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Ultrasound assisted enzymatic conversion of non edible oil to methyl esters
Ultrasonics Sonochemistry 21 (2014) 1374–1381
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Ultrasound assisted enzymatic conversion of non edible oil to methyl esters Sanket H. Jadhav, Parag R. Gogate ⇑ Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 3 December 2013 Accepted 18 December 2013 Available online 3 January 2014 Keywords: Ultrasound Hydrolysis Esterification Methyl ester Process intensification
a b s t r a c t Conventional and ultrasound-assisted hydrolysis and subsequent esterification of Nagchampa oil under mild operating conditions have been investigated with an objective of intensification of methyl esters production using a sustainable approach. The effect of ratio of reactants, temperature, enzyme loading, pretreatment of enzyme (using ultrasonic irradiations) on the hydrolysis and esterification reaction has been studied. Optimum conditions for hydrolysis were observed to be 1:1 weight ratio of oil: water for Lip Z and 1:3 for Lip 2 enzymes, enzyme loading of 400 units for Lip Z and 800 mg for Lip 2 enzymes and reaction time of 6 h. In the case of esterification reaction, optimum conditions obtained were oil to methanol molar ratio of 1:2, enzyme loading of 1000 mg and reaction time of 20 h. Use of pretreated enzyme (using ultrasonic irradiations) was found to increase the extent of esterification reaction from 75% to 92.5%. It was observed that use of ultrasound in the reaction significantly intensified the esterification reaction with time requirement reducing from 20 h for conventional stirring based approach to only about 7.5 h in the presence of ultrasound. The extent of esterification obtained with sonicated enzyme also increased to 96% from 75% with unsonicated enzyme. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction With increasing demand of fragrances in cosmetic industry, it has become a necessity to design and develop fragrance chemicals which originate from sustainable sources. Some of the suggested sources include renewable liquids coming from biological raw material which can prove to be good substitutes in the cosmetics sector. Nagchampa oil is an essential oil used in fragrance industry, but owing to the high acid value, it has limited stability. The stability of such essential oils can be significantly improved by converting it to its methyl ester. Methyl ester can be produced from oils through stepwise hydrolysis-esterification reactions or transesterification reactions. A large variety of plants that produce non-edible oils can be considered for methyl esters production [1]. Non-edible oils from sources such as neem, mahua, karanja, babassu, Jatropha, microalgae, Camelina sativa, Nagchampa etc. are easily available in many parts of the world, and are very cheap compared to edible oils. Also, for growing non-edible oil crops, less fertilizer, herbicides and insecticides are required compared to edible oil crops [2]. Methyl ester production can be carried out with acid catalyst or by using biocatalysts. The main advantages of employment of lipases as catalyst for methyl ester production are mild reaction conditions and easy recovery of glycerol without purification. ⇑ Corresponding author. Tel.: +91 22 33612024; fax: +91 22 33611020. E-mail address: [email protected] (P.R. Gogate). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.12.018
Additionally, free fatty acid content in the oil can be entirely converted to methyl esters with no soap formation, thereby increasing the methyl ester yields [3]. These features of the lipase enzymes allows the direct usage of materials with high free fatty acids (FFA) or high water content such as non-edible oils, waste cooking oils and industrial waste oil for methyl ester synthesis. Lipase enzyme has been widely used for hydrolysis of fats [4], esterification and transesterification [5]. In spite of numerous advantages, enzymatic processes have drawbacks such as low reaction rate, high enzyme cost in comparison to acid and alkali catalyst and low enzyme stability in the presence of excess methanol [6,7]. Besides the enzyme related problems, process of production of methyl esters faces various problems related to the immiscible nature of the reactants causing poor mass transfer rate. This problem is responsible for longer reaction time and low reaction rate leading to an energy intensive process. Ultrasound can eliminate the problem of poor immiscibility between reactants as ultrasonic energy can emulsify the reactants offering much higher interfacial area for reaction also possibly reducing the catalyst requirement, reaction time and reaction temperature. Ultrasound action in methyl ester production is primarily based on the emulsification of the immiscible liquid reactants by microturbulence generated by cavitation bubbles [8]. Gole and Gogate [9] performed experiments on methyl ester formation from non-edible Nagchampa oil and found that temperature and reaction time required for esterification, as well as the transesterification stages, are substantially
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lower in the case of sonochemical reactors, compared to the conventional heating method. Liu et al. [10] used ultrasound cavitation for lipase catalyzed hydrolysis and reported that around 2-fold intensification was obtained. The present work reports the ultrasound assisted synthesis of methyl esters from high-acid-value Nagchampa oil using the two-stage approach of hydrolysis followed by esterification. The objective of first stage was to achieve hydrolysis to considerable extent in order to get acids in sufficient quantity for esterification to methyl esters in the next step of esterification. The effect of different operating parameters such as reaction temperature, ratio of reactants, catalyst concentration and sonication of enzyme has been investigated. Two types of approaches for ultrasound based synthesis were followed in the work viz. enzyme was initially treated with ultrasound and then used in the reaction whereas in the second approach ultrasound was used throughout the reaction period. 2. Materials and methods
Fig. 1. Experimental setup for enzyme sonication A: Generator, B: Beaker introduced in ice bath, C: Ultrasound Probe, E: Temperature Indicator, F: Stand.
2.1. Materials Candida antarctica Lipase Enzyme standard was received as a gift sample from Novozyme, Denmark. Tributyrin, sodium dihydrogen phosphate and disodium hydrogen phosphate were procured from Himedia laboratories, Mumbai. Ultrasonic horn used in the experiments was procured from Dakshin, Mumbai having variable power output up to 220 W, and frequency of 20 kHz. The raw Nagchampa oil used for hydrolysis was procured from M/s Sanjay Shirsat Oil Mill (Vengurla, Dist: Sindhudurg, Maharashtra, India). Oil was initially filtered to remove traces of particles and mud. Table 1 gives the typical composition of oil consisting of 25% saturated acid (stearic and palmitic) and 72.7% unsaturated acid (oleic, linoleic, and linolenic). The initial saponification value of the oil was found to be 156 mg KOH/g of oil. Methanol, potassium hydroxide and oxalic acid (required for standardization and titration) were procured from S.D. Fine chemicals Pvt. Ltd., Mumbai. Distilled water was used as a reactant for hydrolysis and was prepared from the laboratory scale distillation unit. All the chemicals were used as received from the supplier, unless otherwise specified. 2.2. Experimental procedure 2.2.1. Sonication of enzyme Sonication was done in direct mode with 100 ml volume of 3 mg/ml enzyme solution subjected to ultrasonic irradiations using a ultrasound probe. The ultrasound probe was dipped in solution to 0.5 cm height and initial parameters were chosen as duty cycle of 66% and frequency of 20 kHz and power of 50 W. The setup used for enzyme sonication studies has been shown in Fig. 1. 2.2.2. Hydrolysis The hydrolysis reaction was carried out using lipase from two different sources namely Candida rugosa and C. antarctica. The objective was to achieve hydrolysis to considerable extent so as
Table 1 Fatty acid composition of Nagchampa oil. Fatty acid
Composition (%)
Palmitic, C16:0 Stearic, C18:0 Oleic, C18:1 Linoleic, C18:2 Linolenic, C18:3
12 13 34.1 38.3 0.3
