Intensification of enzyme catalysed synthesis of hexyl acetate using sonication

2.1 Experimental setup

The experimental set-up consisted of mechanically agitated flat bottom glass reactor of 50 cm³ capacity, equipped with four baffles and a six-blade turbine impeller. The entire reactor assembly was immersed in a thermostatic sonication bath, which was maintained at the desired temperature with an accuracy of ±1°C. In order to obtain maximum cavitation intensity; the position of the reaction vessel was maintained at 2 cm above from the bottom of ultrasonic bath [24]. A typical reaction mixture consisted of different molar ratios of hexanol and triacetin diluted with hexane as a solvent. The reaction was carried out at various temperatures, enzyme amounts, ultrasound power, agitation and duty cycle. Dodecane was used as internal standard. Samples free from the catalyst particles were withdrawn periodically from the reaction mass and analysed by gas chromatography. A very small amount of samples were withdrawn so that it will not affect the reaction concentration. All experiments were performed three times to check the reproducibility and average values have been reported with the standard deviation.

2.2 Analysis

Analysis of liquid sample was carried out by using a gas chromatograph GC 8610 equipped with flame ionization detector using 4 m×3.8 m stainless steel column packed with 10% SE-30 stationary phase. Nitrogen was used as carrier gas at the flow rate of 42 ml/min. Column oven, injector and detector temperature were set at 70°C, 300°C, and 300°C, respectively.

The temperature programme was as follows: 70°C; 17°C/min up to 180°C; 17°C/min up to 220°C. Dodecane was used as internal standard. The percent conversion was calculated based on area curve of limiting reactant as follows:

% Conversion=[[(A0/I0)−(A/I)]/(A0/I0)]×100

Whereas A₀, A=area under curve of limiting reactant at time t=0 and t=t min, I₀, I=area under curve of internal standard at time t=0 and t=t min.

2.3 Determination of enzyme activity

The activity of biocatalyst lipozyme RM IM was determined by using oleic acid, n- butanol in iso-octane and distilled water [22]. Activity of catalyst was analysed through titrimetric method against alcoholic NaOH. One µmole of oleic acid consumed in the esterification reaction per min per mg lipase is expressed as a unit of enzyme activity. The initial activity of the catalyst was identified as 9.5 U/g.

3.1 Effect of molar ratio

Experiments were carried out in order to determine the effect of substrate ratio on the progress of a reaction. Reactions were carried at 50°C temperature, 1% (w/v) enzyme amount, 80% duty cycle, 100 W power, 100 rpm in hexane solvent. Concentration of Hexanol & triacetin was varied from 0.01 M to 0.03 M, respectively and vice versa from triacetin to Hexanol from 0.01 M to 0.03 M, respectively. It was found that higher conversion was obtained at the stoichiometric concentration of the reactions. While, the excess concentration of both the reactants decreases the conversion in some extent (Figure 1). Initially the reaction progresses very fast up to 60 min and further reduces with the time giving steady conversion rate. The conversions obtained at excess alcohol concentrations i.e. molar ratio of 1:1, 2:1, 3:1 and for excess triacetin concentrations i.e. molar ratio of 1:2, 1:3 are 42%, 24%, 21% and 35%, 30%, respectively. The increase in concentration of alcohol had a negative effect on conversion and possible reason for this behavior is that higher concentration of alcohol may bind the active sites of enzyme [25]. Also, an increase in triacetin content leads to increase in acetic acid concentration, which had a negative effect on enzyme activity [10]. Similar kind of alcohol inhibition was reported in the synthesis of octyl acetate in absence of ultrasound [26].

Figure 1:

Effect of molar concentration of reactants on conversion at hexane 3 ml, enzyme loading 1% (w/v), temperature 50°C, agitation speed 100 rpm, power 100 W, duty cycle 80% and frequency 25 kHz.

3.2 Effect of temperature

Temperature plays a significant role in enzymatic reaction since it has an impact on the solubility of solvent, enzyme activity and cavitation intensity. Thus experiments were performed by varying the temperature in the range of 40~60°C and results are depicted in Figure 2. It is found that all temperatures gives almost similar conversion till 1 h. However, the conversion obtained further varies significantly with temperature and after 4 h maximum conversion was observed for 50°C and 60°C. An increase in temperature reduces the viscosity of the reaction mixture and enhances the reactant solubility, improves diffusion which further increases the interaction between the active site of the enzyme and substrate. But in the case of enzyme catalysed reaction an increase in temperature also affects the activity of enzyme which reduces the conversion and selectivity. Although the conversions obtained after 4 h were similar for temperatures 50°C and 60°C, the reaction rate decreases for 60°C after 3 h as compared with 50°C. This is possibly due to loss in enzyme activity at high temperature [23]. Hence, 50°C temperature is considered as optimum for this reaction. The rate constant of the reaction is calculated by assuming first order reaction kinetics. The first order rate equation is represented as,

r=k[A]x

Figure 2:

Effect of temperature on conversion at hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 1% (w/v), agitation speed 100 rpm, power 100 W, duty cycle 80% and frequency 25 kHz.

