Environmental quality is fundamental for the well being of human life. Environmental risk assessment and analysis have a crucial role for guaranteeing the safety of the population, especially in intensive urbanized and industrialized areas, such as the Campania region (Italy). In Italy, after the Legislative Decree 152/2006, the environmental risk assessment has become mandatory for contaminated lands such as brownfields and dismantled industrial activities. Despite the importance and fragility of the soil, it is rarely taken into account as a resource to be preserved. Moreover, due to the heterogeneity of many variables involved in the process, the environmental and human health risk assessments are used at the scale of a site, while the regional evaluation is very difficult, and keeps the administration sceptical in taking it into account for land planning and decision-making processes. During the last years, some international methodologies for ecological and human health risk assessment have been developed by national environmental agencies in many industrialized countries. An idea recently took place regarding the protection of soils: the monitoring of soil should be limited only to risk areas, as opposed to monitoring systems covering the entire territory. Focusing monitoring in selected areas would help to define priorities, increase efficiency of monitoring activities and reduce the monitoring costs. This is the reason why many methodologies for preliminary and simplified risk assessment of (potentially) contaminated sites, for the prioritization and planning of soil remediation and protection programmes, were developed. The only lack that these methodologies have is the absence of the spatial analysis. The aim of this research was to implement the human health risk assessment. For the purposes of the study we used 3535 topsoil samples, collected across the whole regional territory. The concentrations of 53 elements have been determined at Bureau Veritas Analytical Lab. Ltd (Vancouver, Canada), by means of an Aqua Regia extraction followed by a combination of ICP-MS and ICP-AES methods. After the statistical and cartographic elaboration of these regional dataset, we integrated one of the model of relative risk assessment for single contaminated sites with spatial analysis procedures. In order to support the spatial assessment of contaminated sites at the regional scale, the most suitable tool is the Geographic Information System (GIS). A new approach was applied, in order to assess/rank environmental risk by using geospatial analysis in a GIS platform to adapt a European-wide accepted methodology for the preliminary assessment of human health risks at single contaminated sites to a regional scale. The developed regional risk assessment methodology can be used by the regional administrations to select at regional level the "problem or priority areas". The identification of the problem areas is necessary for developing an efficient monitoring system and to produce a ranking of the risky areas to be used as a reference in determining the development of intervention plans, for better addressing the resources dedicated to the environmental remediation of widely contaminated regions. The methodology chosen as a reference for the risk assessment procedures is the PRA.MS (Preliminary risk assessment model for the identification of problem areas for soil contamination in Europe, 2005). Following the PRA.MS guidelines, a conceptual model for the human health risk assessment for the Campania region has been based on four different exposure routes: 1) dispersion of contaminants in groundwater, 2) dispersion in surface water, 3) dispersion in air, 4) direct contact with the contaminated media (soils). The source, the pathway and the receptor for each of the exposure route are scored on the base of a quantitative or qualitative analysis of some featuring characteristics (parameters). A total of 14 representative parameters were chosen, basing on the available regional data for Campania. Starting from these parameters values, the information is aggregated to higher levels in several steps, adopting a mixed additive and multiplicative algorithm, up to the overall risk score. The final risk map is classified according four classes of risk. This map was useful to identify the problem areas, characterized by a higher risk, where more detailed analysis had to be carried out. The identified area was located in a wide territory in the center of Campania region, between Avellino and Salerno municipalities, an high populated zone, where intensive agricultural and industrial activities are well developed. A high density survey has been carried out in this priority area; the concentrations of 53 elements have been measured on 102 new topsoil samples, followed by statistic and cartographic elaboration of the 15 potentially toxic elements (Sb, As, Be, Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, Sn, Tl, V, Zn) for which the Italian legislation (D.Lgs. 152/06) established the trigger and action limits, by defining the contamination threshold values (CSC) for the amount of metals in topsoils or waters. The new methodology of GIS-based human health risk assessment has been again applied, in order to select sites of particular significance in terms of danger for the resident population. Some small areas characterized by high risk have been identified. New in situ measurements and classic follow up risk assessment should be here carried out to confirm if remediation actions are needed to guaranteeing the safety of the population. Other works has been contemporaneously elaborated during the PhD. Six months have been spent at the Geological Survey of Finland (GTK), where a work on a humus samples dataset have been carried out, under the supervision of Dr. Timo Tarvainen. Moreover a new Geochemical Atlas will be completed based on inorganic elements and organic compounds concentrations in marine sediments samples, collected in the Gulfs of Naples and Salerno.
