href="#ulink_4cc781b7-870c-5e91-ac89-32afacd10dbd">Table 1.8.
Another way of using compound lipases is cloning and expression of two or more different lipase coding genes from different organism in a host organism. But cloning and effective expression of lipases from host organism is a difficult and cumbersome process, but it has also provided better results. For example, according to Guan et al. [184], Pichia pestoris was selected as a host organism to express two lipase coding genes cloned from two different organisms, one of them was R. miehei (source of 1,3- specific lipase) and the other was Penicillium cyclopium (source of non-specific lipase). Extract containing these two lipases when applied for biodiesel production to transesterify soybean oil at 30°C with 4:1 alcohol-to-oil molar ratio resulted in 99.7% yield after 24 h. In another research, a special recombinant Aspergillus oryzae whole-cell biocatalyst was created that used to co-express two different lipases genes, one of these two lipases was derived from Fusarium heterosporum (FHL) and the other one was mono and diacyl glycerol lipase B. Use of that whole-cell recombinant Aspergillus oryzae biocatalyst resulted in 98% methyl ester yield [179]. According to Yan et al. [216], recombinant Pichia pestoris was developed that displayed two lipases, i.e., T. lanuginosus lipase (TLL) and C. antarctica lipase B (CALB) from different sources on its surface. This whole-cell biocatalyst co-expressing both lipases produced 95.4% biodiesel yield under optimum conditions. Apart from ILs advantages, there is a problem with the biodiesel recovery because during continuous biodiesel removal reaction moves backward and affects the resultant yield. So, to avoid this problem SC-CO2 has been suggested along with ILs. SC-CO2 is very effective in recovering biodiesel because ester molecules have good solubility in it. IL-SC-CO2 combination not only provides easy recovery but also prevent glycerol inhibition effect. SC-CO2 saturated with (substrate) oil is introduced into the reaction system and this creates two phases because of immiscibility in each other. SC-CO2 can diffuse through IL (ionic liquid) phase bringing substrate with it, reaches the enzyme active site and makes the reaction easily possible. After enzyme activity when biodiesel is formed, biodiesel esters become soluble in SC-CO2 phase, So, in this way, biodiesel becomes separate from ILs. Glycerol (by-product of the reaction) does not dissolve into SC-CO2 so it makes another separate layer and then glycerol can be easily taken out in pure form. SC-CO2 containing biodiesel is then processed to recover bio-diesel from it by depressurization [217, 218]. In this way, two phase system due to IL-SC-CO2 combination enable to recover biodiesel in good quality. IL-SC-CO2 combination system was first used to extract naphthalene using [bmim] [PF6] as IL [219]. Enzymatic biodiesel was produced with IL-SC-CO2 system by transesterification of triolein using Novozyme 435 as biocatalyst and methanol as acyl acceptor. In IL-SC-CO2 system, 12 different ILs were used and there were two different temperature conditions, i.e., 60°C and 85°C. After 6 h, the resultant biodiesel yield obtained was more than 98% that made this reaction a successful one [220].
Table 1.8 Some examples to produce biodiesel using combination of lipases.
| Combination of lipases | Immobilized on | Substrate | Acyl acceptor | Yield | Reference |
|---|---|---|---|---|---|
| Thermomyces lanuginosus lipase and Rhizomucor miehei lipase. | Lewatit VP OC 1600 | Soybean oil | Ethanol | 90% | [207] |
| TLL immobilized on acrylic resin, RML immobilized on anion-exchange resin | Palm oil | Ethanol | 80% | [208] | |
| Pseudomonas fluorescens and Candida rugosa | Accurel PE-100 microporous polypropylene powder | Used palm oil | Ethanol | >67% | [209] |
| Immobilized Pseudomonas fluorescens lipase followed by immobilized Pseudomonas cepacia lipase | macroporous polypropylene | Soybean oil | Methanol | 98% | [210] |
| Combined use of Lipozyme TL IM and Novozym 435 | Lipozyme TL IM immobilized on acrylic resin and Novozym 435 immobilized on microporous resin | waste cooking sunflower oil | Methanol | 99% | [211] |
| Novozym 435 (CALB) and Lipozyme RM-IM (RML) | RML immobilized on an anion-exchange resin, and CALB immobilized on a macroporous resin | Soybean oil | Ethanol | 80% | [212] |
| Canola oil | Methanol | >95% | [213] | ||
| Combination of Rhizopus arrhizus lipase and Candida antarctica lipase B | microporous polypropylene Accurel MP1000 | Triolein | Ethanol | 96% | [214] |
| Candida rugosa lipase followed by Novozym 435 | Macroporous acrylic resin | Acid oil, product of vegetable oil | Methanol | 91% | [215] |
1.11.2 Microwave and Ultrasonic-Assisted Reaction
Microwave employment in the chemical or biochemical reaction is very clean, economic, fast and energy efficient method. Use of microwave is very helpful in substrate preparation, extraction, and biodiesel production. Microwave irradiation assists the reaction by providing direct energy to the reactants, and hence, the loss of energy becomes minimal. This feature makes it economic than using other conventional methods of heating to make the reaction possible [221]. Easy energy transfers and use makes the enzyme to perform better and its easy separation leads to cost reduction. So, this environment friendly method provides energy efficient, faster and cost-effective reaction. There have some research investigations been done to analyze how effective it would be to use microwave. Macauba oil transesterification was performed using Novozyme 435 and Lipozyme IM in the presence of ethanol as acyl acceptor. Activities of these enzymes were checked and compared in the presence and absence of microwave. Before microwave assistance enzymatic activity values of Novozyme 435 and Lipozyme IM were 0.09 and 0.08, respectively. But after microwave presence activity value were 0.6 and 0.4, respectively. Results were clear that microwave assistance enhanced enzymatic activity about one order of magnitude [222, 223]. In another experiment, soybean oil and sunflower oil was transesterified using Novozyme 435 and ethanol to
