**3.3 Lipases immobilization by covalent attachment**

Covalent attachment is a result of a chemical reaction between the active amino acid residues outside the active catalytic and binding site of the enzyme, and the active functionalities of the carrier (Cao, 2005). Although drastic and complicated, and strongly affected by the carriers' properties, covalent attachment is the most efficient technique for enzyme immobilization. Some carriers used for covalent lipase immobilization are displayed in Table 3.


Table 3. Carrier used for lipases immobilization by covalent attachment

Yücel (2011) reports a method for *Thermomyces lanuginosus* lipase covalent binding on polyglutaraldehyde-activated olive pomace powder. The technique is cost effective, because of the low price of the support and because of the strong covalent bond formed, leading to enzyme stabilization without loss of activity, allowing the multiple reuse of the enzyme. Immobilized lipase was stable for 10 batches of pomace oil transesterification retaining more than 80% residual activity.

Among the other naturally occurring materials, chitosan is considered as appropriate for enzyme binding. Its membrane forming and adhesion ability, high mechanical strength and facility of forming insoluble in water thermally and chemically inert films make it suitable for lipase immobilization. For instance, *Candida rugosa* type VII lipase was fixed onto chitosan beads using a binary method consisting in the follows: (i) lipase

The Immobilized Lipases in Biodiesel Production 405

support particles. The fixation was achieved spontaneously during batch cultivation. The applied technique (Atkinson et al., 1979) offers numerous advantages over other methods: the particles are reusable and mechanically resistant; additional reagents, aseptic handling of particles, and preproduction of cells are not necessary. It has been demonstrated that *Rhysopus oryzae* cells immobilized within biomass support particles can be used as low cost

Enzymatic approach to biodiesel production offers several advantages over the chemical catalysis currently applied. It is more efficient because of the enzyme specificity and selectivity, involves less energy consumption because of the mild reaction conditions, and is environmentally friendly because of the limited release of side products or wastes. Catalyst immobilization presents a number of additional benefits, such as repeated use of the enzymes, enhancement of their thermal and operational stability, localization of the interaction, effective control of the reaction parameters, etc., thus reducing the production cost and making the enzyme biodiesel synthesis an attractive alternative to other technologies. The present review provides an overview on the techniques applied for lipases

Adinarayana, K.; Bapi Raju, K. V. V. S. N.; Zargar, M. I. ; Devi, R. B.; Lakshmi, P. J. & Ellaiah,

fermentation by newly isolated *Aspergillus* species, *Ind. J. Biotechnol*., 3, 65-69 Ahn, K.; Ye, S.; Chun, W.; Rah, H. & Kim, S. (2010). Yield and component distribution of

Akoh, C.; Chang, Shu-Wei; Lee, Guan-Chiun & Shaw, Jei-Fu (2007). Enzymatic approach to

ASTM D6751: American Society for Testing and Materials standard specification for

Atkinson, B.; Black, G. M.; Lewis, P. J. S. & Pinches, A. (1979). Biological particles of given

Bajaj, A.; Lohan, P.; Jha, P. & Mehrotra, R. (2010). Biodiesel production through lipase catalyzed transesterification: An overview. *J. Mol. Catal. B: Enzymatic*, 62, 9-14 Bisen, P.; Sanodiya, B.; Thakur, G.; Baghel, R. & Prasad, G. B. K. S. (2010). Biodiesel

Cao, L. (2005). *Carrier-bound immobilized enzymes. Principles, application and design*. Wiley-

Chahinian, H.; Vanot, G.; Ibrik, A.; Rugani, N.; Sarda, L. & Comeau, L. C. (2000). Production

a partial acylglycerol lipase. *Biosci. Biotechnol. Biochem*., 64, 215-222

size, shape, and density for use in biological reactors. *Biotechnol. Bioeng*., 21, 193-200

production with special emphasis on lipase-catalyzed transesterification. *Biotechnol.* 

of extracellular lipases by *Penicillium cyclopium* purification and characterization of

biodiesel production. J. *Agric. Food Chem*. 55, 8995-9005

biodiesel fuel (B 100) blend stock for distillate fuels

VCH, ISBN-10: 3-527-31232-3, Weinheim

P. (2004). Optimization of process parameters for production of lipase in solid-state

biodiesel by methanolysis of soybean oil with lipase-immobilized mesoporous silica. *Microporous and Mesoporous Materials* doi:10.1016/j.micromeso.2010.11.008 (in

catalyst for biodiesel production.

