**2. State of the art in the domain of biodiesel preparation by enzymatic transesterification of vegetable oils**

Other advantages of using lipases in biodiesel production are: (a) ability to work in very different media which include biphasic system, and monophasic system, (b) they are robust and versatile enzymes that can be produced in bulk because of their extracellular nature in most manufacturing system, (c) when the lipase is used in a packed bed reactor, no separation is necessary after transesterification, and (d) higher thermo stability and shortchain alcohol-tolerant capabilities of lipase make it very convenient for use in biodiesel production (Ghaly *et all*, 2010). Until now the biodiesel manufactured by chemical catalysis is cheaper than the same product obtained by enzymatic catalysis, but in case of considering the pollution suppressing costs needed after the chemical process performing, the costs of both reaction' types could be comparable.

Enzymatic transesterification can be done with crude or purified vegetable oils, free fatty acids, residual grease from food industry or of animal origin, and residual vegetable oils from fry cooking. Beside methanol and ethanol one can also use as acyl acceptors the propanol, iso-propanol, butanol and iso-butanol. Many microorganisms, bacteria, yeasts or fungi can produce useful lipases for transesterification. Of these microorganisms, *Candida antarctica*, *Candida rugosa*, *Pseudomonas cepacia*, *Pseudomonas fluorescens*, *Rhizomucor miehei*, *Rhizopus chinensis*, *Rhizopus oryzae* and *Thermomyces lanuginosa* have produced the most effective lipases, able to perform the biotransformation with high yields. The combination of two or more lipases can increase the conversion in order to lower the cost. A combination of *Candida antarctica* and *Thermomyces lanuginosa* lipases was used to obtain a 95% conversion in methanolysis using a tert-butanol solvent. From the many lipases it is recommended to use those with reduced region specificity, but with higher substrate specificity.

**Criterion Alkaline catalysis process Enzymatic proces** 

The now-a-day technological progress regarding the enzymatic transesterification is demonstrated by the realisation of 2 industrial pilots in China (Moore 2008a, 2008b; Uthoff *et all*, 2009) to apply this advanced methodology, though the biodiesel manufacture price still remains higher than the diesel price no matter the transesterification route, due to the raw materials high prices (Bisen *et all*, 2010). Developments to meet the economical framework are needed, including: (a) the introduction of the enzymatic transesterification of plant oils as a part from a comprehensive technology of complete valorisation of the vegetable oil, meaning the application of the bio refinery concept; (b) the increase of the available vegetable oil quantity with limited interference with the vegetable oils' food use; (c) the

**2. State of the art in the domain of biodiesel preparation by enzymatic** 

Other advantages of using lipases in biodiesel production are: (a) ability to work in very different media which include biphasic system, and monophasic system, (b) they are robust and versatile enzymes that can be produced in bulk because of their extracellular nature in most manufacturing system, (c) when the lipase is used in a packed bed reactor, no separation is necessary after transesterification, and (d) higher thermo stability and shortchain alcohol-tolerant capabilities of lipase make it very convenient for use in biodiesel production (Ghaly *et all*, 2010). Until now the biodiesel manufactured by chemical catalysis is cheaper than the same product obtained by enzymatic catalysis, but in case of considering the pollution suppressing costs needed after the chemical process performing, the costs of

Enzymatic transesterification can be done with crude or purified vegetable oils, free fatty acids, residual grease from food industry or of animal origin, and residual vegetable oils from fry cooking. Beside methanol and ethanol one can also use as acyl acceptors the propanol, iso-propanol, butanol and iso-butanol. Many microorganisms, bacteria, yeasts or fungi can produce useful lipases for transesterification. Of these microorganisms, *Candida antarctica*, *Candida rugosa*, *Pseudomonas cepacia*, *Pseudomonas fluorescens*, *Rhizomucor miehei*, *Rhizopus chinensis*, *Rhizopus oryzae* and *Thermomyces lanuginosa* have produced the most effective lipases, able to perform the biotransformation with high yields. The combination of two or more lipases can increase the conversion in order to lower the cost. A combination of *Candida antarctica* and *Thermomyces lanuginosa* lipases was used to obtain a 95% conversion in methanolysis using a tert-butanol solvent. From the many lipases it is recommended to

use those with reduced region specificity, but with higher substrate specificity.

