**4.1.2 Biodiesel**

300 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

alcoholate catalysts to incorporate fatty acids randomly. This reaction produces a complete positional randomization of acyl groups in TAGs. In enzymatic interesterification the final structure of TAGs is controlled and a desired acyl group can be guided into TAGs using nonspecific, regiospecific (*sn*-1,3- or 2- specific) and fatty acid specific lipases as catalysts. This results in products with predictable composition. Enzymatic interesterification is becoming a more attractive method to convert cheap oils such as olive pomace oil, soya bean oil, rape seed oil, lard, tallow, etc. to high-value-added products and modified fats (An et al., 2007; Liua et al., 1997; Macrae, 1983; Miller et al., 1991; Pomier et al., 2007; Xu, 2003). Furthermore, enzymatic interesterification has milder reaction conditions and produces less waste than the chemical alternative. In addition, the same immobilized enzyme can be used many times (Akoh et al., 1998; Marangoni & Rousseau, 1995; Willis et al., 1998; Willis & Marangoni, 2002). Therefore, intensive research has aimed at replacing chemical

There are three types of interesterification reactions: acidolysis, which is the reaction between an ester and a fatty acid, alcoholysis, the reaction between an ester and an alcohol, and transesterification, the reaction of an ester with another ester, also called ester-ester exchange (Macrae, 1983; Xu, 2003). Production of structured lipids and biodiesel has been

Structured lipids are novel modified TAGs produced by the incorporation of desirable fatty acids at specific positions or by changing the position of the fatty acids on the glycerol backbone. These processes allow for specific characteristics to be obtained such as melting behavior, functionality, and metabolism. Lipases, especially those which are *sn-*1,3 specific, are used for this purpose because these enzymes can make changes at *sn*-1 and *sn*-3

Cocoa butter (CB) has a narrow melting range due to its unique TAG composition. This melting behavior is critical. The steepness of the melting profile (% solid fat as a function of time) has an impact on flavor release and crystallization. The high price of cocoa butter has prompted the industry search for CB alternatives. CB equivalents (CBEs) can be produced from palm oil and exotic fats by means of fractionation. Enzymatic synthesis of CBEs from cheap oils and fats using *sn*-1,3 specific lipases is also an alternative method. CB-like fats could be produced which have even more desirable properties than natural CB. Ciftci et al. (2010) used olive pomace oil for the production of CB-like fat using *sn*-1,3 specific lipase. They interesterified refined olive pomace oil, palmitic acid and stearic acid at a molar ratio of 1:2:6, respectively, at 45°C using a pack bed reactor filled with *sn*-1,3 specific lipase. They reported that the CB-like fat could replace CB up to 30% without significantly changing the physical and chemical properties of the product. Chang et al. (1990) also produced CB-like fat by enzymatic interesterification of fully hydrogenated cotton seed and olive oils. The

melting point of their CB-like fat was 39°C; close to 36°C, the melting point of CB.

Any lipid containing medium-chain and long-chain unsaturated fatty acids might be useful for certain applications and functionalities. Nunes et al. (2011) produced structured lipids containing medium-chain fatty acids at *sn*-1,3 position and long-chain unsaturated fatty acids at the *sn*-2 position by acidolysis of virgin olive oil and caprylic or capric acids using

interesterification with enzymatic interesterification.

the major topics of enzymatic interesterification studies.

positions by keeping *sn*-2 ester group position unchanged.

**4.1.1 Structured lipids** 

Biodiesel can be obtained from vegetable oils, animal fats, recycled grease, or algae and can be produced by the reaction of TAGs with methanol (methanolysis). Lipase-catalyzed methanolysis is more attractive than conventional base-catalyzed method since the glycerol produced as a by-product can easily be recovered and the purification process for fatty acid methyl esters (FAMEs) is relatively simple. In the oil and fat industry, conversion of waste edible oil and soapstock (a by-product generated in alkali refining of vegetable oils) to biodiesel has attracted a great deal of attention (Azócar et al., 2010; Safieddin Ardebili et al., 2011; Singaram, 2009). Unlike the conventional chemical routes for synthesis of diesel fuels, biocatalytic routes permit one to carry out the interesterification of a wide variety of oil feedstocks in the presence of excess FFAs.