to get acids in sufficient quantity for esterification to methyl esters in the next step. During experiments four important factors were considered and optimized in order to get maximum hydrolysis. The effect of ratio of oil to water, time of hydrolysis, temperature of hydrolysis and enzyme loading has been investigated. Initially reaction time was optimized by taking readings every one hour interval up to 8 h and acid values of the samples were analysed using titration. The temperature was optimized by checking the acid value at different temperatures in the range of 25–55 °C for optimized reaction time. Effect of reactant ratio was then investigated over the range 1:1–1:4 at optimized time and temperature. Catalyst concentration was optimized between 100 and 500 activity units for both C. rugosa (Lip Z) and C. antarctica (CALB Lip 2) lipase. All the reactions were carried out for 30 ml volume. After optimization of the entire range of parameters, final run was done to get maximum hydrolysis at larger scale of 200 ml to check the scale up aspects. 2.2.3. Esterification Next step of methyl ester synthesis after hydrolysis is esterification. This step converts free fatty acids into methyl esters using methanol. In order to prevent the progress of reaction in reverse direction i.e. hydrolysis which results in lowering the ester yield, molecular sieves were added in the reaction mixture in order to remove the water produced in the reaction. Parameters optimized were same as described for the hydrolysis reaction. First the reaction time was optimized by taking samples after every 4 h for a total reaction time as 28 h. The samples were analyzed to get the optimum time for minimum acid value which signifies maximum methyl ester yield. The molar ratio of oil to methanol was also optimized. This is a critical step in the process as excess methanol kills the enzyme and less methanol being a limiting reactant does not drive the reaction in the forward direction in an efficient way. Molar ratio was optimized over the ratio of 1:1–1:4. Finally the catalyst loading was also optimized. As the enzyme (CALB Lip 2) used for this step was an immobilized enzyme, the loading was taken in terms of weight of dried immobilized beads. The total volume of the mixture was made to 40 ml in each experiment using hexane. All the samples were analyzed for the determination of acid value. 2.2.4. Hydrolysis and esterification with sonication Hydrolysis and esterification both were done with sonication at 50 W with same reaction mixture in order to check the effect of
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mass transfer on the reaction system. Two different runs were taken with sonication in which one enzyme used was unsonicated immobilized enzyme (USIE) and other was sonicated immobilized enzyme (SIE). The runs were taken at 50 W to assure the stability of enzyme and the reaction mixture used was at optimized reaction conditions. 2.3. Analysis 2.3.1. Lipase assay Normal acid base titration assay was performed to find the amount of butyric acid produced from hydrolysis of tributyrin using lipase [11]. For the analysis, 1062 ll of 50 mM phosphate buffer (pH 7.0) was mixed with 250 ll of enzyme solution and 188 ll tributyrin. Mixture was stirred continuously for 5 min using cyclo mixer. After that indicator was added and mixture was titrated against methanolic NaOH till end point change from colourless to faint pink was observed. 2.3.2. Acid value determination Acid value of oil gives an idea about the amount of free fatty acids in the oil. Acid value can be a measure of how much the oil has been hydrolysed and also indicate the suitability for the process of methyl ester formation. 1.0 g of oil was dissolved in 100% ethanol. 0.05 M alcoholic KOH and 0.1 M oxalic acid were prepared and KOH standardization was done by titrating it with oxalic acid. Standardized alcoholic KOH was then used to titrate the dissolved sample of oil. The titrations were repeated thrice and mean reading was taken to find out the acid value which is expressed in terms of mg of KOH required to neutralize 1.0 g of oil. Acid value (AV) was calculated by using Eq. (1):
AV ¼
56:1 Normality of KOH titer reading : Weight of oilðgÞ
ð1Þ
2.3.3. Saponification value determination The procedure involves the use of excess 0.7 N alcoholic KOH, which catalyzes the saponification i.e. release of the free fatty acids from the glycerol backbone. The unreacted KOH is then back-titrated with standardized hydrochloric acid (HCl) using phenolphthalein as the indicator. 0.5 N HCl is standardized by using 0.1 N oxalic acid. The amount and normality of the HCI used for neutralization can then be used to calculate the saponification value. Saponification value is calculated in terms of mg KOH/g of oil. Sap: value ¼
ðTiter of HCl for blank Titer for HCl sampleÞ Normality of HCl 56:1 Weight of oilðgÞ
ð2Þ
with time. It is clear from the Fig. 2 that acid value increases with an increase in the reaction time, reaches a maximum in 6 h and then marginally decreases. The profile matches with some of the literature illustrations [12]. The decrease in acid value after 6 h can be attributed to the inhibition of the enzyme by glycerol, which is being formed as the product of reaction [13]. The glycerol formed in the reaction mixture gets coated on the enzyme surface to block the sites where substrate binds [14]. Also the concentration of fatty acids might prove to be responsible for product driven inhibition of the reaction [15]. It was also observed that use of Lip Z enzyme results in higher yields as compared to the CALB Lip 2. Lip Z and CALB Lip 2 both are 1,3 specific lipases which give different reaction kinetics due to the structural differences. If we see the 3-D structure of both Lip Z and CALB Lip 2, there is a difference between the two in the fact that the lid conformation of CALB Lip 2 is only half open at interphase which needs extra activation energy to make the reaction happen whereas other form of lipase show complete lid opening at the interphase [16]. It can be concluded that the difference in reaction kinetics is due to different accessibility of substrates to the enzyme active site. 3.1.2. Effect of ratio of oil to water (aqueous enzyme solution) The ratio of oil to aqueous solution has been considered in accordance to weight and the obtained results for the variation in acid value with ratio have been shown in Fig. 3(a) and (b). In case of Lip Z, maximum acid value was obtained for 1:1 weight ratio of oil: water but for CALB Lip 2, maximum acid value was obtained at 1:3 ratio. In both the cases, further increase in reactant ratio resulted in decrease in acid value. This can be attributed to the phase separation happening in the case of higher ratios. Enzyme always remains at the interphase of the oil water mixture but it has more tendency to get dissolved in water. During chemical hydrolysis water taken is in excess so that it won’t behave as a limiting reactant, but during enzymatic hydrolysis the reactant ratio selected should be very stringent in order to control the concentration of enzyme at interphase [17]. This is the reason why there is a decrease in the yield of reaction with CALB Lip 2 for further increase in quantum of water. Similar results have been obtained by Rathod and coworkers for transesterification of waste cooking oil [18]. 3.1.3. Effect of enzyme loading Catalyst or enzyme if used in optimum amount can give good reaction yield but if used in excess this may not give better results but create problems with subsequent separation and higher costs of treatment. With this objective the effect of enzyme loading has been investigated and the obtained results have been shown
3. Results and discussion 3.1. Hydrolysis Acid value gives the amount of free fatty acids in the oil. Increase in the acid value of the oil is an indication of hydrolysis of the oil. Enzymatic methods take a long time to hydrolyze the oil than chemical methods like acid hydrolysis or alkaline hydrolysis and hence it is necessary to investigate the effect of important operating parameters. Two enzymes used for hydrolysis were C. rugosa lipase (Lip Z) and C. antarctica lipase (CALB Lip 2). The reason for selecting two enzymes was to compare the efficacy of two enzymes to maximize hydrolysis. 3.1.1. Effect of time on hydrolysis The samples taken at different interval of times were analyzed for the acid value. Fig. 2 shows the profile of change in acid value
Fig. 2. Effect of time on hydrolysis for Lip Z and CALB Lip 2.
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100
AV (mg KOH/g oil)
90
90.822
80
73.275
70 60
54.235
50
47.913
40 30 20 10 0 1:1
1:2
1:3
1:4
Reactant ratio Fig. 3a. Effect of reactant ratio on hydrolysis with Lip Z (wt/wt) (enzyme loading of 400 units, time 6 h).
70
63.667
AV (mg KOH/g oil)
60 50
47.866
60.036
47.714
40
Fig. 4b. Effect of catalyst loading on hydrolysis with CALB Lip 2 (reactant ratio 1:3, time 6 h).
Fig. 4(b) shows that if the weight of beads is increased above 800 mg, the overall yield decreases. This happens because there is a saturation observed in enzyme available which leads to slower rate of reaction [19]. This again indicates that at high enzyme concentrations, the interfacial area between the oil and the aqueous phase limits the initial rate of reaction. For hydrolysis, optimized parameters for both the enzymes have been given in Table 2.
30 20 10 0
1:1
1:2
1:3
1:4
Reactant ratio Fig. 3b. Effect of reactant ratio on hydrolysis with CALB Lip 2 (enzyme loading 800 mg, time 6 h).
in Fig. 4. It has been observed that if the enzyme activity (units/g of oil) is increased above 400 units of Lip Z, detrimental results are obtained showing enzyme inhibition due to competitive nature of enzymes towards the substrate. For immobilized CALB Lip 2, the catalyst loading was defined in terms of weight of beads.
3.1.4. Effect of sonicated enzyme When the sonicated CALB Lip 2 was used for the hydrolysis reaction, considerable increase in the yield was observed. The obtained results are shown in the Fig. 5. The reasons for increase in the yield may be attributed to the change in the enzyme structure due to the use of ultrasonic irradiations which was maintained by immobilization. The results indicate that, ultrasound affected the overall activity of the sonicated enzyme leading to enhanced acid value till optimum time. The screening effect on enzyme may be attributed to the deposition of viscous oil on the surface of the beads which increases the further mass transfer resistance [20] and results in lower acid value after the optimum time as discussed earlier. The maximum AV obtained for USIE is 79.43 mg KOH/g of oil and for SIE is 96.5 mg KOH/g of oil. This shows that there is a definite increase in the yield of free fatty acids due to sonication of enzyme. Table 3 shows the comparison between unsonicated and sonicated enzymes.