 Free fatty acids from the vegetable oils Saponification products Methylic esters Water from the raw material Reaction interference No influence Methylic esters yield Normal Higher Glycerol recovery Difficult Easy Methylic esters purification Repeated washing No need Catalyst preparation price Cheap Relatively high Table 1. Comparison between the alkaline catalysis and the enzymatic method for biodiesel

Reaction temperature 60-70oC 30-40oC

preparation (Bajaj *et all*, 2010)

possible preparation of methanol from natural resources.

**transesterification of vegetable oils** 

both reaction' types could be comparable.

The reaction can be realised either in organic solvents, or in solvent-free media (where there are only the substrates' mixture). Normally in organic solvents' systems the lipases can catalyse the biotransformation when the alcohol is added stepwise at the beginning (a "batch" system), by comparison with the free-solvent media, where the alcohol is added several times for maintaining a certain molar ratio with the oil concentration.

The **key factors affecting the enzymatic transesterification** are presented in the Figure 1.

Fig. 1. Key factors of influence on the enzymatic transesterification (Antczak *et all*, 2009)

There are two categories of enzymatic biocatalysts: (1) extracellular lipases (i.e. the enzyme has previously been recovered from the cultivation broth and then purified) especially from the microbial producers *Candida rugosa*, *Candida utilis*, *Candida antarctica* and *Pseudomonas cepacia*, generally bacteria and yeasts; (2) intracellular lipases which still remain either inside or attached to the cellular wall; in both cases the enzymes are immobilized directly or together with the whole cell and this use can eliminate downstream operations and assure the enzyme recycling.

The **extracellular lipases** are mostly produced by bacteria and yeasts and the large scale production of these lipases should be economical, fast, easy and efficient. Unfortunately, the cost of specific separation and purification operations is high enough. Still the majority of immobilized lipases that are commercially available are extracellular. The most commonly used is: Novozym 435 which is the lipase from *Candida antarctica*. Meanwhile the bacteria and yeasts can probably form growth associated lipases, in a first stage, linked to the cellular membranes, then released into the cultivation medium as extracellular enzymes.

When preparing the **intracellular lipases** the costly step of purification can be eliminated and this has led to using whole cells as biocatalysts. After the intracellular production of lipases the direct use of fungal cells immobilized within porous biomass support particles as a whole biocatalyst represents an attractive process for bulk production of biodiesel (Fjerbaek *et all*, 2009)

The main criteria to choose between the two lipase types can be: (a) the bacteria and yeasts strains which biosynthesise extracellular lipases, can be considered as recommended producers based on the cultivation conditions, namely easy to apply and reproducible

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 415

Innova 40 at 300 rpm; temperature of 30oC; Erlenmeyer flasks of 500 mL with 150 mL medium. Before their cultivation for enzyme formation the microorganisms were grown on liquid media to develop preinoculum and inoculum stages of 24 hours duration, using an inoculation volume of 5-10 % V/V. Several cultivation media, specific for the studied strains, were tested and both the cellular growth and enzymatic activity were

**Yeasts:** *Yarrowia sp. / Candida lipolytica ATCC 8661, Candida sp. DG 8, Pseudozyma aphidis DSM* 

Glucose: 4 g/L Glucose: 10 g/L KH2PO4: 5 g/L Malt extract: 3.78 g/L Peptone: 0.5 g/L Peptone : 10 g/L (NH4)2SO4: 1 g/L Peptone: 5 g/L Yeast extract: 5 g/L Yeast extract: 10 g/L Yeast extract: 10 g/L Tween 80: 4.33 g/L

KH2PO4: 1 g/L Rapeseed oil: 5 g/L Rapeseed oil: 20 g/L Rapeseed oil: 33.7 g/L

The growth characteristics were evaluated by measuring OD500; the lipase activity was determined by using the volumetric method (Tcacenco *et all*, 2010), considering one unit of lipase activity as corresponding to 1 μmol of fatty acid obtained by the hydrolysis of the triglycerides from the rapeseed / olive oil, the reaction conditions being: temperature of

