**4. Results and discussion**

### **4.1. Lipase screening**

Firstly, lipase screening was performed to find the lipase that has the best catalytic activity in the transesterification of sunflower oil. The most active lipase was then used in further transesterification studies. Two lipases, *Thermomyces lanuginosus* and *Candida antarctica* lipase A, were screened for their transesterification activity. The screening results for the tested lipases are presented in Figures 3 and 4. As can be seen from the figures, among the tested lipases, *T. lanuginosus* lipase showed the highest activity in the transesterification reaction of sunflower oil with methanol at 30 o C. *C. antarctica* lipase A showed very little conversion in the transesterification reaction with both ethanol (44%) and methanol (28%). After 10 h of reaction with *T. lanuginosus* lipase, the product contained 91.3% of methyl esters, 2% mono‐ glycerides and diglycerides, and 6.7% triglycerides (Figure 3). However, 28% methyl esters, 5% monoglycerides and diglycerides and 67% triglycerides were obtained with *C. antarctica* lipase A using methanol (Figure 4). FIGURE 3

**Figure 3.** Immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol, 30 o C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; TG: triglyceride; DG: diglyceride; MG: mono‐ glyceride)

#### FIGURE 4 **4.2. Effect of reaction parameters on transesterification**

### *4.2.1. Effect of alcohol type*

0 10 20

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

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

Firstly, lipase screening was performed to find the lipase that has the best catalytic activity in the transesterification of sunflower oil. The most active lipase was then used in further transesterification studies. Two lipases, *Thermomyces lanuginosus* and *Candida antarctica* lipase A, were screened for their transesterification activity. The screening results for the tested lipases are presented in Figures 3 and 4. As can be seen from the figures, among the tested

maintained at a constant temperature (30 o

28 Biofuels - Status and Perspective

liquid chromatography (HPLC) [50].

**4. Results and discussion**

**4.1. Lipase screening**

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

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

C). Substrate mixture (oil and alcohol) was contin‐

30 40 50 60 70 80 90 100 **Total Ester (%) TG FAEE** It is well known that excessive short-chain alcohols such as methanol might inactivate lipase seriously. However, at least three molar equivalents of methanol are required to complete conversion to its corresponding methyl esters of the oil [26, 30]. Experiments were performed to determine the yield of methyl or ethyl esters by varying the alcohol type using *T. lanugino‐ sus* lipase. The reaction was carried out to avoid enzyme inactivation by adding methanol stepwise. The molar ratio of sunflower oil to alcohol was kept constant in the concentration of 1:3 for both methanol and ethanol. Results are summarized in Figure 5. As was expected, all alcohol types resulted in an increase in the yield of esters. However, the formation of esters reached a maximum level using methanol. The biodiesel conversions were about 91.3% of methyl esters and 82.8% of ethyl esters for methanol and ethanol, respectively.

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**Reaction time (h)**

**FAME**

FIGURE 3

**Total Ester (%)**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**TG**

**FAME**

**Reaction time (h)**

**Figure 4.** Immobilized *Candida antarctica* lipase A catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol and ethanol, 30 o C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; FAEE: Fatty acid ethyl es‐ ter; TG: triglyceride; DG: diglyceride; MG: monoglyceride) FIGURE 5

FIGURE 6 **Figure 5.** Effect of alcohol types on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol and ethanol, 30 o C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; FAEE: Fatty acid ethyl ester; TG: triglyceride; DG: diglyceride; MG: monoglyceride)

#### *4.2.2. Effect of water concentration* 100

90

The effect of water content was examined in the range of 0-2 g and at constant molar ratio of oil to methanol with sunflower oil. The reactions were carried out according to the reaction setup described earlier. The results presented in Figure 6 indicated that water was not required to activate the *T. lanuginosus* lipase. A maximum ester yield (81%) could be achieved at without water reaction conditions. Water concentration in reaction mixture is a characteristic and one 40 50 60 70 80 **Total Ester (%) FAME +%2 water Without water**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**TG**

**Reaction time (h)**

**TG**

**FAEE**

**FAME**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**Reaction time (h)**

**Figure 6.** Effect of water content on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol, 30 o C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; TG: triglyceride; DG: diglyceride; MG: monoglyceride)

of the most important factors affecting lipase-catalyzed transesterification reaction rate and yield of biodiesel synthesis [12, 30, 32, 33]. Fukuda et al. [15] reported that the presence of excess water in the reaction mixture reduces the transesterification reaction rate.