3.2. Esterification Esterification is conversion of free fatty acids into esters. Methyl ester can be produced by esterification of mixture of fatty acids or complex oil with high acid value. In Section 3.1, oil was hydrolyzed to get high acid value so that it will be a good starting material for esterification studies. For a good esterification process, it is necessary to optimize the factors affecting the yield and reaction kinetics.
Table 2 Optimized conditions for hydrolysis.
Fig. 4a. Effect of catalyst loading on hydrolysis with Lip Z (reactant ratio 1:1, time 6 h).
Enzyme
Lip Z
Lip 2
Time (h) Molar ratio Enzyme loading
6 1:1 400 units
6 1:3 800 mg
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Experiments were conducted at different molar ratios from 1:1 to 1:6. Fig. 7 describes the effect of molar ratio on the conversion of acid to esters. Maximum conversion of 72.6% was observed at optimum molar ratio of 1:2 whereas further increase in molar ratio did not result into higher conversion. In the present work, the effect of molar ratio was prominent as when methanol concentration in the reaction system increases, there is not much beneficial effect (contrary to that expected in a typical chemical reaction) which can be possibly attributed to the deactivation of enzyme by methanol. The maximum permissible tolerance limit of methanol for enzyme is 10–20% of the total reaction mixture. The methanol added was pure methanol and thus lower quantity of methanol can be used in the reaction.
Fig. 5. Increase in hydrolysis for sonicated enzyme.
Table 3 Comparison of USIE and SIE for hydrolysis reaction. Enzyme
AV
% Hydrolysis
Intensification fold
Comparison Unsonicated Sonicated
79.147 96.4
38.42 49.305
1.283
3.2.1. Effect of reaction time The effect of time on esterification is shown in Fig. 6. It can be seen from the figure that acid value decreased indicating the progress of the esterification reaction. The acid value becomes almost constant at the end of 20 h of continuous agitation. Further flattening may be due to the formation of water which drives the reaction in the reverse direction to hydrolyze the produced esters again to acids. Hence the optimum reaction time of 20 h was maintained for all further experiments, as beyond 20 h the acid value was marginally changing. 3.2.2. Effect of reactant ratio on esterification of oil Molar ratio is one of the important parameters affecting the progress of the esterification reaction. In previous work by Cerdan et al. [21] the stoichiometric glycerol/FA ratio of 1:3 M was established as optimum for obtaining a high TG yield from glycerol. Initially, use of excessive glycerol increased the esterification because the esterification equilibrium was displaced towards product formation. Similar result was obtained by Ergan and Trani [22] with Lipozyme IM-20 catalyzed reaction.
3.2.3. Effect of enzyme concentration Enzyme concentration is one of the important parameter influencing the rate of reaction and yield of product. Enzymatic synthesis of methyl ester with high amounts of lipase is not economically acceptable in a batch process, in spite of a certain increase of the product yield [23]. In the present work, experiment was conducted using 200 to 1200 mg enzyme loading. As shown in Fig. 8, % conversion increases considerably as the enzyme concentration increases but after 1000 mg of total enzyme loading, the increase in conversion was found to very less. So 1000 mg catalyst can be said to be an optimized concentration of enzyme for the esterification reaction for 40 ml volume. Maximum conversion obtained was 74.60%. The optimized conditions have been listed in Table 4 for esterification system. De et al. [24] obtained 92.7% conversion by using 1:2 molar ratio of oil to alcohol with 10% (w/w) loading of immobilized enzyme. These results are in well agreement with the results obtained in present work.
3.2.4. Effect of sonicated enzyme When sonicated enzyme was used for reaction at optimized parameters, an increase in the extent of esterification was observed as shown in the Fig. 9 where the difference in the kinetics of the esterification for USIE and SIE has been represented. This is due to changed enzyme structure and folding which is responsible for higher accessibility of substrate and increase in the reaction rate. Table 5 gives the quantitative results due to the use of pretreated enzyme. Intensification fold of around 1.24 is obtained which is considerably consistent with earlier results of intensification for hydrolysis which was 1.28. The extent of esterification obtained is 93% with sonicated enzyme and 75% with unsonicated enzyme.
80
72.6
70
% Conversion
60
71.21 63.08
59.45
58.65 47.4
50 40 30 20 10 0
1:1
1:2
1:3
1:4
1:5
1:6
Molar Ratio
Fig. 6. Effect of agitation time on esterification for CALB Lip2.
Fig. 7. Effect of oil: methanol molar ratio on percentage conversion of oil (enzyme loading 1000 mg, time 20 h).
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90 80
71.38
% Conversion
70
61.13
60
74.6
73.44
1000
1200
65.37
47.4
50 40 30 20 10
Fig. 10. Comparison of ultrasound cavitation driven hydrolysis with agitation.
0
200
400
600
800
Catalyst loading (mg) Fig. 8. Effect of enzyme loading on percentage conversion of oil (molar ratio 1:2, time 20 h).
Table 4 Optimized parameters for esterification reaction. Optimized parameters Time Oil: methanol molar ratio Catalyst loading
20 h 1:2 1000 mg
carried out under sonication with both sonicated and unsonicated enzyme. The parameters of ultrasound chosen were on the basis of stability of enzyme towards ultrasonication. The selected parameters were power as 50 W with 66.6% duty cycle. Fig. 10 shows the difference in the yield of hydrolysis reaction and it can be seen that for hydrolysis, same yield is obtained in 20 min at 30 °C instead of 6 h by agitation in an incubator shaker kept at 45 °C. Also, Fig. 11 shows that there is very small difference in the yields between a reaction with sonicated and unsonicated enzyme when used in the reaction assisted by ultrasound. Intensification was also observed in the case of esterification (Fig. 12) where yields obtained in 20 h with agitation at 45 °C were obtained in 7 h 20 min at 30 °C. It was also noticed that sonicated enzyme does not make significant difference when ultrasound was used for esterification. Minimum AV reached for sonicated enzyme under ultrasound was 1.98 mg KOH/g oil whereas it was 3.08 mg KOH/g oil for unsonicated enzyme under ultrasound. Fig. 13 shows the marginal variation found in the case of sonicated enzyme under sonication with respect to unsonicated enzyme under sonication. Tables 6 and 7 show the comparison of yields for hydrolysis and esterification respectively for ultrasound assisted process at 30 °C as compared to agitation at 45 °C. It also shows the marginal increase in the yield with sonicated enzyme with respect to unsonicated enzyme under ultrasound. Similar results can be seen in the literature. Batistella et al. [25] have shown a tremendous decrease in the reaction time to achieve same levels of transesterification of soybean oil with ethanol under the influence of the ultrasound irradiation using two commercial immobilized enzymes. Results showed a promising perspective to the use of
Fig. 9. Increase in esterification for sonicated enzyme.
Table 5 Comparison of USIE and SIE for esterification reaction. Enzyme
AV
% Esterification
Intensification fold
Comparison Unsonicated Sonicated
24.152 6.87
75.037 92.826
1.231
3.3. Effect of sonication on hydrolysis and esterification Use of ultrasound can be an effective intensification approach for many reactions and hence can give higher yields of hydrolysis and esterification in less time, if used throughout the reaction. Surely, the energy dissipation due to the use of ultrasound must be weighed against the benefits that can be obtained in the increased extents of reaction. In the present work, reaction was
Fig. 11. Comparison of USIE and SIE under sonication for hydrolysis.
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substrate to the enzyme due to continuous cavitation which is far more than the small differences of increase in substrate accessibility due to conformational changes in the enzyme. 3.4. Mechanistic details
Fig. 12. Comparison of ultrasound cavitation driven esterification with agitation.