Isolation of extracellular lipase was made by centrifugation (1) and ammonium sulphate precipitation (2): (1) biosynthesis medium was centrifuged at 10 000 rpm for 30 min. at 4 0C. Clear supernatant was treated with benzamidine 2 mM and sodium azide 0.02% to prevent proteolysis and microbial attack and (2) the supernatant is precipitated with ammonium sulphate 30% at 0 0C, then left to stand for 24 hours for achieving precipitation and centrifuged at 10 000 rpm for 30 minutes at 40C. The supernatant is precipitated again with 75% ammonium sulphate. After 24h, the sample is centrifuged again and the resulting product is dissolved in 8 ml TRIS buffer, pH 6.8. This crude enzyme is preserved in the

The growth of both bacteria is low, only *Pseudomonas aeruginosa* (*P. sp.3*) grows more on the medium variant M1b, so the use of both substrates-glucose and oil seems useful. Both bacterium strains have similar small lipase activity levels, the cultivation duration of 24 h being enough for the maximum lipase production, and there is no induction by the rapeseed

M2 for yeasts M3 for yeasts M4 for yeasts

**Bacteria:** *Pseudomonas putida (P. sp. 1) and Pseudomonas aeruginosa (P. sp. 3)* 

Na2SO4: 2 g/L NH4Cl : 5 g/L MgSO4.7H2O: 0.5 g/L

measured.

rapeseed oil)

K2HPO4: 3 g/L MgSO4.7H2O: 0.1 g/L

freezer.

oil (Fig. 2).

**Microorganisms and cultivation media:** 

*70725, and Candida rugosa DSM 70761.* 

Table 2. Cultivation media composition

37ºC, pH=7, duration of 60 minutes.

**3.1.2 Results and discussion** 

1. Bacteria growth and enzyme formation

M1 for bacteria: (variant a: no rapeseed oil; variant b: with 10 mL/L

aerobic bioprocesses; (b) using intracellular lipases slows down the transesterification process due to mass transfer limitations.

**Immobilization of an enzyme** must solve both mass transfer limitations types-internal or external (last case due to formation of an external film layer). Choice of the appropriate lipase immobilization technology is determined by the following objectives: (a) long term enzyme reuse; (b) easy enzyme recovery from the reaction medium; (c) improved activity and thermal, chemical and mechanical stability of the enzyme; (d) potential to run continuous processes. The immobilization support is to be as low cost as possible, condition which is difficult to be observed when the other ones should be fulfilled at the same time (Ghaly *et all*, 2010). Among the great number of immobilization techniques, they can be classified under four general categories: (a) adsorption, (b) cross linking, (c) entrapment and (d) encapsulation. Adsorption seems to be the most attractive, as it is simplest and cheap, retaining high enzyme activity and allowing a good mass transfer, combined or not with the cross linking. The carriers used in adsorption via weak forces include: celite, cellulose, acrylic, silica gel, textile membranes, spherosil, sepharose, sephadex and siliconized glass. The major drawback of the adsorption is the low stability of the enzyme when adsorbed, which determines only limited reuse.

The **stability of the lipase** with low loss of the catalytic activity is the most important characteristic, when used in biodiesel preparation in connection with the enzyme r**ecovery and reuse.**

The most commonly used reactor type for the biodiesel enzymatic preparation is a batchstirred tank reactor, though this biofuel must be considered as a commodity product and therefore produced in continuously operated installations. Possible alternative solutions could be packed bed reactors, fluid beds, expanding bed, recirculation membrane reactors. A wide range of configurations are applicable to perform the transesterification.

As the actual major technical limits of the enzymatic process are still the slower reaction rate by comparison with the alkaline catalysis and the risk of enzyme inactivation, with focus on process design and economy, the researchers calculate the productivity (kg biodiesel/kg enzyme) based on information from different studies and considering a range of enzyme prices from 12 to 185 USD/kg as acceptable, depending on the application characteristics, i.e. per each kg of biodiesel a biocatalyst cost of USD 0.025 could be of economic interest. An increased enzyme life of around 6 years would make enzymes competitive based on productivity again. To this must be added increased reactor costs as enzymes lead to longer space times than alkaline catalysts, but reduced separation costs and low waste water treatment costs will be the benefits.

