Immobilization of Lipase on CaCO3 and Entrapment in Calcium ...

SCIENCE JOURNAL Ubon Ratchathani University

Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 46-51

http://scjubu.sci.ubu.ac.th

Research Article

Immobilization of Lipase on CaCO3 and Entrapment in Calcium Alginate Bead for Biodiesel Production N. Sawangpanya, C. Muangchim, M. Phisalaphong* Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. Received 16/02/10; Accepted 05/06/10

Abstract Biodiesel productions from palm oil by using C. rugosa free and immobilized lipases as biocatalysts were investigated. Three methods of lipase immobilizations were studied: 1) adsorption of lipase on CaCO3 (CRLA), 2) entrapment of lipase in Ca-alginate matrix (CRLE) and 3) entrapment of CaCO3– lipase in Ca-alginate matrix (CRLAE). The effects of operational parameters such as ratio of enzyme to oil, molar ratio of ethanol to oil, operating temperature, bead diameter and shaking speed on transesterification were examined. The optimal conditions for the biodiesel were at the molar ratio of ethanol to palm oil of 9:1, 5 % C. rugosa lipase based on oil weight, 50 oC and 250 rpm. After 48 hours at the optimal condition, the reaction with entrapment of lipase of Ca-alginate matrix (CRLE) exhibited the higher ethyl ester yield (74%) than those of CRLA and CRLAE, whereas ethyl ester yield of 83% was obtained after 24 hours by using the free lipases. Keywords: Alginate, Biodiesel, Calcium carbonate, Immobilization, Lipase.

1. Introduction Due to the limitation of fossil fuels, biodiesel becomes an attractive alternative energy. Biodiesel is a renewable energy. It is clean and environmentally safe. Biodiesel can be obtained from vegetable oils by transesterification with short chain alcohols to form esters. Chemical catalysts are widely developed to improve the reaction rate. Nevertheless, there are several drawbacks *Corresponding author. E-mail address: [email protected]

from using alkali or acid catalyst processes, for example; high energy requirements, difficulties in the recovery of catalyst and glycerol and pollution from waste water. The biocatalyst such as lipase can eliminate the drawbacks of chemical catalysts by producing product of very high purity and offers an environmentally attractive option [1-4]. However, the hurdle to use of lipase for biodiesel fuel production is the cost of biocatalyst. The use of immobilized enzymes could overcome this problem. Immobilizations of lipase can be achieved by adsorption onto support matrices such as particles,

Copyright  2010 Faculty of Science, Ubon Ratchathani University. All Rights Reserved.

N. Sawangpanya et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 46-51

fibers, by entrapping them in gel matrices or by covalent attachment [1]. The enzyme immobilization by adsorption onto a solid support or entrapment in bio-polymer matrix such as calcium alginate remains the most simple and cost-effective. It was suggested that the immobilized lipase facilitates mass transfer by spreading the enzyme on a large surface area and by preventing the enzyme particles from aggregation [4]. The CaCO3 presented the advantages of being non-toxic and lacking of chemical reactivity. Furthermore, this support was selected as a suitable adsorbent leading to high dispersion of the crude R. oryzae lipase in the support and preserving the catalytic activity [2]. Its readsorption is sometimes possible by modification of the pH, followed by binding of new active enzyme [4]. However, ready desorption would also be a major drawback of this immobilization technique if it occurs during the catalyzed reaction. The other physical immobilization of a lipase is its inclusion in an insoluble polymer or entrapment in bio-polymer matrix such as calcium alginate is attractive. The advantage of such an immobilization technique is that the enzyme does not chemically interact with the polymer; therefore, denaturation is possibly avoided [5].

47

from Univar. All substances are the analytical grade. The enzyme immobilization was made on to CaCO3 according to Rosu et al. (1997) with a slight modification. A support powder (2 g) was added to 20 ml of enzymatic solution. The mixture was incubated 1 h at 4 ºC under mild agitation. Afterwards, 10 ml of chilled acetone was added, and the suspension was filtered through a Buchner funnel, the preparation of immobilized lipase was washed two times with another 100 ml aliquot of chilled acetone, dried in vacuum desiccators at room temperature for 1-2 h and stored at 4ºC until use. Entrapment of C. rugosa Lipase Immobilized on CaCO3 in Calcium Alginate Beads (CRLAE). C. rugosa lipase which preimmobilized on CaCO3 by adsorption was entrapped in calcium alginate beads. The sodium alginate solution of 1%w/v and 0.1 M calcium chloride solution was prepared in 0.01 M sodium phosphate buffer (pH 7.0). The immobilized enzyme on CaCO3 was dispersed uniformly in 1% sodium alginate solution and was injected through a syringe into 0.1 M calcium chloride solution from a constant distance. The beads were allowed to harden in calcium chloride solution for an hour.

