Monitoraggio e analisi di segnali ORP, pH, DO finalizzato al controllo del processo di predenitrificazione eseguito su impianto pilota a F.A.

Il lavoro sperimentale condotto nell’ambito della presente tesi è stato svolto su un impianto pilota a flusso continuo con schema predenitro/nitro in scala di laboratorio, alimentato con refluo sintetico ed installato presso i laboratori della Sezione ACS PROT IDR - Gestione Risorse Idriche dell’ENEA (sede di Bologna). Le finalità della sperimentazione erano mirate ad una verifica preliminare sull’utilizzo di segnali indiretti, quali pH, ORP, DO, nel monitoraggio in tempo reale dei processi biologici, mirata alla possibilità di implementare un sistema di controllo automatico di un impianto di depurazione, capace di minimizzare i costi di gestione e in grado di garantire, in ogni caso, elevate efficienze depurative. Nel presente studio sono stati esaminati gli aspetti metodologici e tecnologici alla base dell’automazione di un impianto di trattamento di acque reflue, avendo il duplice obiettivo di garantire una buona qualità dell’effluente e un contestuale risparmio energetico. In particolare si è studiato un problema molto diffuso: il controllo della rimozione dell’azoto in un sistema a fanghi attivi con predenitrificazione per l’ossidazione congiunta di carbonio ed azoto, ponendolo in relazione con la variazione dei tempi di commutazione in un processo SBR. L’obiettivo primario della sperimentazione effettuata è stato quello di portare il sistema in un regime di equilibrio stazionario, in modo da poter definire delle condizioni di regime per le quali, ad un ingresso costante e noto, corrispondesse un’uscita altrettanto costante che mantenesse tali condizioni fino a quando non fosse cambiato l’ingresso e/o le condizioni al contorno. A questo scopo si è provveduto, in primo luogo, allo studio di un influente sintetico con cui alimentare l’impianto, di composizione tale da simulare in tutte le sue caratteristiche un refluo civile reale e, successivamente, alla valutazione di una configurazione impiantistica idonea per il raggiungimento dell’equilibrio dei processi. La prima parte della sperimentazione è stata dedicata all’avviamento dell’impianto, effettuando un monitoraggio continuo dell’intero sistema allo scopo di impostare i parametri operativi e in modo da ottenere condizioni stabili per effettuare le successive sperimentazioni.

Analisi di pH, ORP e DO finalizzata all'utilizzo di segnali indiretti per il monitoraggio e il controllo automatico di impianti di depurazione a fanghi attivi

Con il presente lavoro di tesi si è studiata la possibilità di estrarre informazioni sugli andamenti dei processi biologici dall’osservazione dei segnali di pH, potenziale di ossido riduzione (ORP) e ossigeno disciolto (DO) nelle vasche di processo di un impianto a fanghi attivi a flusso continuo (predenitro/nitro) per verificare la possibilità di utilizzare questo tipo di sonde per lo sviluppo di sistemi per il controllo automatico e la gestione intelligente, eventualmente remota, degli impianti di depurazione. Il lavoro di sperimentazione è stato svolto su un impianto pilota a flusso continuo con schema predenitro – nitro, costruito ed installato presso i laboratori della sede Enea di Bologna (Sezione ACS PROT IDR - Gestione Risorse Idriche). L’obiettivo primario della sperimentazione è stato quello di portare il sistema in uno stato stazionario di equilibrio, così da poter stabilire delle condizioni di funzionamento note e costanti, riscontrabili anche nei segnali di riferimento. Tali condizioni sono state definite prendendo come riferimento un impianto reale noto, funzionante in condizioni di processo costanti e medie, definendo contestualmente test sperimentali che riproducessero condizioni riscontrabili sullo stesso impianto reale. Le condizioni del sistema sono state monitorate costantemente, attraverso il monitoraggio giornaliero dei processi, effettuato con attività di campionamento e di analisi, osservando costantemente i segnali indiretti.