It has been estimated that at least 99 % of the world's microbial biomass exists in form of biofilm, a complex differentiated surface-associated community embedded in a self-produced polymeric matrix enabling microorganisms to develop coordinated and efficient survival strategies. Biofilm formation is a dynamic and cyclical process involving attachment, maturation and a final dispersal phase, and these steps are initiated by a variety of signals. Despite their positive effects in some cases, biofilms can be detrimental in different environmental domains since microorganisms are able to colonize almost all types of surfaces both abiotic and biotic, leading to consequences in terms of social and economic impact. These include human tissues, implantable medical devices, natural aquatic systems, plants, food and industrial lines. Once biofilm is formed, its eradication becomes difficult because its resilience to environmental stresses, disinfectants, and antimicrobial treatments. Plants support a diverse array of microorganisms that exist in form of biofilms. Even if in some cases the association with plants leads to beneficial interactions promoting plant growth, inducing plant defense mechanisms and preventing the deleterious effects of pathogenic microorganisms, in other cases they have a significant negative impact. For instance, in agriculture, plant colonization of fungi and bacteria in form of biofilm is a cause of plant diseases, affecting crop quality and productivity. Indeed, despite the planktonic growth, biofilm lifestyle improves microbial resistance to antimicrobials up to several orders of magnitude, often reducing the possibility of treating biofilm effectively. In addition, due to the worrisome consequences related to the use of these substances on human health and on their persistence in the environment, increasingly regulations are arising to limit antimicrobial application. Furthermore, in addition to the principles of integrated pest management (IPM) embraced by the worldwide legislation aims to recommend alternative approaches to the application of pesticides, an innovative approach could be the use of biocide-free bioactive compounds characterized by novel targets, unique modes of action and properties that are separate from those currently highlighted in the use of antimicrobials. Indeed, the application of non-lethal doses of bio-inspired molecules able to interfere with specific key-steps involved in the biofilm formation process has been suggested as a complementary/alternative strategy to hinder biofilm formation. In addition, this approach also lead to deprive microorganisms of their virulence factors without affecting their viability and decreasing the selection pressure for biocides resistance. In this PhD thesis, the in vitro effects of non-lethal concentrations of several bioactive compounds were evaluated on the biofilm formation of different plant-associated microorganisms. Specifically, the aim of this work was to provide new effective preventive or integrated solutions against bacterial and fungal biofilm formation. In chapter III, the methanol extracts obtained by different plant portions of three seagrass species collected in Vietnam and in India (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) were investigated for their effects in mediating non-lethal interactions on sessile Escherichia coli and Candida albicans cultures taken as models of bacterial and fungal biofilms respectively. The study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Seagrass extracts appeared to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. Results revealed that E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an anti-biofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Methanolic extracts were characterized and major phenolic compounds were identified by MS/MS analysis, showing the unique profile of the E. acoroides leaf extract. In chapter IV, two essential oils (PK and PK-IK) derived from two cultivars of Perilla frutescens, an annual short day plant widely used in therapeutics in the traditional medicine as well as in food preparations in Asian countries. Essential oils were extracted from the leaves and were characterized. Subsequeltly, their ability to affect biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and F. oxysporum have been studied. PK and PK-IK neither inhibited fungal growth nor were they utilized as a carbon energy source. In addition, PK and PK-IK essential oils showed excellent anti-biofilm performances inhibiting conidia germination and reducing conidia adhesion. Furthermore, they revealed a magnificent anti-biofilm effect even during biofilm maturation, affecting biofilm structural development, with a reduction of dried weight, extracellular polysaccharides and proteins. In all cases PK-IK displayed better activity than PK. Thus, the anti-biofilm effects were exploited with a non-lethal mechanism. This research supported the spreading of PK and PK-IK essential oils as biocide-free agents suitable for a preventive or integrative approach for sustainable crop protection. Lastly, in chapter V, a non-lethal concentration of N-Acetylcysteine (NAC) was evaluated on the biofilm formation of Xylella fastidiosa, a phytopathogen bacterium that causes a range of economically important plant diseases worldwide and that has been recently found in Italy in olive plants, where it causes the olive quick decline syndrome (OQSD). NAC is a naturally occurring compound found in several vegetables (including garlic, onion, peppers and asparagus) and it is mostly known in clinical area, in which it is employed at lethal concentrations in the treatment of human diseases due to its ability to reduce bacterial adhesion, inhibit the production of extracellular polysaccharides and promote the dispersion of pre-formed mature biofilms. In this study, N-Acetylcysteine (NAC) was tested for its ability to affect biofilm response of X. fastidiosa CoDiRO strain, mimicking a preventive, a curative and a combination of both approaches. The not-lethal dose 0.08 mg/ml was chosen as representative of plant concentration after its application. NAC did not alter planktonic bacterial growth but promoted biofilm formation in terms of biofilm biomass (above 62 %) and matrix polysaccharides (above 53%) through a ROS-mediated mechanism. Additionally, NAC was not able to destroy X. fastidiosa biofilm when already established on the surface but rather, it was suitable to contain the biofilm infection limiting biofilm dispersal. On the contrary, a combination of both preventive and curative approach has been found promising in biofilm dissolving making it more vulnerable.
