www.elsevier.com/locate/jbiosc

Journal of Bioscience and BioengineeringVOL. xx No. xx, 1e4, 2014NOTE

Lipase catalyzed transesterification of castor oil by straight chain higher alcohols

Deepika Malhotra,1,z Joyeeta Mukherjee,2,z and Munishwar N. Gupta1,*Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India1 and Department of Chemistry,Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India2

Received 7 June 2014; accepted 9 August 2014Available online xxx* CorrespondE-mail add

gmail.com (J. Mz The first tw

1389-1723/$http://dx.doi

Please citeBioeng., (20Biolubricants from Castor oil were produced enzymatically by transesterification with higher alcohols using a lipasemixture of immobilized Mucor miehei (RMIM) and immobilized Candida antarctica lipase B (Novozym 435) under lowwater conditions. The conversions were in the range of 80e95% under the optimized conditions. 2014, The Society for Biotechnology, Japan. All rights reserved.[Key words: Low water enzymology; Enzyme immobilization; Valorization of castor oil; Lipases; Transesterification; Non edible oils]Esters of the medium chain and long chain alcohols find a widevariety of applications in industrial sectors like textiles, plastics,cosmetics and as industrial lubricants (1). The replacement of pe-troleum based lubricants with biodegradable synthetic lubricants isconsidered a desirable goal (2). The oils from the vegetable sourcescan be converted into esters of alcohols by the transesterificationreaction (3) (Fig. S1). While the esters of short chain alcohols suchas methanol and ethanol are used as biodiesel, the esters of higherchain alcohols function as lubricants (4,5). Castor oil, the oil derivedfrom castor bean seeds is considered a non edible oil in view of itstoxic nature. Its use as a starting material for synthesis of bio-lubricants has started attracting attention (6). India is the majorproducer of castor oil in the world, producing about 65% of totalcastor seed production (0.8 million tons) and 51% of castor oil (0.3million tons) (7). The transesterification reaction with vegetableoils can be catalyzed either by chemical catalysts or by enzymes (8).The enzyme catalyzed transesterification for synthesis of bio-lubricants has however attracted less attention. The esters of longstraight chain alcohols as biolubricants have also attracted verylittle attention (3).

The present work describes the use of commercially availablelipases for carrying out transesterification of castor oil with1-hexanol, 1-octanol and 1-dodecanol. So far most of the work ontransesterification with medium chain/long chain alcohols, evenwith chemical catalysts has been carried out by first converting theoil into a methyl or ethyl ester. That involves an additional step. Thepresent study involves direct enzyme catalyzed transesterificationof castor oil with the above mentioned alcohols.

Castor oil (0.5 g) and alcohol (1-hexanol,1-octanol,1-dodecanol)were taken in molar ratio in the range of 1:3e1:8 with n-hexaneing author. Tel.: 91 11 26591503; fax: 91 11 26582282.resses: deepika.2102@gmail.com (D. Malhotra), joyeetamukherjee86@ukherjee), munishwar48@yahoo.co.uk (M.N. Gupta).

o authors contributed equally to this work.

e see front matter 2014, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2014.08.005

this article in press as: Malhotra, D., et al., Lipase catalyzed tran14), http://dx.doi.org/10.1016/j.jbiosc.2014.08.005(5 mL) or without n-hexane (solvent free) in a screw-capped vial.Lipase (5%e7.5% w/w of oil) was added to this reaction mixture andincubated at different temperature (30Ce60C) with a constantshaking at 200 rpm. Aliquots (20 mL in case of solvent free and200 mL in case of hexane) were taken at different time intervals andthe percentage conversion to alkyl ester was determined by car-rying out the GC analysis of the samples (9).

RMIM is the commercially available immobilized preparation ofMucor miehei lipase which has been used fairly extensively in lowwater media (9,10). With castor oil, M. miehei lipase has beenfrequently employed (11). This was chosen for the preparation ofthe fatty acid esters. Solvent free synthesis utilizes substrates assuch, as the reaction media. It allows working with higher con-centration of the substrates, is less expensive and avoids the use ofvolatile organic solvents (12e14). Hence, initially trans-esterification of the castor oil with three different long chain al-cohols 1-hexanol, 1-octanol and 1-dodecanol at different ratios ofoil: alcohol was tried (Fig. 1). Theoretically three moles of alcoholare needed for complete transesterification of the triglyceride. Inthe present case, three times molar excess of the alcohol was foundto give the best results for all the three alcohols. The conversionafter 24 h was found to be 64%, 60% and 47% for 1-hexanol, 1-octanol and 1-dodecanol respectively using 3 M excess of the al-cohols (Fig. 1).

