# Biological Methods in Biodiesel Production and Their Environmental Impact
## 3 Production of Biodiesel by Biological Transesterification Process
In addition, the surface of magnetic nanoparticles can be modified by depositing functional groups thereon (e.g., amino-silane, hydroxyl group) in order to increase stability, enzymatic efficiency, or the possibility of reusing enzymes [56]. Other nanoparticles are also considered for lipase immobilization by adsorption, e.g., silica nanoparticles, metal-organic framework nanoparticles, carbon nanotubes, organic polymers, nanohybrid materials, or modified fibrous materials [43; 57; 58; 59]. Many of the tested lipases have high catalytic abilities, which can be additionally intensified by their various methods of deposition on carriers. However, the enzymatic transesterification process still requires further studies. Such studies include optimization of conditions for the reaction (choice of amount of alcohol and oil, lipase concentration, water content, stirrer speed, time, and temperature) through appropriate reaction modeling and design reactors [43]. In order to improve homogeneity, the rate of diffusion of the reaction mixture, and the stability of the enzyme, as well as to avoid problems connected with the formation of two phases and mass transfer, the two-step process and co-solvent system (with acetonitrile, tert-butanol, tert-pentanol, 1,4-dioxane, n-hexane, n-heptane, supercritical carbon dioxide, tetrahydrofuran, ionic liquids) are also tested [53; 60; 61]. In addition to the work on the selection of enzyme systems or the preparation of new bio-catalysts, other research is also carried out on the possibility of increasing the efficiency of enzymatic biodiesel production. The directions of research on the development of enzymatic transesterification are shown in Figure 1. These works concern, among others:  
* Selection of a co-solvent system in order to improve mass transfer or minimize the inhibitory effect of methanol/glycerol on enzymes. * Development of a two-step transesterification method to increase product quality and process efficiency. * Obtaining by-products of the highest market value in order to minimize the costs of biodiesel production. Enzymatic biodiesel production can be realized in either batch or continuous systems and in different kinds of reactors, e.g., in a tubular reactor with static mixers, simulated moving bed, packed-bed tubular, expanded bed reactor, basket-type stirred tank reactor, as well as membrane, recirculation, and expanding bed reactors [20]. The packed-bed reactor, stirred tank reactor, and fluidized bed reactor are the most widely used reactors in enzymatic biodiesel production, and also in continuous systems. Examples of bioreactor types used in the enzyme-catalyzed process are presented in Figure 2.  
Figure 1: Schematic of approaches to increasing enzymatic biodiesel production [53]. Figure 2: Schematic representation of the main types of bioreactors: **left**—stirred tank reactors, **right**—column reactors [62]. As shown in Figure 2, the reactors are divided into two groups--mechanically stirred and columnar, capable of operating in batch or continuous mode. In the column reactors, the enzyme is immobilized on the bed, which can be packed and remain stationary (the packed-bed reactor) or suspended (the fluidized bed column reactor), and the substrate solution is pumped through it. In mechanically stirred reactors, the catalyst can be present in or separated from the reaction mixture, e.g., placed in a basket through which the mixture flows. The choice of the kind of reactor depends on the form of the immobilized enzyme, mass transfer, reaction kinetics, nature of the substrate, operational requirements of the reaction, catalytic surface per unit volume of reactor, construction of the bioreactor, replacement of the catalyst and regeneration, operational cost of the bioreactor, and mode of operation [62]. Batch reactors are the most commonly used bioreactors in the enzyme-catalyzed transesterification process. Such a process takes quite a long time so tanks with high volume are required.