# Biological Methods in Biodiesel Production and Their Environmental Impact
## 3 Production of Biodiesel by Biological Transesterification Process  
The use of conventional catalysts as acids and bases for biodiesel production leads to the downstream processing connected with by-products' recovery. Transesterification with these catalysts has some disadvantages, such as [20]: low level of free fatty acids (for alkali-catalyzed reactions), requires high temperatures to conduct the process, very difficult process of glycerol purification, transesterification reaction catalyzed by acid needs higher alcohol-to-oil molar ratios, acids cause corrosion of the reactor, soap formation during the base-catalyzed reaction, and purification of methyl esters requires large amounts of water, which influences the operational cost. These disadvantages have led to the investigation of alternative methods of biodiesel production, for example methods that eliminate the adverse effects of chemical catalysts by replacing them with biological ones. Such solutions include catalysis with the participation of enzymes showing substrate specificity in relation to fats. Moreover, replacing chemical with biological catalysts reduces the negative environmental impact of the production technology. This is due to the simplification of the product purification stage and thus energy savings [21]. Lipases are a group of enzymes belonging to the hydrolases. Hydrolases catalyze the decomposition of esters formed by short- and long-chain acids. They show increased activity against esters of glycerol and long-chain fatty acids having more than 10 carbon atoms [22]. Unlike other enzymes, lipases act on water-insoluble substrates. Therefore, the reactions catalyzed by lipases take place at the interfacial border between the lipid phase (substrate) and the water phase in which the enzyme dissolves. In an aqueous medium, lipases catalyze the triglycerides' hydrolysis reaction. For this reason, the water content of the mixture during transesterification is therefore a key parameter and should be kept as low as possible. Otherwise, the thermodynamic equilibrium of the reaction changes from esterification to hydrolysis [23]. Lipases are widely distributed in nature. They are found in seeds and vegetative organs of many plants, in some microorganisms (bacteria and fungi), and in human and animal organisms (liver, pancreas, stomach, and intestine walls). Plant lipases have no industrial application, unlike lipases of microbial and animal origin. Depending on the site of enzyme secretion, we can divide them into extracellular and intracellular. Extracellular lipases require complex purification procedures before being used as a catalyst. In the case of microbial cells producing intracellular lipase, whole cells can be used as catalysts to carry out triglyceride conversion [24]. The decisive factor in the industrial application of lipases is their specificity. Taking this criterion into account, lipases are divided into three groups [25]:  
* Lipases that show broad specificity, i.e., they act on all ester bonds in the triglyceride molecule (e.g., lipases from _Staphylococcus aureus_ and _Candida rugosa_). * Lipases having specificity for positions 1 and 3 in triglycerides (e.g., lipases produced by fungi of the genera _Mucor_ and _Aspergillus_ or by yeast such as _Candida lipolytica_ and _Candida dejormance_). * Lipases having specificity for position 2 in triglycerides (e.g., lipase synthesized by _Galactomyces geotrichum_). Purified lipase can be used commercially in the form of powder. Such enzymes can be used directly in nonaqueous solvent or in a dissolved form in an aqueous medium. Lipases are active over a wide range of pH and temperature. The optimal pH condition of action for plant lipases is 4.5-5.0, and 7.0-8.0 for lipases of animal origin, while the most effective temperature ranges from 35 to 37 \({}^{\circ}\)C. For the microbial lipases, the highest activity falls within the range of pH from 8 to 9 and at 30-40 \({}^{\circ}\)C [25].