**5. Conclusions**

lization process may adsorb the water on cotton cloth in the reaction medium. Fatty acid methyl

**Reaction time (h)**

0 1 2 3 4 5 6 7 8 9 10

**Reaction time (h)**

0 1 2 3 4 5 6 7 8 9 10

0 1 2 3 4 5 6 7 8 9 10

**Reaction time (h)**

C reaction temperature, 10 h reaction time (FAME: Fatty acid methyl ester; TG: triglyceride)

**Canola Oil Waste Cooking Oil Sunflower Oil**

**TG**

**FAME**

**FAME**

**Canola Oil Waste Cooking Oil Sunflower Oil**

**TG**

C, each lasting 10 h. However, immobilized

**Reaction time (h)**

**Figure 8.** Effect of oil types on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3 M

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

lipase decreased its activity dramatically from 91.3% to 61.9% when unwashed with *tert*-

0 1 2 3 4 5 6 7 8 9 10

**Figure 7.** Effect of temperature on immobilized *T. lanuginosus* lipase catalyzed transesterification of sunflower oil, 1:3

ester yields from canola and waste cooking oil were 79.9% and 81%, respectively.

**30 oC 40 oC 50 oC 60 oC** **30 oC 40 oC 50 oC 60 oC**

FIGURE 7

32 Biofuels - Status and Perspective

FIGURE 7

FIGURE 8

M ratio of methanol, 10 h reaction time

**Total Ester (%)**

FIGURE 8

**Total Ester (%)**

**Total Ester (%)** 

**Total Ester (%)** 

*4.2.5. Effect of washing with tert-butanol*

ratio of methanol, 30 o

*tert*-butanol during 10 repeated reactions at 30 o

The method of enzyme immobilization involving polyethyleneimine (PEI)-enzyme aggregate formation was developed and glutaraldehyde was used as a cross-linking agent between free amine groups increasing the enzyme stability. In the present study, a high biodiesel yield was obtained with *T. lanuginosus* lipase immobilized on cotton cloths. To avoid strong methanol inhibition, it was fed 3 steps into reaction medium and a high conversion (91.3%) was achieved. It was also found that glycerol has a significant inhibitory effect on the transesterificaiton reaction. The immobilized enzyme was rinsed with *tert*-butanol to over come this drawback. Additionally, immobilized lipase could be used at least 10 times (100 h) without any activity limitation. Since the immobilization method is very simple and cheap, it could also be used for the immobilization of other enzymes.