Double-stimulus sensitive (temperature and pH) self-assemblable polymers

The rheological properties of block co-polymers in water solution at different pH have been investigated. The block copolymers are based on different architectures containing poly(ethylene glycol), poly(propylene glycol) and different blocks of polymer that change their hydrophobic/hydrophilic behavior as a function of pH. The polymer chains of the starting material were extended at their functional ends with the pH-sensitive units using ATRP; this mechanism of controlled radical polymerization was chosen because of the need to minimize polydispersity and avoid transfer reactions possibly leading to homopolymeric inpurities. The starting material were modified in order to use them as macroinitiator for ATRP. The kinetic of each ATRP reaction has been investigated, in order to be able to synthesize polymers with different degree of polymerization, stopping the reaction when the desired polymers chain length has been reached. We will use polymer chains with different basicity and degree of polymerization to link any possible effect of their presence to the conditions under which they become hydrophobic. It has been shown that the rate of polymerization changes changing the type of macroinitiator and the type of monomer synthesized. The slowest rate of polymerization is the one with the most hindered monomer synthesized using the macroinitiator with the highest molecular weight. The water solubility of the synthesized polymers changes depending on the pH of the solution and on the structure of the polymers. It has been shown using 1H-NMR that some of the synthesized polymers are capable to self-aggregation in water solution. The self-aggregation and the type of aggregation is influenced from the structure of the polymer and from the pH of the solution. Changing the structure of the polymers and the pH it is possible to obtain different type of aggregates in solution. This aggregates differ for the volume occupied from them, and for their hardness. Rheological measurements have been demonstrated that the synthesized polymers are capable to form gel phases. The gelation temperature changes changing the structure of the aggregates in solution and it is possible to correlate the changing in the gelation temperature with the changing in the structure of the polymer.

Changes in the microbial community based on seawater pH variations

Ocean acidification is an effect of the rise in atmospheric CO2, which causes a reduction in the pH of the ocean and generates a number of changes in seawater chemistry and consequently potentially impacts seawater life. The effect of ocean acidification on metabolic processes (such as net community production and community respiration and on particulate organic carbon (POC) concentrations was investigated in summer 2012 at Cap de la Revellata in Corsica (Calvi, France). Coastal surface water was enclosed in 9 mesocosms and subjected to 6 pCO2 levels (3 replicated controls and 6 perturbations) for approximately one month. No trend was found in response to increasing pCO2 in any of the biological and particulate analyses. Community respiration was relatively stable throughout the experiment in all mesocosms, and net community production was most of the time close to zero. Similarly, POC concentrations were not affected by acidification during the whole experimental period. Such as the global ocean, the Mediterranean Sea has an oligotrophic nature. Based on present results, it seems likely that seawater acidification will not have significant effects on photosynthetic rates, microbial metabolism and carbon transport.

Uptake mechanism, intracellular trafficking and endo-lysosomal pH monitoring of polystyrene nanoparticles

What is the intracellular fate of nanoparticles (NPs) taken up by the cells? This question has been investigated for polystyrene NPs of different sizes with a set of molecular biological and biophysical techniques.rnTwo sets of fluorescent NPs, cationic and non-ionic, were synthesized with three different polymerization techniques. Non-ionic particles (132 – 846 nm) were synthesized with dispersion polymerization in an ethanol/water solution. Cationic NPs with 120 nm were synthesized by miniemulsion polymerization Particles with 208, 267 and 603 nm were produced by seeding the 120 nm particle obtained by miniemulsion polymerization with drop-wise added monomer and polymerization of such. The colloidal characterization of all particles showed a comparable amount of the surface groups. In addition, particles were characterized with regard to their size, morphology, solid content, amount of incorporated fluorescent dye and zeta potential. The fluorescent intensities of all particles were measured by fluorescence spectroscopy for calibration in further cellular experiments. rnThe uptake of the NPs to HeLa cells after 1 – 24 h revealed a much higher uptake of cationic NPs in comparison to non-ionic NPs. If the same amount of NPs with different sizes is introduced to the cell, a different amount of particles is present in the cell medium, which complicates a comparison of the uptake. The same conclusion is valid for the particles’ overall surface area. Therefore, HeLa cells were incubated with the same concentration, amount and surface area of NPs. It was found that with the same concentration always the same polymer amount is taking up by cells. However, the amount of particles taken up decreases for the biggest. A correlation to the surface area could not be found. We conclude that particles are endocytosed by an excavator-shovel like mechanism, which does not distinguish between different sizes, but is only dependent on the volume that is taken up. For the decreased amount of large particles, an overload of this mechanism was assumed, which leads to a decrease in the uptake. rnThe participation of specific endocytotic processes has been determined by the use of pharmacological inhibitors, immunocytological staining and immunofluorescence. The uptake of NPs into the endo-lysosomal machinery is dominated by a caveolin-mediated endocytosis. Other pathways, which include macropinocytosis and a dynamin-dependent mechanism but exclude clathrin mediated endocytosis, also occur as competing processes. All particles can be found to some extent in early endosomes, but only bigger particles were proven to localize in late endosomes. No particles were found in lysosomes; at least not in lysosomes that are labeled with Lamp1 and cathepsin D. However, based on the character of the performed experiment, a localization of particles in lysosomes cannot be excluded.rnDuring their ripening process, vesicles undergo a gradual acidification from early over late endosomes to lysosomes. It is hypothesized that NPs in endo-lysosomal compartments experience the same change in pH value. To probe the environmental pH of NPs after endocytosis, the pH-sensitive dye SNARF-4F was grafted onto amino functionalized polystyrene NPs. The pH value is a ratio function of the two emission wavelengths of the protonated and deprotonated form of the dye and is hence independent of concentration changes. The particles were synthesized by the aforementioned miniemulsion polymerization with the addition of the amino functionalized copolymer AEMH. The immobilization of SNARF-4F was performed by an EDC-coupling reaction. The amount of physically adsorbed dye in comparison to covalently bonded dye was 15% as determined by precipitation of the NPs in methanol, which is a very good solvent for SNARF-4F. To determine influences of cellular proteins on the fluorescence properties, a intracellular calibration fit was established with platereader measurements and cLSM imaging by the cell-penetrable SNARF-4F AM ester. Ionophores equilibrated the extracellular and intracellular pH.rnSNARF-4F NPs were taken up well by HeLa cells and showed no toxic effects. The pH environment of SNARF-4F NPs has been qualitatively imaged as a movie over a time period up to 1 h in pseudo-colors by a self-written automated batch program. Quantification revealed an acidification process until pH value of 4.5 over 24 h, which is much slower than the transport of nutrients to lysosomes. NPs are present in early endosomes after min. 1 h, in late endosomes at approx. 8 h and end up in vesicles with a pH value typical for lysosomes after > 24 h. We therefore assume that NPs bear a unique endocytotic mechanism, at least with regards to the kinetic involvedrn