#### **3. Case study: Enzymatic transesterification of the rapeseed oil with yeast lipases**

The chapter presents the research activity done by the authors regarding the rapeseed oil transesterification with yeast lipase, and is structured in three parts: lipase formation in aerobic bioprocessing; lipase recovery and immobilization; enzymatic transesterification with immobilized lipase produced by the yeast *Candida rugosa* DSM 70761.

#### **3.1 Lipase formation**

#### **3.1.1 Materials and methods**

Several bacteria and yeasts from own / international collections were tested for cell growth and enzyme formation, the cultivation conditions being: rotary shaker New Brunswick Innova 40 at 300 rpm; temperature of 30oC; Erlenmeyer flasks of 500 mL with 150 mL medium. Before their cultivation for enzyme formation the microorganisms were grown on liquid media to develop preinoculum and inoculum stages of 24 hours duration, using an inoculation volume of 5-10 % V/V. Several cultivation media, specific for the studied strains, were tested and both the cellular growth and enzymatic activity were measured.

#### **Microorganisms and cultivation media:**

414 Biodiesel – Feedstocks and Processing Technologies

aerobic bioprocesses; (b) using intracellular lipases slows down the transesterification

**Immobilization of an enzyme** must solve both mass transfer limitations types-internal or external (last case due to formation of an external film layer). Choice of the appropriate lipase immobilization technology is determined by the following objectives: (a) long term enzyme reuse; (b) easy enzyme recovery from the reaction medium; (c) improved activity and thermal, chemical and mechanical stability of the enzyme; (d) potential to run continuous processes. The immobilization support is to be as low cost as possible, condition which is difficult to be observed when the other ones should be fulfilled at the same time (Ghaly *et all*, 2010). Among the great number of immobilization techniques, they can be classified under four general categories: (a) adsorption, (b) cross linking, (c) entrapment and (d) encapsulation. Adsorption seems to be the most attractive, as it is simplest and cheap, retaining high enzyme activity and allowing a good mass transfer, combined or not with the cross linking. The carriers used in adsorption via weak forces include: celite, cellulose, acrylic, silica gel, textile membranes, spherosil, sepharose, sephadex and siliconized glass. The major drawback of the adsorption is the low stability of the enzyme when adsorbed,

The **stability of the lipase** with low loss of the catalytic activity is the most important characteristic, when used in biodiesel preparation in connection with the enzyme r**ecovery** 

The most commonly used reactor type for the biodiesel enzymatic preparation is a batchstirred tank reactor, though this biofuel must be considered as a commodity product and therefore produced in continuously operated installations. Possible alternative solutions could be packed bed reactors, fluid beds, expanding bed, recirculation membrane reactors.

As the actual major technical limits of the enzymatic process are still the slower reaction rate by comparison with the alkaline catalysis and the risk of enzyme inactivation, with focus on process design and economy, the researchers calculate the productivity (kg biodiesel/kg enzyme) based on information from different studies and considering a range of enzyme prices from 12 to 185 USD/kg as acceptable, depending on the application characteristics, i.e. per each kg of biodiesel a biocatalyst cost of USD 0.025 could be of economic interest. An increased enzyme life of around 6 years would make enzymes competitive based on productivity again. To this must be added increased reactor costs as enzymes lead to longer space times than alkaline catalysts, but reduced separation costs and low waste water

**3. Case study: Enzymatic transesterification of the rapeseed oil with yeast** 

with immobilized lipase produced by the yeast *Candida rugosa* DSM 70761.

The chapter presents the research activity done by the authors regarding the rapeseed oil transesterification with yeast lipase, and is structured in three parts: lipase formation in aerobic bioprocessing; lipase recovery and immobilization; enzymatic transesterification

Several bacteria and yeasts from own / international collections were tested for cell growth and enzyme formation, the cultivation conditions being: rotary shaker New Brunswick

A wide range of configurations are applicable to perform the transesterification.

process due to mass transfer limitations.

which determines only limited reuse.

treatment costs will be the benefits.