Dottorato di ricerca in Scienze ambientali ; Sistemi agricoli ed ecologici sono intimamente connessi (l'agricoltura gioca un ruolo importante nei modelli ecosistemici) e l'attività agricola concreta in se concetti di gestione e cambiamento ambientale atti alla produzione di beni (in primis gli alimenti). Durante il secolo scorso, l'attività agricola si è intensificata caratterizzandosi sia nella crescente dipendenza da fattori esterni sia nella conversione delle coperture del suolo. Sebbene tale processo abbia incrementato la produttività, la sostenibilità di numerosi agroecosistemi è stata compromessa. Nei Paesi sviluppati la situazione è particolarmente critica e richiede una riorganizzazione del settore agricolo al fine di recuperare la sostenibilità venuta meno. Al fine di affrontare le questioni emergenti connesse con la crescita demografica mondiale e la veemenza tecnologica nella biosfera, assume una fondamentale importanza il quantificare la sostenibilità degli agroecosistemi. La sfida dei ricercatori è quindi quella di bilanciare le questioni bio-fisiche con quelle socio-economiche per promuovere lo sviluppo sostenibile dell'agricoltura. In Europa sono presenti modelli di sviluppo rurale sostenibile ed essi devono essere maggiormente compresi, esplorati e diffusi in quanto magnifici esempi di conoscenza acquisita tramite la tradizione e il sapiente uso del suolo. Il principale obiettivo della presente ricerca è quello di produrre un'indagine ambientale ed economica finalizzata alla progettazione e gestione di agroecosistemi sostenibili. Al fine di perseguire tale obiettivo, è necessario adottare un approccio multidimensionale capace di associare le caratteristiche agroecosistemiche alla gestione sostenibile. Questa ricerca vuole contribuire alla costruzione di una Scienza della Sostenibilità tramite la definizione di soluzioni pratiche capaci di incrementare la sostenibilità agricola, con minime ripercussioni sui livelli produttivi. La ricerca (articolata in vari studi) ha riguardato la valutazione della sostenibilità di alcuni agroecosistemi, a livello gerarchico di paesaggio (Lazio meridionale) e aziendale (Lazio meridionale, Provincia di Viterbo e Isola di Terceira), tramite indicatori di diversità (paesaggio e azienda) e di input/output (azienda). Gli studi condotti a livello di paesaggio hanno analizzato la sostenibilità ambientale in termini di metriche territoriali distinguendo l'ecoregione in base ad alcune caratteristiche (proprietà, altimetria e fitoclima). I risultati forniscono un profilo ecoregionale dell'Italia centrale dove i modelli storici di uso del suolo sono sopravvissuti a testimonianza della capacità umana di bilanciare il proprio sviluppo in base al contesto locale. Anche se i recenti cambiamenti sociali hanno portato a una maggior irruenza antropica e tecnologica sull'ambiente, la tradizione nei modelli d'uso del suolo (tramandata tra le generazioni mediante la cultura, l'educazione e le regolamentazioni locali) ha contribuito nel mitigare l'impatto umano ed ha agire da cuscinetto per la resilienza degli ecosistemi. Gli studi condotti a livello aziendale hanno analizzato la sostenibilità ambientale ed economica, in termini di circolazione dei flussi di materia ed energia, confrontando alcuni regimi gestionali contrastanti (biologico e convenzionale, misto e non misto). In generale, i risultati mostrano migliori prestazioni delle aziende biologiche rispetto alle convenzionali in ragione dell'organizzazione aziendale maggiormente portata al reimpiego della energia-materia prodotta e alla minor richiesta di energie ausiliari esterne provenienti da fonti non rinnovabili. Gli studi confermano il ruolo fondamentale degli allevamenti in quanto componente essenziale a migliorare l'efficienza e la sostenibilità aziendale (tale ruolo non viene sempre riconosciuto in termini sociali ed economici). In situazioni di bassa diversificazione strutturale e forte ascendente politico sul processo decisionale aziendale (come ad esempio sull'isola di Terceira) i costi ambientali dell'agricoltura possono aumentare significativamente. Al fine di informare in maniera appropriata i soggetti (pubblici e privati) coinvolti nel processo decisionale, sono necessarie maggiori risorse di conoscenza e di finanziamenti per misurare e monitorare le condizioni di sostenibilità dell'agricoltura. ; Agricultural and ecological systems are directly connected (agriculture plays an important role in ecosystem patterns) and the agroecosystems convey a high sense of stewardship care and historicity as food providers. During the last century, agriculture activity has intensified worldwide, characterized by an increasing dependence on external inputs and on land cover conversion. Although agriculture intensification has increased productivity, the sustainability of many agroecosystems has been compromised. In developed Countries the situation is particularly critical and requires a reorganization of the agricultural sector which would recover the sustainability failed. The measurement of agroecosystems sustainability has become of supreme importance, now essential to address the obvious problems related to the large population growth and technological vehemence in the biosphere. Defining socio-economical and bio-physical balance is a fundamental challenge for researchers in order to promote the sustainable development in agriculture. In Europe examples of sustainable rural development should be better acknowledged, explored and disseminated as meaningful case studies of traditional knowledge and wise land use. The main objective of the present research is to provide environmental-economic frameworks in order to design and evaluate agroecosystem sustainability. To achieve this objective, a multidimensional approach is needed that combines the feature of the agroecosystems with sustainable management. This research want to be a contribution in building a science of sustainability developing practical ways of improving sustainability in agriculture, with minimal impact to production. This research (containing various studies) has concerned the assessment of agroecosystem sustainability at landscape (Southern Lazio) and farming (Southern Lazio, Viterbo Province, Terceira Island) level based on the use of diversity (landscape and farming studies) and input/output (farming studies) indicators. Landscape level studies have analyzed the environmental sustainability in terms of landscape metrics distinguishing the landscape according to some characteristics (ownership, elevation and phytoclimate). Results provided a profile of an ecoregion in Central Italy, where historical land-use patterns are still alive on the territory and testify the capacity of human beings to develop a balanced relationship with their context of life at local level. Even if recent changes in society trends bring about more demographic pressure and more environmentally-aggressive technological fixes, tradition in land use patterns transferred from generation to generation through culture, education, regulations and action at local level, can help mitigate human