BIOTECHNOLOGICAL EMPLOYMENT OF OLIVE OIL MILL WASTEWATERS FOR WATER REMEDIATION AND POTENTIAL BIODIESEL PRODUCTION
Creators: name: Addorisio, Veria
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2010

description: 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. starkeyi as a source of lipids to be used as a feedstock for the synthesis of II generation biodiesel. 
Finally, we investigated the effect of immobilization of two laccases, from the fungi Myceliophthora thermophila and Trametes versicolor, on different supports. These enzymes are able to degrade a wide array of substrates, abundant in olive oil mill wastewaters, such as phenols, aromatic amines, benzenethiols, hydroxyindoles and phenothiazinic compounds. The third aim of this thesis is to evaluate the enhance of operational stability and durability of the enzymes after immobilization. Laccase is a multi-copper oxidase, able to catalyze the one-electron oxidation of a wide array of substrates with the simultaneous reduction of oxygen to water. The low substrate specificity exhibited by laccase and its ability to oxidize priority pollutants has attracted interest for its use in wastewaters treatment and bioremediation. Many laccases from different sources could be considered for biotechnological applications, but laccases from higher plants and bacterial strains are less capable to degrade polymers of phenolic origin, including lignin, than laccases from fungi. In particular, it is well documented that thermophilic fungi may comprise a rich source of thermostable industrial enzymes. Furthermore, the thermal tolerance is an attractive feature for many biotechnological applications of enzymes. In the present thesis we used a fungal laccase (Novozym 51003) from the thermophilic ascomycete Myceliophthora thermophila and a laccase from basidiomycete fungus Trametes versicolor. This latter was reported to have the highest redox potential among laccases; i.e., 785 mV versus the standard hydrogen electrode, which makes this enzyme particularly interesting since high redox potentials correlate with high activity. However, enzymes are soluble and they cannot easily be used for many reaction cycles, are quite unstable under conditions of high temperatures, extreme pHs or use of organic co-solvents or toxic products, because of that the enzymes have to be stabilized to convert the process in a profitable one. The immobilization of two laccases presented in this thesis allowed to obtain heterogeneous catalysts with good perspectives to be used at industrial level in different processes of oxidation of products highly toxic. The use of different techniques of immobilization allowed having catalysts with different activities and stabilities for their use in processes in which different conditions of pH, temperature or the presence of denaturant agents (unfolding agents or organic cosolvents) are required. 
Both the enzymes were able to be purified almost completely in only one step of adsorption-desorption using anionic exchanger supports. The immobilization of laccase from Myceliopthora thermophila was performed using different supports by which catalyst with different activities and stabilities were obtained. Non aminated enzyme only could be immobilized on supports capable to immobilize through the richest place in negative charges as aminated or glutaraldehyde supports. The amination of this laccase was performed in a very simple way, so the immobilization of the aminated enzyme allowed obtaining derivatives more stable than that obtained with the non aminated enzyme. The most stable derivatives in all assayed inactivation conditions were those where the aminated enzyme was immobilized on heterofunctional supports activated with iminodiacetic acid (IDA) and glyoxyl groups. These derivatives were immobilized through the richest place in positive charges and were more than 30 times more stable than soluble enzyme in some of the assayed conditions; in addition they resulted slightly more stable against the presence of organic cosolvent or in the presence of unfolding reagents (urea). 
As regards Trametes versicolor laccase, the enzyme was first aminated and than immobilized on glyoxyl supports obtaining derivatives with different stabilization factors depending of the incubation conditions during the immobilization process. The best stabilizations were obtained after incubation at pH 10 where it was possible to obtain stabilization factors over 70 times if compared with the stability of the soluble enzyme or the correspondent one point immobilized derivative (cyanogen bromide support). In addition the best derivatives were at least twice more stable against organic solvents. 
In conclusion, in the present thesis different strategies aimed at a biotechnological employment of olive oil mill wastewaters have been proposed, in order to remediate water polluted from pesticide and, at the same time, to valorize this waste using it for potential biodiesel production. Moreover, among the technologies developed to disposing of OMW, the use of laccases immobilized on new generation supports is an innovative and great important strategy.