Fig. 13. Comparison of USIE and SIE under sonication for esterification.
environmentally benign technique to produce methyl ester as appreciable reaction yields were obtained under mild conditions, irradiation power (100 W), temperature (60–70 °C), in a relatively short reaction time (4 h). It is clear from the above results that sonicated enzyme does lead to an increase in the reaction yields due to the effects of cavitation on the structure of the enzyme. It also increases the accessibility of substrate to the enzyme active site due to more open conformation. One more important observation that can also be made is that when a reaction is driven with high turbulence as in the case of ultrasound cavitation, there is not much difference in the yields obtained for sonicated and unsonicated enzyme. This can be attributed to the extreme increase in the mass transfer of
It is important to explain the results related to the effect of sonication on the rates of hydrolysis and esterification giving a mechanistic insight. The mild shock waves emitted by cavitation bubbles cause rapid movement of substrate towards the enzyme in the reaction mixture and helps in eliminating the mass transfer resistances commonly hampering the progress of the enzymatic reactions. In addition, the high velocity micro-streaming generated by ultrasound causes better mixing in the reaction mass and thus helps in accelerating the progress of the reaction leading to reduction in required time for the conversion of oil to methyl ester [26,27]. From the results presented in Fig. 10, it can be clearly seen that for hydrolysis reaction, similar progress of reaction is obtained in 20 min at 30 °C instead of 6 h using the conventional approach of agitation in an incubator shaker kept at 45 °C. The combination of enzyme with sonication resulted in higher yield of methyl ester than conventional agitation process, which can be attributed to the enhanced convection generated by sonication that boosts the mass transfer characteristics of the system [28]. From the results presented in Fig. 7, it is clear that for the molar ratio of oil to methanol as 1:2, highest yield of methyl ester was obtained. For molar ratio of 1:1, high level of micro-turbulence is generated by the cavitation bubbles in methanol in the vicinity of the interface resulting in dispersion of methanol in the oil phase, but the RO-radical production is limited due to a smaller volume of methanol and also the equilibrium limitations are not overcome due to the stoichiometric proportions, which puts a limit on the yield of methyl ester. With increasing the molar ratio to 1:2, the equilibrium of the reaction shifts in the forward direction and also there is greater generation of the RO- radicals increasing the overall rate of the esterification reaction. The uniform dispersion of methanol in oil due to the ultrasonic irradiations also results in higher available interfacial area for the reaction. Due to combination of these favorable factors, the yield of the esterification reaction is intensified. For higher molar ratios, dispersion of methanol in oil may not be complete due to a rather large volume of methanol. As a result, the interfacial area for the reaction reduces further and so does the overall yield of methyl ester [29,30]. 4. Conclusions The current work examined the feasibility of ultrasound-assisted hydrolysis and esterification reaction for methyl ester production from Nagchampa oil. Ultrasound assisted process showed considerable reduction in reaction time for getting same amount
Table 6 Summary of hydrolysis at different conditions. Mode of operation
Parameters
Unsonicated enzyme %
Sonicated enzyme %
Agitation Ultrasound assisted
45 °C, 180 rpm, 6 h 30 °C, 50 W, 66% duty cycle, 20 min
38.422 47.616
49.305 48.712
Table 7 Summary of esterification at different conditions. Mode of operation
Parameters
Unsonicated enzyme %
Sonicated enzyme %
Agitation Ultrasound assisted
45 °C, 180 rpm, 20 h 30 °C, 50 W, 66% duty cycle, 7 h and 20 min
75.40 96.78
92.47 97.98
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of yield as compared to the conventional method. For ultrasound assisted hydrolysis of oil, same yield was obtained in 20 min at 30 °C instead of 6 h by agitation in an incubator shaker kept at 45 °C. Similarly, in case of esterification yields obtained in 20 h with agitation at 45 °C were obtained in 7 h 20 min at 30 °C in the presence of ultrasound. Overall, the significant decrease in the reaction time and temperature were seen to be the attractive features of ultrasound-assisted reaction. It has been also established that pretreatment of enzyme using ultrasonic irradiation have a marginal effect in terms of intensification as compared to the continuous use in the actual reactions. References [1] Y. Feng, B. He, Y. Cao, J. Li, M. Liu, F. Yan, X. Liang, Biodiesel production using cation-exchange resin as catalyst heterogeneous, Bioresour. Technol. 101 (2010) 1518–1521. [2] A. Dembirbas, Progress and recent trends in biodiesel fuel, Energy Conserv. Manage. 50 (2009) 14–34. [3] N. Lukovic, Z. Knezevic-Jugovic, D. Bezbradica, Biodiesel fuel production by enzymatic transesterification of oils: recent trends, challenges and future perspectives, in: Dr. M. Manzanera (Ed.), Alternative Fuel (2011) ISBN: 978953-307-372-9. [4] O.P. Narula, Treatise on Fats, Fatty Acids and Oleochemicals, vol. 1, Industrial Consultants, New Delhi, 1994 (J-5/1–J-5/5). [5] A. Mustranta, P. Forsell, K. Poutanen, Applications of immobilized lipases to transesterification and esterification reactions in non aqueous systems, Enzyme Microb. Technol. 15 (1993) 133–139. [6] A. Bajaj, P. Lohan, P.N. Jha, R. Mehrotra, Biodiesel production through lipase catalyzed transesterification: an overview, J. Mol. Catal. B: Enzym. 62 (2010) 9–14. [7] L. Fjerbaek, K.V. Christensen, B. Norddahl, A review of the current state of biodiesel production using enzymatic transesterification, Biotechnol. Bioeng. 102 (2009) 1298–1315. [8] V. Veljkovic, J. Avramovic´, O. Stamenkovic, Biodiesel production by ultrasoundassisted transesterification: state of art and the perspectives, Renewable Sustainable Energy Rev. 16 (2012) 1193–1209. [9] V.L. Gole, P.R. Gogate, Intensification of synthesis of biodiesel from nonedible oils using sonochemical reactors, Ind. Eng. Chem. Res. 51 (2012) 11866–11874. [10] Y. Liu, Q. Jin, L. Shan, Y. Liu, W. Shen, X. Wang, The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system, Ultrason. Sonochem. 15 (2008) 402–407. [11] R. Staubmann, I. Ncube, G.M. Gubitz, W. Steiner, J.S. Read, Esterase and lipase activity in Jatropha curcas L. seeds, J. Biotechnol. 75 (1999) 117–126. [12] N.A. Serri, A.H. Kamarudin, S.N. Abdul Rahaman, Preliminary studies for production of fatty acids from hydrolysis of cooking palm oil using C. Rugosa lipase, J. Phys. Sci. 19 (2008) 79–88.
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[13] Q.L. Chang, J.Y. Chen, Effect of lipase catalyzed hydrolysis of sunflower oil in AOT/Isooctane reverse micelle, Bioprocess Eng. 13 (1995) 157–159. [14] B.D. Ribeiro, A.M. Castro, M.A.Z. Coelho, D.M.G. Freire, Production and use of lipases in bioenergy: a review from the feedstocks to biodiesel production, Enzyme Res. (2011) 1–16. [15] C. Tao, B. He, Process study on enzymatic hydrolysis of crambe oil for erucic acid isolation, Am. Soc. Agric. Biol. Eng. 49 (2006) 167–174. [16] F. Bordes, S. Barbe, P. Escalir, L. Mourey, I. Andre, A. Marty, S. Tranier, Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase, Biophys. J. 99 (2010) 22252. [17] D. Goswami, S. De, J.K. Basu, Effect of process variables and additives on mustard oil hydrolysis by porcine pancreas lipase, Braz. J. Chem. Eng. 29 (2012) 449–460. [18] S. Hingu, P.R. Gogate, V.K. Rathod, Synthesis of biodiesel from waste cooking oil using sonochemical reactors, Ultrason. Sonochem. 17 (2010) 827–832. [19] I.M. Noor, M. Hasan, K.B. Ramachandran, Effect of operating variables on the hydrolysis rate of palm oil by lipase, Process Biochem. 39 (2003) 13–20. [20] H. Noureddini, X. Gao, R.S. Philkana, Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil, Bioresour. Technol. 96 (2005) 769–777. [21] E. Cerdan, A.R. Medina, A. Gimenez, M.J. Gonzalez, E.M. Grima, Synthesis of polyunsaturated fatty acid-enriched triglycerides by lipase-catalyzed esterification, J. Am. Oil Chem. Soc. 75 (1998) 1329–1337. [22] F. Ergan, M. Trani, Effect of lipase specificity on triglyceride synthesis, Biotechnol. Lett. 13 (1991) 19–24. [23] N. Ognjanovi, D. Bezbradica, Z. Knezevi, Optimization of the production of biodiesel by a commercial immobilized lipase in a solvent-free system using a response surface methodology, J. Serb. Chem. Soc. 73 (2008) 147– 156. [24] B.K. De, D.K. Bhattacharyya, C. Bandhu, Enzymatic synthesis of fatty alcohol esters by alcoholysis, J. Am. Oil Chem. Soc. 76 (1999) 451–453. [25] L. Batistella, L.A. Lerin, P. Brugnerotto, A.J. Danielli, C.M. Trentin, A. Popiolski, H. Treichel, J.V. Oliveira, D. Oliveira, Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system, Ultrason. Sonochem. 19 (2012) 452–458. [26] S. Khanna, S. Jaiswal, A. Goyal, V.S. Moholkar, Ultrasound enhanced bioconversion of glycerol by Clostridium pasteurianum: a mechanistic investigation, Chem. Eng. J. 200–202 (2012) 416–425. [27] S. Khanna, A. Goyal, V.S. Moholkar, Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support, Biotechnol. Bioeng. 110 (2013) 1637–1645. [28] R.S. Malani, S. Khanna, V.S. Moholkar, Sonoenzymatic decolourization of an azo dye employing immobilized horse radish peroxidase (HRP): a mechanistic study, J. Hazard. Mater. 256–257 (2013) 90–97. [29] A. Kalva, T. Sivasankar, V.S. Moholkar, Physical mechanism of ultrasound-assisted synthesis of biodiesel, Ind. Eng. Chem. Res. 48 (2009) 534–544. [30] H.A. Choudhury, R.S. Malani, V.S. Moholkar, Acid catalyzed biodiesel synthesis from Jatropha oil: mechanistic aspects of ultrasonic intensification, Chem. Eng. J. 231 (2013) 262–272.