RMIM has been used up to 50C in low water media (9,10).Hence, the above reactionwith the three alcohols was performed at30C, 40C and 50C. The highest temperature of 50C gave thebest performance with all the three alcohols (69%, 63% and 56% for1-hexanol, 1-octanol and 1-dodecanol respectively) and waschosen for all further reactions (Fig. S2). It is noteworthy thatconversions obtained in 24 h with the different alcohols followedthe trend 1-hexanol> 1-octanol> 1-dodecanol. The specificity of alipase preparation is known to depend upon the chain length of thealcohol (15).

Castor oil has considerably high viscosity as compared to othervegetable oils (16). While solvent free synthesis do have theAll rights reserved.

sesterification of castor oil by straight chain higher alcohols, J. Biosci.

FIG. 1. Effect of molar ratio of castor oil: alcohol on solvent free synthesis of the fatty acid esters. (A) 1-Hexanol, (B) 1-octanol, and (C) 1-dodecanol were taken in molar ratio of 1:3(open triangles), 1:4 (closed triangles), 1:6 (open squares), and 1:8 (closed squares). Reaction was carried out at 40C at 200 rpm using RMIM (5% w/w of oil).

2 MALHOTRA ET AL. J. BIOSCI. BIOENG.,advantages like greenness, high molarity of substrates can be dis-solved and cost reduction, it was decided to examine the effect ofreducing the mediumviscosity by adding hexane. Moreover, higherconversions are reported when transesterification of triglycerideswith alcohols are carried out in hexane as solvent as compared tosolvent free conditions (17). So, it was necessary to examineFIG. 2. Effect of additional RMIM/Novozym 435 on synthesis of the fatty acid esters. Synthehexanol (B), 1-octanol, and (C) 1-dodecanol in molar ratio of 1:3 in the presence of hexane as(containing RMIM- 5% w/w of oil) at 4th hour. Symbols are represented as follows: closed sqw/w of oil); closed circles, RMIM (5% w/w of oil) Novozym 435 (2.5% w/w of oil).

Please cite this article in press as: Malhotra, D., et al., Lipase catalyzed tranBioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.005whether the use of solvent outweighed the advantages of solventfree system in the present instance. The choice of hexane as thesolvent was made because transesterification of oils/fatty acids hasfrequently been carried out using hexane as a reaction medium (5).Table S1 shows the results of the effect of adding hexane. With allthe three alcohols, both initial rates and % conversion in 24 h weresis of the fatty acid esters was carried out at 50C, 200 rpm with castor oil and (A) 1-the solvent. Additional RMIM/Novozym 435 (2.5% w/w of oil) was added to the reactionuares, only RMIM (5% w/w of oil); open triangles, RMIM (5% w/w of oil) RMIM (2.5%

sesterification of castor oil by straight chain higher alcohols, J. Biosci.

VOL. xx, 2014 NOTE 3found to improve significantly. The fact that this increase washighest in the case of 1-dodecanol (the most viscous alcohol)indicated that the viscosity factor was very important.

TheM.miehei lipase is a 1, 3-specific lipase (10,15). Although acylmigration during transesterification does result in>66% conversion(18) it was decided to try Novozym 435 as well. This well knownindustrial preparation of lipase is an immobilized form of Candidaantarctica lipase B and has been found to be non-specific for all thethree positions of the triglyceride in many transesterification re-actions involving triglycerides (9). Just like RMIM, 5% (w/w of oil)Novozym 435 was used. Both enzyme preparations showed com-parable performance with all the three alcohols reaching 79% and80%, 72% and 71%, 67% and 69% conversion in 24 h with RMIM andNovozym 435 for 1-hexanol, 1-octanol and 1-dodecanol respec-tively (Fig. S3).

