**4.1 Ionic liquids**

*Ionic liquids*: 1-butyl-3-methyl-imidazolium hexafluoro phosphate [bmim]PF6, 1-butyl-3 methyl-imidazolium tetrafluoro-borate [bmim]BF4, tributyl tetradecyl phosphonium dodecilbenzol sulfonate (Cyphos-201), trihexil tetradecyl phosphonium bis- (2,2,4 trimetilpentil)-phosphate (Cyphos-104), tetradecyl phosphonium trihexil decanoate (Cyphos-105) trihexyl tetradecyl phosphonium hexafluoride, phosphate (Cyphos-110) (IoLiTec GmbH, Germany)

*Reaction mixture composition*: Under the pre-investigations, the reaction mixture was the follow: 6.36 mmol of oleic acid, 36.95 mmol of isoamyl alcohol, 304.35 mmol n-hexane and 50 mg Novozym 435 lipase. In the course of the main experiment: 1.23 mmol of ionic liquid, 0.16 mmol oleic acid, 1.41 mmol isoamyl-alcohol were used. The reaction mixtures were shaken with an intensity of 150 rpm at 40, 50 and 60 º C temperatures for 4 hours.

#### **4.2 Effect of ionic liquid**

As it well known ionic liquids are not only green solvents, they can have catalytic effect. Our aim was to investigate separately the enzyme stabilization effect, therefore preliminary experiments were necessary. Through these the most important investigated criterion was to verify if the ionic liquid itself had catalytic effect for the reaction without enzyme. Basic criterion was to be work in a two-phase reaction, where separation is easier, since the application of a new solvent can be avoid (Eckstein et al., 2004). Two of the investigated

Kinetic model for description of the enzymatic esterification of oleic acid and i-amyl alcohol was – according to our best knowledge – elaborated and presented in the first time in literature. The model is based on the ping-pong bi-bi mechanism and not only product, but substrate inhibition is taken into account, as well. The model fitted well to the experimental data (checked by r2 values), though the measurements covered an extremely wide

Ionic liquids can be suitable media for biocatalytic synthesis because of their enzyme stabilization effect, reusability and negligible vapour pressure (Yang & Pan, 2005). Many enzymes, especially lipases showed higher activity and greater selectivity in ionic liquids than in organic solvents (Jain et al., 2006). The stability of *Candida antarctica* lipase B (CALB) in ionic liquid was found to be higher than in organic solvent (Romero et al., 2005). The main advantages of ionic liquid + enzyme system are milder, lower reaction temperature comparing with conventional chemical synthesis, it needs less energy investment, can be better-controlled, attended with less side-reaction, furthermore the product more readily separable. In this work the purpose was to find a utilization of fusel oil where biolubricants

In organic solvents low conversions were achieved, therefore it became necessary to develop a better method, which is energy efficient, able to achieve higher conversion, furthermore environmental friendly, however waste materials may be as natural substrates. Our aim is to optimize the parameters of production, enhance the yield improving the advantages of

*Ionic liquids*: 1-butyl-3-methyl-imidazolium hexafluoro phosphate [bmim]PF6, 1-butyl-3 methyl-imidazolium tetrafluoro-borate [bmim]BF4, tributyl tetradecyl phosphonium dodecilbenzol sulfonate (Cyphos-201), trihexil tetradecyl phosphonium bis- (2,2,4 trimetilpentil)-phosphate (Cyphos-104), tetradecyl phosphonium trihexil decanoate (Cyphos-105) trihexyl tetradecyl phosphonium hexafluoride, phosphate (Cyphos-110)

*Reaction mixture composition*: Under the pre-investigations, the reaction mixture was the follow: 6.36 mmol of oleic acid, 36.95 mmol of isoamyl alcohol, 304.35 mmol n-hexane and 50 mg Novozym 435 lipase. In the course of the main experiment: 1.23 mmol of ionic liquid, 0.16 mmol oleic acid, 1.41 mmol isoamyl-alcohol were used. The reaction mixtures were

As it well known ionic liquids are not only green solvents, they can have catalytic effect. Our aim was to investigate separately the enzyme stabilization effect, therefore preliminary experiments were necessary. Through these the most important investigated criterion was to verify if the ionic liquid itself had catalytic effect for the reaction without enzyme. Basic criterion was to be work in a two-phase reaction, where separation is easier, since the application of a new solvent can be avoid (Eckstein et al., 2004). Two of the investigated

shaken with an intensity of 150 rpm at 40, 50 and 60 º C temperatures for 4 hours.

concentration range.

**4. Reaction in ionic liquid** 

can be manufactured in ionic liquid.

enzyme+ionic liquid system.

(IoLiTec GmbH, Germany)

**4.2 Effect of ionic liquid** 

**4.1 Ionic liquids** 

ionic liquids (Cyphos-105 and Cyphos-110) were mixed with the substrates, so they were not investigated further. Henceforth the reactions were carried out at 50 °C, the ester yield was followed by GC and the decreasing oleic acid concentration using titrimetry. The percentage esterification was calculated from the values obtained for the blank and the test samples. The further phosphonium-type ionic liquids, Cyphos-201 and Cyphos-104 without the presence of enzyme greatly catalyze the process as it shown on Fig. 7.

