L-amino acids affect the hydrogenase activity and growth of Ralstonia eutropha H16

Ralstonia eutropha H16 is a chemolithoautotrophic bacterium with O₂-tolerant hydrogenase (Hyds) enzymes. Hyds are expressed in the presence of gas mixtures (H₂, O₂, CO₂) or under energy limitation and stress conditions. O₂-tolerant Hyds are promising candidates as anode biocatalysts in enzymatic fuel cells (EFCs). Supplementation of 0.5% (w/v) yeast extract to the fructose-nitrogen (FN) growth medium enhanced H₂-oxidizing Hyd activity ~ sixfold. Our study aimed to identify key metabolites (L-amino acids (L-AAs) and vitamins) in yeast extract that are necessary for the increased synthesis and activity of Hyds. A decrease in pH and a reduction in ORP (from + 240 ± 5 mV to - 180 mV ± 10 mV values) after 24 h of growth in the presence of AAs were observed. Compared to the FN-medium control, supplementation of 7.0 μmol/ml of the L-AA mixture stimulated the growth of bacteria ~ 1.9 to 2.9 fold, after 72 h. The whole cells' H₂-oxidizing Hyd activity was not observed in control samples, whereas the addition of L-AAs, mainly glycine resulted in a maximum of ~ 22 ± 0.5 and 15 ± 0.3 U, g CDW^-1 activity after 24 h and 72 h, respectively. Our results suggest a correlation between ORP, pH, and function of Hyds in R. eutropha H16 in the presence of key L-AAs. L-AAs used in small amounts can be proposed as signaling molecules or key components of Hyd maturation. These results are important for the optimization of O₂-tolerant Hyds production as anode biocatalysts.