**3.2.2 Results and discussion**

The final activities and isolation yields obtained when the crude lipases were separated from the cultivation medium by precipitation with ammonium sulphate are presented in the Table 3. High efficient lipases isolation was done by precipitation of the cultivation medium of *Yarrowia lipolytica* yeast with (NH4)2SO4, a yield of 95% was got for both variants (24 hr. and 28 hr.), while when the same procedure was applied for *Candida rugosa* samples the isolation yields were lower: 62% for 24 hr. extract and only 29.6% for 28 hr. extract.


Table 3. The final activities and isolation yields determined for the crude lipases separated from the cultivation media of the yeasts strains *Candida rugosa* DSM 70761 and *Yarrowia (Candida lypolitica)* ATCC 8661

At a first step the preliminary transesterification results obtained by thin layer chromatography demonstrated both lipases have high enough catalysis activities.

Legend:

A2-lipase from *Candida rugosa DSM 70761*/M2; C1-lipase from *Candida rugosa DSM 70761*/M3; D1 lipase from *Yarrowia lipolytica/*M2; M - Control, ester of oleic acid

Fig. 4. Thin layer chromatography of the products obtained by the transesterification

immobilization of *Aspergillus niger* lyophilized lipase (Fluka), 0.1 g of lipase immersed in 0.5 M phosphate buffer, pH 5.6 was added to this mixture. Then 2.5 mL 50% glutaraldehyde dissolved in 25 mL double distilled water was added. The mixture rested for 30 minutes at 4 0 C. 0.25 g sodium borohydride was added in portions, during 15 minutes, with ice pieces to low the temperature, and finally the mixture was filtrated in vacuum. The immobilized product thus obtained was washed with double distilled water and 0.5 M phosphate buffer, pH 5.6. Lipase activity and immobilization yield were evaluated for each application.

The final activities and isolation yields obtained when the crude lipases were separated from the cultivation medium by precipitation with ammonium sulphate are presented in the Table 3. High efficient lipases isolation was done by precipitation of the cultivation medium of *Yarrowia lipolytica* yeast with (NH4)2SO4, a yield of 95% was got for both variants (24 hr. and 28 hr.), while when the same procedure was applied for *Candida rugosa* samples the

> **Initial activity (UEA)**

1. *Candida rugosa,* 24 hr 20 3 820 14 2 368 62.0 2. *Candida rugosa,* 28 hr 800 289 600 560 85 721 29.6 3. *Yarrowia lipolytica,* 24 hr 20 2 320 14 2 204 95.0 4. *Yarrowia lipolytica,* 28 hr 800 85 200 560 80 940 95.0 Table 3. The final activities and isolation yields determined for the crude lipases separated from the cultivation media of the yeasts strains *Candida rugosa* DSM 70761 and *Yarrowia* 

At a first step the preliminary transesterification results obtained by thin layer

A2-lipase from *Candida rugosa DSM 70761*/M2; C1-lipase from *Candida rugosa DSM 70761*/M3; D1-

Fig. 4. Thin layer chromatography of the products obtained by the transesterification

lipase from *Yarrowia lipolytica/*M2; M - Control, ester of oleic acid

chromatography demonstrated both lipases have high enough catalysis activities.

**Quantity (NH4)2SO4 (g)** 

**Final activity (UEA)** 

**Isolation yield (%)** 

isolation yields were lower: 62% for 24 hr. extract and only 29.6% for 28 hr. extract.

**Extract volume (mL)** 

**3.2.2 Results and discussion** 

**No**. **Strain / duration of bioprocessing** 

*(Candida lypolitica)* ATCC 8661

Legend:

Fig. 5. Immobilization efficiency of the tested lipases (Tcacenco *et all*, 2010)

The experimental results are presented in the Figure 5, obtained with the described immobilization techniques for both crude lipases.

The immobilization techniques, characterized in the following table, were performed in comparison with the immobilization of a lipase from the fungus *Aspergillus niger.* 


Table 4. Applied immobilization techniques

The experimental study regarding the immobilization of lipases gave interesting results: high yield of 99% obtained for the immobilization of *Yarrowia lipolytica* lipase by adsorption on Celite support, good yields of 63.26% for the immobilization of *Candida rugosa* lipase by adsorption on chitosan cross linked with glutaraldehyde and respectively 44 - 49% for the same lipase immobilized by adsorption on Celite or Silicagel. On the contrary the immobilization of *Aspergillus niger* lipase gave unsatisfactory results.