**4. Conclusion** 

immobilization.

**5. References** 

press)

*Lett*., 32, 1019–1030

immobilization onto the hydroxyl groups of chitosan by activation with 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride; (ii) immobilization of additional lipase molecules through their amino groups to chitosan by cross-linking with glutaraldehyde. The immobilized enzyme has been used to catalyse the hydrolysis of soybean oil. Then, the feedstock containing free fatty acids, mono-, di- and triglycerides was esterified with methanol in the presence of an acid catalyst to produce biodiesel. It has been demonstrated that the enzymatic/acid-catalyzed hybrid process uses milder reaction conditions and allows avoiding the inactivation of the immobilized enzyme by polar compounds and increase biodiesel yields.

A new method for the synthesis of hydrophobic microporous matrices for enzyme immobilization, namely styrene–divinylbenzene–polyglutaraldehyde and poly(styrenedivinylbenzene)-polyglutaraldehyde copolymers, applying High Internal Phase Emulsions (HIPE) technique has been developed by Dizge et al. (2008, 2009a, 2009b). *Thermomyces lanuginosus* lipase was successfully attached to the support by covalent binding. According to the authors, the copolymers could be prepared in a short time and in large amounts and shapes. The immobilization efficiency, defined as the ratio of the activity of the immobilized enzyme to the activity of the free enzyme was found to vary from 80% to 89%. The immobilized enzyme retained its activity during 10-15 repeated batch reactions.

Promising results in terms of enzyme thermal and operational stability improvement have been obtained using as supports for lipases immobilization silanized Nb2O5 and SiO2-PVA (Da Rуs et al., 2010), and glutaraldehyde or ethanolamine activated silica gels (Lee and al., 2008; Kumari et al., 2009). However, the stability of the immobilized enzyme depends not only on the chemical/physical nature of the carrier, but also on the binding mode, the binding number, and the position of the binding on the enzyme surface (Cao, 2005), among other. Mendes et al. (2011) and Rodrigues et al. (2010) demonstrate that the multipoint covalent attachment of *Thermomyces lanuginosus* lipase on Toyopearl AF-amino-650M resin and on aldehyde-Lewatit is an efficient strategy for enzyme stabilization. It has been demonstrated that *Thermomyces lanuginosus* lipase immobilized on glyoxyl-resin is between 27 and 31 times more stable than the soluble lipase (Mendes et al., 2011).

Another important issue provided by enzyme immobilization concerns the localization of the interaction in the zone where the maximum concentration of reagents is present and/or at the interface between the immiscible heterogeneous phases, regarding lipases. For this purpose, lipases (from *Candida rugosa* and *Thermomyces lanuginosus*) have been immobilized on magnetic nanostructures (Fe3O4) and localized by application of a magnetic field. In addition, the method favours the simple and fast separation of the enzyme from the reaction mixture. Thus, it allows the intensification of the process and the reduction of the production costs.

