**The Immobilized Lipases in Biodiesel Production**

Margarita Stoytcheva1, Gisela Montero1, Lydia Toscano1, Velizar Gochev2 and Benjamin Valdez1 *1Universidad Autónoma de Baja California, Instituto de Ingeniería, 2Plovdiv University "Paisii Hilendarski, 1México 2Bulgaria* 

#### **1. Introduction**

396 Biodiesel – Feedstocks and Processing Technologies

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The leading standard setting organization ASTM International, formerly ASTM (American Society of Testing and Materials), defines biodiesel as a fuel comprised of mono-alkyl esters of long chain fatty acids (ASTM D6751). It is usually manufactured by triglycerides transesterification with methanol or ethanol in the presence of a catalyst, according to the following reaction:

Recently, biodiesel production by lipase catalyzed transesterification has been suggested as a promising alternative to the conventional chemical catalysis, in spite of the high conversion and reaction rates of the latter (Akoh et al., 2007; Bajaj et al., 2010; Bisen et al., 2010; Demirbas, 2009; Fjerbaek et al., 2009; Fukuda et al., 2001, 2009; Ghaly et al., 2010; Helwani et al., 2009; Jegannathan & Abang, 2008; Man Xi Ao et al., 2009; Marchetti et al., 2007; Ranganathan et al., 2008; Robles-Medina et al., 2009; Semwal et al., 2011). The enzymatic process enables eliminating the drawbacks of the alkali- or acid-catalyzed transesterification, namely: product contamination, wastewater release, free fatty acids and water interferences, and difficult glycerol recovery. Nevertheless, the commercialization of

The Immobilized Lipases in Biodiesel Production 399

& Gupta, 2007), *Penicillium* (Chahinian et al, 2000; Lima et al., 2003; Vardanega et al., 2010), and *Trichoderma* (Kashmiri et al., 2006; Rajesh et al., 2010) are described as the most prospective lipase producers. Only microbial lipases are a matter of practical interest for biodiesel production, because only microbial lipases are produced in industrial scale. The application of the microbial lipases, all together with their immobilization which allows the regeneration and the reuse of the enzyme preparation in several working cycles reduces the production costs, and respectively the final cost of biodiesel. A review on microbial lipase production with emphasis on lipase engineering and use of mathematical models for

Some fungi cultured by the authors as powerful producers of lipase of use in biodiesel

 *Aspergillus awamori Aspergillus niger* 

 *Aspergillus oryzae Penicillium chrysogenum*  Fig. 1. Filamentous fungi belonging to various species of genera *Aspergillus* and *Penicillium*,

The term "enzyme immobilization" was defined at the first Enzyme Engineering Conference held at Hennicker, NH, USA, in 1971. It describes "enzymes physically confined at or

process improvement and control is provided by Treichel et al. (2010).

synthesis are shown in Fig. 1.

considered as prospective lipase producers.

**3. The immobilized lipases in biodiesel production** 

the lipase-catalyzed biodiesel synthesis remains problematic, because of the cost of the enzyme: approximately 1000 USD per kg of Novozym 435 lipase. Therefore, the implementation of strategies, such as enzyme immobilization, for the development of economic and effective enzyme based technologies for biodiesel production is of crucial importance. Enzyme immobilization ensures several issues: repetitive and continuous use of the enzyme and its stabilization, localization of the interaction, prevention of product contamination, reduction of effluent problems and material handling, and effective control of the reaction parameters (D'Souza, 1982). All these aspects are reflected on the production cost.

The present review is intended to provide an overview on the use of immobilized lipases in biodiesel production, the techniques applied for enzyme immobilization, and the factors affecting the process.

#### **2. Lipases mode of action and classification**

Lipases (EC 3.1.1.3 triacylglycerol acylhydrolase) represent a group of water soluble enzymes that originally catalyze the hydrolysis of ester bonds in water insoluble lipid substrates, acting at the interface between the aqueous and the organic phases. This unique heterogeneous reaction is feasible because of: (i) the specific lipases molecule 3D structure consisting of three domains: "contact domain", responsible for distinguishing of substrate surface, "hydrophobic" domain, responsible for extracting of one substrate molecule and its association with the "functional" domain, and "functional" domain, containing the catalytic triad Ser, Hys and Asp/Glu; (ii) the transition from closed to open conformation in the presence of the lipidic phase (Guncheva & Zhiryakova 2011; Panalotov & Verger, 2000). Enzymatic action of lipases on the substrate is a result of a nucleophilic attack on the carbonyl carbon atom from ester groups. Some lipases are also able to catalyze the processes of esterification, interesterification, transesterification, acidolysis, amynolysis and may show enantioselective properties (Hasan et al., 2009). The mechanism of the lipase catalyzed transesterification of triglycerides with an alcohol to produce biodiesel (Fjerbaek et al., 2009) could be presented by the following sequence of reactions:

with: E-enzyme; TG-triglyceride; DT-diglyceride; MG-monoglyceride; G-glycerol; AcEacylated enzyme; FFA-free fatty acid.