Novozym 435 is considerably more expensive than RMIM on aweight basis. Recently, we found that blending of RMIM andNovozym 435 gave better results (as compared to RMIM alone) inthe transesterification of coffee oil (9). Earlier, Lee et al. (19) haveused a combination of 1,3 specific Rhizopus oryzae lipase and non-specific Candida rugosa lipase for obtaining 99% biodiesel in 21 h.To find out whether slowing down of the conversion rate around4 h with RMIM (Fig. 1) was a result of its 1,3-specific nature,additional 2.5% (w/w of oil) Novozym 435 was added after 4 h ofthe reaction. With all the three alcohols, % conversion improvedupon addition of Novozym 435; hexyl, octyl and dodecyl estersconversion increased from 71% to 82%, 65%e77% and 52%e64% in7 h, respectively (Fig. 2). On the other hand, adding additional 2.5%(w/w of oil) RMIM had no significant effect. This indicated thatNovozym 435 helped in conversion of 2-acyl glycerol to the alkylesters. However, it was observed in the case of 1-hexanol that the% conversion did not increase more than 3e4% beyond 7 h evenwhen the reaction was carried up to 24 h (Fig. 2A). From the pointof view of process economy, it was interesting to observe that useof the oil: alcohol in the molar ratio 1:3 gave the best resultsFIG. 3. Effect of additional RMIM/Novozym 435 on synthesis of the fatty acid esters using (Afatty acid esters was carried using castor oil and alcohol in a molar ratio of 1:4 in the presencw of oil); gray bars, RMIM (5% w/w of oil) RMIM (2.5% w/w of oil); closed bars, RMIM (5% wof reaction.

Please cite this article in press as: Malhotra, D., et al., Lipase catalyzed tranBioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.005(Fig. 1). However, with added hexane as a solvent, 1:4 M ratio ofoil: alcohol was found to improve the % conversion to 95% in 48 h(Fig. 3). A careful analysis showed that under solvent free condi-tions, most of the withdrawals of the aliquots for analysis (duringthe time course measurement) were made when less alcohol wasconsumed [the rates were slow under solvent free conditions and% conversions were in the lower range], this left enough availablealcohol for the reaction to continue over 24 h. Hence, under thesolvent free conditions, 1:3 ratio was observed to be best. Withhexane as the reaction medium, rates were much faster, so most ofthe withdrawals of aliquots (which included the alcohol) weremade at time periods at higher % conversions. Hence, not enoughalcohol was left and this slowed down the reaction. Hence, withhexane as the solvent, 4 M excess of the alcohol was needed forobtaining maximum conversion during the time course study. Forthe actual process, 1:3 ratio may be used and this step wouldcontribute towards process economy.

Under these conditions [with additional 2.5% (w/w of oil)Novozym 435 added after 4 h] octyl and dodecyl esters formed after48 h were 90% and 80% respectively (Fig. 3B and C).

In some cases, stepwise addition of alcohol has been reportedto improve conversions in lipase catalyzed transesterificationreactions (17). These effects are interpreted in terms of inhibition oflipases with short chain alcohols (20). With higher alcohols, in thepresent instance, the stepwise addition of alcohols did not help(Fig. S4).

Thus the present work focused more on: (i) use of non-edibleoil from castor beans (as in many developing countries like Indiaedible oils are in short supply and cannot be diverted for thispurpose); (ii) use of less expensive enzyme preparations. It wasfound advantageous to blend RMIM with Novozym 435 to achieve95% conversion with 1-hexanol, 90% with 1-octanol and 80%with 1-dodecanol. These results show considerable promise inproduction of the esters using non-edible vegetable oils for variousapplications including their use as biolubricants.) 1-hexanol, (B) 1-octanol, and (C) 1-dodecanol at higher molar ratio. Synthesis of thee of hexane as the solvent. Symbols are represented as follows: open bars, RMIM (5% w//w of oil) Novozym 435 (2.5% w/w of oil). Additional enzyme was added at 4th hour

sesterification of castor oil by straight chain higher alcohols, J. Biosci.

4 MALHOTRA ET AL. J. BIOSCI. BIOENG.,Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jbiosc.2014.08.005.

We acknowledge financial support from the Government of Indias Departmentof Science and Technology (DST) (Grant No. SR/SO/BB-68/2010) and Department ofBiotechnology (DBT) (Grant No. BT/PR14103/BRB/10/808/2010) are gratefullyacknowledged. JM thanks the Council of Scientific and Industrial Research for theSenior Research Fellowship.References

1. Lacaze-Dufaure, C. and Mouloungui, Z.: Catalysed or uncatalysed esterifica-tion reaction of oleic acid with 2-ethylhexanol, Appl. Catal. A, 20, 223e227(2000).