Olive pomace oil was used by Yucel (2011) for enzymatic production of biodiesel. Yucel (2011) immobilized microbial lipase from *Thermomyces lanuginosus* on olive pomace by covalent binding, and then used this immobilized lipase for the methanolysis of olive pomace oil. Under the optimized conditions for solvent-free reaction, the maximum yield was reported to be 93% at 25 °C after 24 h. Sanchez and Vasudevan (2006) produced biodiesel by transesterification of olive oil triolein with methanol using lipase. They studied the effects of the molar ratio of methanol to triolein, semibatch (stepwise addition of methanol) vs batch operation, enzyme activity, and reaction temperature on overall conversion. Because of the inactivation of the enzyme by insoluble methanol, stepwise methanolysis with a 3:1 methanol to triolein molar ratio and an overall ratio of 8:1 gave the best results.

#### **4.2 Enzymatic deacidification**

One method to reduce the FFA content in fats and oils is to convert the FFAs to TAGs. This is carried out by direct esterification of fatty acids with glycerol.

A reported application of enzymatic deacidification of olive pomace oil is the enzymatic glycerolysis of highly acidic (32%) olive pomace oil (Fadiloglu et al., 2003). FFAs of olive pomace oil were esterified with glycerol using a nonspecific immobilized lipase, reducing

Potential Applications of Green Technologies in Olive Oil Industry 303

Olive mill wastes can be treated with other methods such as composting to produce fertilizers (Ntoulas et al., 2011); using as a culture medium to produce useful microbial biomass (de la Fuente et al., 2011); using as a low-cost fermentation substrate for producing microbial biopolymers for production of polysaccharides and biodegradable plastics (Ntaikou et al., 2009); and as a base-stock for production of biofuels (Rincon et al., 2010).

Molecular distillation, also called short path distillation, has become an important alternative for separation of heat sensitive compounds or substances with very high boiling points. Molecular distillation is characterized by a short time exposure of the distilled liquid to elevated temperature and high vacuum, with a small distance between the evaporator and the condenser (Lutišan et al., 2002). The small distance between the evaporator and the condenser and a high vacuum in the distillation gap results in a specic mass transfer mechanism with evaporation outputs as high as 20–40 gm−2 s−1 (Cvengroš et al., 2000). Due to short residence time and low temperature, distillation of heat-sensitive materials is accomplished without thermal decomposition. Another advantage of the process is the absence of solvents. Therefore, molecular distillation is considered as a promising method in the separation, purication and concentration of natural products (Martins et al., 2006).

Vegetable oil deodorization process produces a distillate rich in high value components such as phytosterols, tocopherols, and fatty acids, depending on the oil or fat. Martins et al. (2006) separated FFAs from soybean oil deodorizer distillate to obtain a tocopherol concentrate, which contained only 6.4% of FFA and 18.3% of tocopherols (from a raw material containing 57.8% of FFA and 8.97% of tocopherols.) The specific processing conditions were an evaporator temperature of 160 °C and a feed ow rate of 10.4 gmin−1. Under these conditions, they achieved 96% FFA elimination and 81% tocopherol recovery. Although molecular distillation is a promising separation and purification method, it is not commonly applied in the olive oil industry. One relevant application is the purification of the structured lipids enzymatically produced from olive oil and caprylic acid (Fomuso & Akoh, 2002). If the advantages and efficiency of the system are further considered, it may be used in the olive oil industry for deacidification and separation of nutraceuticals. The cost of the system and possible alterations in the structure of the oil during the process seem to be serious disadvantages. Therefore, optimization of each particular system is necessary for a

**6. Use of by-products of olive oil industry for waste treatment** 

removal of RR198 textile dye from aqueous solutions (Akar et al., 2009).

