In order to reduce the use of fossil fuels as an energy source, the use of green alternative fuels, such as hydrogen, is being promoted. Of particular interest is the production of biohydrogen, which is based on the decomposition of organic substances (e.g. organic waste) by microorganisms. In this work, the influence of the composition of the culture medium (proportion of fructose and glycerol) on the growth of the bacterial strain Ralstonia eutropha H16, which produces four hydrogenases, promising biocatalysts for the production of biohydrogen, was analyzed. The efficacy of six different agents (EDTA, Triton X-100, SDS, CTAB, PEG and BSA) on the permeabilization of R. eutropha H16 cells was also analyzed to release the desired hydrogenase enzymes at the highest possible concentration. The results show that the highest biomass concentration of 0.470 ± 0.134 g/L was achieved in the experiment with 4 g/L glycerol solution in the culture medium. In addition, the highest protein concentration of 25.536 ± 1.102 mg/ml was released when CTAB was used as a permeabilizing agent. The permeabilizate obtained with Triton X-100 showed the highest enzymatic activity due to the specific nature of the interaction with the surface structures of the cells.