influence on the transport systems, altering the mechanism of pH regulation and the functioning of Na+/H+ antiporter systems.

Physiologische Veränderungen bei der Thylakoidmembranbiogenese und die Funktionsweise des Nukleotidaustauschfaktors GrpE in Cyanobakterien

Bei Wachstum im Dunkeln zeigten sich rudimentäre Thylakoidstrukturen, wobei nach dem Transfer ins Licht ein vollständiges Thylakoidmembransystem erneut ausgebildet wurde. Parallel stieg, der Chlorophyllgehalt pro Zelle und das Verhältnis von Phycobilisomen zu Chlorophyll verschob sich erneut auf die Seite des Chlorophylls. Das bei Wachstum im Dunkeln als Monomer vorliegende PS II, war nicht funktional. Nach dem Transfer ins Licht, war nach etwa acht bis zwölf Stunden ein aktives PS II zu detektieren. Das PS I lag nach der Inkubation im Dunkeln, in geringerer Konzentration aber aktiv als Trimer in den Zellen vor.rnZwei Typ I Signalpeptidasen aus Synechocystis sp. PCC 6803 zeigten Unterschiede im Bezug auf ihre intrazelluläre Lokalisation. Für die Untersuchungen der Lokalisation konnte ein neues System der Fluoreszenzmikroskopie entwickelt und erfolgreich eingesetzt werden. Das LepB1 zeigte einen (auto-) proteolytischen Abbau. Für Untersuchungen zur katalytischen Aktivität wurden Vorläuferproteine als Substrate für LepB2 identifiziert.rnDie Funktionsweise der GrpE-Proteine aus verwandten Cyanobakterien zeigt Unterschiede. Bei beiden GrpE-Proteinen erfolgt der reversible Übergang von einem Dimer hin zu einem Monomer innerhalb eines physiologisch relevanten Temperaturbereichs in einem Schritt. Bei dem Protein aus Synechocystis sp. ist der N-Terminus und bei dem Protein aus dem thermophilen Bakterium Thermosynechococcus der C-Terminus für die Dimerisierung essentiell. rn

Untersuchungen zur Funktion multipler DnaJ-Proteine in dem Cyanobakterium Synechocystis sp. PCC 6803

Sowohl in Synechocystis sp. PCC 6803 als auch in anderen Cyanobakterien konnten multiple DnaJ-Proteine nachgewiesen werden, deren Funktion jedoch noch weitestgehend unverstanden ist. Im Rahmen dieser Arbeit wurden die Funktionen der multiplen DnaJ-Proteine von Synechocystis sp. charakterisiert. Das DnaJ-Protein, Sll0897 gehört aufgrund seiner Domänenstruktur zu den Typ I-Proteinen, Slr0093 und Sll1933 zu den Typ II-Proteinen und Sll0909, Sll1011, Sll1384 und Sll1666 zu den Typ III DnaJ-Proteinen. Durch Komplementationsstudien des E. coli ΔdnaJ-Stammes OD259 konnte eine Komplementation des Wachstumsdefekts bei höheren Temperaturen durch die Proteine Slr0093 und Sll0897 gezeigt werden. In Synechocystis war eine komplette Disruption von sll1933 nicht möglich, weshalb das Protein Sll1933 unter normalen Wachstumsbedingungen essentiell ist. Doppelte Insertionmutationen waren lediglich bei der Kombination der Gene sll0909 und sll1384 möglich. Untersuchungen des Wachstumsverhaltens der dnaJ-Disruptions-stämme unter Hitze- und Kältestressbedingungen zeigten, dass das Protein Sll0897 eine wichtige Funktion bei der Stressantwort in Synechocystis besitzt und unter Hitzestressbedingungen essentiell ist. Eine vollständige Deletion des Gens sll0897 war Synechocystis sp. bereits unter normalen Wachstumsbedingungen nicht möglich. Bei den für ein Wachstum mindestens notwendigen Domänen des Sll0897 handelt es sich um die charakteristische J-Domäne und die Glycin-Phenylalanin-reiche Domäne. Unter Hitzestressbedingungen ist das Volllängen-Protein Sll0897 für ein Wachstum essentiell. rnNeben den in vivo Wachstumsexperimenten wurde eine Methode zur heterologen Expression der sieben DnaJ-Proteine in E. coli und einer nativen Reinigung von Slr0093, Sll0897, Sll0909 und Sll1666 etabliert. Untersuchungen zur Thermostabilität der gereinigten Proteine zeigten für das Slr0093 und Sll1666 einen reversiblen Prozess, wodurch sie auch nach dem Hitzestress noch als Faltungshelfer fungieren können. Bei den Proteinen Sll0897 und Sll0909 ist der Prozess jedoch nicht reversibel, so dass sie nach Hitzestresseinwirkung neu synthetisiert oder durch Chaperoneinwirkung korrekt gefaltet werden müssen. Die Affinitäts-„Pull-Down“ Analysen lieferten keine klaren Hinweise auf die DnaK-Interaktionspartner der Proteine Slr0093, Sll0897, Sll0909 und Sll1666, weshalb weitere Untersuchungen notwendig sind. Mit Hilfe der Gelfiltrationsanalysen konnten die errechneten molaren Massen der Proteine Slr0093 und Sll1666 bestätigt und beide Proteine in einer monomeren Form nachgewiesen werden. Die DnaJ-Proteine Sll0897 und Sll0909 konnten in zwei oligomeren Zuständen detektiert werden. Analysen der ATPase-Aktivität des DnaK2-Proteins alleine und des DnaK2-Proteins zusammen mit den DnaJ-Proteinen Slr0093, Sll0897, Sll0909 und Sll1666 zeigten eine Steigerung der ATP-Hydrolyserate bei der Interaktion von DnaK und DnaJ, wobei Sll0897 die größte Steigerung der ATPase-Aktivität des DnaK2 induzierte.

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

Study of the genes involved in Naphthenic acids (NAs) degradation by Rhodococcus spp.

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.