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Ultrasound assisted enzymatic conversion of non edible oil to methyl esters Sanket H. Jadhav, Parag R. Gogate ⇑ Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 3 December 2013 Accepted 18 December 2013 Available online 3 January 2014 Keywords: Ultrasound Hydrolysis Esterification Methyl ester Process intensification
a b s t r a c t Conventional and ultrasound-assisted hydrolysis and subsequent esterification of Nagchampa oil under mild operating conditions have been investigated with an objective of intensification of methyl esters production using a sustainable approach. The effect of ratio of reactants, temperature, enzyme loading, pretreatment of enzyme (using ultrasonic irradiations) on the hydrolysis and esterification reaction has been studied. Optimum conditions for hydrolysis were observed to be 1:1 weight ratio of oil: water for Lip Z and 1:3 for Lip 2 enzymes, enzyme loading of 400 units for Lip Z and 800 mg for Lip 2 enzymes and reaction time of 6 h. In the case of esterification reaction, optimum conditions obtained were oil to methanol molar ratio of 1:2, enzyme loading of 1000 mg and reaction time of 20 h. Use of pretreated enzyme (using ultrasonic irradiations) was found to increase the extent of esterification reaction from 75% to 92.5%. It was observed that use of ultrasound in the reaction significantly intensified the esterification reaction with time requirement reducing from 20 h for conventional stirring based approach to only about 7.5 h in the presence of ultrasound. The extent of esterification obtained with sonicated enzyme also increased to 96% from 75% with unsonicated enzyme. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction With increasing demand of fragrances in cosmetic industry, it has become a necessity to design and develop fragrance chemicals which originate from sustainable sources. Some of the suggested sources include renewable liquids coming from biological raw material which can prove to be good substitutes in the cosmetics sector. Nagchampa oil is an essential oil used in fragrance industry, but owing to the high acid value, it has limited stability. The stability of such essential oils can be significantly improved by converting it to its methyl ester. Methyl ester can be produced from oils through stepwise hydrolysis-esterification reactions or transesterification reactions. A large variety of plants that produce non-edible oils can be considered for methyl esters production [1]. Non-edible oils from sources such as neem, mahua, karanja, babassu, Jatropha, microalgae, Camelina sativa, Nagchampa etc. are easily available in many parts of the world, and are very cheap compared to edible oils. Also, for growing non-edible oil crops, less fertilizer, herbicides and insecticides are required compared to edible oil crops [2]. Methyl ester production can be carried out with acid catalyst or by using biocatalysts. The main advantages of employment of lipases as catalyst for methyl ester production are mild reaction conditions and easy recovery of glycerol without purification. ⇑ Corresponding author. Tel.: +91 22 33612024; fax: +91 22 33611020. E-mail address: [email protected] (P.R. Gogate). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.12.018
Additionally, free fatty acid content in the oil can be entirely converted to methyl esters with no soap formation, thereby increasing the methyl ester yields [3]. These features of the lipase enzymes allows the direct usage of materials with high free fatty acids (FFA) or high water content such as non-edible oils, waste cooking oils and industrial waste oil for methyl ester synthesis. Lipase enzyme has been widely used for hydrolysis of fats [4], esterification and transesterification [5]. In spite of numerous advantages, enzymatic processes have drawbacks such as low reaction rate, high enzyme cost in comparison to acid and alkali catalyst and low enzyme stability in the presence of excess methanol [6,7]. Besides the enzyme related problems, process of production of methyl esters faces various problems related to the immiscible nature of the reactants causing poor mass transfer rate. This problem is responsible for longer reaction time and low reaction rate leading to an energy intensive process. Ultrasound can eliminate the problem of poor immiscibility between reactants as ultrasonic energy can emulsify the reactants offering much higher interfacial area for reaction also possibly reducing the catalyst requirement, reaction time and reaction temperature. Ultrasound action in methyl ester production is primarily based on the emulsification of the immiscible liquid reactants by microturbulence generated by cavitation bubbles [8]. Gole and Gogate [9] performed experiments on methyl ester formation from non-edible Nagchampa oil and found that temperature and reaction time required for esterification, as well as the transesterification stages, are substantially
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lower in the case of sonochemical reactors, compared to the conventional heating method. Liu et al. [10] used ultrasound cavitation for lipase catalyzed hydrolysis and reported that around 2-fold intensification was obtained. The present work reports the ultrasound assisted synthesis of methyl esters from high-acid-value Nagchampa oil using the two-stage approach of hydrolysis followed by esterification. The objective of first stage was to achieve hydrolysis to considerable extent in order to get acids in sufficient quantity for esterification to methyl esters in the next step of esterification. The effect of different operating parameters such as reaction temperature, ratio of reactants, catalyst concentration and sonication of enzyme has been investigated. Two types of approaches for ultrasound based synthesis were followed in the work viz. enzyme was initially treated with ultrasound and then used in the reaction whereas in the second approach ultrasound was used throughout the reaction period. 2. Materials and methods
Fig. 1. Experimental setup for enzyme sonication A: Generator, B: Beaker introduced in ice bath, C: Ultrasound Probe, E: Temperature Indicator, F: Stand.