In order to improve the immobilization yield of the lipase from the yeast *Candida rugosa*  DSM 70761 on Celite support a supplementary treatment with acetone as organic solvent

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 421

Fig. 7. The effect of the acetone treatment associated to Celite immobilization on the static activity of the immobilized lipase from *Candida rugosa* DSM 70761 (sample 1-no acetone

The static stability for the enzyme from *Candida rugosa* DSM 70761 immobilized on Celite with / without acetone treatment was determined for a long period of time, the results being presented in the Figure 7. The biocatalyst obtained with the lipase from the above mentioned yeast immobilized on Celite 545 by physical adsorption with or without organic medium treatment presented a high static stability, when preserved in freezer. The residual activity was as high as 82% after 1 year and half, and after the first 2 weeks the residual activity was practically unchanged in both cases. These findings were considered as a selection criterion between the lipases from the two studied yeasts, so the lipase produced by *Candida rugosa* DSM 70761 with a better static stability was further used to continue the research. Firstly the biocatalyst prepared by the described procedure with the lipase from the yeast *Candida rugosa* DSM 70761 was tested for its operational activity. The test consisted of using the same biocatalyst quantity in several reaction cycles and measuring the enzymatic activity at the beginning and after each reaction phase. The results are presented

> Final activity (UEA/g)

1. 705.00 606.72 13.34 2. 606.72 549.75 9.40 3. 549.75 481.36 12.44 4. 431.36 413.97 14.00

Table 6. Evolution of the operational activity of the lipase from *Candida rugosa* DSM 70761

The results from the table indicate a biocatalyst half time of 5-6 reaction cycles, because after

Activity loss (%)

treatment; sample 2-with acetone treatment)

Initial activity (UEA/g)

4 cycles the residual activity was 58.71%.

in the following table.

immobilized on Celite 545

No. of cycles


was done, the obtained results in comparison with the control procedure (without acetone adding) are presented in the following table.

Table 5. The immobilization yields of the lipase from the yeast *Candida rugosa* DSM 70761 on Celite support with / without acetone treatment

The acetone treatment had as consequence a big improvement of the immobilization yield on Celite from 49% to 97% in case of the lipase from *Candida rugosa* DSM 70761. It seems that the system hydration degree highly increases due to the support treatment with organic solvent, which determines a better adsorption of the enzyme. The improved procedure to get the immobilized biocatalyst was further applied in the research regarding the immobilized enzyme characteristics: static activity, operational activity, and transesterification performance.

The immobilized lipases from both yeasts *Yarrowia lipolytica* and *Candida rugosa* prepared by Celite adsorption were preserved in a freezer at -18°C and tested for static stability at different time duration. Results are presented in the Figure 6.

Fig. 6. Static stability determination by yield of the residual activity for the Celite adsorption immobilized lipases from *Yarrowia lipolytica* and *Candida rugosa* 

The results demonstrated a higher static time stability for the Celite adsorption immobilized lipase from the yeast *Candida rugosa* DSM 70761, with 73% residual activity after more than 1 month, by comparison with only 48% residual activity for the immobilized lipase from the yeast *Yarrowia lipolytica* ATCC 8661.

was done, the obtained results in comparison with the control procedure (without acetone

Final

activity

*Yarrowia lipolyitica Candida rugosa*

yield (%) UEA/mL Total activity UEA/mL Total

activity Immobilization

Initial activity

1. Celite 545 800 362.0 289 600 840.2 142 841.7 49.32 2. Celite 545 + acetone 1150 182.6 210 000 2537.2 204 246.2 97.26 Table 5. The immobilization yields of the lipase from the yeast *Candida rugosa* DSM 70761 on

The acetone treatment had as consequence a big improvement of the immobilization yield on Celite from 49% to 97% in case of the lipase from *Candida rugosa* DSM 70761. It seems that the system hydration degree highly increases due to the support treatment with organic solvent, which determines a better adsorption of the enzyme. The improved procedure to get the immobilized biocatalyst was further applied in the research regarding the immobilized enzyme characteristics: static activity, operational activity, and

The immobilized lipases from both yeasts *Yarrowia lipolytica* and *Candida rugosa* prepared by Celite adsorption were preserved in a freezer at -18°C and tested for static stability at

adding) are presented in the following table.