This study exploits the idea on developing a new enzyme carrier by adsorption of lipase on CaCO3 followed by the entrapment in calcium alginate beads (CRLAE) for application in biodiesel production using purified palm oil and 95% (v/v) ethanol as substrates. The result was compared with those using free enzymes, immobilized enzymes in conventional calcium alginate beads (CRLE) and enzymes immobilized by adsorption on CaCO3 (CRLA).

Lipase Hydrolysis Activity. The hydrolysis activity was assayed titrimetrically using olive oil emulsion method [7]. The substrate was prepared by mixing 50 ml of olive oil with 50 ml of Arabic gum solution (7%w/v). The reaction mixture consisting of 20 ml of emulsion, 2 ml of 0.1 M sodium phosphate buffer, pH 7.0 and immobilized lipase (200 mg) was incubated for 12 h at 37 °C. The liberated fatty acid was titrated with 0.05 N potassium hydroxide solution using Phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of 2. Materials and Methods enzyme that produced 1 μmol of free fatty Enzyme and Chemicals. Candida rugosa acids per min under the assay conditions. lipase (EC 3.1.1.3) was received as a gift sample from Amano Pharmaceuticals, Japan. 3. Results and Discussion Virgin olive oil was purchased from local market. Carbonate of calcium (CaCO3) was


48

Immobilization of Lipase on CaCO3 and Entrapment in Calcium Alginate Bead for Biodiesel Production

Hydrolytic activity (U)

Biodiesel production from purified palm oil enzymes slightly decreased. Therefore, the and 95% (v/v) ethanol by using C. rugosa in immobilization in CRLE, CRLA and CRLAE forms of free and immobilized lipases as carriers could provide good heat resistance. biocatalysts was studied. The influence of operating conditions on enzyme activity was 150 investigated using lipase-CaCO3 immobilized 120 in calcium alginate beads (CRLAE) in comparison to that using free enzyme, and 90 immobilized enzymes adsorbed on CaCO3 60 (CRLA) and entrapped in calcium alginate bead (CRLE). 30

Effect of Temperature. The effect of temperature on the activity of free enzymes and immobilized enzymes was investigated by using olive oil as a substrate at pH 7.0 in the temperature range of 37-60 °C as shown in Figure 3. The activity of the lipase increased with the increase of the operating temperature up to 50°C. The maximum activity of both free and immobilized enzymes appeared at 50°C. However, above 50°C, the enzyme activity of the free enzyme was significantly decreased with the increasing temperature, while the enzyme activity of the immobilized

0 0

2

4

6

8

10

12

lipase quantity (wt% of oil)

Figure 1. Effect of C. rugosa lipase quantity on hydrolytic activity, at pH 7.0, 50°C, 250 rpm and reaction time of 12 h. 100.00 Ethyl Ester Yield (%)

Effect of Lipase Quantity. The effect of lipase quantity on the alcoholysis of purified palm oil was investigated by varying free lipase quantities at 1%, 3%, 5% and 10 % based on palm oil weight with a reaction temperature of 50°C. The results are presented in Figures 1 and 2. The hydrolytic activity and ethyl ester content were increased by increasing lipase quantity up to 5%. The highest conversion was obtained when 5-10 % C. rugosa lipase based on oil weight was used. The maximum hydrolytic activity and ethyl ester yield were about 120 U and 83.4 %, respectively. According to Kose et al. (2001), the highest ethyl ester formation (82.6%) was obtained by using 30% C. antarctica lipase based on cotton seed oil weight with an operation temperature at 50°C. Awang et al. (2007) reported the highest conversion of 79.5% when 10% (w/w) of C. rugosa lipase concentration was used for esterification of oleic acid and oleyl alcohol in hexane. From the result of this study, C. rugosa lipase at 5% (by wt. of oil) was applied for the further study.

