Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study

These days the interest of fuels preparing from sustainable natural resources is continuously increasing due to the rising prices of the fossil fuels and the political instability in the oil producing countries. The fuels manufacturing from local vegetal resources can sustain the every country’ prosperity, including rural, agricultural, economically disadvantaged regions. Nowadays only the bioethanol and the biodiesel are already produced at industrial level from sustainable raw materials. The biodiesel is manufactured by the chemically catalysed transesterification of the triglycerides from the vegetable oils, rapeseed oil in Europe and soya oil in USA. As the methanol is often used as alcohol reagent, the reaction is consequently named methanolysis. The most applied catalysts are alkalines (especially NaOH) or mineral acids. So the biodiesel represents the methyl esters of the fatty acids from the vegetable oils. The present diesel engines can normally use a mixture of diesel with 5% v/v biodiesel. Biodiesel contains virtually no sulfur or aromatics, and use of biodiesel in a conventional diesel engine results in substantial reduction of unburned hydrocarbons, carbon monoxide and particulate matter. The production and use of biodiesel, compared to petroleum diesel, resulted in a 78.5% reduction in carbon dioxide emissions. Moreover, biodiesel has a positive energy balance. The chemical transesterification applied at industrial level has important advantages, but also limitations: in spite of the high conversion yields and the short reaction duration, the global transformation is energetically intensive, the glycerol recovery is difficult, the alkaline catalyst must be separated, the wastewaters are to be treated by a rather complex procedure, and both the free fatty acids and water can badly influence the reaction. These unfavourable situations can be diminished by performing the enzymatic transesterification on conditions that: (a) the immobilised lipase used as biocatalyst must be as cheap as possible; (b) one can obtain the economic efficiency of the whole biotransformation process similar to that characteristic to the chemical process, these objectives being presented function of the research methodology and results. The comparison between the chemical way and the enzymatic way is presented in the Table 1.


Introduction
These days the interest of fuels preparing from sustainable natural resources is continuously increasing due to the rising prices of the fossil fuels and the political instability in the oil producing countries.The fuels manufacturing from local vegetal resources can sustain the every country' prosperity, including rural, agricultural, economically disadvantaged regions.Nowadays only the bioethanol and the biodiesel are already produced at industrial level from sustainable raw materials.The biodiesel is manufactured by the chemically catalysed transesterification of the triglycerides from the vegetable oils, rapeseed oil in Europe and soya oil in USA.As the methanol is often used as alcohol reagent, the reaction is consequently named methanolysis.The most applied catalysts are alkalines (especially NaOH) or mineral acids.So the biodiesel represents the methyl esters of the fatty acids from the vegetable oils.The present diesel engines can normally use a mixture of diesel with 5% v/v biodiesel.Biodiesel contains virtually no sulfur or aromatics, and use of biodiesel in a conventional diesel engine results in substantial reduction of unburned hydrocarbons, carbon monoxide and particulate matter.The production and use of biodiesel, compared to petroleum diesel, resulted in a 78.5% reduction in carbon dioxide emissions.Moreover, biodiesel has a positive energy balance.The chemical transesterification applied at industrial level has important advantages, but also limitations: in spite of the high conversion yields and the short reaction duration, the global transformation is energetically intensive, the glycerol recovery is difficult, the alkaline catalyst must be separated, the wastewaters are to be treated by a rather complex procedure, and both the free fatty acids and water can badly influence the reaction.These unfavourable situations can be diminished by performing the enzymatic transesterification on conditions that: (a) the immobilised lipase used as biocatalyst must be as cheap as possible; (b) one can obtain the economic efficiency of the whole biotransformation process similar to that characteristic to the chemical process, these objectives being presented function of the research methodology and results.The comparison between the chemical way and the enzymatic way is presented in the Table 1.
Table 1.Comparison between the alkaline catalysis and the enzymatic method for biodiesel preparation (Bajaj et all, 2010) The now-a-day technological progress regarding the enzymatic transesterification is demonstrated by the realisation of 2 industrial pilots in China (Moore 2008a(Moore , 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 possible preparation of methanol from natural resources.

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.
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 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 recovery 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.

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.

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 30 o C; 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.The growth characteristics were evaluated by measuring OD 500 ; 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 0 C. 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 0 C, then left to stand for 24 hours for achieving precipitation and centrifuged at 10 000 rpm for 30 minutes at 4 0 C. 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.

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).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.

