## 4 Discussion  
With each passing year, the generation and accumulation of by-products from various industrial processes poses a greater hazard to the natural environment. Waste glycerol generation during biodiesel production has become an inspiration for many scientists to develop methods for it's effective  
Figure 11: Efficiency of the bioconversion of waste glycerol by free or immobilized cell extracts from _G. oxydans_ in the medium with different initial contents of the substrate. Figure 10: Efficiency of the bioconversion of waste glycerol by free or immobilized cells of _G. oxydans_ in the medium with different initial contents of the substrate. [30 g\(\cdot\)L\({}^{-1}\)]. utilization. Planning and conducting processes for the valorization of industrial wastes, including glycerol, could bring many benefits. It not only enables removing wastes from the ecosystem, but also creates the possibility of obtaining new, industrially valuable compounds, such as dihydroxyacetone. Production of DHA using live whole cells requires their early preparation (including refreshing, proliferation, enzyme activation, and biomass washing, etc.) and maintaining the metabolic activity of cells at a high level under optimal conditions for their growth (pH 5.0, 28 \({}^{\circ}\)C). The disadvantage in using whole cells is in providing complex fermentation media, which is essential for DHA production. Replacement of whole cells by a cell extract allows the process to be run under optimal conditions for glycerol dehydrogenase (pH 7.5, 23 \({}^{\circ}\)C), which catalyzes the oxidation of glycerol to DHA. Immobilization of the biocatalysts facilitates its easy separation from the reaction medium, allows the reaction to become independent from the presence of free, whole, live cells of _G. oxydans_, and also creates an opportunity to reuse the catalyst (gel beads) in subsequent production cycles, which could reduce the cost of the whole process in the future [26]. This study describes microbial conversion of a waste glycerol fraction to DHA with biocatalysts from _G. oxydans_. The highest DHA concentration of 20 g\(\cdot\)L\({}^{-1}\) was obtained after 72 h of biotransformation with free _G. oxydans_ cells in the medium containing an initial waste glycerol concentration of 30 and 50 g\(\cdot\)L\({}^{-1}\). In this case, process efficiency ranged from 70% to 77%, whereas DHA productivity was about 0.3 g\(\cdot\)L\({}^{-1}\cdot\)h\({}^{-1}\). Immobilization of whole, live cells of _G. oxydans_ had a significant impact on the course of the bioconversion process, as it significantly reduced DHA productivity and reaction efficiency. The use of free cell extract resulted in a higher DHA production than in the reaction with the immobilized ones. However, DHA concentrations obtained in these cell extracts were still lower as compared to the reactions with live cells. Reaction efficiency was highest in the medium containing free cell extract as the biocatalyst (up to 98%). The presented data showed that immobilization could, to some extent, reduce DHA production. Presumably, the weight of wet biomass used in this study (1 g of wet biomass per each variant of utilization media) could have an impact on the course of the reaction. At this condensation of the biological material, substrate competition for active sites of the enzyme was so high that it inhibited the reaction. It may also be hypothesized that the number of available active sites was too low to enable conversion of the whole amount of glycerol present in the production media. In turn, immobilization could hinder the gas exchange between the substrate and the cells entrapped in the carrier (or between the substrate and a cell extract). The spatial structure of the alginate carrier and size of alginate beads could also, to some extent, reduce the rate of product diffusion to the reaction medium.