80.00 60.00 40.00 20.00 0.00 1

3

5

10

Lipase quantity (wt% of oil)

Figure 2. Effect of C. rugosa lipase quantity on ethyl ester yield with molar ratio of ethanol to palm oil 9:1, reaction temperature of 50°C, 250 rpm and reaction time of 24 h. Effect of Molar Ratio of Ethanol to Palm Oil. The effect of molar ratio of ethanol to palm oil on ethyl ester yield was determined under reaction temperature of 50°C. The ethyl ester yield in Figure 4 was obtained after the reaction was carried out for 24 h. The ethyl ester yield increased from about 47% to 83 % as the molar ratio increased from 3:1 to 9:1 and considerably decreased after that. In the enzymatic catalysis in aqueous medium, the nature of the organic solvent influences the activity and the stability of the enzymes considerably. Highly polar and hydrophilic solvents are capable of solubilizing large


49



150 120 90 60 30 0 20

30

40

50

60

70

Temperature (C)

Figure 3. Effect of temperature on the hydrolytic activity of free and immobilized C. rugosa lipases at pH 7.0 and 12 h; ♦, free lipase; *, CRLA; ∆, CRLE; □, CRLAE.

Ethyl ester yield (%)

100.00 80.00

accelerated with the increase of shaking speed up to about 250 rpm. The external mass transfer limitation was not so significant when the rotating speed was greater than 250 rpm. At 250 rpm, the final ethyl ester yield of 79.6 % was obtained, which was only slightly less than that of shaking speed at 300 rpm (81.9%). Although some minor variation from the result of the previous study was observed, the result of this study clearly demonstrated the rise of reaction rate with increasing shaking speed up to 250 rpm. It could be explained that at high shaking speed, the reaction system was thoroughly emulsified and the interfacial area was increased evidently. Consequently, the reaction was facilitated because the collision probability between lipase and substrate was improved greatly [11]. 100 Ethyl Ester Yield (%)

amounts of water. The removing of essential water from the enzymes could cause significant loss of the catalytic activity [10]. The result demonstrated the optimal molar ratio of ethanol to reactants for catalytic transesterification of palm oil at 9:1.

80 60 40 20 0 0

60.00

4

8

12

16

20

24

Time (hr)

40.00 20.00 0.00 1 3:1

2 6:1

3 9:1 (a)

4 12:1

5 15:1

Figure 5. Effect of shaking speed on ethyl ester yield by using the free lipases (5% based on oil weight). The molar ratio of ethanol to palm oil was 9:1 at the reaction temperature of 50°C, 24 h; ●, 150; ∆, 200; ■, 250; *, 300.

Figure 4. Effect of molar ratio of ethanol to palm oil on ethyl ester yield by using free Effect of Diameter of Immobilized Bead. The lipase at 5% by wt of oil, reaction effect of bead diameter on the activity of temperature of 50°C, 250 rpm and reaction immobilized lipase was investigated by varying time of 24 h. the diameter of bead at 1.7, 2 and 4 mm with 1% sodium (Na)-alginate concentration Effect of Shaking Speed. Figure 5 presents the (Figure 6). The activity of CRLAE was effect of shaking speed on soluble lipase significantly lower than that of CRLE. The mediated transesterification of purified palm Na-alginate concentration and bead diameter oil at 50˚C, ethanol:oil molar ratio at 9:1 with for the maximum activity (184.17 U) were using 5% free lipase (by wt of oil). It was 1% w/v and 1.7 mm, respectively. Nafound that the final conversion yield was alginate ≥ 1.5 % w/v could cause the strong


50

Immobilization of Lipase on CaCO3 and Entrapment in Calcium Alginate Bead for Biodiesel Production

Ethyl Ester Yield (%)

limitation in internal diffusion of the bead and CaCO3-lipase entrapped in Ca-alginate (data not shown). It was found that the (CRLAE) (42.7%), respectively. activity of immobilized lipase was decreased with the increase of bead diameter. The 100.00 diffusion interference in hydrolytic reaction over the calcium alginate bead intruded to 80.00 slow the rate of reaction. Moreover, it was 60.00 found that the lack of buffer solution in the 40.00 bead could be the cause of the lower lipase activity. 20.00


0.00 0

200

6

12

18

24

30

36

42

48

Time (hr)

150

Figure 7. The ethyl ester yield by using the different techniques of immobilized lipase.♦, free lipase; ○, CRLE;▲, CRLA; □, CRLAE; *, CaCO3.

