Cultivation of Rhodopseudomonas palustris for Hydrogen Production

In this study, Rhodopseudomonas palustris (NCIMB 1774) was used as model organism. Cells were precultured in Van Niels medium—a fast-growing medium containing (per 1 L): 1 g K₂HPO₄, 0.5 g MgSO₄, 10 g yeast extract and the balance deionised water (du Toit and Pott 2021 (link)). After autoclaving (121 °C, 20 min), 10 mL of sterile glycerol (4 M) was added to the medium aseptically (du Toit and Pott 2021 (link)). Bacterial cells were suspended in the medium and grown anaerobically in 500 mL Schott bottles under argon atmosphere. Temperature was maintained at 35 °C (± 0.2 °C) and light intensity was calibrated to 200 W m⁻² (± 20 W m⁻²) in the wavelength range of 500–1100 nm (tungsten filament incandescent light, Eurolux^©, South Africa) (Bosman et al. 2022a (link)) using a handheld spectrophotometer (RGB Photonics, Qmini VIS–NIR). Culturing time was approximately five days to allow cells to reach the mid-logarithmic phase.
All growth and hydrogen production experiments were conducted using a Rhodospirillaceae medium containing (per 1 L): 0.6 g K₂HPO₄, 1.7 g KH₂PO₄, 0.02 g MgSO₄·7H₂O, 0.005 g CaCl₂·2H₂O, 0.4 g NaCl, 0.3 g Na₂S₂O₃, 0.0005 g ferric citrate, 0.0002 g para-aminobenzoic acid, and 1 mL of trace element solution containing (per 1 L): 70 mg ZnCl₂, 100 mg MnCl₂·4H₂O, 60 mg H₃BO₃, 200 mg CoCl₂·6H₂O, 20 mg CuCl₂·2H₂O, 20 mg NiCl₂·6H₂O, and 40 mg NaMoO₄·2H₂O (Pott et al. 2014 (link)). The medium was autoclaved (121 °C, 20 min) and the pH measured at 7.2. Also aseptically added to the medium after autoclaving was a vitamin solution containing (per 1 L): 1.2 g thiamine HCl and 0.01 g cyanocobalamin (filter-sterilized), and lastly 10 mL of 5 M sterile glycerol (final concentration of 50 mM) and 5 mL of sterile glutamic acid (final concentration of 10 mM) were added to the medium aseptically (Pott et al. 2014 (link)).