Expanding the scope of poly(ethylene glycol)s for bioconjugation to squaric acid-mediated PEGylation and pH-sensitivity

Poly(ethylene glycol) (PEG) is used in a broad range of applications due to its unique combination of properties and is approved use in formulations for body-care products, edibles and medicine. This thesis aims at the synthesis and characterization of novel heterofunctional PEG structures and the establishment of diethyl squarate as a suitable linker for the covalent attachment to proteins. Chapter 1 is an introduction on the properties and applications of PEG as well as the fascinating chemistry of squaric acid derivatives. In Chapter 1.1, the synthesis and properties of PEG are described, and the versatile applications of PEG derivatives in everyday products are emphasized with a focus on PEG-based pharmaceuticals and nonionic surfactants. This chapter is written in German, as it was published in the German Journal Chemie in unserer Zeit. Chapter 1.2 deals with PEGs major drawbacks, its non-biodegradability, which impedes parenteral administration of PEG conjugates with polyethers exceeding the renal excretion limit, although these would improve blood circulation times and passive tumor targeting. This section gives a comprehensive overview of the cleavable groups that have been implemented in the polyether backbone to tackle this issue as well as the synthetic strategies employed to accomplish this task. Chapter 1.3 briefly summarizes the chemical properties of alkyl squarates and the advantages in protein conjugation chemistry that can be taken from its use as a coupling agent. In Chapter 2, the application of diethyl squarate as a coupling agent in the PEGylation of proteins is illustrated. Chapter 2.1 describes the straightforward synthesis and characterization of squaric acid ethyl ester amido PEGs with terminal hydroxyl functions or methoxy groups. The reactivity and selectivity of theses activated PEGs are explored in kinetic studies on the reactions with different lysine and other amino acid derivatives, followed by 1H NMR spectroscopy. Further, the efficient attachment of the novel PEGs to a model protein, i.e., bovine serum albumin (BSA), demonstrates the usefulness of the new linker for the PEGylation with heterofunctional PEGs. In Chapter 2.3 initial studies on the biocompatibility of polyether/BSA conjugates synthesized by the squaric acid mediated PEGylation are presented. No cytotoxic effects on human umbilical vein endothelial cells exposed to various concentrations of the conjugates were observed in a WST-1 assay. A cell adhesion molecule - enzyme immunosorbent assay did not reveal the expression of E-selectin or ICAM-1, cell adhesion molecules involved in inflammation processes. The focus of Chapter 3 lies on the syntheses of novel heterofunctional PEG structures which are suitable candidates for the squaric acid mediated PEGylation and exhibit superior features compared to established PEGs applied in bioconjugation. Chapter 3.1 describes the synthetic route to well-defined, linear heterobifunctional PEGs carrying a single acid-sensitive moiety either at the initiation site or at a tunable position in the polyether backbone. A universal concept for the implementation of acetal moieties into initiators for the anionic ring-opening polymerization (AROP) of epoxides is presented and proven to grant access to the degradable PEG structures aimed at. The hydrolysis of the heterofunctional PEG with the acetal moiety at the initiating site is followed by 1H NMR spectroscopy in deuterium oxide at different pH. In an exploratory study, the same polymer is attached to BSA via the squarate acid coupling and subsequently cleaved from the conjugate under acidic conditions. Furthermore, the concept for the generation of acetal-modified AROP initiators is demonstrated to be suitable for cholesterol, and the respective amphiphilic cholesteryl-PEG is cleaved at lowered pH. In Chapter 3.2, the straightforward synthesis of α-amino ω2-dihydroxyl star-shaped three-arm PEGs is described. To assure a symmetric length of the hydroxyl-terminated PEG arms, a novel AROP initiator is presented, who’s primary and secondary hydroxyl groups are separated by an acetal moiety. Upon polymerization of ethylene oxide for these functionalities and subsequent cleavage of the acid-labile unit no difference in the degree of polymerization is seen for both polyether fragments.