### *4.2.3. Effect of reaction temperature*

FIGURE 5

FIGURE 6

**Total Ester (%)**

Experiments were performed to determine the effect of temperature on catalytic activity of immobilized *T. lanuginosus* lipase in transesterification reaction. Temperatures in the range of 30-60 o C were examined with results shown in Figure 7. It was found that the enzyme lost its activity dramatically when temperature was increased above 40 o C. Optimal temperature observed for biodiesel production was 30 o C. Studies of biodiesel production performed with immobilized lipase under laboratory conditions have generally indicated use of temperatures between 30-40 o C [20, 26, 34-37]. On the other hand a number of studies have used temperatures between 40-50 o C [5, 9]. Biodiesel yields of enzymes have increased with increasing of tem‐ perature, but enzymes have denatureted and decreased in efficiency in most temperatures [26].

### *4.2.4. Effect of oil type*

*4.2.2. Effect of water concentration*

**Total Ester (%)**

FIGURE 6

M ratio of methanol and ethanol, 30 o

FIGURE 3

FIGURE 4

30 Biofuels - Status and Perspective

ter; TG: triglyceride; DG: diglyceride; MG: monoglyceride) FIGURE 5

Fatty acid ethyl ester; TG: triglyceride; DG: diglyceride; MG: monoglyceride)

**Total Ester (%)**

**Total Ester (%)**

and ethanol, 30 o

**Total Ester (%)**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**TG**

**FAME**

**TG**

**FAME**

**+%2 water**

C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; FAEE:

**TG**

**FAEE**

**FAME**

**FAEE**

**FAME**

**Reaction time (h)**

**TG**

0 1 2 3 4 5 6 7 8 9 10

**Figure 4.** Immobilized *Candida antarctica* lipase A catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol

**MG+DG**

**Reaction time (h)**

C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; FAEE: Fatty acid ethyl es‐

The effect of water content was examined in the range of 0-2 g and at constant molar ratio of oil to methanol with sunflower oil. The reactions were carried out according to the reaction setup described earlier. The results presented in Figure 6 indicated that water was not required to activate the *T. lanuginosus* lipase. A maximum ester yield (81%) could be achieved at without water reaction conditions. Water concentration in reaction mixture is a characteristic and one

**Without water**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**Reaction time (h)**

0 1 2 3 4 5 6 7 8 9 10

**MG+DG**

**Reaction time (h)**

**Figure 5.** Effect of alcohol types on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3

The results depicted in Figure 8 shows that sunflower oil provided the highest methyl ester yield (91.3%) in reactions with methanol, among sunflower, canola, and waste cooking oil. However, the initial reaction rate was higher for canola oil and waste cooking oil than sunflower oil. Free fatty acids formed soaps with alkali salts when alkali-catalyzed process was used to produce biodiesel from waste cooking oils. Use of waste cooking oil in the production of biodiesel with immobilized lipase to cotton cloth has been effective enough in providing substantial methyl ester yield. Since hydrophilic feature of carrier used in immobi‐ FIGURE 7

> 60 70

100

**Total Ester (%)**

0 10 0 1 2 3 4 5 6 7 8 9 10 FIGURE 8 **Figure 7.** Effect of temperature on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol, 10 h reaction time

lization process may adsorb the water on cotton cloth in the reaction medium. Fatty acid methyl ester yields from canola and waste cooking oil were 79.9% and 81%, respectively. FIGURE 8 **Reaction time (h)** 80 90 **FAME**

**Canola Oil**

**Figure 8.** Effect of oil types on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M ratio of methanol, 30 o C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; TG: triglyceride)

#### *4.2.5. Effect of washing with tert-butanol*

The effect of washing with *tert*-butanol of immobilized cotton cloths during transesterification reaction of sunflower oil and 1:3 molar ratio of oil to methanol is presented in Figure 9. It was shown that immobilized lipase decreased its activity from 91.3% to 77.5% when washed with *tert*-butanol during 10 repeated reactions at 30 o C, each lasting 10 h. However, immobilized lipase decreased its activity dramatically from 91.3% to 61.9% when unwashed with *tert*- butanol. Activity of lipase increased by washing of immobilized cotton cloths with *tert*-butanol of before methanol addition, since the washing process removed hydrophilic glycerol that occurred during reaction, which couldn't be limited to the diffusion of substrate to lipase molecule. The methanol migrates from the reaction mixture to glycerol layer, and the lipase is inactivated by higher concentration of methanol in the glycerol layer [35]. FIGURE 9

**Figure 9.** Effect of washing with *tert*-butanol on immobilized *T. lanuginosus* lipase catalyzed transesterification of sun‐ flower oil, 1:3 M ratio of methanol, 30 o C reaction temperature, 10 h reaction time, 10th repeated use (FAME: Fatty acid methyl ester; TG: triglyceride; DG: diglyceride; MG: monoglyceride)
