**4. 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.

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 425

This research was financially supported by the Romanian National Agency for Research

Antczak M. S., Kubiak A., Antczak T, & Bielecki S. (2009), Enzymatic biodiesel synthesis –

Bajaj A., L. Purva, Jha P. N., & Mehrotra R., (2010), Biodiesel production through lipase

Bisen P. S., Sanodiya B. S., Thakur G. S., Baghel R. K., & Prasad G. B. K. S., (2010),

Chirvase A. A., Ungureanu C., Tcacenco L., Radu N., (2010), Determination of Yeast

Moore A., (2008a), Biofuels are dead: long live biofuels (?) – Part one, *New Biotechnology*, Vol.

Moore A., (2008b), Biofuels are dead: long live biofuels (?) – Part two, *New Biotechnology*,

Tcacenco L., Chirvase A. A.,Berteanu E., (2010), The preparation and immobilization of

*Biotechnological Letters,* Vol. 15, No. 5, pp. 5631-5639, ISSN 1224-5984

Biodiesel *Revista de Chimie*, Vol. 61, No.9, pp. 866-868, ISSN 0034-7752 Fjerbaek L., Christensen K.V., & Norddahl B., (2009), A Review of the Current State of

*Bioengineering,* Vol. 102, No. 5, April 1, pp. 1298-1315, ISSN 1097-0290 Ghaly A. E., Dave D., Brooks, & Budge S., (2010), Production of Biodiesel by Enzymatic

Key factors affecting efficiency of the process, *Renewable Energy*, Vol. 34, 1No. 4,

catalyzed transesterification: An overview, *Journal of Molecular Catalysis B*:

Biodiesel production with special emphasis on lipase-catalyzed transesterification, *Biotechnological Letters*, Vol. 32, No. 10, pp. 1019-1030, ISSN

Strains Characteristics as Lipase Providers for Enzymatic Transesterification to

Biodiesel Production Using Enzymatic Transesterification, *Biotechnology and* 

Transesterification: Review, *American Journal of Biochemistry and Biotechnology*, Vol.

some yeast lipases for rapeseed oil transesterification to biodiesel, *Romanian* 

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.

pp.1185–1194, ISSN 0960-1481

6, No 2, pp. 54-76, ISSN 1553-3468

25, No. 1, pp. 6-13, ISSN 1871-6784

Vol. 25, No. 2/3, pp. 96-101, ISSN 1871-6784

under the Second National Program, Project 61-032/2007.

*Enzymatic,* Vol. 62, No. 1, pp. 9–14, ISSN 1381-1169

**5. Acknowledgment** 

0141-5492

**6. References** 

optimization procedures to be applied for each technological phase, comprising enzyme


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.

#### **5. Acknowledgment**

This research was financially supported by the Romanian National Agency for Research under the Second National Program, Project 61-032/2007.

#### **6. References**

424 Biodiesel – Feedstocks and Processing Technologies

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

3. The molar ratio of the substrates used in the biotransformation of vegetable oils to

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

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

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

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

biodiesel must be determined for each studied system: alcohol – oil – lipase.

cultivation parameters were optimized for both yeasts' lipases formation.

more developed specificity for the substrate.

immobilized by adsorption on Celite or Silicagel.

still 58.7%.


**21** 

*Argentina* 

**Adsorption in Biodiesel Refining - A Review** 

Biodiesel is a petrodiesel substitute composed of a mixture of fatty acid methyl esters obtained by the transesterification of plant oils or animal fats with short chain alcohols such as methanol or ethanol. Despite its natural origin biodiesel is technically fully compatible with petroleum diesel, requiring virtually no changes in the fuel distribution system or the Diesel motor. Its production and use have increased significantly in many countries and are in nascent status in many others. Other advantages of biodiesel compared to petrodiesel are reduction of most exhaust emissions, biodegradability, higher flash point, inherent lubricity

Literature on the refining of biodiesel is abundant but concentrates almost exclusively on the transesterification steps for transforming fats and oils into esters of short alcohols and fatty acids. In this sense in the last years the most important advances in the reaction technology have been the development of continuous heterogeneous transesterification reactors (Bournay et al., 2005; Portilho et al., 2008) and the design of new robust non-catalytic processes for multifeedstock operation (Saka & Kusdiana, 2001; Saka & Minami, 2009). In the case of the refining operations downstream and upstream the transesterification reactors the biodiesel literature is however scarce. Two are the reasons for this: (i) Feedstock pretreatment in the case of biodiesel is a mature technology developed decades ago for the production of edible oil. (ii) After natural triglycerides are converted into fatty acid methyl esters, the product mixture needs little chemical adjustment since many properties of these

Some reports on post-reactor biodiesel refining have dealt with classical and simple techniques of purification, e.g. water washing (Karaosmanoglu et al., 1996). Others have indicated that adsorption technologies are particularly suited for the refining of biodiesel (Yori et al. 2007; Mazzieri et al., 2008; Manuale et al. 2011). In order to elucidate the role of adsorption processes in the refining of biodiesel, this review studies some theoretical and practical aspects related to the functioning, design and operation of adsorbers and their

The objectives of Diesel fuel refining operations are aimed at improving the fuel combustion performance, maximizing the power delivered to the motor, increasing the engine life and reducing the emission of noxious compounds. The relevant properties involved are cetane

and domestic origin (Chang et al., 1996; Romig & Spataru, 1996; Wang et al., 2000).

esters are ideal for the functioning of Diesel motors.

application to the purification of biodiesel product and feedstocks.

**2. The needs for refining of petrodiesel and biodiesel fuels** 

**1. Introduction** 

Carlos Vera, Mariana Busto, Juan Yori, Gerardo Torres, Debora Manuale, Sergio Canavese and Jorge Sepúlveda *INCAPE (FIQ, Universidad Nacional del Litoral-CONICET),* 

Uthoff S., Broker D., Steinbuchel A., (2009), Current state and perspectives of producing biodiesel-like compounds by biotechnology, *Microbial Biotechnology*, Vol. 2, No. 5, pp. 551-565, ISSN 1751-7915