**and reuse.**

**lipases** 

**3.1 Lipase formation** 

**3.1.1 Materials and methods** 

**Bacteria:** *Pseudomonas putida (P. sp. 1) and Pseudomonas aeruginosa (P. sp. 3)*  **Yeasts:** *Yarrowia sp. / Candida lipolytica ATCC 8661, Candida sp. DG 8, Pseudozyma aphidis DSM 70725, and Candida rugosa DSM 70761.* 


Table 2. Cultivation media composition

The growth characteristics were evaluated by measuring OD500; the lipase activity was determined by using the volumetric method (Tcacenco *et all*, 2010), considering one unit of lipase activity as corresponding to 1 μmol of fatty acid obtained by the hydrolysis of the triglycerides from the rapeseed / olive oil, the reaction conditions being: temperature of 37ºC, pH=7, duration of 60 minutes.

Isolation of extracellular lipase was made by centrifugation (1) and ammonium sulphate precipitation (2): (1) biosynthesis medium was centrifuged at 10 000 rpm for 30 min. at 4 0C. Clear supernatant was treated with benzamidine 2 mM and sodium azide 0.02% to prevent proteolysis and microbial attack and (2) the supernatant is precipitated with ammonium sulphate 30% at 0 0C, then left to stand for 24 hours for achieving precipitation and centrifuged at 10 000 rpm for 30 minutes at 40C. The supernatant is precipitated again with 75% ammonium sulphate. After 24h, the sample is centrifuged again and the resulting product is dissolved in 8 ml TRIS buffer, pH 6.8. This crude enzyme is preserved in the freezer.

#### **3.1.2 Results and discussion**

1. Bacteria growth and enzyme formation

The growth of both bacteria is low, only *Pseudomonas aeruginosa* (*P. sp.3*) grows more on the medium variant M1b, so the use of both substrates-glucose and oil seems useful. Both bacterium strains have similar small lipase activity levels, the cultivation duration of 24 h being enough for the maximum lipase production, and there is no induction by the rapeseed oil (Fig. 2).

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 417

Fig. 3. The maximum specific rate and the lipase activity of the yeasts for the experimental variants Ai-Di (i=1, 2); (a) max specific growth rate (µ-1); (b) lipase activity (UAE/mL)

The techniques by physical adsorption were chosen due to the fact they are simple and

The crude lipase obtained from *Yarrowia lipolytica* and *Candida rugosa* yeasts after the precipitation with 70% ammonium sulphate was dissolved in 0.05 M phosphate buffer, pH 7. Then the adsorbent was added until the limit activity in the supernatant is reached, respectively: for *Yarrowia lipolytica* 2.5 g silicagel G at 800 mL extract, 22 g of Celite in the same volume of extract and for *Candida rugosa* 11 g Celite at 800 ml extract. Adsorption

30 mL chitosan 1% solution was prepared by adding 2mL CH3COOH p.a. , 19.8 mL 0.5 N NaOH by heating to 50 0C and stirring for 10 minutes to complete dissolution of chitosan. 0.5 mL 25% of glutaraldehyde was added dropwise under high stirring. Microspheres thus obtained were filtered and washed with H2O dist. and 0.05 M phosphate buffer, pH 7. 1g wet chitosan microspheres were used for immobilization; they were suspended in 2 mL 0.05 M phosphate buffer, pH 7 and mixed with 2mL solution of lipase (*Candida rugosa*) obtained by solving the crude enzyme precipitated with ammonium sulphate into 0.05 M

1g wet chitosan particles was obtained by injecting 25 mL solution of 3% chitosan into 250 mL solution of NaOH 1N and C2H5OH 26%. The chitosan particles were suspended into 3 mL 0.75% carbodiimide solution, prepared in 0.05 M phosphate buffer, pH 6, 25 0C. After 10 minutes of activation, the particles were washed with distilled water and transferred to 10 mL 1% lipase solution immersed in 0.05 M phosphate buffer, pH 6. The adsorption duration was

duration was approx. 2 hours at ambient temperature and under mechanical stirring.

phosphate buffer, pH 7, 1:5 (w / v) ratio. The mixture was stirred for 1 hour at 37 0C.

60 minutes; then the immobilized enzyme was washed 3 times with distilled water.