100 50 0 0 1 2 3 4 Diameter of alginte beads (mm)

Figure 6. Effect of bead diameter on the hydrolytic activity of immobilized C. rugosa lipase with 1% Na-alginate concentration; (), CRLE carrier; (▲), CRLAE carrier with buffer solution; (), CRLAE carrier prepared without buffer solution. Ethyl Ester Yields of Biodiesel by Catalytic Transesterification. The optimum reaction conditions from the previous study were employed for transesterification using immobilized lipase. The ethyl ester yields of biodiesel by catalytic transesterification of purified palm oil are shown in Figure 7. The ethyl ester yield by using the immobilized lipase was lower than that of the free lipase (83.4%). After 48 hours, the entrapment of C. rugosa lipase in Ca-alginate matrix (CRLE) showed the higher ethyl ester yield (74.2 %) than that of the immobilization of C. rugosa lipase adsorbed on CaCO3 (CRLA) (57.6 %)

4. Conclusions Biodiesel productions from purified palm oil and 95 % (v/v) ethanol by using C. rugosafree and immobilized lipases as biocatalysts were investigated. The optimal conditions were at the molar ratio of ethanol to palm oil of 9:1 using 5% wt (by oil) lipase, controlled at temperature 50 °C, 250 rpm and reaction time 24 h, with the yields of ethyl ester at 83.4% whereas the biodiesel production by the immobilized lipase in CRLE, CRLA and CRLAE resulted in ethyl ester yields of about 74.2, 57.6 and 42.7, respectively after 48 hours at the optimal condition. Acknowledgements This work was financially supported by the Thailand Research Fund (TRF) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) under grant number MRG-WII 515E011.



51

References [1] Carneiro-da-Cunha, M.G., Rocha, J.M.S., Garcia, F.A.P., & Gil, M.H. (1999). Lipase immobilization on to polymeric membranes. Biotechnol Tech. 13, 403-9. [2] Ghamgui, H., Karra-Chaabouni, M., & Gargouri, Y. (2004). 1-Butyloleate synthesis by immobilized lipase from Rhizopus oryzae: a comparative study between nhexane and solvent-free system. Enzyme and Microbial Technology. 35, 355-63. [3] Hertzberg, S., Kvittingen, L., Anthonsent, T., & Gudmund, T. (1992). Alginate as immobilization matrix and stabilizing agent in a twophase liquid system: Application in lipasecatalysed reactions. Enzyme and Microbial Technology.14, 42-7. [4] Wahlgren, M., & Arnebrant, T. (1991). Protein adsorption to solid surfaces.TIBTECH. 9, 201. [5] Villeneue, P. (2000). Customizing lipases for biocatalysis :a survey of chemical, physical and molecular biological approaches. J. Molecular Catalysis B :Enzymatic. 9, 113-48. [6] Rosu, R., Uozaka, Y., Iwasaki, Y., & Yamane, T. (1997).Repeated use of immobilized lipase for monoacylglycerol production by solid-phase glycerolysis of olive oil. J. Am. Oil Chem. Soc.91, 445-50.

[7] Yadav, G.D., & Jadhav, S.R. (2005). Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium. Microporous and Mesoporous Mat. 86, 215-22. [8] Kose, O., Tuter, M., & A yse Aksoy, H. (2002). Immobilized Candida Antarctica lipase-catalyzed alcoholysis of cotton seed oil in a solvent-free medium. Bioresource Technology, 83, 125–9. [9] Awang, R., Ghazuli Rafaei, M., & Baari, M. (2007). Immobilization of lipase from Candida rugosa on palm-based polyurethane foam as a support material. American Journal of Biochemistry and Biotechnology, 163-6. [10] de A. Vieire, A.P., da Silva, M.A.P., & Langone, M.A.P. (2006). Biodiesel production via esterification reactions catalyzed by lipase. Latin American Applied Research, 36, 283-8. [11] Xin, C., Wei, D., & Liu, D. (2008). Effect of several factors on soluble lipase mediated biodiesel preparation in the biphasic aqueous -oil systems. J. Microbiol Biotechnol, 24, 2097-102.