2.1. Materials Candida antarctica Lipase Enzyme standard was received as a gift sample from Novozyme, Denmark. Tributyrin, sodium dihydrogen phosphate and disodium hydrogen phosphate were procured from Himedia laboratories, Mumbai. Ultrasonic horn used in the experiments was procured from Dakshin, Mumbai having variable power output up to 220 W, and frequency of 20 kHz. The raw Nagchampa oil used for hydrolysis was procured from M/s Sanjay Shirsat Oil Mill (Vengurla, Dist: Sindhudurg, Maharashtra, India). Oil was initially filtered to remove traces of particles and mud. Table 1 gives the typical composition of oil consisting of 25% saturated acid (stearic and palmitic) and 72.7% unsaturated acid (oleic, linoleic, and linolenic). The initial saponification value of the oil was found to be 156 mg KOH/g of oil. Methanol, potassium hydroxide and oxalic acid (required for standardization and titration) were procured from S.D. Fine chemicals Pvt. Ltd., Mumbai. Distilled water was used as a reactant for hydrolysis and was prepared from the laboratory scale distillation unit. All the chemicals were used as received from the supplier, unless otherwise specified. 2.2. Experimental procedure 2.2.1. Sonication of enzyme Sonication was done in direct mode with 100 ml volume of 3 mg/ml enzyme solution subjected to ultrasonic irradiations using a ultrasound probe. The ultrasound probe was dipped in solution to 0.5 cm height and initial parameters were chosen as duty cycle of 66% and frequency of 20 kHz and power of 50 W. The setup used for enzyme sonication studies has been shown in Fig. 1. 2.2.2. Hydrolysis The hydrolysis reaction was carried out using lipase from two different sources namely Candida rugosa and C. antarctica. The objective was to achieve hydrolysis to considerable extent so as
Table 1 Fatty acid composition of Nagchampa oil. Fatty acid
Composition (%)
Palmitic, C16:0 Stearic, C18:0 Oleic, C18:1 Linoleic, C18:2 Linolenic, C18:3
12 13 34.1 38.3 0.3
to get acids in sufficient quantity for esterification to methyl esters in the next step. During experiments four important factors were considered and optimized in order to get maximum hydrolysis. The effect of ratio of oil to water, time of hydrolysis, temperature of hydrolysis and enzyme loading has been investigated. Initially reaction time was optimized by taking readings every one hour interval up to 8 h and acid values of the samples were analysed using titration. The temperature was optimized by checking the acid value at different temperatures in the range of 25–55 °C for optimized reaction time. Effect of reactant ratio was then investigated over the range 1:1–1:4 at optimized time and temperature. Catalyst concentration was optimized between 100 and 500 activity units for both C. rugosa (Lip Z) and C. antarctica (CALB Lip 2) lipase. All the reactions were carried out for 30 ml volume. After optimization of the entire range of parameters, final run was done to get maximum hydrolysis at larger scale of 200 ml to check the scale up aspects. 2.2.3. Esterification Next step of methyl ester synthesis after hydrolysis is esterification. This step converts free fatty acids into methyl esters using methanol. In order to prevent the progress of reaction in reverse direction i.e. hydrolysis which results in lowering the ester yield, molecular sieves were added in the reaction mixture in order to remove the water produced in the reaction. Parameters optimized were same as described for the hydrolysis reaction. First the reaction time was optimized by taking samples after every 4 h for a total reaction time as 28 h. The samples were analyzed to get the optimum time for minimum acid value which signifies maximum methyl ester yield. The molar ratio of oil to methanol was also optimized. This is a critical step in the process as excess methanol kills the enzyme and less methanol being a limiting reactant does not drive the reaction in the forward direction in an efficient way. Molar ratio was optimized over the ratio of 1:1–1:4. Finally the catalyst loading was also optimized. As the enzyme (CALB Lip 2) used for this step was an immobilized enzyme, the loading was taken in terms of weight of dried immobilized beads. The total volume of the mixture was made to 40 ml in each experiment using hexane. All the samples were analyzed for the determination of acid value. 2.2.4. Hydrolysis and esterification with sonication Hydrolysis and esterification both were done with sonication at 50 W with same reaction mixture in order to check the effect of
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mass transfer on the reaction system. Two different runs were taken with sonication in which one enzyme used was unsonicated immobilized enzyme (USIE) and other was sonicated immobilized enzyme (SIE). The runs were taken at 50 W to assure the stability of enzyme and the reaction mixture used was at optimized reaction conditions. 2.3. Analysis 2.3.1. Lipase assay Normal acid base titration assay was performed to find the amount of butyric acid produced from hydrolysis of tributyrin using lipase [11]. For the analysis, 1062 ll of 50 mM phosphate buffer (pH 7.0) was mixed with 250 ll of enzyme solution and 188 ll tributyrin. Mixture was stirred continuously for 5 min using cyclo mixer. After that indicator was added and mixture was titrated against methanolic NaOH till end point change from colourless to faint pink was observed. 2.3.2. Acid value determination Acid value of oil gives an idea about the amount of free fatty acids in the oil. Acid value can be a measure of how much the oil has been hydrolysed and also indicate the suitability for the process of methyl ester formation. 1.0 g of oil was dissolved in 100% ethanol. 0.05 M alcoholic KOH and 0.1 M oxalic acid were prepared and KOH standardization was done by titrating it with oxalic acid. Standardized alcoholic KOH was then used to titrate the dissolved sample of oil. The titrations were repeated thrice and mean reading was taken to find out the acid value which is expressed in terms of mg of KOH required to neutralize 1.0 g of oil. Acid value (AV) was calculated by using Eq. (1):
AV ¼
56:1 Normality of KOH titer reading : Weight of oilðgÞ
ð1Þ
2.3.3. Saponification value determination The procedure involves the use of excess 0.7 N alcoholic KOH, which catalyzes the saponification i.e. release of the free fatty acids from the glycerol backbone. The unreacted KOH is then back-titrated with standardized hydrochloric acid (HCl) using phenolphthalein as the indicator. 0.5 N HCl is standardized by using 0.1 N oxalic acid. The amount and normality of the HCI used for neutralization can then be used to calculate the saponification value. Saponification value is calculated in terms of mg KOH/g of oil. Sap: value ¼
ðTiter of HCl for blank Titer for HCl sampleÞ Normality of HCl 56:1 Weight of oilðgÞ
ð2Þ
with time. It is clear from the Fig. 2 that acid value increases with an increase in the reaction time, reaches a maximum in 6 h and then marginally decreases. The profile matches with some of the literature illustrations [12]. The decrease in acid value after 6 h can be attributed to the inhibition of the enzyme by glycerol, which is being formed as the product of reaction [13]. The glycerol formed in the reaction mixture gets coated on the enzyme surface to block the sites where substrate binds [14]. Also the concentration of fatty acids might prove to be responsible for product driven inhibition of the reaction [15]. It was also observed that use of Lip Z enzyme results in higher yields as compared to the CALB Lip 2. Lip Z and CALB Lip 2 both are 1,3 specific lipases which give different reaction kinetics due to the structural differences. If we see the 3-D structure of both Lip Z and CALB Lip 2, there is a difference between the two in the fact that the lid conformation of CALB Lip 2 is only half open at interphase which needs extra activation energy to make the reaction happen whereas other form of lipase show complete lid opening at the interphase [16]. It can be concluded that the difference in reaction kinetics is due to different accessibility of substrates to the enzyme active site. 3.1.2. Effect of ratio of oil to water (aqueous enzyme solution) The ratio of oil to aqueous solution has been considered in accordance to weight and the obtained results for the variation in acid value with ratio have been shown in Fig. 3(a) and (b). In case of Lip Z, maximum acid value was obtained for 1:1 weight ratio of oil: water but for CALB Lip 2, maximum acid value was obtained at 1:3 ratio. In both the cases, further increase in reactant ratio resulted in decrease in acid value. This can be attributed to the phase separation happening in the case of higher ratios. Enzyme always remains at the interphase of the oil water mixture but it has more tendency to get dissolved in water. During chemical hydrolysis water taken is in excess so that it won’t behave as a limiting reactant, but during enzymatic hydrolysis the reactant ratio selected should be very stringent in order to control the concentration of enzyme at interphase [17]. This is the reason why there is a decrease in the yield of reaction with CALB Lip 2 for further increase in quantum of water. Similar results have been obtained by Rathod and coworkers for transesterification of waste cooking oil [18]. 3.1.3. Effect of enzyme loading Catalyst or enzyme if used in optimum amount can give good reaction yield but if used in excess this may not give better results but create problems with subsequent separation and higher costs of treatment. With this objective the effect of enzyme loading has been investigated and the obtained results have been shown
3. Results and discussion 3.1. Hydrolysis Acid value gives the amount of free fatty acids in the oil. Increase in the acid value of the oil is an indication of hydrolysis of the oil. Enzymatic methods take a long time to hydrolyze the oil than chemical methods like acid hydrolysis or alkaline hydrolysis and hence it is necessary to investigate the effect of important operating parameters. Two enzymes used for hydrolysis were C. rugosa lipase (Lip Z) and C. antarctica lipase (CALB Lip 2). The reason for selecting two enzymes was to compare the efficacy of two enzymes to maximize hydrolysis. 3.1.1. Effect of time on hydrolysis The samples taken at different interval of times were analyzed for the acid value. Fig. 2 shows the profile of change in acid value
Fig. 2. Effect of time on hydrolysis for Lip Z and CALB Lip 2.
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100
AV (mg KOH/g oil)
90
90.822
80
73.275
70 60
54.235
50
47.913
40 30 20 10 0 1:1
1:2
1:3
1:4
Reactant ratio Fig. 3a. Effect of reactant ratio on hydrolysis with Lip Z (wt/wt) (enzyme loading of 400 units, time 6 h).
