BIOTECHNOLOGICAL EMPLOYMENT OF OLIVE OIL MILL WASTEWATERS FOR WATER REMEDIATION AND POTENTIAL BIODIESEL PRODUCTION

Olive oil mill wastewaters (OMW) are dark-colored wastes characterized by high values of COD (chemical oxygen demand) and BOD (biological oxygen demand). OMW contains high amounts of organic and inorganic compounds. The first category includes mostly sugars, polyphenols, organic acids, proteins, fatty substances, mixed phenol-polysaccharide polymers, polyalcohols, cellulose and hemicellulose, pectins and tannins. The inorganic substance, present in lower amount, includes mainly potassium and, to a lesser degree, sodium, calcium and magnesium as cations, and chlorides, phosphates and sulphates as anions. OMW, for their composition, are particularly dangerous for soil and waters if dispersed unprocessed because of phenolic compounds and long-chain fatty acids which have phytotoxic and antimicrobial properties. So, the aim of this thesis is to develop integrate strategies aimed at valorisation and/or disposing of OMW. In order to obtain by-products to reuse in environmental technology processes the recovery of organic matter from this waste was performed. Polymerin, the high molecular organic fraction of OMW, is a polyelectrolyte with humic-like properties and can be used as a potential bio-filter for decontamination of polluted wastewaters because exhibited very interesting sorption capacities for cationic and anionic heavy metals, ionic or ionisable pesticides and hydrophobic organic compounds. The first aim of this thesis is the use of polymerin for the decontamination of wastewaters contamined by pesticides. A study was performed on the sorption capacity of polymerin towards the cyhalofop acid (CyA), a new generation aryloxyphenoxypropionic herbicide, and compare it with a mineral matrix, the ferrihydrite, and an organo-mineral one, the ferrihydrite-polymerin complex. Moreover, we performed another comparative study on sorption capacity of polymerin and two mesoporous mineral matrices, Al2O3 and Fe2O3 towards the 4-chloro-2-methylphenoxyacetic acid (MCPA), a phenoxyacetic herbicide, and 2-choloro-4,6-bis(ethylamino)-s-triazine (simazine), a chlorotriazinic one. Cyhalofop-butyl (CyB), 2-[4-(4-cyano-2-fluoro-phenoxy)phenoxy]propanoic acid, butyl ester (R), recently introduced from Dow AgroSciences, is used for the post-emergence control of grasses in rice, mainly against barnyard grass (Echinochloa species) and silver top (Lepthochloa fusca species). The ester presents a low water solubility, but it hydrolyzes rapidly into its corresponding derivative 2-?4-(4-cyano-2-fluorophenoxy) phenoxy] propionic acid (CyA), which is much more soluble and presents the effective herbicide action. Moreover, CyA is the effective chemical compound occurring in water after its application on the crop because it originates by the rapid hydrolysis of its corresponding butyl ester form. Among the different pollutants commonly found in soil and waters, MCPA deserves a particular interest. It is a post emergence phenoxy acid herbicide extensively used in agriculture to control annual and perennial weeds in cereals, grasslands, trees, and turf. It is very soluble (273.9 mg/L in water, at neutral pH), highly mobile, and can leach from soil and it is suspected for mutagen and carcinogen properties. This compound has been found in well water in some countries and is classified by the U.S. Environmental Protection Agency (EPA) as a potential groundwater contaminant. Simazine is a synthetic s-triazine herbicide widely used for pre-emergence control of broad-leaf weeds and annual grasses in agricultural and non-crop fields. Simazine is the second most commonly detected pesticide in surface and groundwaters in the United States, Australia and Europe. It is persistent in the environment up to eight months and not easily degraded by microbes. Due to the carcinogenic potential of s-triazines, simazine presence in water is of increasing concern. Effects of pH, contact time, initial concentration and sorbent dosage on the sorption of the herbicides were investigated. The most efficient sorbent for cyhalofop acid showed to be ferrihydrite followed by ferrihydrite-polymerin complex and polymerin; while for MCPA and simazine the collected data evidenced the greater sorption efficiency of Al2O3 with respect to Fe2O3 and polymerin. In particular, cyhalofop acid bonds to ferrihydrite by a combination of ionic and ion-dipole bonds, the ferrihydrite-polymerin complex by ionic bonds and polymerin by hydrogen ones. Sorption of MCPA on polymerin occurred by the formation of H-bonds while for simazine by hydrogen and ionic bonds. Moreover, MCPA is assumed to be bond to Al2O3 and Fe2O3 by a combination of ionic and ion-dipole interactions. Simazine is sorbed on Fe2O3 by hydrogen bonds and because of the high acidity of this matrix also by electrostatic interactions, while on Al2O3 sorption occurred only by hydrogen bonds. So, the highest superficial surface area of Al2O3 than Fe2O3 and the presence of secondary small pores at boundary of micropores region has a positive influence in the uptake of simazine and MCPA. Simulated wastewaters contaminated with cyhalofop acid were completely purified by two sorption cycles on ferrihydrite and five cycles on the ferrihydrite-polymerin complex, whereas the same wastewaters maintained a constant residue even after five sorption cycles on polymerin. The same experiment carried out for MCPA and simazine indicated that Al2O3 allowed the total removal of MCPA by four sorption cycles and a removal of 84% of simazine after only two sorption cycles, whereas on Fe2O3 MCPA was removed for 92% after five cycles and simazine for 69% after five cycles. Cyclic sorption experiments on polymerin revealed that no total removal was possible for both pesticides. An ideal sorbent should have a high surface area (i.e., high density of sorption sites), uniformly accessible pores and physical and/or chemical stability. It is believed that the sorption capacity of a sorbent is largely determined by the surface area available which increases with decreasing the particle size although the pores size distribution is also decisive for an optimal sorption process. Therefore, with the introduction of nanoscaled oxide materials, the pollutant removal efficiency can be increased dramatically. So, mineral matrices can be used as sorbents for a fast and highly efficient removal of ionic and ionisable pesticides and as a suitable filters for the decontamination of point sources. Moreover, Al2O3 can be regenerated by incineration method and could be considered for small-scale treatment systems and industrial scale. A further innovative strategy, aimed to enhance the valorisation of OMW, concerns their use as grow media for oleaginous microorganisms, for potential biodiesel production, so this use could be a convenient alternative to avoid the negative environmental impact associated with its discharge. The demand for fatty acid methyl esters (FAMEs) as diesel fuel (biodiesel) has increased significantly. Microorganisms can accumulate high levels of lipids and do not require arable land. Therefore, the microbial lipids can potentially be used as raw material for biodiesel production using the common way to produce FAMEs in the biodiesel industry. The second aim of this thesis is to investigate the potential of Lipomyces starkeyi, an oleaginous yeast able to survive and proliferate in the presence of olive oil mill wastewaters, as a source of lipids. L. starkeyi proved to store large amounts of lipids, showing only a minimal reutilization of the stored ones. It was grown in the presence of undiluted OMW, without external organic supplements, producing a significant reduction of both the total organic carbon (TOC) and the total phenol content. The OMW treated by L. starkeyi showed a significant increase of the germination index. The preliminary dilution of OMW enhanced the reduction of polluting components of OMW, leading to a complete TOC removal, as well as to lower levels of residual phenols. The activities of extracellular lipases and esterases significantly increased in the course of the OMW fermentation. A significant increase in lipid yield was observed in L. starkeyi in the course of the OMW treatment, particularly enhanced when the feedstock was preliminarily diluted. The fatty acid distribution showed a prevalence of oleic acid, demonstrating the potential of L.