#### **3.4 Cells immobilization**

The technological and economic advantages of immobilized cells over immobilized enzymes are well known (D'Souza, 1982): higher operational stability, higher yields of enzyme activity after immobilization, greater resistance to environmental perturbations, greater potential for multistep processes, and lower effective enzyme cost (enzyme purification and extraction are avoided). Despite of these benefits, only few investigations on the use of immobilized cells in biodiesel synthesis are reported until now (Fukuda et al., 2009; Hama et al., 2006, 2007; Li et al., 2007; Oda et al., 2005; Tamalampudi et al., 2008). The research efforts were focused on the immobilization no more than of *Rhysopus oryzae* within porous biomass support particles. The fixation was achieved spontaneously during batch cultivation. The applied technique (Atkinson et al., 1979) offers numerous advantages over other methods: the particles are reusable and mechanically resistant; additional reagents, aseptic handling of particles, and preproduction of cells are not necessary. It has been demonstrated that *Rhysopus oryzae* cells immobilized within biomass support particles can be used as low cost catalyst for biodiesel production.

#### **4. Conclusion**

404 Biodiesel – Feedstocks and Processing Technologies

immobilization onto the hydroxyl groups of chitosan by activation with 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride; (ii) immobilization of additional lipase molecules through their amino groups to chitosan by cross-linking with glutaraldehyde. The immobilized enzyme has been used to catalyse the hydrolysis of soybean oil. Then, the feedstock containing free fatty acids, mono-, di- and triglycerides was esterified with methanol in the presence of an acid catalyst to produce biodiesel. It has been demonstrated that the enzymatic/acid-catalyzed hybrid process uses milder reaction conditions and allows avoiding the inactivation of the immobilized enzyme by

A new method for the synthesis of hydrophobic microporous matrices for enzyme immobilization, namely styrene–divinylbenzene–polyglutaraldehyde and poly(styrenedivinylbenzene)-polyglutaraldehyde copolymers, applying High Internal Phase Emulsions (HIPE) technique has been developed by Dizge et al. (2008, 2009a, 2009b). *Thermomyces lanuginosus* lipase was successfully attached to the support by covalent binding. According to the authors, the copolymers could be prepared in a short time and in large amounts and shapes. The immobilization efficiency, defined as the ratio of the activity of the immobilized enzyme to the activity of the free enzyme was found to vary from 80% to 89%. The

Promising results in terms of enzyme thermal and operational stability improvement have been obtained using as supports for lipases immobilization silanized Nb2O5 and SiO2-PVA (Da Rуs et al., 2010), and glutaraldehyde or ethanolamine activated silica gels (Lee and al., 2008; Kumari et al., 2009). However, the stability of the immobilized enzyme depends not only on the chemical/physical nature of the carrier, but also on the binding mode, the binding number, and the position of the binding on the enzyme surface (Cao, 2005), among other. Mendes et al. (2011) and Rodrigues et al. (2010) demonstrate that the multipoint covalent attachment of *Thermomyces lanuginosus* lipase on Toyopearl AF-amino-650M resin and on aldehyde-Lewatit is an efficient strategy for enzyme stabilization. It has been demonstrated that *Thermomyces lanuginosus* lipase immobilized on glyoxyl-resin is between

Another important issue provided by enzyme immobilization concerns the localization of the interaction in the zone where the maximum concentration of reagents is present and/or at the interface between the immiscible heterogeneous phases, regarding lipases. For this purpose, lipases (from *Candida rugosa* and *Thermomyces lanuginosus*) have been immobilized on magnetic nanostructures (Fe3O4) and localized by application of a magnetic field. In addition, the method favours the simple and fast separation of the enzyme from the reaction mixture. Thus, it allows the intensification of the process and the reduction of the

The technological and economic advantages of immobilized cells over immobilized enzymes are well known (D'Souza, 1982): higher operational stability, higher yields of enzyme activity after immobilization, greater resistance to environmental perturbations, greater potential for multistep processes, and lower effective enzyme cost (enzyme purification and extraction are avoided). Despite of these benefits, only few investigations on the use of immobilized cells in biodiesel synthesis are reported until now (Fukuda et al., 2009; Hama et al., 2006, 2007; Li et al., 2007; Oda et al., 2005; Tamalampudi et al., 2008). The research efforts were focused on the immobilization no more than of *Rhysopus oryzae* within porous biomass

immobilized enzyme retained its activity during 10-15 repeated batch reactions.