70
63.667
AV (mg KOH/g oil)
60 50
47.866
60.036
47.714
40
Fig. 4b. Effect of catalyst loading on hydrolysis with CALB Lip 2 (reactant ratio 1:3, time 6 h).
Fig. 4(b) shows that if the weight of beads is increased above 800 mg, the overall yield decreases. This happens because there is a saturation observed in enzyme available which leads to slower rate of reaction [19]. This again indicates that at high enzyme concentrations, the interfacial area between the oil and the aqueous phase limits the initial rate of reaction. For hydrolysis, optimized parameters for both the enzymes have been given in Table 2.
30 20 10 0
1:1
1:2
1:3
1:4
Reactant ratio Fig. 3b. Effect of reactant ratio on hydrolysis with CALB Lip 2 (enzyme loading 800 mg, time 6 h).
in Fig. 4. It has been observed that if the enzyme activity (units/g of oil) is increased above 400 units of Lip Z, detrimental results are obtained showing enzyme inhibition due to competitive nature of enzymes towards the substrate. For immobilized CALB Lip 2, the catalyst loading was defined in terms of weight of beads.
3.1.4. Effect of sonicated enzyme When the sonicated CALB Lip 2 was used for the hydrolysis reaction, considerable increase in the yield was observed. The obtained results are shown in the Fig. 5. The reasons for increase in the yield may be attributed to the change in the enzyme structure due to the use of ultrasonic irradiations which was maintained by immobilization. The results indicate that, ultrasound affected the overall activity of the sonicated enzyme leading to enhanced acid value till optimum time. The screening effect on enzyme may be attributed to the deposition of viscous oil on the surface of the beads which increases the further mass transfer resistance [20] and results in lower acid value after the optimum time as discussed earlier. The maximum AV obtained for USIE is 79.43 mg KOH/g of oil and for SIE is 96.5 mg KOH/g of oil. This shows that there is a definite increase in the yield of free fatty acids due to sonication of enzyme. Table 3 shows the comparison between unsonicated and sonicated enzymes.
3.2. Esterification Esterification is conversion of free fatty acids into esters. Methyl ester can be produced by esterification of mixture of fatty acids or complex oil with high acid value. In Section 3.1, oil was hydrolyzed to get high acid value so that it will be a good starting material for esterification studies. For a good esterification process, it is necessary to optimize the factors affecting the yield and reaction kinetics.
Table 2 Optimized conditions for hydrolysis.
Fig. 4a. Effect of catalyst loading on hydrolysis with Lip Z (reactant ratio 1:1, time 6 h).
Enzyme
Lip Z
Lip 2
Time (h) Molar ratio Enzyme loading
6 1:1 400 units
6 1:3 800 mg
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Experiments were conducted at different molar ratios from 1:1 to 1:6. Fig. 7 describes the effect of molar ratio on the conversion of acid to esters. Maximum conversion of 72.6% was observed at optimum molar ratio of 1:2 whereas further increase in molar ratio did not result into higher conversion. In the present work, the effect of molar ratio was prominent as when methanol concentration in the reaction system increases, there is not much beneficial effect (contrary to that expected in a typical chemical reaction) which can be possibly attributed to the deactivation of enzyme by methanol. The maximum permissible tolerance limit of methanol for enzyme is 10–20% of the total reaction mixture. The methanol added was pure methanol and thus lower quantity of methanol can be used in the reaction.
Fig. 5. Increase in hydrolysis for sonicated enzyme.
Table 3 Comparison of USIE and SIE for hydrolysis reaction. Enzyme
AV
% Hydrolysis
Intensification fold
Comparison Unsonicated Sonicated
79.147 96.4
38.42 49.305
1.283
3.2.1. Effect of reaction time The effect of time on esterification is shown in Fig. 6. It can be seen from the figure that acid value decreased indicating the progress of the esterification reaction. The acid value becomes almost constant at the end of 20 h of continuous agitation. Further flattening may be due to the formation of water which drives the reaction in the reverse direction to hydrolyze the produced esters again to acids. Hence the optimum reaction time of 20 h was maintained for all further experiments, as beyond 20 h the acid value was marginally changing. 3.2.2. Effect of reactant ratio on esterification of oil Molar ratio is one of the important parameters affecting the progress of the esterification reaction. In previous work by Cerdan et al. [21] the stoichiometric glycerol/FA ratio of 1:3 M was established as optimum for obtaining a high TG yield from glycerol. Initially, use of excessive glycerol increased the esterification because the esterification equilibrium was displaced towards product formation. Similar result was obtained by Ergan and Trani [22] with Lipozyme IM-20 catalyzed reaction.
3.2.3. Effect of enzyme concentration Enzyme concentration is one of the important parameter influencing the rate of reaction and yield of product. Enzymatic synthesis of methyl ester with high amounts of lipase is not economically acceptable in a batch process, in spite of a certain increase of the product yield [23]. In the present work, experiment was conducted using 200 to 1200 mg enzyme loading. As shown in Fig. 8, % conversion increases considerably as the enzyme concentration increases but after 1000 mg of total enzyme loading, the increase in conversion was found to very less. So 1000 mg catalyst can be said to be an optimized concentration of enzyme for the esterification reaction for 40 ml volume. Maximum conversion obtained was 74.60%. The optimized conditions have been listed in Table 4 for esterification system. De et al. [24] obtained 92.7% conversion by using 1:2 molar ratio of oil to alcohol with 10% (w/w) loading of immobilized enzyme. These results are in well agreement with the results obtained in present work.
3.2.4. Effect of sonicated enzyme When sonicated enzyme was used for reaction at optimized parameters, an increase in the extent of esterification was observed as shown in the Fig. 9 where the difference in the kinetics of the esterification for USIE and SIE has been represented. This is due to changed enzyme structure and folding which is responsible for higher accessibility of substrate and increase in the reaction rate. Table 5 gives the quantitative results due to the use of pretreated enzyme. Intensification fold of around 1.24 is obtained which is considerably consistent with earlier results of intensification for hydrolysis which was 1.28. The extent of esterification obtained is 93% with sonicated enzyme and 75% with unsonicated enzyme.
80
72.6
70
% Conversion
60
71.21 63.08
59.45
58.65 47.4
50 40 30 20 10 0
1:1
1:2
1:3
1:4
1:5
1:6
Molar Ratio
Fig. 6. Effect of agitation time on esterification for CALB Lip2.
Fig. 7. Effect of oil: methanol molar ratio on percentage conversion of oil (enzyme loading 1000 mg, time 20 h).
S.H. Jadhav, P.R. Gogate / Ultrasonics Sonochemistry 21 (2014) 1374–1381
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90 80
71.38
% Conversion
70
61.13
60
74.6
73.44
1000
1200
65.37
47.4
50 40 30 20 10
Fig. 10. Comparison of ultrasound cavitation driven hydrolysis with agitation.
0
200
400
600
800
Catalyst loading (mg) Fig. 8. Effect of enzyme loading on percentage conversion of oil (molar ratio 1:2, time 20 h).
Table 4 Optimized parameters for esterification reaction. Optimized parameters Time Oil: methanol molar ratio Catalyst loading
20 h 1:2 1000 mg
carried out under sonication with both sonicated and unsonicated enzyme. The parameters of ultrasound chosen were on the basis of stability of enzyme towards ultrasonication. The selected parameters were power as 50 W with 66.6% duty cycle. Fig. 10 shows the difference in the yield of hydrolysis reaction and it can be seen that for hydrolysis, same yield is obtained in 20 min at 30 °C instead of 6 h by agitation in an incubator shaker kept at 45 °C. Also, Fig. 11 shows that there is very small difference in the yields between a reaction with sonicated and unsonicated enzyme when used in the reaction assisted by ultrasound. Intensification was also observed in the case of esterification (Fig. 12) where yields obtained in 20 h with agitation at 45 °C were obtained in 7 h 20 min at 30 °C. It was also noticed that sonicated enzyme does not make significant difference when ultrasound was used for esterification. Minimum AV reached for sonicated enzyme under ultrasound was 1.98 mg KOH/g oil whereas it was 3.08 mg KOH/g oil for unsonicated enzyme under ultrasound. Fig. 13 shows the marginal variation found in the case of sonicated enzyme under sonication with respect to unsonicated enzyme under sonication. Tables 6 and 7 show the comparison of yields for hydrolysis and esterification respectively for ultrasound assisted process at 30 °C as compared to agitation at 45 °C. It also shows the marginal increase in the yield with sonicated enzyme with respect to unsonicated enzyme under ultrasound. Similar results can be seen in the literature. Batistella et al. [25] have shown a tremendous decrease in the reaction time to achieve same levels of transesterification of soybean oil with ethanol under the influence of the ultrasound irradiation using two commercial immobilized enzymes. Results showed a promising perspective to the use of
Fig. 9. Increase in esterification for sonicated enzyme.