Celite support with / without acetone treatment

> 19 days

immobilized lipases from *Yarrowia lipolytica* and *Candida rugosa* 

 33 days

 60 days

Fig. 6. Static stability determination by yield of the residual activity for the Celite adsorption

The results demonstrated a higher static time stability for the Celite adsorption immobilized lipase from the yeast *Candida rugosa* DSM 70761, with 73% residual activity after more than 1 month, by comparison with only 48% residual activity for the immobilized lipase from the

(mL)

different time duration. Results are presented in the Figure 6.

No. Support Lipase

transesterification performance.

**Residual activity, %**

yeast *Yarrowia lipolytica* ATCC 8661.

Fig. 7. The effect of the acetone treatment associated to Celite immobilization on the static activity of the immobilized lipase from *Candida rugosa* DSM 70761 (sample 1-no acetone treatment; sample 2-with acetone treatment)

The static stability for the enzyme from *Candida rugosa* DSM 70761 immobilized on Celite with / without acetone treatment was determined for a long period of time, the results being presented in the Figure 7. The biocatalyst obtained with the lipase from the above mentioned yeast immobilized on Celite 545 by physical adsorption with or without organic medium treatment presented a high static stability, when preserved in freezer. The residual activity was as high as 82% after 1 year and half, and after the first 2 weeks the residual activity was practically unchanged in both cases. These findings were considered as a selection criterion between the lipases from the two studied yeasts, so the lipase produced by *Candida rugosa* DSM 70761 with a better static stability was further used to continue the research. Firstly the biocatalyst prepared by the described procedure with the lipase from the yeast *Candida rugosa* DSM 70761 was tested for its operational activity. The test consisted of using the same biocatalyst quantity in several reaction cycles and measuring the enzymatic activity at the beginning and after each reaction phase. The results are presented in the following table.


Table 6. Evolution of the operational activity of the lipase from *Candida rugosa* DSM 70761 immobilized on Celite 545

The results from the table indicate a biocatalyst half time of 5-6 reaction cycles, because after 4 cycles the residual activity was 58.71%.

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 423

b. "Semi-batch" enzymatic transesterification with methanol and without organic solvent, characterised by the same reaction conditions, except the fact that the methanol is added two times, each addition realizing a ratio between alcohol and the triglycerides

c. Batch enzymatic transformation in hexane characterized by: vegetable oil concentration 0.09M; methanol concentration 0.09 M; biocatalyst concentration 5000 UEA / 100 mL reaction medium; reaction temperature of 370C; mixing of 250 rpm; reaction total

The most important transesterification results are presented in the Figure 8.

Fig. 8. The vegetable oils transesterification yield for the 3 experimental models: a1-batch model without solvent, with soya oil; a2-batch model without solvent, with rapeseed oil; b-semibatch model without solvent, with soya oil; c1-batch model in hexane

The transesterification was realised with the following biotransformation yields: 56-67 % for the experimental variant a, 69% for the variant b, and 56-63% for the variant c; these results can be improved by adequate optimization procedures to be applied for each technological phase, comprising enzyme obtaining in aerobic bioprocess, lipase immobilization and

1. The main objectives of the research to replace the actual chemical transesterification with the enzymatic process are: (a) the preparation of cheap and stable immobilized lipases; (b) the realization of biotransformation systems characterized by the biocatalyst long use in many reaction cycles. One of the raisons to choose between extracellular or intracellular lipases is the immobilization of extracellular enzymes by physical adsorption, a low price technology, but imposing to improve the shorter duration. Moreover these lipases are normally biosynthesized by bacteria or yeasts, easier to cultivate in aerobic bioprocess than the intracellular lipases producing'

with soya oil; c2-batch model in hexane with rapeseed oil

substrate of 4:1.

duration of 24 hr.

**3.3.2 Results and discussion** 

transesterification performing.

**4. Conclusions** 

fungi.