impact and operate as a cultural buffer for ecosystem resilience. Farming level studies have analyzed the environmental and economical sustainability in terms of energy and material fluxes circulation comparing groups of farms in contrasting management regimes (organic vs. conventional; mixed vs. non-mixed). In general terms, results shows a diffuse better performance of organic farms respect to conventional ones because their organization was based on increased re-use of on-farm produced energy-matter flow and reduced demand of external inputs of non renewable energy-matter sources. The studies have confirmed the fundamental role of livestock as crucial agroecosystem component that improves the efficiency and sustainability of farms (this role is not yet acknowledged by society in economic terms). In situations of low structural diversification and strong policy ascendancy on farm decision making processes, the agricultural environmental costs may be enhanced significantly (eg.: Terceira Island). More intellectual and financial resources for measuring and monitoring sustainability conditions in agriculture are necessary, in order to appropriately inform decision making processes at both institutional and individual level.
The twentieth century (characterized by the gruesome and haze of horror of two World Wars, the Cold Wars-CW, dictatorships, civil wars, genocides, etc.) has seen a great transformation in warfare but to the expense of the innocent civilians and yet in the full view of regulatory internationally recognized war-laws. So, if at one point in history, civilian populations hardly suffered war directly, the order of the state of affairs has now changed. Many civilians perish simply because warlords so desire; extremes of violence, killings and destruction of property is predominantly preferred. As if that is not enough, the indifference of the majority of the public in tranquil zones of the world towards the fate of the civilians in zones under by fire kind of provide implicit licenses to violence planners to do whatever it takes to "win". Consequently, great numbers of survivors are seen trying to escape from situations of assured death to that of probable death. It is against this background that we feel moved to take on this dissertation. Bearing in mind the generally complex and challenging contemporary conflicts that acutely breeds volatile security environments (for civilians), our thesis is that there needed to be an increased, noteworthy and continued applicable innovation of approaches to civilian protection. To be precise, as a strategy to sustainable peace, we have aspired after a world where the United Nations Peacekeeping Department (UNPKD) is not singly considered the sole custodian of the concept of civilian protection but (based on contexts and cases) as one but a leader among other stakeholders (local and foreign) able and ready to contribute to the common-pool of operational arenas. Thinking about these other stake holders, we have in this work stood by those that: firstly, move towards more civilian-centered operations that are; secondly, carried out by (a mixture of grassroots and international) unarmed civilians by means of; thirdly, engages nonviolent approaches and practices that in themselves anticipate the basic constituents of successive bottom-up Peacemaking (PK) and Peacebuilding (PB) in the hic et nunc of their Peacekeeping (PK) initiatives and applications. All these basics, in our view, do not just add up to drawing a continuous line that intersects the just mentioned Three Approaches to Peace (PK, PM, PB) coined by Johan Galtung way back in 1975; they also open avenues to sustainability. The thesis is taken on through three different parts; each subdivided into two chapters. With due attention to intrastate contemporary violent conflicts, the first part tries to demonstrate the reason why in PK there has been indeed need for rethinking the protection of civilians (PoC) and/or for enriching the methods until now employed in bringing it about. In the first chapter of the part, we kind of gave a sketchy attention to the historical journey that the patterns of violent conflicts in relation to the fate of non-combatants have made. It emerges that, unlike in the past, the pattern of contemporary violent conflict, especially with reference to the CW (especially in third-world countries) and post-CW periods, have become severely complex to handle. Wars have continued and proved to be very hot especially on the populations on the periphery; on those who are minimally directly concerned with and honestly ignorant of its objectives. In the period in question, these innocent men, women and children are more than ever struck hard not just by its direct consequences but also the indirect ones and their hopes are constantly put at the brink of mere survival and of the grave. Mores so the lucky ones who manage to escape these snares, continue to unwaveringly hope for bread, freedom, justice and peace, instead of iron that kills and destroys. In chapter II of the same part, looking at the commitments borne by the UN right from its early years in keeping, initially, the interstate and successively also the intrastate peace (of those tormented by reign of violence and terror), we acknowledge the strides gradually taken along the years. These strides has better late than never embraced a multidimensional point in time where civilian protection counts as a primacy. Accordingly, we recognize that the UN military PK is certainly capable of reducing the level of tension in conflicts but we also negated that, by so doing, it is able to guarantee a durable peace not only because of the application of the non-peaceful means which is limited to separating the conflicting parties but also because it lacks the strategic concern of fostering an active citizenship which is a basic ingredient to democratic populace. In Part Two, we have concentrated on the vision and the peculiar picture of the practitioners of the alternative way, particularly; the Nonviolent Peaceforce (NP) which operates on a benchmark of bottom-up strategic empowerment of local civilian unarmed and nonviolent efforts by international unarmed and nonviolent civilians to protect civilians, prevent, reduce and stop violent conflicts. The first chapter of this second part begins by singling out some of the nuts and bolts (Like: The centrality of sustainability; strategic, local and multilevel capacity and relational empowerment and mediation for peace; conflict transformation as the adequate language; nonviolence and nonpartisanship as a