Relazioni fra regime dei venti e pH in un "campo di fumarole" a CO2 sui fondali costieri dell'isola d'Ischia (Italy).

Il problema dell'acidificazione degli oceani, conseguente ai cambiamenti climatici, è un processo ancora poco conosciuto. Per comprendere questo fenomeno, possono essere utilizzati degli ambienti naturalmente acidificati, considerati laboratori a cielo aperto. Lo scopo di questo lavoro di tesi è stato quello di utilizzare le fumarole presenti nell'isola di Ischia, per approfondire le dinamiche dei processi di acidificazione e per analizzare l'eventuale interazione tra pH e condizioni meteorologiche. I dati utilizzati, forniti dalla Stazione Zoologica “Anton Dohrn” di Napoli, erano serie di pH e di vento rilevate in continuo, in due aree, nord e sud rispetto all'isolotto del Castello Aragonese, e in tre stazioni lungo un gradiente di acidificazione. Tutto il lavoro è stato svolto a step, dove il risultato di un'analisi suggeriva il tipo e il metodo analitico da utilizzare nelle analisi successive. Inizialmente i dati delle due serie sono stati analizzati singolarmente per ottenere i parametri più salienti delle due serie. In seguito i dati sono stati correlati fra loro per stimare l'influenza del vento sul pH. Globalmente è stato possibile evidenziare come il fenomeno dell'acidificazione sia correlato con il vento, ma la risposta sembra essere sito-specifica, essendo risultato dipendente da altri fattori interagenti a scala locale, come la geomorfologia del territorio, le correnti marine e la batimetria del fondale. È però emersa anche la difficoltà nel trovare chiare correlazioni fra le due serie indagate, perché molto complesse, a causa sia della numerosa quantità di zeri nella serie del vento, sia da una forte variabilità naturale del pH, nelle varie stazioni esaminate. In generale, con questo lavoro si è dimostrato come utilizzare tecniche di analisi delle serie storiche, e come poter utilizzare metodi di regressione, autocorrelazione, cross-correlation e smoothing che possono integrare i modelli che prendono in considerazione variabili esogene rispetto alla variabile di interesse.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Effects of pH and acid concentration on erosive dissolution of enamel, dentine, and compressed hydroxyapatite

The aims of this study were to determine the effects of pH and acid concentration on the dissolution of enamel, dentine, and compressed hydroxyapatite (HA) in citric acid solutions (15.6 and 52.1 mmol l(-1) ; pH 2.45, 3.2, and 3.9), using a pH-stat system. After an initial adjustment period, the dissolution rates of enamel and HA were constant, while that of dentine decreased with time. The dissolution rate increased as the pH decreased, and this was most marked for enamel. To compare substrates, the rate of mineral dissolution was normalized to the area occupied by mineral at the specimen surface. For a given acid concentration, the normalized dissolution rate of HA was always less than that for either dentine or enamel. The dissolution rate for dentine mineral was similar to that for enamel at pH 2.45 and greater at pH 3.2 and pH 3.9. The concentration of acid significantly affected the enamel dissolution rate at pH 2.45 and pH 3.2, but not at pH 3.9, and did not significantly affect the dissolution rates of dentine or HA at any pH. The variation in response of the dissolution rate to acid concentration/buffer capacity with respect to pH and tissue type might complicate attempts to predict erosive potential from solution composition.