Fig. 7. Oleate ester concentration in case of using different phosphonium-type ionic liquids (reaction time: 180 min)

For imidazolium-cation containing ionic liquids literary data show that these types of ionic liquids are the most suitable for esterification, transesterification reactions (Moniruzzaman at al., 2010). Both investigated imidazolium-type ionic liquids were successfully applied in earlier experiments, where natural aroma esters production was the aim. In case of [bmim]BF4 there were no significant differences in the detected oleate concentration if enzyme was added or not. For [bmim]PF6 - without the presence of the enzyme only negligible product formation was observed, in the presence of enzyme, higher concentrations were achieved than in the experiments where n-hexane was used as solvent.

Therefore for further investigations this ionic liquid was chosen. In case of ionic liquids for water solubility cations are responsible. Comparing the same cation having [bmim]BF4 and [bmim]PF6 shows that while the first not miscible with water, the later has an unlimited solubility in water. Thus, the hydrophilic [bmim]BF4 ionic liquid often distracts the absorbed water layer from the surface of the enzyme which should be necessary for the active conformation. Therefore the enzyme is deactivated (van Rantwijk & Sheldon, 2007). Further advance that in case of using [bmim]PF6 side-reactions were not observed.

#### **4.3 Acid/alcohol molar ratio**

The optimal parameters of the batch production were determinated using experimental design software application. In doing so Statistica 8.0 program was applied. Based on earlier studies substrate molar ratio, amount of enzyme and ionic liquid were chosen as key factors. Each factor was prepared in two levels: -1 for low level and +1 for high level.

Time [h]) 0 5 10 15 20 25 30

The yield-time functions in all cases show the saturation curve. Increasing the temperature equilibrium yield was not changed, but shorter time was necessary to achieve it. As it shown neither at 80 °C became the enzyme deactivation significant, but increasing up from 70 °C

Our investigations have proven that an ester type biolubricant could be prepared from fusel oils and oleic acid by lipase enzyme in solvent-free system. Compared to the product obtained by acid catalysis, in the biolubricant there was no trace of oleic acid since complete conversion was achieved by continuous water removal by pervaporation. Our product was then tested in an acute toxicological procedure by zebrafishes, which has verified the assumption: the biolubricant is not toxic for the living water, so it is considered as an

The tribological study has shown that the features of the biolubricant are similar to the DB 32 type synthetic reference lubricating oil, so it can be applied as a low viscosity lubricant, suitable even for special publication processes, where lubricant loss may accur. Although the biological degradation of the product has not been studied, it was manufactured from initial compounds having biological origin (oleic acid from plant oils and alcohols from a by-product in bioalkohol production), thus it is considered as a completely environmental-

The kinetic model containing the parameters determined can be used in the particular enzymatic esterification reaction for calculations of the optimal conditions of various aspects, like highest yield, lowest acid residue, lowest amount of enzyme, shortest reaction time…etc. Moreover we are planning to apply the model for bioreactor design to realise

Our work has showenn that an ester type biolubricant could be prepared from isoamylalcohol and oleic acid by lipase enzyme in ionic liquid two phase system. Compared to the product obtained in solvent free system, higher conversion in shorter time was achieved. Despite the lack of water removal in the biolubricant there was no trace of oleic

Fig. 9. Influence of reaction temperature on the synthesis of isoamyl-oleate

did not shorten the reaction time till achieving the equilibrium.

continuous enzymatic i-amyl oleate synthesis by lipase.

70°C 60°C 80°C 133

Yield [%]

**5. Conclusions** 

friend product.

environmental safe product.

0

20

40

60

80

100

120

Concrete values were applied in a design matrix. It is evident that increasing reaction temperature enhances the reaction rate, that is way that its affect will be investigated later separately.

During the experiments different, software-defined combinations of the previously selected values of experimental parameters were investigated. Other parameters were fixed: temperature 50 °C, 150 rpm shaking intensity, 5 hours reaction time. Water content of the reaction mixture was also followed as an important parameter of esterification reactions which may shift the equilibrium, but there was not detectable concentration change using Carl-Fischer titration. The aim was to find the optimal parameter values of the isoamyloleate production. The results are shown on Fig. 8.

As it shown, increasing amount of ionic liquid results in higher oleate yield, which gives evidence for the advantageous enzyme stabilization effect. Complex investigation of the three chosen key factors shows that the highest ester conversion was obtained in the case of application the highest acid/alcohol molar ratio, amount of enzyme and ionic liquid. Relatively high yield was obtained also around medium values, around the center points.

Fig. 8. Influence of the acid/alcohol molar ratio and amount of ionic liquid on the synthesis of isoamyl oleate

There was no inhibition effect observed neither in 1:16 acid/alcohol molar ratio. As none of the point of parameters could be an optimal value, because the highest yield belonging to the highest values, therefore further investigations will necessary to find the optimal values.

#### **4.4 Effect of temperature**

In case of using conventional reaction media it is well known that increasing reaction temperature results the same yield in shorter time, but till a limit due to thermal deactivation of enzyme. Beside of the structure stabilization effect of ionic liquids the enzyme can be resist in the active conformation at higher temperatures (van Rantwijk & Sheldon, 2007). For these investigations that mixture was chosen, in which the highest oleate yield was obtained. The results are shown on Fig. 9.

Fig. 9. Influence of reaction temperature on the synthesis of isoamyl-oleate

The yield-time functions in all cases show the saturation curve. Increasing the temperature equilibrium yield was not changed, but shorter time was necessary to achieve it. As it shown neither at 80 °C became the enzyme deactivation significant, but increasing up from 70 °C did not shorten the reaction time till achieving the equilibrium.