27 and 31 times more stable than the soluble lipase (Mendes et al., 2011).

polar compounds and increase biodiesel yields.

production costs.

**3.4 Cells immobilization** 

Enzymatic approach to biodiesel production offers several advantages over the chemical catalysis currently applied. It is more efficient because of the enzyme specificity and selectivity, involves less energy consumption because of the mild reaction conditions, and is environmentally friendly because of the limited release of side products or wastes. Catalyst immobilization presents a number of additional benefits, such as repeated use of the enzymes, enhancement of their thermal and operational stability, localization of the interaction, effective control of the reaction parameters, etc., thus reducing the production cost and making the enzyme biodiesel synthesis an attractive alternative to other technologies. The present review provides an overview on the techniques applied for lipases immobilization.

#### **5. References**


The Immobilized Lipases in Biodiesel Production 407

Ghaly, A. E.; Dave, D.; Brooks, M. S. & Budge, S. (2010). Production of biodiesel by enzymatic transesterification: Review. *Am. J. Biochem. Biotechnol*., 6, 54-76 Guncheva, M. & Zhiryakova, D. (2011). Catalytic properties and potential applications of

Hama, S.; Tamalampudi, S.; Fukumizu, T.; Miura, K.; Yamaji, H.; Kondo, A. & Fukuda, H.

Hama, S.; Yamaji, H.; Fukumizu, T.; Numata, T.; Tamalampudi, S.; Kondo, A. Noda, H. &

Hasan, F.; Shah, A. & Hameed, A. (2009). Methods for detection and characterization of

Helwani, Z.; Othman, M. R.; Aziz, N.; Fernando, W. J. N. & Kim, J. (2009). Technologies for

Hiol, A.; Jonzo, M. D.; Rugani, N.; Druet, D.; Sarda, L. & Comeau, L. C. (2000). Purification

Jegannathan, K.; Jun-Yee, L.; Chan, E. & Ravindra, P. (2009). Design an immobilized lipase

Jegannathan K. Abang, S. (2008). Production of biodiesel using immobilized lipase-A critical

Jegannathan, K.; Jun-Yee, L.; Chan, E. & Ravindra, P. (2010). Production of biodiesel from

Ji, Q.; Xiao, S.; He, B. & Liu, X. (2010). Purification and characterization of an organic

Karanam, S. K. & Medicherla, N. R. (2008). Enhanced lipase production by mutation

Kashmiri, M. A.; Adnan, A.; Butt, B. W. (2006). Production, purification and partial characterization of lipase from *Trichoderma viride*. *African J. Biotechnol*., 5, 878-882 Khor, G.; Sim, J.; Kamaruddin, A. & Uzir, M. (2010). Thermodynamics and inhibition studies

Kumari, A.; Mahapatra, P.; Garlapati, V. & Banerjee, R. (2009). Enzymatic transesterification of Jatropha oil. *Biotechnology for Biofuels*, 2:1, doi:10.1186/1754-6834-2-1 Lee Jong Ho; Dong Hwan Lee; Jung Soo Lim; Byung-Hwan Um; Chulhwan Park; Seong

Li, N.; Zong, M.; Wu, H. (2009). Highly efficient transformation of waste oil to biodiesel by immobilized lipase from *Penicillium expansum*. *Process Biochemistry*, 44, 685–688

strain isolated from palm fruit. *Enzyme Microb. Technol*., 26, 421–30

review. *Critical Reviews in Biotechnology*, 28, 253–264

biodiesel production. *J. Mol. Catal. B: Enzymatic*, 66, 264–269

induced *Aspergillus japonicus*. *African J. Biotechnol*., 7, 2064-2067

lipases: A comprehensive review*. Biotechnol. Adv.,* 27, 782-798

(2006). Lipase localization in *Rhizopus oryzae* cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel-fuel production. *J.* 