2. Hama, S. and Kondo, A.: Enzymatic biodiesel production: an overview ofpotential feedstocks and process development, Bioresour. Technol., 135,386e395 (2013).

3. Encinar, J. M., Gonzlez, J. F., and Pardal, A.: Transesterification of castor oilunder ultrasonic irradiation conditions. Preliminary results, Fuel Process.Technol., 103, 9e15 (2012).

4. Shah, S., Sharma, S., and Gupta, M. N.: Biodiesel preparation by lipase-catalyzedtransesterification of Jatropha oil, Energy Fuels, 18, 154e159 (2004).

5. Robles-Medina, A., Gonzalez-Moreno, P. A., Cerdan, L. E., andMolina-Grima, E.: Biocatalysis: towards ever greener biodiesel production,Biotechnol. Adv., 27, 398e408 (2009).

6. Salimon, J. and Salih, N.: Chemical modification of oleic acid oil forbiolubricant industrial applications, Aust. J. Basic Appl. Sci., 4, 1999e2003(2010).

7. Sharma, S. M.: Castor, pp. 447e472, in: Vegetable oils, sources and enhance-ment research in India. Jain Brothers, New Delhi (2003).

8. Hsu, A. F., Jones, K., Foglia, T. A., and Marmer, W. N.: Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel,Biotechnol. Appl. Biochem., 36, 181e186 (2002).Please cite this article in press as: Malhotra, D., et al., Lipase catalyzed tranBioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.0059. Banerjee, A., Singh, V., Solanki, K., Mukherjee, J., and Gupta, M. N.: Combi-protein coated microcrystals of lipases for production of biodiesel from oil fromspent coffee grounds, Sustain. Chem. Process., 1, 14 (2013).

10. Gitlsen, T., Svensson, I., Adlercreutz, P., Mattiasson, B., and Nilsson, J.: High-oleic-acid rapeseed oil as starting material for the production of confectionaryfats via lipase-catalyzed transesterification, Ind. Crops Prod., 4, 167e171(1995).

11. Ghosh, M. and Bhattacharyya, D. K.: Enzymatic interesterification of blends ofcastor oil and some oils rich in saturated fatty acids, Eur. J. Lipid Sci. Technol.,104, 214e216 (1999).

12. Baron, A. M., Barouh, N., Barea, B., Villeneuve, P., Mitchell, D. A., andKrieger, N.: Transesterification of castor oil in a solvent-free medium using thelipase from Burkholderia cepacia LTEB11 immobilized on a hydrophobic sup-port, Fuel, 117, 458e462 (2014).

13. Kumari, V., Shah, S., and Gupta, M. N.: Preparation of biodiesel by lipasecatalysed transesterification of high free fatty acid containing oil fromMadhucaindica, Energy Fuels, 21, 368e372 (2007).

14. Singh, V., Solanki, K., and Gupta, M. N.: Process optimization for biodieselproduction, Recent Pat. Biotechnol., 2, 130e143 (2008).

15. Kapoor, M. and Gupta, M. N.: Lipase promiscuity and its biochemical appli-cations, Process Biochem., 47, 555e569 (2012).

16. Jeong, G. T. and Park, D. H.: Optimization of biodiesel production from castoroil using response surface methodology, Appl. Biochem. Biotechnol., 156,431e441 (2009).

17. Fukuda, H., Kondo, A., and Noda, H.: Biodiesel fuel production by trans-esterification of oils, J. Biosci. Bioeng., 92, 405e416 (2001).

18. Du, W., Xu, Y. Y., Liu, D. H., and Li, Z. B.: Study on acyl migration in immo-bilized lipozym TL-catalyzed transesterification of soybean oil for biodieselproduction, J. Mol. Catal. B: Enzym., 37, 68e71 (2005).

19. Lee, D. H., Kim, J. M., Shin, H. Y., Kang, S. W., and Kim, S. W.: Biodieselproduction using a mixture of immobilized Rhizopus oryzae and Candida rugosalipases, Biotechnol. Bioprocess Eng., 11, 522e525 (2006).

20. Du, W., Xu, Y., Liu, D., and Zeng, J.: Comparative study on lipase-catalysedtransformation of soybean oil for biodiesel production with different acylacceptors, J. Mol. Catal. B: Enzym., 30, 125e129 (2004).sesterification of castor oil by straight chain higher alcohols, J. Biosci.