Extracellular lipases are separated from broth containing lipase producing cells and after purification used as a catalyst in biodiesel production processes [55]. The way to purify extracellular lipases depends upon its structure and source organism [80]. Mostly extracellular lipases are used in the immobilized form for transesterification than as free lipases because of the low conversion rate and costly process [11]. Literature is full of different methods as well as materials used for immobilization of extracellular lipases. Main methods for immobilization involve cross linking, carrier binding and entrapment while the most commonly used materials for immobilization include silica, magnetic particles, and nanofibers or nanoparticles for carrier binding, alginate beads, gels, and silicon polymers for entrapment and glutaraldehyde for cross-linking [47]. The use of a suitable solvent in case of extracellular lipase is a key factor for high yield in transesterification as the use of unrelated solvent or absence of solvent results in very low yield [102, 165]. The use of extracellular lipase is also adapted because the use of intracellular lipase results in difficulties of extraction and purification of the final product [171]. Extracellular lipases are obtained from Candida guilliermondii, Burkholderia glumae, Pseudomonas aeruginosa, and Yarrowia lipolytica [176–179]. Table 1.4 indicated a comparison between intracellular and extracellular lipases.
Table 1.4 Comparison between intracellular and extracellular lipase.
| Intracellular lipase | Extracellular lipase |
|---|---|
| Present inside the cell or linked to its walls (cell bound lipase) | Separated from cells producing it |
| No need of isolation and purification steps | Complex isolation and purification are required before using it for biodiesel production |
| Low conversion rate | High conversion rate |
| Not analyzed by direct sampling | Analyzed by direct sampling |
| Direct immobilization of lipase producing cells (whole-cell immobilization | Purification is required before immobilization |
| Biodiesel production is cost-effective | Biodiesel production is costly |
1.9 Recombinant Lipases for Cost-Effective Biodiesel Production
In order to produce economically feasible and cost-effective enzymatic biodiesel production, various methods have been used. For example, improvement in purification procedures, use of better bioreactors, various immobilization techniques, use of easy to handle solvents and solution, finding novel organisms that can produce more stable and effective lipases. These genetic manipulations enable recombinant organism to overexpress active lipases. A lot of work has been done to produce recombinant lipases from recombinant organisms by deriving desired gene from another specie or organism to meet our demands. According to Huang et al. [173], a good conversion of microalgal oil into FAME and FAEE was obtained (<90%) when recombinant lipase from Rhizomucor miehei was expressed in Pichia pastoris, in the presence of methanol and ethanol as acyl acceptor, respectively. That recombinant lipase enzyme was called Lipase GH2 and it was used as free enzyme for catalysis. Aspergillus oryzea has been used majorly for lipase expression as manipulating organism. Adachi et al. [174] created thermostable and solvent tolerant whole-cell biocatalysts that worked as robust biocatalyst for bio-diesel production. Geobacilus thermocatenulatus was used as a source organism from which thermostable lipase (BTL2) gene was obtained and then introduced into Aspergillus oryzea (r-BTL). Immobilized whole-cell r-BTL was then used to convert palm oil into FAME production and the resultant yield obtained was 100%. Whole-cell biocatalyst tolerated 40-50°C temperature and 30% (v/v) solvents such as methanol, DMC, and acetone. These results proved the thermostability and solvent tolerance of Aspergillus oryzea (r-BTL). Lipase from B68 strain of Pseudomonas fluorescence (lipB68) was transformed into E. coli BL21 which resulted in 92% yield upon transesterification. The amazing thing about this experiment was use of that novel psychrophilic lipase (lipB68) that catalyzed reaction at 20°C and energy consumption became reduced in the reaction system
