# Biological Methods in Biodiesel Production and Their Environmental Impact
## 3 Production of Biodiesel by Biological Transesterification Process
Moreover, research is conducted on the microorganisms (e.g., microalgae) as a biomass rich in lipids [47; 48; 49]. Algae are promising sources in biodiesel production because of the potential high oil content and fast generation of biomass. However, the big problem \begin{table}
\begin{tabular}{c c c c} \hline
**Parameter** & **Alkali-Catalyzed Process** & **Acid-Catalyzed Process** & **Enzyme-Catalyzed Process** \\ \hline Reaction temperature & (60–70) \({}^{\circ}\)C & (70–120) \({}^{\circ}\)C & (20–40) \({}^{\circ}\)C \\ \hline Reaction time & (1–8) h & (3–50) h & (4–72) h \\ \hline Biodiesel yield & (86–99)\% & 97\% & (86–100)\% \\ \hline Alcohol to oil ratio & (3.3–6):1 & (20–30):1 & (4–6):1 \\ \hline Free acids in raw material & Products of saponification & Esters & Esters \\ \hline Water in raw material & \multirow{2}{*}{Affects negatively} & \multirow{2}{*}{-} & Depends on the type and form of lipase and the presence of organic solvent \\ \cline{3-3} Glycerol separation & & & \\ \cline{3-3} Purification of product & Washing & Washing & Not necessary \\ \hline Cost of catalyst & Low & Low & High \\ \hline Energy consumption & High & High & Lower than in alkali- and acid-catalyzed processes \\ \hline Corrosion & Low & High & Low \\ \hline \end{tabular}
\end{table} 
Table 1: Comparison of transesterification methods with the use of different types of catalysts [7; 8; 11; 20; 25; 26; 37]. in algae cultivation is proper temperature for their optimal growing (about 15-30 \({}^{\circ}\)C), which may be a significant obstacle to the common use of this type of raw material for fuel production, especially in the countries with a cold climate. In these kinds of countries, the algae should be cultivated in special kinds of bioreactors using energy to maintain the required temperature and access to light. Due to the high costs of enzymes, which limit the widespread use of biocatalytic transesterification technology, the work carried out concerns the possibility of obtaining cheaper and more effective biocatalysts. The research is carried out in the fields of:  
* Selection of new enzyme sources (new microorganisms such as bacteria, fungi, yeast). Researchers have conducted tests on, for example: _Pseudomonas, Candida rugose, Burkholderia, Thermomyces lanuginose, Candida Antarctica_, and _Aspergillus niger_[43; 50]. Research is also conducted on the direction of the evolution of microorganisms and their rational design in order to increase the tolerance of lipases produced by them to temperatures or the type of solvent [51]. * Using enzymes used so far in other areas, e.g., phospholipes commonly used, inter alia, in the food industry, in fine chemistry or in the production of aromas and fragrances [34; 52], and improvement of enzyme production and quality (increase in catalytic efficiency, substrate specificity, thermostability, etc.) by DNA recombination of microorganisms [37; 51]. * Improvement of enzyme efficiency (combining several types of lipase [53]). Phospholipids are inhibitors of transesterification because they interfere with lipase. Studies curried out in [54] indicated that it is possible to obtain biodiesel from crude oil by the degumming-transesterification process, in which the phospholipes are used with a soluble lipase. * Durability of enzyme immobilization on different materials. The kind of material used depends on the immobilization method. For example, the most commonly used materials for immobilizing lipase by adsorption are silica gel, activated carbon, resins, cellulose, etc. In recent years, there has been a growing interest in the use of nanomaterials to immobilize lipases. Magnetic nanoparticles are of particular interest due to their interesting properties, such us: large surface area, high force of adsorption, low toxicity, and easy separation under the magnetic field [55].