Fukuda, H. (2007). Biodiesel-fuel production in a packed-bed reactor using lipaseproducing *Rhizopus oryzae* cells immobilized within biomass support particles.

production of biodiesel focusing on green catalytic techniques: A review. *Fuel* 

and characterization of an extracellular lipase from a thermophilic *Rhizopus oryzae*

enzyme for biodiesel production. *J. Renewable and Sustainable Energy*, 1, 063101-1 -

palm oil using liquid core lipase encapsulated in -carrageenan. *Fuel*, 89, 2272–2277

solvent-tolerant lipase from *Pseudomonas aeruginosa* LX1 and its application for

of lipozyme TL IM in biodiesel production via enzymatic transesterification.

Woo Kang & Seung Wook Kim (2008). Optimization of the process for biodiesel production using a mixture of immobilized *Rhizopus oryzae* and *Candida rugosa*

Bacillus lipases. *J. Mol. Catalysis B: Enzymatic*, 68, 1-21

*Bioscience Bioeng*., 101, 328-333

*Biochem. Eng. J*., 34, 273-278

063101-8

*Processing Technology*, 90, 1502–1514

*Bioresource Technology*, 101, 6558–6561

lipases*. J. Microbiol. Biotechnol*., 18, 1927–1931


Cheirsilp, B.; H-Kittikuna, A. & Limkatanyu, S. (2008). Impact of transesterification

Chen, Y.; Xiao, B.; Chang, J.; Fu, Y.; Lv, P. & Wang, X. (2009). Synthesis of biodiesel from

Da Rуs, P.; Silva, G.; Mendes, A.; Santos, J. & Castro, H. (2010). Evaluation of the catalytic

De Paola, M. G.; Ricca, E.; Calabrò, V.; Curcio, S. & Iorio, G. (2009). Factor analysis of

Demirbas, A. (2009). Progress and recent trends in biodiesel fuels. *Energy Conversion and* 

Dizge, N. & Keskinler, B. (2008). Enzymatic production of biodiesel from canola oil using

Dizge, N.; Keskinler, B. & Tanriseven, A. (2008). Covalent attachment of microbial lipase

Dizge, N.; Keskinler, B. & Tanriseven, A. (2009a). Biodiesel production from canola oil by

Dizge, N.; Aydiner, C.; Imer, D.; Bayramoglu, M.; Tanriseven, A. & Keskinler, B. (2009b).

D'Souza, S. F. (1989). Immobilized cells: techniques and applications. *Indian J. Microbiol*., 29,

Dussán, K.; Giraldo, O. & Cardona, C. (2007). Application of magnetic nanostructures in

Dussan, K.; Cardona, C.; Giraldo, O.; Gutiérrez, L. & Pérez, V. (2010). Analysis of a reactive

Fjerbaek, L.; Christensen, K. & Norddahl, B. (2009). A Review of the Current State of

Foresti, M. L. & Ferreira M. L. (2004). Ethanol pretreatment effect and particle diameter

Fukuda, H.; Kondo, A. & Noda, H. (2001). Biodiesel fuel production by transesterification of

Fukuda, H.; Kondo, A. & Tamalampudi, S. (2009). Bioenergy: Sustainable fuels from biomass by yeast and fungal whole-cell biocatalysts. *Biochem. Eng. J*., 44, 2–12

immobilized lipase. *Biomass and Bioenergy*, 32, 1274–1278

copolymer. *Biochem. Eng. J.*, 44, 220–225

*Bioresource Technology*, 100, 1983–1991

Copenhagen, 16-20 September 2007

oils. *J. Bioscience Bioeng*., 5, 405-416

nanostructures. *Bioresource Technology,* 101, 9542–9549

glutaraldehyde. *Colloids and Surfaces B: Biointerfaces*, 66, 34–38

lipase. *Biochem. Eng. J.,* 42, 261–269

*and Management*, 50, 668–673

*Technology*, 100, 5126–513

*Management*, 50, 14–34

5508–5516

83-117

1298-1315

*Surf. Sci*., 238, 86–90

mechanisms on the kinetic modeling of biodiesel production by immobilized

waste cooking oil using immobilized lipase in fixed bed reactor. *Energy Conversion* 