philosophy) that make Unarmed Civilian Protection (UCP) stands out faithfully to the above stated aspirations. Without giving importance to the chronological specifics and with a particular reference to the assessment of the practicality of the project that, on a later date, would organizationally become the NP, an extensive attention is paid to the vicissitudes that surrounded the founding of this UCP protection agency and especially to the foundations of the formative elements entailed. Chapter II does not only build on the findings and stimuli of Chapter I, it supersedes it and makes real a new and distinct reality. Herein, a unique place is devoted to the formative components reserved to the practitioners as a strategy for guaranteeing the competencies and high professionalism needed for the successful execution of field strategies attached to the NP UCP objectives, principles, key methods and practices. Through the analysis of the UCP Training Course entitled "Strengthening Civilian Capacities to Protect Civilians; A joint UNITAR- Nonviolent Peaceforce online Course" the chapter tries to show how the activities of the organization intrinsically flow from its very being; from elements which define it. And this is illustrated in how the very life of the NP UCP is blended with its formative spirit and content; a sort of transformative training that seeks to promote transformative operational frameworks that applicable to situations and contexts. The third part of the work is an applied one. It is dedicated to our chosen case study, namely, NP's intervention in the longtime violence-stricken Republic of South Sudan; in a country which (Thomas Hobbes would say) has once again reverted to its natural state; a harsh reality of hand to mouth living and a never ending search for sustenance in an ambiance virtually challenging to change. In chapter I, the pragmatic implementation of NP UCP in strengthening the local civilians' capacity, security and sense of safety in situation of violent conflict is marked out. Here, some concrete instances of this intervention are presented to exemplify the claim that a multiple base of actors (UCPs, the inviting civil society and/or local NGOs of an UCP presence and local partners) can sustainably and strategically provide the PoC work that for a long time was and is still largely entrusted to the military. And at the end of the day PK, PM, PB resources are considered to consist in not only financial and material supports, but also, and (in the same way) importantly, the socio-cultural resources of the affected people. And in this way people in conflict settings are seen as resources rather than recipients. Even though we evidently confirmed that the alternative way counts exceptionally big in strategically promoting, developing, and implementing sustainable unarmed civilian PK as a tool for preventing, reducing and stopping violence and protecting civilians in situations of violent conflict, we also acknowledge that it is not without challenges. These are actually what chapter two of the part extensively dwells on. The second chapter is instead dedicated (at length) to looking at the challenges that NP faces not only with regard to its missions lands but also in general. We have gone about this in the form of a comprehensive assessment and in some humble recommendations are advanced. Among these challenges we have particularly paid attention to issues like: The meager UCP funding and the dominant top-down mentality; the violent bully character of some major world power wielders; the need for more practitioners to carry out UCP; the dynamicity and complexity of conflict nature as a challenge; conflict prevention challenges like delays in capturing the signs of time so as to effectively intervene; the presence of spoilers as a challenge; the challenge of effective sustainable credibility. Recommendations proposed include among others: Investing in systematic reflections on the extent of the progress and failures so far registered in efforts to involve the UN, regional bodies and other donor agencies or individuals in the cause of NP (UCP) and reflecting on the philosophy that underpins the reasons why financial assistance to UCP and NP in particular is founded; more emphasis on the already existing engagement with political leaders and other influential people and embarking on popular campaigns to propagate a concretely evidenced knowledge of the feasibility of the alternative way, instituting and investing in "School Project" (dedicated to preferably to high schools) within the NP Advocacy and Outreach office and insisting on the positives of volunteers' contribution; enriching a little more the content of the just elaborated online UCP training course; etc. Hereafter, the general conclusion of our dissertation will be drawn. A profound acknowledgement of the UNPK pivotal role with its actual multidimensional fronts in PK basically intended as PoC specifically in the contemporary intrastate violent conflicts. It is also observed that, thanks to the appropriate blending of local and international capacities giving priority to the former, UCP's strategic approach to PK (which is not limited to the PoCs but is also anchored to preventing, reducing and ending not just those that are already on but also lays for standing up to the future possible ones) could be counted on. Thus far, it is on one hand, admissible that, despite all the challenges that there may be, NP (UCP) mechanisms is already proffering a great deal to this end, and on the other, it is evident that it can and should still do more. The ability of its interventions to stand the test of time and to stand up to the future conflicts (i.e. its sustainability) resides in a time which is not yet at hand and in the continuous involvement and inventiveness of many. As per now, if the Italian proverb "Il buongiorno si vede dal mattino" (Meaning: You can tell how something will go by how it begins) holds, then it is, up till now, realistic to count on NP as one of the most outstanding Bottom-up UCP organizations in the PoC in (selected) contemporary violent conflict situations. All that is needed is the building and the consolidation of international interest and support for UCP that presents the hope and reality of alternatives to over dependence on armed intervention; alternatives that chances the revitalization of local communities and the restoration of the social fabrics and capital of the affected people.