Table 5 Comparison of USIE and SIE for esterification reaction. Enzyme
AV
% Esterification
Intensification fold
Comparison Unsonicated Sonicated
24.152 6.87
75.037 92.826
1.231
3.3. Effect of sonication on hydrolysis and esterification Use of ultrasound can be an effective intensification approach for many reactions and hence can give higher yields of hydrolysis and esterification in less time, if used throughout the reaction. Surely, the energy dissipation due to the use of ultrasound must be weighed against the benefits that can be obtained in the increased extents of reaction. In the present work, reaction was
Fig. 11. Comparison of USIE and SIE under sonication for hydrolysis.
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substrate to the enzyme due to continuous cavitation which is far more than the small differences of increase in substrate accessibility due to conformational changes in the enzyme. 3.4. Mechanistic details
Fig. 12. Comparison of ultrasound cavitation driven esterification with agitation.
Fig. 13. Comparison of USIE and SIE under sonication for esterification.
environmentally benign technique to produce methyl ester as appreciable reaction yields were obtained under mild conditions, irradiation power (100 W), temperature (60–70 °C), in a relatively short reaction time (4 h). It is clear from the above results that sonicated enzyme does lead to an increase in the reaction yields due to the effects of cavitation on the structure of the enzyme. It also increases the accessibility of substrate to the enzyme active site due to more open conformation. One more important observation that can also be made is that when a reaction is driven with high turbulence as in the case of ultrasound cavitation, there is not much difference in the yields obtained for sonicated and unsonicated enzyme. This can be attributed to the extreme increase in the mass transfer of
It is important to explain the results related to the effect of sonication on the rates of hydrolysis and esterification giving a mechanistic insight. The mild shock waves emitted by cavitation bubbles cause rapid movement of substrate towards the enzyme in the reaction mixture and helps in eliminating the mass transfer resistances commonly hampering the progress of the enzymatic reactions. In addition, the high velocity micro-streaming generated by ultrasound causes better mixing in the reaction mass and thus helps in accelerating the progress of the reaction leading to reduction in required time for the conversion of oil to methyl ester [26,27]. From the results presented in Fig. 10, it can be clearly seen that for hydrolysis reaction, similar progress of reaction is obtained in 20 min at 30 °C instead of 6 h using the conventional approach of agitation in an incubator shaker kept at 45 °C. The combination of enzyme with sonication resulted in higher yield of methyl ester than conventional agitation process, which can be attributed to the enhanced convection generated by sonication that boosts the mass transfer characteristics of the system [28]. From the results presented in Fig. 7, it is clear that for the molar ratio of oil to methanol as 1:2, highest yield of methyl ester was obtained. For molar ratio of 1:1, high level of micro-turbulence is generated by the cavitation bubbles in methanol in the vicinity of the interface resulting in dispersion of methanol in the oil phase, but the RO-radical production is limited due to a smaller volume of methanol and also the equilibrium limitations are not overcome due to the stoichiometric proportions, which puts a limit on the yield of methyl ester. With increasing the molar ratio to 1:2, the equilibrium of the reaction shifts in the forward direction and also there is greater generation of the RO- radicals increasing the overall rate of the esterification reaction. The uniform dispersion of methanol in oil due to the ultrasonic irradiations also results in higher available interfacial area for the reaction. Due to combination of these favorable factors, the yield of the esterification reaction is intensified. For higher molar ratios, dispersion of methanol in oil may not be complete due to a rather large volume of methanol. As a result, the interfacial area for the reaction reduces further and so does the overall yield of methyl ester [29,30]. 4. Conclusions The current work examined the feasibility of ultrasound-assisted hydrolysis and esterification reaction for methyl ester production from Nagchampa oil. Ultrasound assisted process showed considerable reduction in reaction time for getting same amount
Table 6 Summary of hydrolysis at different conditions. Mode of operation
Parameters
Unsonicated enzyme %
Sonicated enzyme %
Agitation Ultrasound assisted
45 °C, 180 rpm, 6 h 30 °C, 50 W, 66% duty cycle, 20 min
38.422 47.616
49.305 48.712
Table 7 Summary of esterification at different conditions. Mode of operation
Parameters
Unsonicated enzyme %
Sonicated enzyme %
Agitation Ultrasound assisted
45 °C, 180 rpm, 20 h 30 °C, 50 W, 66% duty cycle, 7 h and 20 min
75.40 96.78
92.47 97.98
S.H. Jadhav, P.R. Gogate / Ultrasonics Sonochemistry 21 (2014) 1374–1381
of yield as compared to the conventional method. For ultrasound assisted hydrolysis of oil, same yield was obtained in 20 min at 30 °C instead of 6 h by agitation in an incubator shaker kept at 45 °C. Similarly, in case of esterification yields obtained in 20 h with agitation at 45 °C were obtained in 7 h 20 min at 30 °C in the presence of ultrasound. Overall, the significant decrease in the reaction time and temperature were seen to be the attractive features of ultrasound-assisted reaction. It has been also established that pretreatment of enzyme using ultrasonic irradiation have a marginal effect in terms of intensification as compared to the continuous use in the actual reactions. References [1] Y. Feng, B. He, Y. Cao, J. Li, M. Liu, F. Yan, X. Liang, Biodiesel production using cation-exchange resin as catalyst heterogeneous, Bioresour. Technol. 101 (2010) 1518–1521. [2] A. Dembirbas, Progress and recent trends in biodiesel fuel, Energy Conserv. Manage. 50 (2009) 14–34. [3] N. Lukovic, Z. Knezevic-Jugovic, D. Bezbradica, Biodiesel fuel production by enzymatic transesterification of oils: recent trends, challenges and future perspectives, in: Dr. M. Manzanera (Ed.), Alternative Fuel (2011) ISBN: 978953-307-372-9. [4] O.P. Narula, Treatise on Fats, Fatty Acids and Oleochemicals, vol. 1, Industrial Consultants, New Delhi, 1994 (J-5/1–J-5/5). [5] A. Mustranta, P. Forsell, K. Poutanen, Applications of immobilized lipases to transesterification and esterification reactions in non aqueous systems, Enzyme Microb. Technol. 15 (1993) 133–139. [6] A. Bajaj, P. Lohan, P.N. Jha, R. Mehrotra, Biodiesel production through lipase catalyzed transesterification: an overview, J. Mol. Catal. B: Enzym. 62 (2010) 9–14. [7] L. Fjerbaek, K.V. Christensen, B. Norddahl, A review of the current state of biodiesel production using enzymatic transesterification, Biotechnol. Bioeng. 102 (2009) 1298–1315. [8] V. Veljkovic, J. Avramovic´, O. Stamenkovic, Biodiesel production by ultrasoundassisted transesterification: state of art and the perspectives, Renewable Sustainable Energy Rev. 16 (2012) 1193–1209. [9] V.L. Gole, P.R. Gogate, Intensification of synthesis of biodiesel from nonedible oils using sonochemical reactors, Ind. Eng. Chem. Res. 51 (2012) 11866–11874. [10] Y. Liu, Q. Jin, L. Shan, Y. Liu, W. Shen, X. Wang, The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system, Ultrason. Sonochem. 15 (2008) 402–407. [11] R. Staubmann, I. Ncube, G.M. Gubitz, W. Steiner, J.S. Read, Esterase and lipase activity in Jatropha curcas L. seeds, J. Biotechnol. 75 (1999) 117–126. [12] N.A. Serri, A.H. Kamarudin, S.N. Abdul Rahaman, Preliminary studies for production of fatty acids from hydrolysis of cooking palm oil using C. Rugosa lipase, J. Phys. Sci. 19 (2008) 79–88.
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