**1. Introduction**

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Complete utilisation of by-products from agricultural and food industrial production is an important task both from economical and environmental aspects. There are numerous possibilities for manufacture of bioproducts by biotechnological processes, among them biofuels attracted the greatest attention (Watanabe et al., 2000). Industrial by-products can be, however, processed by several biotechnological methods, for example their utilisation as a food additive, which requires intensive research and development work (Filipini & Hogg, 1997, Demirbas, 2000).

Fusel oil is a by-product of distilleries, its average composition is 10% ethanol, 13% npropanol, 15% i-butanol, 51% i-amyl-alcohol, 11% miscellaneous alcohols and water. Nowadays fusel oil is usually burned to cover the energy demand of the distilleries. Researches have been carried out to utilise it as an additive to improve octane number in gasoline or for production of natural flavours and lubricants (Özgülsün & Karaosmanoglu, 1999).

Esterification of fusel oil with oleic acid using sulphuric acid catalyst was studied by Turkish researchers and bio-lubricant—according to ASTM (American Society Testing and Materials) standard—was manufactured for industrial purposes. Pure, natural lubricants manufactured by environmental-safe processes, however, have gained more and more attention recently, since they do not contain toxic compounds and are biological degradable. The demands against a bio-lubricant are that it should provide maximal protection during its usage, do not pollute the environment and do not accumulate (Özgülsün et al., 2000).

Unfortunately the used lubricants are usually deposited in the environment, endangering our planet. To solve the problem lubricants should be manufactured from plant oil derivatives. There are several industrial application possibilities for fatty acid esters, as natural compounds. Oleic acid (cis-9-octadecenoic acid) is one of the most important fatty acids in nature, it can be obtained from plant oils (Bélafi-Bakó et al., 1994), its esters produced by enzyme catalysis can be applied as lubricant (Linko et al., 1998).

Modern enzymology has achieved improvements in the development and application of lipase as catalyst. New immobilisation techniques make possible to use enzymes in industrial processes in a similar way to the classical catalysis for heterogeneous reactions.

n-propyl laurate obtained in the standard test corresponding to the esterification of lauric

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The fusel oil was provided as a gift by Distillery Győr (Hungary). All the other chemicals used in analysis were of analytical grade and purchased from Reanal Ltd. (Hungary) and

Two different procedures were used for ester production. Firstly, synthesis of esters was carried out in shaking flasks (150 rpm) containing 25 ml solution of several alcohols and oleic acid mixture with different molar ratios, different temperatures and various amounts of enzyme by using New Brunswick Scientific (USA) shaking incubator to study the esterification kinetics. The starting time of the reaction was the addition of the enzyme. In the other procedure a 200 ml round flask reactor was thermostated and connected with a pervaporation unit using hydrophilic membrane for continuous removal of water produced. The reaction mixture was circulated through the pervaporation unit by a peristaltic pump. The vacuum pump, manometer and the cooled traps were connected to the pervaporation unit. The laboratory scale pervaporation unit was purchased from Carbone Lorraine (Germany) and it was jacketed later. The membrane surface area was 2.0\*10-2 m2. The membranes used for the pervaporation experiments (PERVAP 2201, PERVAP 2202, CMC-VP-43) were

Aliquots of the reaction mixture were withdrawn periodically and the residual acid content was assayed by titrating against potassium hydroxide (0.1 M) using phenolphthalein as an indicator and ethanol as a quenching agent. The percentage esterification was calculated from the values obtained for the blank and the test samples. The fusel oil esters were confirmed by chromatographic analyses of the samples using a Hewlett Packard Model 5890 Series II GC equipped with a flame ionisation detector and a 30 m HP-FFAP capillary column. The percentage esterification calculated by both GC analysis (which showed product formation) and titrimetry (which showed acid consumption) were found to be in good agreement. The water content of the reaction mixture was measured by Mettler DL 35

The esterification reaction of oleic acid with the fusel oil fraction occurs as follows:

oleic acid + fraction of fusel oil = oleate esters + water In this reversible reaction, the molar ratio of reactants, temperature, enzyme and removal of water from the reaction mixture are the variables affecting the conversion and the reaction

Water level is critical for enzymes and affects enzyme action in various ways: by influencing enzyme structure via noncovalent bonding and disruption of hydrogen bonds; by facilitating reagent diffusion; and by influencing the reaction equilibrium. Too low water content usually reduces enzyme activity. High water content can also decrease reaction rates by aggregating enzyme particles and causing diffusional limitations. The optimal amount of

acid with n-propyl alcohol, after 15 min at atmospheric pressure.

provided by GFT (Germany) and Celfa (Switzerland).

Sigma Chemical Co. (USA).

automatic Karl-Fischer titrator

**2.2 Results** 

**2.2.1 Water content** 

water is often within a narrow range.

rate.

For example, esters produced from long-chain fatty acids (12–20 carbon atoms) and shortchain alcohols (3–8 carbon atoms) have been used increasingly in the food, detergent, cosmetic and pharmaceutical industries. Esters prepared from the reaction of long-chain acids with long-chain alcohols (12–20 carbon atoms) also have important applications as plasticizers and lubricants (Zaidi et al., 2002, Dossat et al., 2002). The direct effect of the ester group on the physical properties of a lubricant is to lower the volatility and raise the flash point.

Compared with conventional chemical synthesis from alcohols and carboxylic acids using mineral acids as a catalyst, the use of enzymes such as lipases to produce these high valueadded fatty acid esters in solvent-free media may offer many significant advantages (Yadav & Lathi, 2003). These include the use of any hydrophobic substrate, higher selectivity, milder processing conditions and the ease of product isolation and enzyme reuse. The ecological properties of oleochemical esters have been intensively studied within the last couple of years. In general, their aquatic toxicity is very low or almost negligible. For the aquatic compartment the fish, daphnia, algae and bacteria are the most relevant test organisms and standardized test methods, such as laid down in the OECD methods 201–210 (Willing, 1999).

Esterification reactions by lipase in non-conventional media have been studied in our laboratory for long (Gubicza et al., 2003). Enzymatic esterification of fatty acids and ingredients of fusel oil was studied by (Gulati et al., 2003) using lipase from *Aspergillus tereus*. They found that in n-hexane solvent the alcohols were able to react with the fatty acids (miristic acid, palmitic acid, stearic acid), except oleic acid. Using other lipase preparations (*Candida antarctica, Candida rugosa, Rhizomucor miehei*, *Porcine pancreas*), however, made it possible the successful oleic acid esterification with similar low molecular weight alcohols. Description of the correct kinetics on the particular esterification reaction is even more difficult due to the various possible inhibition effects.

In our earlier work natural aroma esters were produced by enzymatic esterification in organic solvents and in solvent-free media (Gubiza, 2000). In this work the purpose was to find a utilisation of fusel oil, where bio-lubricants can be manufactured. The alcohol compounds of fusel oil were esterified with oleic acid using enzyme catalysis in nonconventional, solvent-free media (section 2) and in ionic liquid (section 4), moreover the kinetics of the reaction was described (section 3).