Where, r is rate of the reaction, k is rate constant of the reaction and [A] is the concentration of triacetin.

In the linearized form it can be written as,

ln r=ln k+x ln [A]

The intercept in the plot of ln r versus ln [A] gives the value of rate constant k. By considering this value of k value of activation energy can be calculated with the help of Arrhenius equation, lnk=−EaRT+lnA

Where, E_a is the activation energy, R is the gas constant, T is the Temperature and A is the frequency factor. The value of the activation energy obtained is −569 kJ/mol. The large negative value indicates major influence of ultrasonication on the rate of reaction. From the value of activation energy, thermodynamic parameters of the reaction can be calculated using some basic thermodynamic correlations. Thermodynamic parameters such as change in enthalpy (ΔH), change in entropy (ΔS) and change in Gibbs free energy (ΔG) are important to determine the feasibility of the reaction. The values obtained for ΔH, ΔS and ΔG were represented in Table 1. Thermodynamic parameters show that the reaction is exothermic in nature as ΔH has negative value, spontaneous due the negative value of Gibbs free energy and the negative ΔS value shows that the reaction is thermodynamically feasible. A good feasibility of the process is observed as the parameters obtained are in agreement with the reported literature [27].

3.3 Effect of power

Ultrasound power affects the cavitation intensity which further affects the rate of reaction. Ultrasound power enhances the rate of reaction by increasing relative movement between catalyst molecule and substrate to improve attachment of the substrate to the catalyst surface through diffusion process and detachment of the product formed from the catalyst by strong mechanical forces. To determine the effect of power, experiments were conducted at ultrasound power of 40, 60, 80, and 100 W at 80% duty cycle keeping all other parameters constant to determine optimum power input value. Figure 3 shows that an increase in power increases the conversion till 60 W due to higher power dissipation and then decreases with further increase in power. At 40, 60, 100, and 140 W the power dissipation was calculated as 36, 42.7, 57.2, and 72.7 W, respectively (performing the calorimetric analysis at 25 kHz). With higher input power, not only the power dissipation was maximum but also denaturation of enzyme occurred leading to lowering the conversion [28]. Thus the power of 60 W was taken as optimum for further set of reactions.

Figure 3:

Effect of sonication power on conversion at hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 1% (w/v), temperature 50°C, agitation speed 100 rpm, duty cycle 80% and frequency 25 kHz.

3.4 Effect of enzyme loading

Enzyme loading is one of the crucial parameters affecting the conversion as well as the economy of the process. Experiments were performed with the enzyme loading ranging from 0.5~5% (w/v) and the results obtained are shown in Figure 4. The conversion was better with the addition in enzyme concentration up to 4% enzyme loading. The conversion obtained at 4 % (w/v) enzyme loading was 81%. While further increase in the enzyme at 5% fetched conversion of 82% with slight rise during the initial rate that is comparable to the data obtained with enzyme loading of 4%. The rise in reaction rate by increase in enzyme loading is due to the rapid formation of enzyme-substrate complex at sufficient enzyme loading. However, excess enzyme loading leads to the problem of mixing by an increase in viscosity. Similarly, when used in excess the active surface area of the catalyst may get reduced by blocking the active sites with the enzyme in the vicinity. Hence enzyme loading of 4% (w/v) was observed as the optimized condition and continued for further experimentation.

Figure 4:

Effect of enzyme loading on conversion at hexanol:triacetin ratio 1:1, hexane 3 ml, temperature 50°C, agitation speed 100 rpm, power 60 W, duty cycle 80% and frequency 25 kHz.

3.5 Effect of duty cycle

Continuous exposure of ultrasound to enzyme causes its degradation and therefore, periodic exposure of ultrasound is generally preferred for its better utilization. Duty cycle refers to the exposure of reaction medium to the ultrasound waves in one cycle. The duty cycle % can be varied by changing on/off timing of ultrasound. Experiments were carried out in order to find the optimum duty cycle with respect to higher conversion. Experiments with different duty cycles in the range of 60~80% were conducted and results are reported in Figure 5. It has been observed that conversion increases with a duty cycle till 70% and it gives less conversion at 80% duty cycle. The rate of reaction was almost similar for initial 1 h for all three duty cycles studied. However, the rate of reaction was decreased for 80% duty cycle compared to 70% duty cycle. Lower conversion at higher duty cycle may be due to denaturation of enzyme by increasing the exposure time to sonication waves. Extensive cavitation at higher duty cycle affects the protein structure due to imparted mechanical shear [23]. Thus 70% duty cycle is considered as optimum for further reactions.

