**3. Experimental**

### **3.1. Material**

Commercial powder lipase from *Thermomyces lanuginosus* (fungal lipase, lyophilized, specific activity of 1400 U/mg solid) and *Candida antarctica* lipase A (specific activity of 2500 U/mg solid) were purchased from Codexis Inc. (Pasadane, CA). Sunflower oil and canola oil were supplied by a local company; waste cooking oil (WCO) was used following filtration to remove particles remaining in the oil after domestic use. Polyethyleneimine [PEI; (C2H5N)n] as 50% (w/v) (average molecular weight 750.000) and glutaraldehyde (GA, acidic aqueous solution as 25% (w/v)) were obtained from Sigma (USA) and AppliChem (Darmstadt, Germany), respectively. All other chemicals were of analytical reagent grade and used without further purification.

### **3.2. Experimental procedure**

### *3.2.1. Lipase immobilization*

stable than plant and animal lipases and are available in bulk at lower cost compared to lipases of other origin. Yeasts lipases are easy to handle and grow compared to bacterial lipases. Among the yeast lipases, *Candida rugosa* has gained good commercial importance. The most commonly used biocatalyst for biodiesel production are the microbial lipases that are pro‐ duced by a number of fungal, bacterial, and yeast species [40]. Free enzymes are far cheaper than immobilized lipases. They can be purchased in an aqueous solution composed of the enzyme solution plus nothing more than a stabilizer to prevent enzyme denaturation (e.g. glycerol or sorbitol) and a preservative to inhibit microbial growth (e.g. benzoate) [49].

Immobilization of lipases was carried out using entrapment, physical adsorption, ion ex‐ change, and crosslinking. Carriers for lipase immobilization include polyurethane foam, silica, sepabeads, cellulosic nanofibers. Based on the criteria for selecting the immobilization technique and carrier dependings on the source of lipase, the type of reaction system (aqueous, organic solvent or two-phase system), and the bioreactor type (batch, stirred tank, membrane reactor, column and plug-flow) can be designed. The literature is replete with various lipase producing microorganisms, enzyme immobilization methods, and physical carriers. The challenge will be to select a carrier and immobilization technique that will allow maximum lipase activity, retention, and stability on the oil substrate. Among the immobilization method, adsorption technique is the simplest and most widely used technique for lipase immobiliza‐ tion. Adsorption method consists of bonding the lipase to the immobilization support surface through weak forces such as van der Waals or hydrophobic interactions. However, the main disadvantage of this technique is enzyme desorption from the support due to low bond

In recent years, whole-cell immobilized lipases have been studied for biodiesel production. This method is cheaper as it does not require the enzyme purification and isolation steps from fermentation broth. The efficiency of the transesterification process could be increased by using microbial cells that produce intra-cellular lipase as whole-cell biocatalysts [40, 46]. Filamentous fungi have been identified as robust whole-cell biocatalysts for biodiesel production: among these *Rhizopus* and *Aspergillus* have been most widely used [42]. There are several recent works reporting the utilization of bacteria, yeast and fungi as whole-cell biocatalysts in biodiesel

Commercial powder lipase from *Thermomyces lanuginosus* (fungal lipase, lyophilized, specific activity of 1400 U/mg solid) and *Candida antarctica* lipase A (specific activity of 2500 U/mg solid) were purchased from Codexis Inc. (Pasadane, CA). Sunflower oil and canola oil were supplied by a local company; waste cooking oil (WCO) was used following filtration to remove particles

*2.4.2. Immobilized biocatalysis*

26 Biofuels - Status and Perspective

*2.4.3. Whole-cell biocatalysis*

process [27].

**3.1. Material**

**3. Experimental**

strength between the enzyme and the support [40].

Figure 1 shows multi-layer immobilization of *T. lanuginosus* lipase on cotton cloth by aggre‐ gation with polyethyleneimine (PEI). 1 mL of PEI solution (pH=11), containing 2 mg of PEI, was added to each 0.1 g piece of cotton cloth. The PEI solution volume was at a sufficient level to completely wet the cloth. After adsorption of PEI, 50 mg of enzyme (5 mL of 10 mg/mL enzyme solution) was added. Upon the addition of enzyme to PEI-adsorbed cotton, a "milky" turbid solution was formed. The flasks were put into a shaker-incubator (Heidolf Unimax 1010, Germany) at 150 rpm at room temperature (25±1 o C) for 5 min. The white turbidity disappeared within 5 min and the coupling solution was completely clarified. The clarified coupling solution was slowly decanted and PEI-enzyme coated cottons were dipped in a cold GA solution (2.5% (w/v), pH= 3.5) for cross-linking at 5 min. The cross-linked cottons were washed with distilled water and potassium phosphate buffer (1 M, pH= 7). It is important to note that there was no washing step until the completion of GA cross-linking [25, 39]. Based on the degree of immobilization tests, about 80-90% of the enzyme was immobilized in this proce‐ dure. The actual enzyme loading was determined as 180 mg of *T. lanuginosus* lipase per 1 g of cotton cloth.

**Figure 1.** The procedure for PEI-multilayer lipase immobilization on cotton cloth fibrils

### *3.2.2. Biodiesel production with immobilized lipase-catalyzed transesterification*

Production of biodiesel by enzymatic catalyzed transesterification from various vegetable oils was studied in a packed bed reactor (Figure 2). A small piece of immobilized cotton cloth (1 g) was placed in the glass column reactor (1 cm diameter x 12 cm height) with a water jacket maintained at a constant temperature (30 o C). Substrate mixture (oil and alcohol) was contin‐ uously recirculated throughout the immobilized enzyme reactor with a peristaltic pump at a flow rate of 50 mL/min by adding of alcohol in three-steps. Immobilized cotton cloths were washed by *tert*-butanol before adding of alcohol to each reaction medium. Reaction was continued for 10 h. Samples were taken from the flask at appropriate time intervals and analyzed for fatty acid methyl esters (FAMEs) and glyceride contents by high performance liquid chromatography (HPLC) [50].

**Figure 2.** Schematic diagram of the enzymatic column reactor used in the transesterification reaction