Dottorato di ricerca in Biotecnologie degli alimenti ; Lo smaltimento delle acque di vegetazione dei frantoi oleari costituisce, attualmente, uno dei principali problemi dal punto di vista ambientale, specialmente nei paesi del Mediterraneo dove si concentra la maggior parte della produzione mondiale di olio di oliva. Le acque di vegetazione sono tra i reflui agro-industriali a più alto tasso inquinante a causa del loro elevato carico organico, caratterizzato soprattutto da composti fenolici e polifenolici ad elevata azione antimicrobica e fitotossica. La purificazione biologica delle acque di vegetazione è particolarmente difficile poiché questo refluo presenta solidi in sospensione e un elevato carico organico, in particolare polifenoli con attività biostatica e/o biocida, che riduce fortemente le prestazione degli impianti di depurazione. Di conseguenza, l'impianto deve prevedere due o più stadi di trattamento che rendono la depurazione complessa e costosa. Attualmente, la normativa vigente consente la pratica dello spandimento delle acque di vegetazione sui terreni agrari; nonostante questa risulti, al momento, essere la soluzione migliore sia dal punto di vista pratico che economico, trova attuazione solo se si ha disponibilità di terreni sufficientemente vicini su cui spargere il refluo e comunque deve essere applicata in maniera controllata dal momento che gli eventuali effetti positivi o negativi sulla composizione, sulla carica microbica e la fertilità del terreno sono ancora oggi oggetto di studio. Inoltre, la migrazione di alcuni composti negli strati più bassi del terreno potrebbe causare la contaminazione di eventuali falde acquifere sottostanti con conseguenze per la salute dell'uomo. Negli ultimi anni sono state proposte soluzioni alternative finalizzate a sfruttare questo refluo, in quanto ricco di composti utili. La valorizzazione delle AV mediante il loro impiego per l'ottenimento di prodotti a medio o alto valore aggiunto attraverso processi fisico-chimici o fermentativi, riveste notevole interesse scientifico. Nelle AV sono presenti una grande varietà di biomolecole come acidi organici, polialcoli, zuccheri semplici e complessi e lipidi che le rendono una possibile base per i processi fermentativi. In virtù del contenuto residuo di lipidi, le AV potrebbero rappresentare un ottimo candidato come terreno liquido di crescita per la produzione di lipasi microbiche. Lo scopo della presente tesi di dottorato è stato quello di mettere a punto un processo fermentativo per la valorizzazione delle AV mediante produzione microbica di enzimi, in particolare enzimi lipolitici, ottenendo al contempo un abbattimento, o quanto meno una riduzione, del loro potere inquinante. Esiste una vasta bibliografia in cui viene presa in esame la produzione di lipasi da numerose specie microbiche tra cui Penicillium e Candida e sia il terreno che il processo fermentativo per la produzione di questo enzima è stato ampiamente ottimizzato. Nella maggior parte dei casi, una buona produzione di lipasi microbica prevede l'utilizzo di terreni sintetici piuttosto complessi che sicuramente incidono in maniera significativa sul prezzo finale del prodotto. Inoltre, negli ultimi anni anche la produzione di preparati enzimatici commerciali contenenti lipasi di origine microbica ha avuto un notevole sviluppo. Sigma, Amano, Roche, Novo Nordisk, etc., forniscono preparati lipolititici con varie composizioni e proprietà catalitiche utilizzati in diversi settori: industria alimentare, farmaceutica, dei detergenti e per la produzione di biodiesel. L'innovazione che dovrebbe introdurre questo lavoro è l'opportunità di produrre lipasi microbiche di possibile interesse industriale utilizzando un substrato costituito da un refluo agro-industriale. Con questa idea, si è cercato di mettere a punto un terreno di produzione a basso costo che permettesse di ottenere buoni livelli di attività e contemporaneamente un abbattimento del carico inquinante del refluo finale. In prima battuta, è stato effettuato uno screening di microrganismi (Geotrichum candidum, NRRL 552, 553; Rhizopus sp, ISRIM 383; Rhizopus arrhizus, NRRL 2286; Rhizopus oryzae, NRRL 6431; Aspergillus oryzae, NRRL 1988, 495; Aspergillus niger, NRRL 334; Candida cylindracea, NRRL Y-17506; Penicillium citrinum, NRRL 1841, 3754, ISRIM 118) in grado di crescere sulle acque di vegetazione producendo lipasi. Le produzioni più elevate di enzima sono state ottenute, in condizioni non-ottimizzate, dopo 168 h con Geotrichum candidum NRRL 553 (0,521 U/ml) e Candida cylindracea (0,460 U/ml). Inoltre, livelli di produzione molto interessanti sono stati raggiunti dopo 72 h con i ceppi di Penicillium citrinum (0,365, 0,320 e 0,375 U/ml per NRRL 1841, NRRL 3754 e ISRIM 118, rispettivamente). Questi ceppi sono stati selezionati per valutare, in via preliminare, l'effetto di alcuni fattori sulla produzione di lipasi quali tipologia di AV, utilizzo di vari oli come induttori di attività e impiego di diverse fonti di azoto. Per quanto riguarda la produzione di lipasi da P. citrinum NRRL 1841 su AV, l'attività è stata influenzata in maniera marcata dal tipo di fonte di azoto ma non era aumentata in maniera significativa dall'aggiunta di oli. Nel caso della produzione di lipasi da C. Cylindracea NRRL Y-17506, il cloruro di ammonio e l'olio di oliva rappresentavano rispettivamente la fonte di azoto e l'induttore più adatto; infatti questo ceppo cresciuto in condizioni parzialmente ottimizzate produceva 9,48 U/ml di attività lipolitica dopo 264 h di fermentazione. Successivamente, la produzione di lipasi da P. citrinum NRRL 1841, utilizzando il terreno a base di AV, è stata ottimizzata in beuta valutando l'effetto del pH iniziale, della concentrazione di azoto e di estratto di lievito secondo un approccio multi-fattoriale. La combinazione ottimizzata dal modello è stata la seguente: pH 6,15, 2,7 g/l NH4Cl e 1,1 g/l YE. La produzione massima raggiunta è stata di 1,242 U/ml. Con il terreno così ottimizzato, al fine di ottenere informazioni sul possibile trasferimento di scala del processo, sono stati condotti altri esperimenti in reattori da banco. Allo scopo, sono stati impiegati due tipi di sistemi, un bioreattore ad agitazione meccanica (STR) e uno ad agitazione pneumatica (Air-lift). In entrambi i casi, l'attività lipolitica extracellulare aveva raggiunto il suo picco massimo dopo 192 h di fermentazione. Tuttavia, il massimo di attività è stato significativamente più alto in STR che in Airlift (0,700 vs 0,420 U/ml, rispettivamente). Sebbene tutti i ceppi studiati sono stati in grado di crescere sulle acque di vegetazione e produrre a livelli significativi attività lipolitica, una particolare attenzione è stata riservata a C. cylindracea (noto anche come C. rugosa) per il notevole interesse applicativo della lipasi prodotta da questo lievito. Inizialmente, si è cercato di ottimizzare in beuta la composizione del terreno di produzione (concentrazione dell'olio di oliva, effetto del glucosio, aggiunta di surfactanti e di vari fattori di crescita) e di valutare in via preliminare l'effetto sulla crescita cellulare e sull'attività di alcune condizioni colturali quali velocità di agitazione e aerazione. La migliore composizione del terreno di produzione si è confermata essere quella contenente 3 g/l di olio di oliva, 2,4 g/l di NH4Cl e 0,5 g/l di estratto di lievito, senza l'aggiunta di glucosio e Tween 80. Inoltre, con lo scopo di valutare la fattibilità tecnica di un trasferimento di scala del bioprocesso e approfondire la messa a punto del processo fermentativo sono stati condotti una serie di esperimenti in bioreattore da banco ad agitazione meccanica (STR). In particolare, utilizzando il terreno a base di AV ottimizzato, si è cercato di ottimizzare alcuni parametri quali pH, velocità di agitazione e aerazione. Per quanto riguarda l'effetto della velocità di agitazione e dell'aerazione sulla produzione enzimatica, sono state prese in esame tre velocità di agitazione (300, 500 e 700 giri/min), mantenute fisse durante tutta la fermentazione, e in più è stato condotto un esperimento in cui si è cercato di mantenere la concentrazione dell'ossigeno disciolto nel mezzo superiore al 20% di saturazione facendo variare la velocità di agitazione tra 300 e 800 giri/min. Mentre per valutare l'effetto del pH, sono stati condotti degli esperimenti a pH 6,5 fisso confrontando la produzione con quella ottenuta a pH libero e a pH mantenuto inferiore a 6,5. La massima produzione di lipasi da C. cylindracea è stata ottenuta in bioreattore lavorando a pH libero e ad una velocità di agitazione costante di 500 giri/min (18,50 U/ml) o ad una velocità di agitazione variabile tra 300 e 800 giri/min in modo da assicurare un valore di ossigeno disciolto nel brodo superiore al 20% di saturazione (18,70 U/ml); in quest'ultimo caso, inoltre, la comparsa del picco massimo è stata anticipata nel tempo favorendo così la produttività oraria del bioprocesso. Per quanto riguarda i reattori a 300 e 700 giri/min, la produzione enzimatica è stata di 2,54 e 11,65 U/ml, rispettivamente. Infine, messo a punto il bioprocesso di produzione della lipasi da C. cylindracea coltivata su un terreno a base di AV, si è cercato di identificare il profilo enzimatico del campione grezzo così ottenuto, dal momento che, come è noto dalla letteratura, questo lievito è in grado di produrre fino a sette isoforme ad attività lipolitica. A tale scopo sono stati condotti degli esperimenti di isoelettrofocalizzazione (IEF) analitica. Nel gel sono stati caricati un campione di lipasi commerciale (Tipo VII, Sigma) e due campioni grezzi ottenuti da C. cylindracea coltivata sul terreno a base di AV, prelevati a due tempi fermentativi diversi e corrispondenti ai due picchi di attività lipolitica raggiunti durante le prove in STR (I° e II° picco di massima attività, 48esima e 192esima ora, rispettivamente). Dai risultati ottenuti, è stato osservato che il campione grezzo era costituito da più isoenzimi con attività lipolitica e che il profilo isoenzimatico aveva una sola banda in comune con quello della lipasi commerciale (Typo VII, Sigma) a cui è stato assegnato pI 4,7. Per quanto riguarda il campione prelevato alla 48esima ora, sono state osservate anche una banda piuttosto intensa a pI 5,1 e una tripletta di bande più deboli a pIs di 5,06, 5,0 e 4,9. Durante la fermentazione il profilo isoenzimatico del campione aveva subito delle modifiche: infatti, alla 192esima ora, le bande a pIs 5,1, 5,0 e 4,9 erano scomparse, mentre era comparsa una banda di attività intensa a cui è stato assegnato un pI di 4,5. Infine, in entrambi i campioni grezzi è stata rilevata una banda tenue a pI 3,8. In conclusione, i buoni livelli di attività enzimatica raggiunti dimostrano la fattibilità tecnica di un processo fermentativo finalizzato alla valorizzazione dei reflui oleari mediante la produzione di lipasi, che può avere promettenti utilizzi in varie applicazioni industriali. Comunque, ulteriori fasi di scale-up del processo sono ancora necessarie al fine di poter effettuare una valutazione sulla fattibilità economica del processo. ; The olive mill wastewater (OMW) disposal is, currently, one of the main environmental problems in all olive-oil producing countries, especially in the Mediterranean area. In fact, for its high organic load, phenolic fraction with phytotoxic effects and antimicrobial activity, the OMW is a highly polluted agro-industrial effluent. The biological treatment can be very difficult since solid residues, high organic load and phenols may strongly reduce the depuration efficiency. Consequently, a possible process should include several technological options, physical, chemical and biological, as well as combinations thereof, thus resulting in increased process costs. At the moment, the Italian legislation allows land spreading of untreated olive mill wastewater that is the best economical solution. Application on agriculture soils is a practice which solves partially the problem of OMW disposal. Positive and negative effects on soil composition and fertility are still under study, so that OMW application must be strictly controlled. Land spreading, in fact, may cause serious negative environmental impact regarding, for instance, groundwater contamination. In the last years, alternative solutions have been proposed in view of the use this waste as a source of valuable compounds. Several recent research studies have reported the possibility of OMW valorization to obtain products of actual or potential industrial interest. The presence in OMW of a wide range of biomolecules such as organic acids, polyalcohols, simple and complex sugars and lipids makes it a potential basis for fermentation processes. In this way, OMW could be a putative candidate as a potentially suitable liquid growth medium for the production of microbial lipases by virtue of its residual lipid content. For these reasons, the objective of the present PhD thesis was to assess the suitability of OMW as growth medium for the production of lipases and to set up a related fermentation process that might lead, at the same time, to a low polluting load final effluent. A large number of microbial strains have been screened for lipase production belonging to several fungal genera, Candida and Penicillium in particular. In literature, numerous methods