3. Cross-linking with glutaraldehyde and reduction with sodium borohydride A mixture was prepared from 0.5 g chitosan, 1.041 mL 2M acetic acid, 25 mL distilled water and 1.041 mL of 1M sodium acetate, maintained on water bath at 500C with stirring. For the

(a) (b)

cheap, so the price of the immobilized biocatalyst is expected to be low. a. Lipase immobilization by adsorption on silicagel or celite support

b. Lipase immobilization by adsorption on chitosan support

1. Cross-linking with glutaraldehyde:

2. Cross-linking with carbodiimide:

(Chirvase *et all*, 2010)

**3.2 Lipase immobilization 3.2.1 Materials and methods** 

(a) (b)

Fig. 2. Growth and lipase activity of bacteria *Pseudomonas putida* (*P. sp.1*) and *Pseudomonas aeruginosa* (*P. sp. 3*) on cultivation medium M1, a and b; (a) growth (OD); (b) lipase formation

	- A1: *Candida rugosa* DSM 70761 on M1, 48 h
	- A2: *Candida rugosa* DSM 70761 on M2, 48 h
	- B1: *Pseudozyma aphidis* DSM 70725 on M1, 48 h
	- B2: *Pseudozyma aphidis* DSM 70725 on M2, 48 h
	- C1: *Candida rugosa* DSM 70761 on M3, 48 h
	- C2: *Pseudozyma aphidis* DSM 70725 on M3, 48 h
	- D1: *Yarrowia (Candida lypolitica)* ATCC 8661 on M2, 24 h
	- D2: *Candida sp. DG 8* on M3, 24 h

For the cultivation medium M2 the growth rate for the yeasts *Candida rugosa* DSM 70761 and *Candida lypolitica* ATCC 8661 were higher and close enough: variant D1 *Yarrowia lipolytica* with the specific growth rate of 0.2 h-1 ; variant A2 *Candida rugosa* with the specific growth rate of 0.15 h-1. But the final enzyme activity was higher for the second yeast: *Candida rugosa* final enzymatic activity of 289.0 UAE/mL by comparison with *Yarrowia lipolytica* enzymatic activity of 106.0 UAE/mL. At the same time the growth and lipase activity of both yeasts were much higher than those of the studied bacteria. So the immobilization study was to be performed with these already mentioned yeasts. In a first step, the preliminary transesterification results, obtained by thin layer chromatography, demonstrated that both lipases have high enough catalysis activities. After the confirmation of the transesterification capacity, it was of interest to develop appropriate immobilization techniques for these lipases, so to be able to use the immobilized enzymes in several cycles of biotransformation.

Fig. 3. The maximum specific rate and the lipase activity of the yeasts for the experimental variants Ai-Di (i=1, 2); (a) max specific growth rate (µ-1); (b) lipase activity (UAE/mL) (Chirvase *et all*, 2010)

#### **3.2 Lipase immobilization 3.2.1 Materials and methods**

416 Biodiesel – Feedstocks and Processing Technologies

*P. sp.1 on M1a P. sp.1 on M1b P.sp.3 on M1a P. sp.3 on M1b*

(a) (b)

D1: *Yarrowia (Candida lypolitica)* ATCC 8661 on M2, 24 h

Fig. 2. Growth and lipase activity of bacteria *Pseudomonas putida* (*P. sp.1*) and

*Pseudomonas aeruginosa* (*P. sp. 3*) on cultivation medium M1, a and b; (a) growth (OD);

For the cultivation medium M2 the growth rate for the yeasts *Candida rugosa* DSM 70761 and *Candida lypolitica* ATCC 8661 were higher and close enough: variant D1 *Yarrowia lipolytica* with the specific growth rate of 0.2 h-1 ; variant A2 *Candida rugosa* with the specific growth rate of 0.15 h-1. But the final enzyme activity was higher for the second yeast: *Candida rugosa* final enzymatic activity of 289.0 UAE/mL by comparison with *Yarrowia lipolytica* enzymatic activity of 106.0 UAE/mL. At the same time the growth and lipase activity of both yeasts were much higher than those of the studied bacteria. So the immobilization study was to be performed with these already mentioned yeasts. In a first step, the preliminary transesterification results, obtained by thin layer chromatography, demonstrated that both lipases have high enough catalysis activities. After the confirmation of the transesterification capacity, it was of interest to develop appropriate immobilization techniques for these lipases, so to be able to use the immobilized enzymes

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

(b) lipase formation

**O p tica l d en sity**

> 0 5 10 15 20 25 30 **Time, hours**

2. Yeasts growth and lipase formation

D2: *Candida sp. DG 8* on M3, 24 h

in several cycles of biotransformation.