According to the origin lipases are plant, animal and microbial. The mostly used lipases in biodiesel production are of bacterial and fungal origin, such as: *Candida antarctica* (Novozym 435), *Candida Rugosa* (Lipase AY), *Pseudomonas cepacia* (Lipase PS)*, Pseudomonas fluorescens*  (lipase AK), *Pseudomonas aeruginosa*, and *Thermomyces lanuginose* (Lipozime TL), among other. The catalytic properties and potential applications of *Bacillus* lipases are extensively reviewed by Guncheva & Zhiryakova (2011). Among the available lipase producing microorganisms, filamentous fungi belonging to various species of genera *Aspergillus* (Adinarayana et al., 2004; Karanam, & Medicherla, 2008), *Rhizopus* (Hiol et al., 2000; Shukla

the lipase-catalyzed biodiesel synthesis remains problematic, because of the cost of the enzyme: approximately 1000 USD per kg of Novozym 435 lipase. Therefore, the implementation of strategies, such as enzyme immobilization, for the development of economic and effective enzyme based technologies for biodiesel production is of crucial importance. Enzyme immobilization ensures several issues: repetitive and continuous use of the enzyme and its stabilization, localization of the interaction, prevention of product contamination, reduction of effluent problems and material handling, and effective control of the reaction parameters (D'Souza, 1982). All these aspects are reflected on the production

The present review is intended to provide an overview on the use of immobilized lipases in biodiesel production, the techniques applied for enzyme immobilization, and the factors

Lipases (EC 3.1.1.3 triacylglycerol acylhydrolase) represent a group of water soluble enzymes that originally catalyze the hydrolysis of ester bonds in water insoluble lipid substrates, acting at the interface between the aqueous and the organic phases. This unique heterogeneous reaction is feasible because of: (i) the specific lipases molecule 3D structure consisting of three domains: "contact domain", responsible for distinguishing of substrate surface, "hydrophobic" domain, responsible for extracting of one substrate molecule and its association with the "functional" domain, and "functional" domain, containing the catalytic triad Ser, Hys and Asp/Glu; (ii) the transition from closed to open conformation in the presence of the lipidic phase (Guncheva & Zhiryakova 2011; Panalotov & Verger, 2000). Enzymatic action of lipases on the substrate is a result of a nucleophilic attack on the carbonyl carbon atom from ester groups. Some lipases are also able to catalyze the processes of esterification, interesterification, transesterification, acidolysis, amynolysis and may show enantioselective properties (Hasan et al., 2009). The mechanism of the lipase catalyzed transesterification of triglycerides with an alcohol to produce biodiesel (Fjerbaek et al., 2009)

E + DG E-DG MG + AcE biodiesel + E

with: E-enzyme; TG-triglyceride; DT-diglyceride; MG-monoglyceride; G-glycerol; AcE-

According to the origin lipases are plant, animal and microbial. The mostly used lipases in biodiesel production are of bacterial and fungal origin, such as: *Candida antarctica* (Novozym 435), *Candida Rugosa* (Lipase AY), *Pseudomonas cepacia* (Lipase PS)*, Pseudomonas fluorescens*  (lipase AK), *Pseudomonas aeruginosa*, and *Thermomyces lanuginose* (Lipozime TL), among other. The catalytic properties and potential applications of *Bacillus* lipases are extensively reviewed by Guncheva & Zhiryakova (2011). Among the available lipase producing microorganisms, filamentous fungi belonging to various species of genera *Aspergillus* (Adinarayana et al., 2004; Karanam, & Medicherla, 2008), *Rhizopus* (Hiol et al., 2000; Shukla

cost.

affecting the process.

**2. Lipases mode of action and classification** 

could be presented by the following sequence of reactions:

E + TG E-TG DG + AcE AcE-alcohol

E + MG E-MG G + AcE E + FFA

H2O alcohol

alcohol

acylated enzyme; FFA-free fatty acid.

& Gupta, 2007), *Penicillium* (Chahinian et al, 2000; Lima et al., 2003; Vardanega et al., 2010), and *Trichoderma* (Kashmiri et al., 2006; Rajesh et al., 2010) are described as the most prospective lipase producers. Only microbial lipases are a matter of practical interest for biodiesel production, because only microbial lipases are produced in industrial scale. The application of the microbial lipases, all together with their immobilization which allows the regeneration and the reuse of the enzyme preparation in several working cycles reduces the production costs, and respectively the final cost of biodiesel. A review on microbial lipase production with emphasis on lipase engineering and use of mathematical models for process improvement and control is provided by Treichel et al. (2010).

Some fungi cultured by the authors as powerful producers of lipase of use in biodiesel synthesis are shown in Fig. 1.

*Aspergillus awamori Aspergillus niger* 

*Aspergillus oryzae Penicillium chrysogenum* 

Fig. 1. Filamentous fungi belonging to various species of genera *Aspergillus* and *Penicillium*, considered as prospective lipase producers.