Lipase immobilization 3.2.1 Materials and methods
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.b.Lipase immobilization by adsorption on chitosan support 1. Cross-linking with glutaraldehyde: 30 mL chitosan 1% solution was prepared by adding 2mL CH 3 COOH p.a. , 19.8 mL 0.5 N NaOH by heating to 50 0 C 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 H 2 O 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 0 C.
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 C 2 H 5 OH 26%.The chitosan particles were suspended into 3 mL 0.75% carbodiimide solution, prepared in 0.05 M phosphate buffer, pH 6, 25 0 C.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 50 0 C with stirring.For the 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.

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 (NH 4 ) 2 SO 4 , 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.

No.
Strain  The experimental results are presented in the Figure 5, obtained with the described immobilization techniques for both crude lipases.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.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.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 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%.
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 substrate of 4:1.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 37 0 C; mixing of 250 rpm; reaction total duration of 24 hr.

Results and discussion
The most important transesterification results are presented in the Figure 8.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 transesterification performing.

Conclusions
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' fungi.
2. The lipases with advanced specificity are not useful in the transesterification to produce biodiesel; the most recommended are the lipases with reduced region specificity, but more developed specificity for the substrate.3. The molar ratio of the substrates used in the biotransformation of vegetable oils to biodiesel must be determined for each studied system: alcohol -oil -lipase.4. The rapeseed oil is of interest as raw material in the transesterification, as it is largely produced by the European agriculture and also in Romania, and at the same time it is used in the alkaline catalysed transformation.But in USA the soya oil is in charge.5.The aerobic bio processing of several bacteria and yeasts from Romanian research collections or from international collections demonstrated that two yeasts, Candida rugosa DSM 70761 and Yarrowia lipolytica ATCC 8661 produced lipases characterized by high activity in simple and short duration cultivation.The media composition and the cultivation parameters were optimized for both yeasts' lipases formation.6.The immobilisation techniques by physical adsorption were studied for the lipases from the above mentioned yeasts.First of all the extracellular lipases from the yeasts Candida rugosa DSM 70761 and Yarrowia lipolytica ATCC 8661 can be easily separated in the liquid fraction by centrifugation and further on the crude enzymes can be obtained by ammonium sulphate precipitation.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.7. As the lipase from the yeast Candida rugosa DSM 70761 was immobilized on Celite 545 support with yields of 49 -63%, and higher yields are obtained for the immobilization of the lipase from Yarrowia lipolytica ATCC 8661, and the immobilization procedure is easy and low price, the laboratory experimental model was developed on this support.8.In order to improve the immobilization of the lipase of Candida rugosa DSM 70761, a treatment with acetone as organic solvent was introduced and this operation had as consequence a big increase of the immobilization yield on Celite from 49% to 97%. 9.A higher static stability was determined 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.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 for the first biocatalyst.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.10.This biocatalyst operational stability was also tested and the immobilized enzyme half time was of about 5-6 reaction cycles, as after 4 reaction cycles the residual activity was still 58.7%.11.Three experimental models were considered to perform the transesterification: (a) batch enzymatic transesterification with methanol and without organic solvent; (b) "semibatch" enzymatic transesterification with methanol and without organic solvent; (c) batch enzymatic transformation in hexane.The reaction yields were good enough for all the tested experimental models and for both -soya and rapeseed oils, the results variation being in the range of 56 -69%.They can be improved by adequate optimization procedures to be applied for each technological phase, comprising enzyme obtaining in aerobic bioprocess, lipase immobilization and transesterification.Further research work is to be developed in two directions: (1) the use of the glycerol formed as by product in the transesterification process, especially as C source in several other bioprocesses; (2) as beside this product there are several others, the most important future research direction will be the technical application of the bio refinery concept realised for the vegetable oils extracted from many plants specific to each geographical area.A possible future bio refinery will integrate physical, chemical, and biological procedures for the biodiesel preparation, conversion of solid residue with high carbohydrates or protein content; glycerol use, the whole application being characterised by both high economic efficiency and reduction of solid or liquid residues.

Fig. 2 .
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 2. Yeasts growth and lipase formation Experimental variants: 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 70761and 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. 4 .Fig. 5 .
Fig. 4. Thin layer chromatography of the products obtained by the transesterification

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

Fig. 7 .
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)

Fig. 8 .
Fig. 8.The vegetable oils transesterification yield for the 3 experimental models: a 1 -batch model without solvent, with soya oil; a 2 -batch model without solvent, with rapeseed oil; b-semibatch model without solvent, with soya oil; c 1 -batch model in hexane with soya oil; c 2 -batch model in hexane with rapeseed oil

Table 2 .
Cultivation media composition

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; D1lipase from Yarrowia lipolytica/M2; M -Control, ester of oleic acid

Table 4 .
The immobilization techniques, characterized in the following table, were performed in comparison with the immobilization of a lipase from the fungus Aspergillus niger.Applied immobilization techniquesThe 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 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

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