A1: *Candida rugosa* DSM 70761 on M1, 48 h A2: *Candida rugosa* DSM 70761 on M2, 48 h B1: *Pseudozyma aphidis* DSM 70725 on M1, 48 h B2: *Pseudozyma aphidis* DSM 70725 on M2, 48 h C1: *Candida rugosa* DSM 70761 on M3, 48 h C2: *Pseudozyma aphidis* DSM 70725 on M3, 48 h

Experimental variants:

The techniques by physical adsorption were chosen due to the fact they are simple and cheap, so the price of the immobilized biocatalyst is expected to be low.

a. Lipase immobilization by adsorption on silicagel or celite support

The crude lipase obtained from *Yarrowia lipolytica* and *Candida rugosa* yeasts after the precipitation with 70% ammonium sulphate was dissolved in 0.05 M phosphate buffer, pH 7. Then the adsorbent was added until the limit activity in the supernatant is reached, respectively: for *Yarrowia lipolytica* 2.5 g silicagel G at 800 mL extract, 22 g of Celite in the same volume of extract and for *Candida rugosa* 11 g Celite at 800 ml extract. Adsorption duration was approx. 2 hours at ambient temperature and under mechanical stirring.

	- 1. Cross-linking with glutaraldehyde:

30 mL chitosan 1% solution was prepared by adding 2mL CH3COOH p.a. , 19.8 mL 0.5 N NaOH by heating to 50 0C and stirring for 10 minutes to complete dissolution of chitosan. 0.5 mL 25% of glutaraldehyde was added dropwise under high stirring. Microspheres thus obtained were filtered and washed with H2O dist. and 0.05 M phosphate buffer, pH 7. 1g wet chitosan microspheres were used for immobilization; they were suspended in 2 mL 0.05 M phosphate buffer, pH 7 and mixed with 2mL solution of lipase (*Candida rugosa*) obtained by solving the crude enzyme precipitated with ammonium sulphate into 0.05 M phosphate buffer, pH 7, 1:5 (w / v) ratio. The mixture was stirred for 1 hour at 37 0C.

2. Cross-linking with carbodiimide:

1g wet chitosan particles was obtained by injecting 25 mL solution of 3% chitosan into 250 mL solution of NaOH 1N and C2H5OH 26%. The chitosan particles were suspended into 3 mL 0.75% carbodiimide solution, prepared in 0.05 M phosphate buffer, pH 6, 25 0C. After 10 minutes of activation, the particles were washed with distilled water and transferred to 10 mL 1% lipase solution immersed in 0.05 M phosphate buffer, pH 6. The adsorption duration was 60 minutes; then the immobilized enzyme was washed 3 times with distilled water.

3. Cross-linking with glutaraldehyde and reduction with sodium borohydride A mixture was prepared from 0.5 g chitosan, 1.041 mL 2M acetic acid, 25 mL distilled water and 1.041 mL of 1M sodium acetate, maintained on water bath at 500C with stirring. For the

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 419

**123456**

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

comparison with the immobilization of a lipase from the fungus *Aspergillus niger.* 

immobilization techniques for both crude lipases.

source

DSM 70761

<sup>3</sup>*Aspergillus niger* lyophilized lipase *(*Fluka)

No Lipase

<sup>1</sup>*Candida rugosa,* 

<sup>4</sup>*Candida rugosa,* 

<sup>5</sup>*Candida rugosa,* 

<sup>6</sup>*Yarrowia lipolytica* 

Table 4. Applied immobilization techniques

The experimental results are presented in the Figure 5, obtained with the described

The immobilization techniques, characterized in the following table, were performed in

<sup>2</sup>*Aspergillus niger* (Fluka) Chitosan adsorption and cross-linking with

DSM 70761 Adsorption on Celite 545

DSM 70761 Adsorption on Silicagel G

ATCC 8661 Adsorption on Celite 545

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

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

immobilization of *Aspergillus niger* lipase gave unsatisfactory results.

Immobilization technique

Chitosan adsorption and cross-linking with glutaraldehyde

carbodiimide

Chitosan adsorption, cross-linking with glutaraldehyde and granulation with sodium borohydride

**Immobilization yield (%)**

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.
