# Biological Methods in Biodiesel Production and Their Environmental Impact
## 3 Production of Biodiesel by Biological Transesterification Process
The authors of [26] isolated the LipB68 enzyme, which enables the transesterification process to be carried out with 92% efficiency within 12 h. This enzyme has a unique optimum temperature of 20 \({}^{\circ}\)C.  
The use of enzymes in biodiesel production has a lot of advantages, such as [20]: the possibility to work under different environments (aqueous and nonaqueous), the possibility of enzyme production in bulk, ease of adaptation of enzymes, no separation is required when transesterification is conducted in a packed-bed reactor, and the good thermalstability of lipases enables to carry out the process at elevated temperatures. Lipases also have their drawbacks. Under the influence of methanol, they are deactivated, so the efficiency of the process may be low. The problem can be prevented by gradual methanol dosing. As a result, the conversion efficiency of oil to biodiesel is around 98% [27]. The inhibitory effect of methanol on the process can also be minimized by using the addition of tert-butanol as a solvent. The authors of [28] studied enzymatic production of biodiesel in t-butanol solvent with the use _Candida antarctica_ lipase as a catalyst. It has been stated that for a reaction mixture containing 32.5% t-butanol, 13.5% methanol, 54% oil, and 0.017 g of enzyme (g oil)\({}^{-1}\), the yield of biodiesel was 97% (after 24 h at 50 \({}^{\circ}\)C). A compound that also strongly inhibits the enzymatic transesterification process is glycerin (by-product of the reaction). To avoid this effect, glycerin should be removed from the reaction medium, for example by washing the immobilized enzyme with isopropanol after each transesterification cycle [29]. Another way to remove glycerin is dialysis into the aqueous phase, e.g., using a flat-sheet membrane module [30]. The presence of phospholipids in the oil may also inhibit the process. The authors of [31] studied methanolysis of soybean oil with the use of _Candida antarctica_ lipase. They stated that crude oil did not undergo the transesterification process while degummed oil did. The inhibition was caused by the interaction of the phospholipids with lipase, so before the process the oil should be degummed. Other disadvantages of the lipase-catalyzed process are the high costs of the enzymes (caused by costs of separation and purification) and their poor stability. These drawbacks can be partially overcome by immobilization of enzymes, i.e., the binding of the enzymes to a solid support that is insoluble in the reaction medium, without affecting their catalytic properties. Immobilization of the enzyme allows for easier separation of the catalyst (e.g., by filtration, centrifugation) from the reaction mixture after the end of the process. Immobilization also prolongs the catalytic activity of the enzyme, which enables its re-use in subsequent reaction cycles [32]. This process stabilizes and strengthens the entire protein structure, which increases the resistance of the enzyme to the adverse effects of pH, temperature, or chemicals. Additionally, immobilized enzymes retain high activity and catalytic functions in organic solvents, which is not possible with the use of a soluble form of this catalyst. Binding the enzymes to the substrate allows the reaction to be carried out in various reactors (batchwise, continuous), because it is possible to constantly supply the substrates and collect the reaction products [33]. The factor determining the division of immobilization methods is the type of interactions between the catalyst and the carrier. There are five main methods: adsorption, covalent binding, entrapment, encapsulation, and cross-linking. The choice of the method depends on several factors, such as the type of carrier, the type of enzyme, the nature of the catalyzed reaction, or the required stability of the immobilized catalyst [20].