properties of *Burkholderia cepacia* lipase immobilized on non-commercial matrices to be used in biodiesel synthesis from different feedstocks. *Bioresource Technology*, 101,

transesterification reaction of waste oil for biodiesel production. *Bioresource* 

onto microporous styrene-divinylbenzene copolymer by means of poly

using lipase immobilized onto hydrophobic microporous styrene-divinylbenzene

Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer.

biotechnological processes: Biodiesel production using lipase immobilized on magnetic carriers. *Proceedings of European Congress of Chemical Engineering* (ECCE-6),

extraction process for biodiesel production using a lipase immobilized on magnetic

Biodiesel Production Using Enzymatic Transesterification. *Biotechnol Bioeng*., 102, 5,

issues on the adsorption of *Candida rugosa* lipase onto polypropylene powder. *Appl.* 


The Immobilized Lipases in Biodiesel Production 409

Noureddini, H.; Gao, X. & Philkana, R. S. (2005). Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. *Bioresource Technology*, 96, 769–777 Panalotov, I. & Verger, R. (2000). *Physical Chemistry of Biological Interfaces.* Marcel Dekker

Powel, L. W. (1996). In *Industrial Enzymology*, T. Godfrey and S. West, Editors, 2nd ed.

Rajesh, E. M.; Arthe, R.; Rajendran, R.; Balakumar, C.; Pradeepa, N. & Anitha, S. (2010).

Ranganathan, S.; Narasimhan, S. & Muthukumar, K. (2008). An overview of enzymatic

Robles-Medina, A.; González-Moreno, P.A.; Esteban-Cerdán, L. & Molina-Grima, E. (2009).

Rodrigues, R. & Fernandez-Lafuente, F. (2010). Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process. *J. Mol. Catal. B: Enzymatic*, 64, 1–22 Rodrigues, R.; Pessela, B.; Volpato, G.; Fernandez-Lafuente, R.; Guisan, J. & Ayub, M. (2010).

Sakai, S.; Yuping Liu, Y.; Yamaguchi, T.; Watanabe, R.; Kawabe, M. & Kawakami, K. (2010).

Salis, A.; Pinna, M.; Monduzzi, M. & Solinas, V. (2008). Comparison among immobilised

Salis, A.; Bhattacharyya, M.; Monduzzi, M. & Solinas, V. (2009). Role of the support surface

Semwal, S.; Arora, A.; Badoni, R. & Tuli. D. (2011). Biodiesel production using

Séverac, E.; Galy, O.; Turon, F.; Pantele, C.; Condoret, J-S.; Monsan, P. & Marty, A. (2011).

Shah, S. & Gupta, M. (2007). Lipase catalyzed preparation of biodiesel from Jatropha oil in a

Shao, P.; Meng, X.; He, J. & Sun, P. (2008). Analysis of immobilized *Candida rugosa* lipase

Shukla, P. & Gupta, K. (2007). Ecological screening for lipolytic molds and process

Tamalampudi, S.; Talukder, M.; Hama, S.; Numata, T.; Kondo, A. & Fukuda, H. (2008).

production of biodiesel. *Bioresource Technology*, 99, 3975–3981

*Thermomyces lanuginosus*. *Process Biochemistry*, 45, 1268–1273

polyacrylonitrile fibers. *Bioresource Technology*, 101, 7344–7349

synthesis. *J. Mol. Catal. B: Enzymatic*, 57, 262–269

heterogeneous catalysts. *Bioresour. Technol.*, 102, 2151-2161

solvent free system. *Process Biochemistry*, 42, 409–414

production parameters. *EJEAFChe*, 9, 1177-1189

Investigation of lipase production by *Trichoderma reesei* and optimization of

Biocatalysis: Towards ever greener biodiesel production. *Biotechnology Advances*, 27,

