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Transesterification reaction for biodiesel production is done with both extracellular and intracellular lipases. Preference for their use is dependent upon either we want simple upstream processes as in case of intracellular lipase or high enzymatic conversion as in case of extracellular lipase. But either we use intracellular lipase or extracellular lipase there is no need for some downstream processes including separation and recycling. Further, both immobilized lipase (extracellular) and immobilized whole-cell lipase (intracellular) are proved to have highly efficient when compared with free lipase used for transesterification [165]. Some experimental studies for both intracellular and extracellular lipases are given in Table 1.5. Intracellular lipases are the enzymes present inside the cells or linked to the walls of cells producing it known as whole-cell biocatalysts. They are not purified or separated from their cells and used as a whole-cell for transesterification (whole-cell biocatalyst) or immobilized (whole-cell immobilization) [55]. Rhizopus and Aspergillus which are filamentous fungi are most widely used as whole-cell biocatalyst for transesterification process [166]. As the main issue related to biodiesel production at large scale is cost and the use of intracellular lipase for transesterification resolves this problem like the use of intracellular lipase is considered cost-effective because of the elimination of costly processes of lipase isolation and purification before immobilization which are required in case of extracellular lipase [167]. Intracellular lipase producing cells or whole-cell biocatalysts are directly employed for immobilization without separation and purification steps for lipase enzymes [55]. Porous biomass support particles (BSPs) are mostly used for whole-cell immobilization. BSPs was developed by Atkinson et al. [168] and used by many scientists and each scientist provide an efficient way of immobilization on it giving out a high yield of biodiesel. A study to check the lipolytic activities of Bacillus species using intracellular as well as extracellular lipase showed higher intracellular lipase activity than extracellular lipase activity [169]. Reported whole-cell biocatalysts are Aspergillus oryzae, Burkholderia cepacia, filamentous fungus Rhizopus chinensis, R. oryzae, and Enterococcus faecium [170–174]. Aspergillus oryzae used as whole-cell biocatalyst exhibited 98.1% relative stability after the fourth batch and produced more than 97% FAME in 32 hours. Extracellular lipases are the purified form mainly fungal and bacterial cells for their use in transesterification process.

Table 1.1 Some of the commonly used bacterial lipases for biodiesel production.

Enzyme	Immobilized on	Substrate	Acyl acceptor	Yield	Reference
Burkholderia cepacia Lipase	Hydrophobic silica monolith	Jatropha oil	Methanol	95%	[125]
Hybrid matrix of alginate and κ-carrageenan	Jatropha curcas L. oil	Ethanol	100%	[126]
κ-carrageenan	Palm oil	Methanol	100%	[127]
Modified attapulgite	Jatropha oil	Methanol	94%	[128]
SiO2-PVA	Babassu oil	Ethanol	100 %	[129, 130]
SiO2-PVA	Babassu oil	Ethanol	100%	[131]
Nb2O5	Babassu oil	Ethanol	74.1%	[132]
Pseudomonas cepacia Lipase	Epoxy-acrylic resin	Waste vegetable oil	Ethanol	46–47%	[133]
Phyllosilicate solgel matrix	restaurant grease	Methanol and Ethanol	98%	[134]
Fe3O4 nanoparticle biocomposite	Soybean oil	Methanol	>88%	[135]
Accurel	Madhuca indica	Ethanol	96%	[136]
Protein-coated microcrystals	Soybean oil	Ethanol	98.93%	[137]
Celite	Jatropha oil	Ethanol	98%	[89]
Pseudomonas fluorescence	Octyl-silica resin	Babassu oil	Ethanol	97.5%	[85]
Hydrophobic sol-gel	Soybean oil	Methanol	65%	[138]
Porous kaolinite particles	Triolein	Methanol and Ethanol	90%	[139]
Asymmetric membrane	Triolein	Methanol	80%	[140]

Table 1.2 Some of the commonly used fungal lipases for biodiesel production.

Enzyme	Immobilized on	Substrate	Acyl acceptor	Yield	Reference
Candida antartica Lipase	Activated textile cloth	Waste cooking oil	Methanol	91.08%	[157]
Polyurethane foam	Soybean oil	Ethanol	81%	[158]
Acrylic resin	Sunflower oil	Ethyl acetate	92.7%	[78]
Soybean oil	Methanol	83.31%	[95]
Candida rugosa Lipase	Microporous bio silica-polymer	Scenedesmus quadricauda microalgal oil	Methanol	96.4%	[59]
Poly(styrenemethacrylic acid) microsphere	Soybean oil	Methanol	86%	[159]
within an activated carbon as support	Palm oil	Methanol	70%	[160]
Thermomyces lanuginosus Lipase	Olive pomace	Pomace oil	Methanol	93%	[161]
Phyllosilicate sol-gel matrix	Grease	Ethanol	80-90%	[162]
Mesoporous polyhydroxybutyrate particles (PHB)	Oleic acid	Methanol and Ethanol	90%	[163]
Toyopearl AF-amino-650M resin	Babassu oil	Ethanol	86.6%	[164]

Table 1.3 Some examples commercial lipases commonly used for biodiesel production.

Enzyme	Substrate	Acyl acceptor	Reaction yield	Reference
Novozyme 435	Chlorella sp. KR-1	Dimethyl carbonate and methanol mixture	90%	[145]
Sunflower oil	Methanol, Absolute ethanol, 1-propanol	>90%	[146]
Oleic acid	Ethanol, n-propanol, and n-butanol	>90%	[147]
Crude soybean oil	Methanol	94%	[148]
Soybean oil	Ethyl acetate	63.3 %	[149]
Soybean oil	Ethanol		[150]
Lipozyme TL IM	Soybean oil	Methanol	>90%	[151]
Crude palm oil	Methanol	96.15%	[90]
Waste cooking oil	Methanol	92.8%	[152]
Palm oil	Oleyl alcohol	79.54%	[153]
Corn oil	Methanol	92%	[154]
Lipozyme RM IM	Crude rapeseed oil	Monoacylglycerol	90%	[155]
Sunflower oil	Methanol	>80%	[82]
Castor oil	Ethanol	98%	[3]
Soybean oil deodorizer distillate	Ethanol	>88%	[156]

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