Figure 5:

Effect of duty cycle on conversion at hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 4% (w/v), temperature 50°C, agitation speed 100 rpm, power 60 W and frequency 25 kHz.

3.6 Effect of speed of agitation

Earlier, it is reported that the only use of ultrasound is not sufficient to suspend the solid catalyst in the reaction mixture even a mild agitation is needed to improve the conversion. Hence, in order to determine the optimum value of speed of agitation, it is varied between 0~300 rpm by keeping other conditions at optimum (Figure 6). It was found that conversion is increased with an increase in speed up to 100 rpm and beyond that there is no significant increase in conversion. Ultrasound can improve mass transfer only when the enzyme is suspended in the reaction mixture. Thus when agitation speed is increased the more enzyme particles are suspended in reaction mixture which further increased the conversion. Once all the enzyme particles are suspended in reaction mixture there is no change in reaction conversion after 100 rpm. Similar results for the dependence of agitation speed in ultrasound assisted lipase catalysed reaction have been reported earlier [29].

Figure 6:

Effect of agitation speed on conversion at hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 4% (w/v), temperature 50°C, power 60 W, duty cycle 70% and frequency 25 kHz.

3.7 Enzyme reusability

It is necessary to reuse the catalyst after the reaction, as immobilized enzyme plays crucial role in economic profitability in biocatalytic reaction. Thus reusability study is important for the economic feasibility of a reaction. Reusability study was performed by using optimum parameters. After each cycle immobilized enzyme was filtered, washed with acetone, then kept in a desiccator at room temperature (30±2°C) for overnight, and then reused for the next cycle. The results obtained after six cycles are shown in Figure 7. It shows that conversion was decreased with an increase in reusability cycle. Due to continuous decrease in enzyme activity fall in percent conversion up to 36% was seen after sixth cycle. The loss in activity and a decrease in percent conversion was due to the possibility of physical damage during each cycle and the ultrasonic irradiation which may lead to the breakage of bonding between enzyme and supporting media.

Figure 7:

Reusability study performed at hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 4% (w/v), temperature 50°C, agitation speed 100 rpm, duty cycle 70% and frequency 25 kHz.

3.8 Comparison with conventional methods

To compare the results obtained under ultrasound and conventional stirring method, three experiments were carried out under optimized conditions for enzyme catalysed transesterification. Transesterification in the presence of ultrasonication, reactions assisted with sonication but without stirring and conventional stirring methods were compared to detect the efficiency of the experimentation methodologies. It was observed that the conversion of hexyl acetate when ultrasound coupled with stirring gives the conversion of 85% as compared to 67% and 60% for conventional stirring method and only ultrasound without stirring after 4 h, respectively. Figure 8 gives the comparison between enzyme catalyzed synthesis in the presence and in absence of ultrasound. This clearly shows that the rate of reaction in the presence of ultrasound is very high and almost 85% conversion is obtained in 4 h while conventional stirred could only produce 68% at a prolonged time of 9 h. Thus, it can be concluded that in the case of ultrasonication coupled with stirring gives higher conversion compared with ultrasonication alone and conventional stirring method. Also, the value of the activation energy for conventional process reported in the literature is positive and found to be in the range of 5~45 kJ/mol [30], [31] and for ultrasound assisted process it shows negative value, describing very less amount of energy required to overcome the energy barrier of transition state to form a product. This might be happened due to the cavitation phenomenon which produces adequate turbulence and mixing of the reaction mass. On the other hand, in order to understand the energy effectiveness of the ultrasound, the preliminary calculation for power required are done and it is found that ultrasonic irradiation requires nearly 20% extra power than conventional stirring method. However, considering the large difference in time requirement for conventional stirring method, this method sounds technically feasible in case of process intensification and ambient operating conditions. Further, various researchers are currently working on the scale up of sonochemical reactors, which suggest the use of multiple transducers in a reactor having uniform power distribution over a wide area [32].

Figure 8:

Comparison between Sonication & Conventional method (Sonication Condition- hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 4% (w/v), temperature 50°C, agitation speed 100 rpm, duty cycle 70% and frequency 25 kHz) (conventional method condition- hexanol:triacetin ratio 1:1, hexane-3 ml, enzyme loading 4% (w/v), temperature 50°C, agitation speed, 400 rpm).