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from

Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun

lipases on macroporous polypropylene toward biodiesel synthesis. *J. Mol. Catal. B:* 

on the loading and the activity of *Pseudomonas fluorescens* lipase used for biodiesel

Selection of CalB immobilization method to be used in continuous oil transesterification: Analysis of the economical impact. *Enzyme and Microbial* 

catalyzed preparation of biodiesel from rapeseed soapstock. *Food and Bioproducts* 

optimization for lipase production from *Rhizopus oryzae* KG-5. *J Appl. Sci. Environ.* 

Enzymatic production of biodiesel from Jatropha oil: A comparative study of

Inc., New York

398–408

Stockton Press, NY

*Enzymatic*, 54, 19–26

*Technology*, 48, 61–70

*Processing*, 8 6, 283–289

*Sanit.,* 2, 35–42


Li, Q. & Yan, Y. (2010). Production of biodiesel catalyzed by immobilized *Pseudomonas* 

Li, W.; Du, W. & Liu, D. (2007). Rhizopus oryzae IFO 4697 whole cell catalyzed methanolysis

Li, Z.; Deng, L.; Lu, J.; Guo, X.; Yang, Z. & Tan, T. (2010). Enzymatic synthesis of fatty acid

Lima, V. M. G.; Krieger, N.; Sarquis, M. I. M.; Mitchell, D. A.; Ramos, L. P. & Fontana, J. D.

Lu, J.; Nie, K.; Xie, F.; Wang, F. & Tan, T. (2007). Enzymatic synthesis of fatty acid methyl

Lu, J.; Deng, L.; Zhao, R.; Zhang, R.; Wang, F. & Tan, T. (2010). Pretreatment of immobilized

Macario, A.; Moliner, M; Corma, A. & Giordano, G. (2009). Increasing stability and

Man Xi Ao, Sini Mathew & Obbard, J. (2009). Biodiesel fuel production via transesterification of oils using lipase biocatalyst. *GCB Bioenergy*, 1, 115–125 Marchetti, J. M.; Miguel, V. U. & Errazu, A.F. (2007). Possible methods for biodiesel

Mendes, A.; Giordano, R. C.; Giordano, R. L. C. & Castro, H. (2011). Immobilization and

Meunier, S. & Legge, R. (2010). Evaluation of diatomaceous earth as a support for sol–gel immobilized lipase for transesterification. *J. Mol. Catal. B: Enzymatic*, 62, 54–58 Moreno-Parajàn, J. C. & Giraldo, L. (2011). Study of immobilized candida rugosa lipase for

Naranjo, J.; Córdoba, A.; Giraldo, L.; García, V. & Moreno-Parajàn, J. C. (2010). Lipase

Oda, M.; Kaieda, M.; Hama, S.; Yamaji, H.; Kondo, A.; Izumoto, E. & Fukuda, H. (2005).

Orçaire, O.; Buisson, P. & Pierre, A. (2006). Application of silica aerogel encapsulated lipases

the synthesis of biodiesel fuel. *J. Mol. Catal. B: Enzymatic*, 66, 166–171 Nassreddine, S.; Karout, A.; Christ, M. & Pierre, A. (2008). Transesterification of a vegetal oil

migration in biodiesel-fuel production. *Biochem. Eng. J*., 23, 45–51

production. *Renewable and Sustainable Energy Reviews*, 11, 1300-1311

87, 3148–3154

1370

*Process Biochemistry*, 42, 1481–1485

*J. Mol. Catal. B: Enzymatic*, 62, 15–18

*Enzymatic*, 68, 109–115

*Chemistry*, 4, 55–62

*Enzymatic*, 42, 106–113

*Applied Catalysis A: General*, 344, 70–77

*Journal of Chemical Engineering*, 18, 870-875

*aurantiogriseum. Food Technol. Biotechnol.,* 41, 105–110

system. *Microporous and Mesoporous Materials*, 118, 334–340

*cepacia* lipase from Sapium sebiferum oil in micro-aqueous phase. *Applied Energy*,

of crude and acidified rapeseed oils for biodiesel production in tert-butanol system.

