Enzymatic reactions play an important role in numerous industrial processes, e.g., in food production, pharmaceuticals and the production of biofuels. However, a major challenge when using enzymes in industrial applications is maintaining their stability and activity, especially under harsh operating conditions. To solve this problem, enzyme immobilization techniques have been developed. Immobilization involves fixing the enzymes on solid supports, which increases their stability, enables their reusability and facilitates the easy separation of reaction mixtures. In addition, immobilized enzymes are ideal for continuous flow systems such as millireactors, where they allow better control of reaction conditions, improving efficiency and product consistency. Glucose dehydrogenase is an important enzyme in biotechnology, particularly in biosensors and the production of biofuels, as it catalyzes the oxidation of glucose to gluconolactone, reducing NAD⁺ to NADH. However, like many other enzymes, it tends to lose activity over time. The immobilization of glucose dehydrogenase in a millireactor provides a controlled environment that increases the stability and activity of the enzyme. The aim of this study was to investigate the effects of different immobilization strategies on the performance of glucose dehydrogenase in a 3D printed millireactor. The enzyme was immobilized in alginate gel in three immobilization strategies: as beads, on the bottom surface, and on both the top and bottom surfaces of the millireactor. The results showed that the application of the enzyme on both surfaces improved the glucose conversion two-fold compared to immobilization in beads and four-fold compared to immobilization only on the bottom surface. The dual-surface enzyme immobilization strategy showed the highest efficiency, achieving the highest conversion of 95.76 ± 1.01% (τ = 131 min) and NADH productivity of 0.166 ± 0.01 mmol/(L·min) (τ = 7.11 min) combined with operational stability over five days. Effective diffusion rates comparable to those of aqueous solutions confirmed the suitability of alginate gels for biocatalysis. These advancements highlight the potential of this modular and scalable platform for various biotechnological applications.

Due to environmental pollution and the depletion of fossil fuels, there is growing interest in the development and use of biofuels as environmentally friendly alternatives. One of the most promising biofuels is biohydrogen, hydrogen produced through sustainable processes using microorganisms such as bacteria and algae. One of the most interesting bacteria for hydrogen production is Ralstonia eutropha H16, known for its ability to produce oxygen-tolerant hydrogenases. These enzymes play a crucial role in biohydrogen metabolism and production. The aim of this work was to determine the optimal conditions (reactor type and synthetic medium composition) for the cultivation of R. eutropha H16. The culture media contained different concentrations of fructose and glycerol (mono- or double-substrate cultivation) and the experiments were carried out in a batch reactor. The initial experiments were carried out with 4 g/L fructose or glycerol in the culture medium at pH 7, T = 30 °C, and 120 rpm. The mathematical model, consisting of the growth kinetics (described by the Monod’s model) and the corresponding mass balances, was proposed. The developed model was validated using two independent experiments with different initial substrate concentrations: 2 g/L glycerol and fructose in one medium and 4 g/L fructose and 1 g/L glycerol in the second. In order to propose the optimal cultivation procedure for future research, the mathematical model simulations were performed for different reactor types (batch, fed-batch, and continuous stirred tank reactors) and different initial substrate concentrations. The most successful experiment was the one with 4 g/L glycerol, where γ_X = 0.485 ± 0.001 g/L of biomass was achieved. Further calculations showed that the most biomass would be produced at higher glycerol concentrations (at γ_G = 6.358 g/L, γ_X = 1.311 g/L should be achieved after 200 h of cultivation) and when using a fed-batch reactor (γ_X = 0.944 g/L after 200 h of cultivation).

Hydrogenases are key enzymes in microbial energy metabolism, catalyzing the reversible conversion between molecular hydrogen and protons. Among them, [NiFe]-hydrogenases are particularly attractive for biocatalytic applications due to the oxygen tolerance of several members of this class and their ability to couple hydrogen oxidation with redox cofactor regeneration. In this study, a recombinant soluble [NiFe]-hydrogenase from Cupriavidus necator H16 was successfully expressed in Escherichia coli BL21 (DE3), purified, and characterised with a focus on its applicability for NAD⁺ regeneration. Unlike previous studies that primarily used native C. necator extracts or complex maturation systems, this work provides the first quantitative demonstration that an aerobically purified recombinant soluble [NiFe]-hydrogenase expressed in E. coli can function effectively as an NAD⁺ regeneration catalyst and operate within multi-enzymatic cascade reactions under application-relevant conditions. The crude recombinant enzyme displayed a volumetric activity of 0.273 ± 0.024 U/mL and a specific activity of 0.018 ± 0.002 U/mg_cells in the hydrogen oxidation assay, while purification yielded a specific activity of 0.114 ± 0.001 U/mg with an overall recovery of 79.2%. The enzyme exhibited an optimal temperature of 35 °C and a pH optimum of 7.00. Thermal stability analysis revealed rapid deactivation at 40 °C (k_d = 0.4186 ± 0.0788 h⁻¹, t_1/2 ≈ 1.7 h) and substantially slower deactivation at 4 °C (k_d = 0.1141 ± 0.0139 h⁻¹, t_1/2 ≈ 6.1 h). Batch NADH oxidation experiments confirmed efficient cofactor turnover and high specificity towards NADH over NADPH. Finally, integration of the hydrogenase into a one-pot two-enzyme glucose oxidation system demonstrated its capacity for in situ NAD⁺ regeneration, although the reaction stopped after approximately 5 min due to acidification from gluconic acid formation, highlighting pH control as a key requirement for future process optimization.