The use of by-products of the olive oil industry for waste treatment is another green approach. Solid olive wastes were used for water purification by El-Hamouz et al. (2007). The solid olive residue was processed to yield relatively high-surface area active carbon after extraction of the oil from the residue. The resulting carbon was used to reversibly adsorb chromate ions from water, aiming at a purication process with reusable active carbon. In another study, olive pomace was used as reactive dye biosorbent material for the

Vlyssides et al. (2004) developed an integrated pollution prevention method which decreased wasterwater production 50% from the 3-phase olive oil extraction process. The

**5. Molecular distillation** 

successful industrialization.

the acidity of the oil to 2.36%. In another study, the FFA content of high acidic (31.6%) degummed and dewaxed olive oil was reduced to 3.7%.

#### **4.3 Bioremediation**

Bioremediation, generally classified as *in situ* or *ex situ*, is the use of microorganism metabolism to remove pollutants. *In situ* bioremediation involves treating the contaminated material at the site, while *ex situ* involves the removal of the contaminated material to be treated elsewhere. Some examples of bioremediation technologies are phytoremediation, bioventing, bioleaching, landfarming, composting, bioaugmentation, rhizofiltration, and biostimulation (Shukla et al., 2010). Besides being cost effective, bioremediation can result in the complete mineralization of the pollutant, considered a permanent solution of the pollution problem. Furthermore, it is a non-invasive technique, leaving the ecosystem intact. Bioremediation can deal with lower concentrations of contaminants where cleanup by physical or chemical methods would not be feasible. Unfortunately, it presents some major drawbacks which still limit the application of these techniques, including long processing times and less predictable results compared to conventional methods (Perelo, 2010).

The disposal of OMW is predominantly carried out via land spreading or by means of evaporation ponds, although a wide number of chemical and biological decontamination and valorisation technologies have been reported. The two-phase centrifugation system, as an alternative ecological approach for olive oil production, drastically reduces the water consumption during the process. This system generates olive oil plus a semi-solid waste, known as the two-phase olive-mill waste (Morillo et al., 2009).

Ramos-Cormenzana et al. (1996) performed aerobic biodegradation on OMW by using bacterium *Bacillus pumilus* to reduce the phenol content. They reported 50% reduction in phenol content using *Bacillus pumilu*s. The detoxification of OMW following inoculation with *Azotobacter vinelandii* (strain A) was performed for two successive 5-day-period cycles in an aerobic, biowheel-type reactor, under non-sterile conditions by Ehaliotis et al. (1999). The authors indicated that the phytotoxicity of the processed product was reduced by over 90% at the end of both cycles. However, aerobic bacteria cannot generally biodegrade complex phenolic compounds which are responsible for the dark color of OMW. Fungi, compared to bacteria, are more effective at degrading both simple and complex phenolic compounds presenting in olive mill wastes. This is due to the presence of compounds analogous to lignin monomers, which are more easily degraded by wood-rotting fungi (Garća Garća et al., 2000).

Demirer et al. (2000) generated biogas containing about 77% methane by anaerobic bioconversion of OMW (57.5 L methane per liter of wastewater). Ammary (2005) treated OMW using a lab scale anaerobic sequencing batch reactor, achieving more than 80% COD removal at 3 d hydraulic retention time. Anaerobic bioconversion has some advantages compared to aerobic processes: (a) high organic load feeds are used, (b) low nutrient requirements are necessary, (c) small quantities of excess sludge are usually produced, and (d) a combustible biogas is generated. However, the nutrient imbalance of OMW, mainly due to its high C/N ratios, low pH and the presence of biostatic and inhibitory substances, cause a problem. Not quite clear Rephrase An additional problem of two-phase olive-mill waste is its high consistency making its transport, storage and handling difficult (Morillo et al., 2009).

Olive mill wastes can be treated with other methods such as composting to produce fertilizers (Ntoulas et al., 2011); using as a culture medium to produce useful microbial biomass (de la Fuente et al., 2011); using as a low-cost fermentation substrate for producing microbial biopolymers for production of polysaccharides and biodegradable plastics (Ntaikou et al., 2009); and as a base-stock for production of biofuels (Rincon et al., 2010).