methyl esters from crude rice bran oil with immobilized *Candida sp*. 99-125. *Chinese* 

(2003). Effect of nitrogen and carbon sources on lipase production by *Penicillium* 

esters from lard with immobilized *Candida sp*. 99-125. *Process Biochemistry*, 42, 1367–

*Candida sp*. 99-125 lipase to improve its methanol tolerance for biodiesel production.

productivity of lipase enzyme by encapsulation in a porous organic–inorganic

stabilization of microbial lipases by multipoint covalent attachment on aldehyderesin affinity: Application of the biocatalysts in biodiesel synthesis. *J. Mol. Catal. B:* 

biodiesel fuel production from palm oil by flow microcalorimetry. *Arabian Journal of* 

supported on granular activated carbon and activated carbon cloth as a catalyst in

with methanol catalyzed by a silica fibre reinforced aerogel encapsulated lipase.

Facilitatory effect of immobilized lipase-producing *Rhizopus oryzae* cells on acyl

in the synthesis of biodiesel by transesterification reactions. *J. Mol. Catal. B:* 


**20** 

*Romania* 

**Progress in Vegetable Oils Enzymatic** 

*2The National Institute for Research & Development in Biological Sciences,* 

Ana Aurelia Chirvase1, Luminita Tcacenco2,

Nicoleta Radu1 and Irina Lupescu3

**Transesterification to Biodiesel - Case Study** 

*1The National Institute for Research & Development in Chemistry and Petrochemistry,* 

These days the interest of fuels preparing from sustainable natural resources is continuously increasing due to the rising prices of the fossil fuels and the political instability in the oil producing countries. The fuels manufacturing from local vegetal resources can sustain the every country' prosperity, including rural, agricultural, economically disadvantaged regions. Nowadays only the bioethanol and the biodiesel are already produced at industrial

The biodiesel is manufactured by the chemically catalysed transesterification of the triglycerides from the vegetable oils, rapeseed oil in Europe and soya oil in USA. As the methanol is often used as alcohol reagent, the reaction is consequently named methanolysis. The most applied catalysts are alkalines (especially NaOH) or mineral acids. So the biodiesel represents the methyl esters of the fatty acids from the vegetable oils. The present diesel

Biodiesel contains virtually no sulfur or aromatics, and use of biodiesel in a conventional diesel engine results in substantial reduction of unburned hydrocarbons, carbon monoxide and particulate matter. The production and use of biodiesel, compared to petroleum diesel, resulted in a 78.5% reduction in carbon dioxide emissions. Moreover, biodiesel has a

The chemical transesterification applied at industrial level has important advantages, but also limitations: in spite of the high conversion yields and the short reaction duration, the global transformation is energetically intensive, the glycerol recovery is difficult, the alkaline catalyst must be separated, the wastewaters are to be treated by a rather complex procedure, and both the free fatty acids and water can badly influence the

These unfavourable situations can be diminished by performing the enzymatic transesterification on conditions that: (a) the immobilised lipase used as biocatalyst must be as cheap as possible; (b) one can obtain the economic efficiency of the whole biotransformation process similar to that characteristic to the chemical process, these objectives being presented function of the research methodology and results. The comparison between the chemical way and the enzymatic way is presented in the Table 1.

engines can normally use a mixture of diesel with 5% v/v biodiesel.

**1. Introduction** 

level from sustainable raw materials.

positive energy balance.

reaction.

*3The National Institute for Research & Development in Pharmaceutical Chemistry,* 

immobilized-whole cell and commercial lipases as a biocatalyst. *Biochem. Eng. J*., 39, 185–189

