• recalcitrant
• fungi
• fungal degradation
• biodegradation
• batch tests
• tannins removal

Bioremediation, defined as the controlled use of organisms to remove pollutants from the environment represents a cost effective and environmentally sustainable method of cleaning up polluted matrices. Fungi have proven to be extremely efficient in degrading complex polymers, which are inaccessible to most prokaryotes. The ability of fungi to degrade lignin, a complex organic polymer diffused in many plant cells, has been known for decades and is closely associated with the production of non-specific extracellular enzymes. Considering the similarity between the chemical structure of numerous recalcitrant molecules and lignin, the use of fungi has been the subject of numerous studies, also considering their possible combination with bacteria, for the purification of wastewater.
The present thesis encloses five experiments in which fungi were employed for the degradation of recalcitrant compounds, within a sterile and/or not-sterile environment, or to verify their ability to attach to different types of supports.
The first experiment involved the use of the fungal candidate Aspergillus tubingensis, a cosmopolitan ascomycete, used in previous experiments on the degradation of tannins carried out by the University of Florence, at CUOIODEPUR wastewater treatment plant. The objective of the experiment, performed in flasks, was to verify whether the introduction of bacterivorous organisms, such as ciliates and rotifers could reduce bacterial load and enhance the removal of the natural tannin Quebracho (recalcitrant compound). The experiment has shown that such introduction reduces the bacterial load, increasing the stability of the fungal system. However, general efficiency parameters, such as chemical oxygen demand, which is commonly used, provide little information about the degradation process of the recalcitrant. Based on these results, a second experiment was designed, using fungi for the degradation process. In this case, the study focused on a molecule of non-natural origin, which, according to the literature, is difficult to be biodegraded by bacteria and easily identifiable by High Pressure Liquid Chromatography (HPLC). Nonylphenol is defined as priority hazardous by the European Community. Although banned, in several countries nonylphenol is still commonly employed in industries as non-ionic surfactant. The purpose of this second experiment, carried out in a controlled environment, was to verify whether a fungal strain could successfully degrade and / or bio-absorb the selected molecule. Several tests were performed in controlled environment. Firstly, four fungal strains were screened and Tyromyces chioneus, a fungus belonging to the "White-Rot" group, originally isolated from Brady Road landfill leachate, was selected for its ability to grow on a synthetic medium (prepared with agar and NaCl) added with nonylphenol. Then, other batch tests were performed using the selected strain in suspended form to establish its optimal co-substrate concentration. Finally, the main test, was conducted in non-sterile conditions for 21 days, to verify the presence of a reduction of nonylphenol concentration and if such eventual depletion was due to biodegradation and/or biosorption. The analyses were performed, through HPLC on both liquid and suspended samples (biomass). Although both the method and the experimental design have room for improvement, decreases in the concentration of nonylphenol in the liquid have been observed. At the same time significant concentrations of nonylphenol in the fungal biomass, that could suggest biosorption, were not found.
The third and fourth experiments were part of the European Project “MNET17/ENER-1143 project FUNCELL”, which is aimed at using fungi for the remediation of recalcitrant molecules, such as tannery and other cellulosic wastewaters. One of the main goals of FUNCELL is the set-up of a pilot-scale bioreactor in which fungi could exploit their potential for the degradation of recalcitrant molecules/effluents. In view of this goal, several batch tests were performed to verify the ability of four different fungi to attach to different type of carriers (i.e. with or without cellulose) in sterile conditions. The results showed a strong affinity of basidiomycetes with cellulose. Starting from these results, other experiments have been designed to test the compatibility of two fungal strains (a basidiomycete and an ascomycete) simulating the conditions designed for pilot-scale experiments (FUNCELL). From these last experiments, it is clear that the two fungi are compatible and that, at least in batch condition, this combination is associated with a slight reduction in removal efficacy compared to the conditions in which they were not associated.
At present, most of the inocula for field research with fungi are conveyed by a physical and not or not completely biodegradable support impregnated with mycelia, as in the tests described in the present thesis. Considering that, an alternative and extremely promising method for the inoculum could be represented by the encapsulation of fungi mycelia directly into microspheres of alginate, that are biodegradable and provide, simultaneously, protection and a slow release mechanism for inocula. The objective of this test was to determine whether mycelial homogenate of Bjerkandera adusta would have survived encapsulation in calcium alginate and to determine the vitality of encapsulated mycelia after the encapsulation and the storage. Starting from the latter test, other experiments could be designed to verify the effectiveness of this type of immobilization in the removal of recalcitrant compounds.