for lipolytic enzyme production are published and medium composition and cultural conditions have been fully optimised. Neverthless, the most frequently used medium is a chemical defined and complex one, significantly affecting the final product costs. Besides, in the last years, a whole range of microbial lipase preparations has been developed. Sigma, Amano, Roche, Novo Nordisk, etc., provide lipolytic preparations with various compositions and catalytic proprierties employed in areas such as detergent pharmaucetic and food industries and biodiesel production. Our innovative approach consists in the trial of producing microbial lipases using an agroindustrial-waste based medium. Our basic idea, in fact, was that of developing a low cost production medium. Firstly, 12 fungal strains belonging to well-known lypolytic species (Geotrichum candidum, NRRL 552, 553; Rhizopus sp, ISRIM 383; Rhizopus arrhizus, NRRL 2286; Rhizopus oryzae, NRRL 6431; Aspergillus oryzae, NRRL 1988, 495; Aspergillus niger, NRRL 334; Candida cylindracea, NRRL Y-17506; Penicillium citrinum, NRRL 1841, 3754, ISRIM 118) were screened for their ability to grow on undiluited OMW and to produce extracellular lipase activity. The highest lipase productions were obtained under non-optimized conditions after 168 h with Geotrichum candidum NRRL 553 (0.521 U/ml) and Candida cylindracea (0.460 U/ml). Interesting production levels were also achieved after 72 h with strains of Penicillium citrinum (0.365, 0.320 and 0.375 U/ml for NRRL 1841, NRRL 3754 and ISRIM 118, respectively). These strains were then selected to study the effect of culture conditions, such as OMW typology, nitrogen sources and inducers, on the enzyme production. With regard to the lipase production by P. citrinum NRRL 1841, the enzyme activity was significantly influenced by nitrogen addition; on the other hand, the addition of oils resulted in a marked increase in biomass without affecting, however, lipase production. Lipase production by C. cylindracea NRRL Y-17506 was significatly favored by ammonium salts and oil addition. This strain growth in OMW medium containing ammonium chloride and olive oil led to an activity peak of 9.48 U/ml after 264 hours of fermentation. In order to optimise lipase production by P. citrinum in OMW-based medium, the combined effect of three variables (i.e, concentration of NH4Cl, yeast extract and initial pH) was assessed using a multi-factorial design with 'optimizer' function of 'Modde 5.0' program. The optimised combination by the model was as follows: pH 6.15, 2.7 g/l NH4Cl e 1.1 g/l extract yeast. The maximum lipase activity was 1.242 U/ml after 192 hour of fermentation. To gain information on the possible up-scaling of the process, further experiments were performed in 3-l laboratory-scale reactors. Specifically, pneumatically agitated (Airlift) and mechanically agitated (STR) reactors were employed using the optimised OMW-based medium. In both cases, the extracellular lipase peaked 192 h after inoculation. Howewer, the maximum activity was significatly higher in STR with respect to the Airlift (0.700 vs 0.420 U/ml, respectively). Of all strains, C. cylindracea appeared to be particularly interesting and was, therefore, used as the model microorganism to further investigate the feasibility of an OMW substrate. Firstly, the optimisation of medium composition was assessed in shaken cultures. In particular, the effects on the lipase production of olive oil concentration (1, 3, 5 e 10 g/l), glucose (5 g/l), Tween 80 (0,5 g/l) and several growth nutrients (yeast extract, malt extract and peptone) addition were studied. The best medium composition was as follows: diluited OMW (1:2), olive oil 3 g/l, NH4Cl 2.4 g/l and yeast extract 0.5 g/l. The glucose and Tween 80 addition negatively affected the production of lipolytic enzyme. Lipase production by C. cylindracea on OMW-optimized medium was subsequently assessed in mechanically agitated bioreactor (STR). To study the agitation influence on enzyme production, a set of experiments was carried out at three impeller speed, 300, 500 and 700 rpm; moreover, an additional experiment was carried out at dissolved oxygen DO > 20% saturation (agitation speed automatically controlled between 300 and 800 rpm). To evaluate the effect of pH, three conditions were compared: free pH; fixed pH (6.5) maintained constant by addition of HCl 4.0 M and NaOH 4.0 M; pH lower than 6.5 controlled with addition of HCl 4.0 M. The maximum lipase productions were obtained with the pH left free to vary, 500 rpm costant agitation speed (18.5 U/ml) and variable agitation speed between 300 and 800 rpm to ensure a dissolved oxygen value upper to 20% (18.7 U/ml); in the latter thesis the onset of enzyme activity was anticipated thus leading to increased bioprocess productivity. At 300 e 700 rpm agitation speed, the maximum lipase productions were 2.54 and 11.65 U/ml, respectively. Finally, to set up the bioprocess of lipase production by C. Cylindracea grown on OMW-based medium, the isoenzymatic profiles of the raw sample was evaluated. This aspect appears to be very interesting since it is known that commercial C. rugosa lipase is a mixture of 3 isoenzymes namenly Lip 1, Lip2 and Lip 3 but the yeast is able to produce up to seven different isoenzymes (Lip 1-Lip 7). Moreover isoenzymatic profiles can depend on media composition and fermentation conditions. With this aim, a set of analitycal isoelectrofocusing experiments were carried out. In the gels, a sample of commercial lipase (Type VII, Sigma) and two raw samples of lipase by C. cylindracea grown on OMW-optimized medium and corresponding to two lipolytic activity peaks (1st and 2nd peak, 48esime and 192esime hour of fermentation, respectively) obtained in STR, were loaded. The results suggest that the raw samples were constituted of more lipolytic isoenzymes with the isoenzymatic profile having only one band in common with that of the commercial lipase (assigned pI 4.7). The sample corresponding to the 1st activity peak showed a strong band at pI 5.1 and a triplette of weak bands at pIs 5.06, 5.0 e 4.9. Moreover, the isoenzymatic profiles changed during fermentation; in fact, the bands at pIs 5.1, 5.0 and 4.9 disappeared and a new strong band at pI 4.5 formed. Finally, in both raw samples a band at pI 3.8 was observed. OMWs valorisation by its use as growth medium for lipase production by C. cylindracea NRRL Y-17506 and P. citrinum NRRL 1841 appears to be possible and promising. Moreover, the investigation for further up-scaling is need to evaluate the economic fattibility of the bioprocess.
