Mycoremediation for degrading hydrocarbon pollutants

Nowadays waste products derived from human life and industrial activities represent a serious problem in the world, due to their difficulty of disposal and recovery. Although in the past little importance has been given to environmental quality, the last two decades have been characterized by an increasing awareness on the topic; nevertheless, pollution from many organic substances such as hydrocarbons, remains a mostly unsolved issue, because these compounds are often recalcitrant and show high levels of toxicity. In recent years, the scientific interest for hydrocarbons is mainly related to their recognized carcinogenic action resulting from the metabolic transformations of these compounds into diol-epoxides, molecules able to bind to DNA and to induce genetic mutations (Man et al.2013). These recalcitrant substances are known to enter the environment frequently and in large scale via several routes (Singh, 2006). One of the major routes is the spill on the ground, often due to neglected maintenance of structures dedicated to production, storage and distribution; as concerns terrestrial environments, these phenomena may determine some harmful effects such as surface water pollution through runoff, groundwater pollution by leachate, evaporative atmospheric pollution, sublimation or wind drift, thus resulting in decreases in crop yields, poisoning through the food chain and impact on the landscape. Another route includes production, storage and transportation which represent a hazard due to accidental releases of petroleum compounds, mostly in marine environments, especially involving bottom sediments. Currently, restoring areas polluted by hydrocarbon products usually requires some chemical, physical, and biological treatments. Among these, biological treatments are more suitable for sustainable and economic applications because traditional remediation techniques for the treatment of hydrocarbon compounds are often limited by application costs and low efficiency. Hence, the present study proposes a sustainable and economic biotechnology aimed at allowing the recovery and disposal of soils and marine sediments contaminated by hydrocarbons. The project represents a response to the need for providing a biotechnological protocol devoted to safeguard the environment andterritory and to allow the reclamation of sites contaminated by substances which have been deemed harmful to human health. As it is known, some fungal species are extremophiles, able to adapt to adverse environmental conditions (both for abiotic factors and for nutrient deficiency) which would be limiting for most living organisms (Gadd, 2007); this feature makes some fungi an important tool to biodegrade pollutant compounds (Kumar et al. 2011). For these reasons, as concerns the hydrocarbon pollution of soils and marine sediments, the present study proposes an alternative solution to the traditional technologies of rehabilitation and environmental remediation, through the study, isolation, identification, and exploitation of those fungi able to degrade these toxic compounds. Several experiments reported in the literature demonstrate that fungi have an ability to metabolize and degrade many hydrocarbon compounds, such as oils, petroleum derivatives, and polycyclic aromatic hydrocarbons (Cerniglia et al. 2010; Harms et al. 2011; Al-Jawhari et al. 2014; Reyes-C\ue9sar et al. 2014; Marco-Urrea et al. 2015). The studies conducted so far provide a general overview about fungal remediation activity, contributing to support the hypothesis of the possible large-scale application of some fungi in the degradation processes of organic pollutants. The research is aimed at assessing the sustainability of these innovative biotechnologies and at deepening the knowledge of the factors that may affect the activity of some fungal species or strains within different matrices contaminated by different types of hydrocarbons. The study is the result of a multidisciplinary approach involving mycologists, geologists, chemists, and oceanographers. The thesis is organized as follows. Chapter 1 presents the hydrocarbons focusing on polycyclic aromatic hydrocarbons (PAHs); it describes the traditional remediation techniques which are currently used and defines the role of fungi in the degradation of recalcitrant organic compounds and in the remediation of contaminated soils and waters (mycoremediation). Chapter 2 details the aims of the thesis, while in Chapter 3 the materials and methods are listed and described. Chapter 4 deals with the mycological characterization of a marine area polluted by hydrocarbon compounds in relation to different substrates (biotic and abiotic), depths and marine currents, to identify and select a pool of fungal species adaptable to extreme polluted environments, which could be used for bioremediation purposes. Chapter 5 shows the results of the investigations on 15 fungal species isolated from a real oily slime and screened to assess their ability to degrade a PAH mixture. The most suitable fungal strains were employed in the in vitro degradation tests. Chapter 6 investigates, through pilot-scale experiments, the role that fungi can play as tools for the remediation of real polluted matrices contaminated by petroleum derivatives. Finally, Chapter 7 outlines the conclusions, showing the significant importance of the fungal exploitation for recovering marine and terrestrial areas contaminated by hydrocarbons. Furthermore, some hints for future works and applications are provided

Non normal logics: semantic analysis and proof theory

We introduce proper display calculi for basic monotonic modal logic,the conditional logic CK and a number of their axiomatic extensions. These calculi are sound, complete, conservative and enjoy cut elimination and subformula property. Our proposal applies the multi-type methodology in the design of display calculi, starting from a semantic analysis based on the translation from monotonic modal logic to normal bi-modal logic

Tool support for reasoning in display calculi

We present a tool for reasoning in and about propositional sequent calculi. One aim is to support reasoning in calculi that contain a hundred rules or more, so that even relatively small pen and paper derivations become tedious and error prone. As an example, we implement the display calculus D.EAK of dynamic epistemic logic. Second, we provide embeddings of the calculus in the theorem prover Isabelle for formalising proofs about D.EAK. As a case study we show that the solution of the muddy children puzzle is derivable for any number of muddy children. Third, there is a set of meta-tools, that allows us to adapt the tool for a wide variety of user defined calculi