Formate-driven H2 production by whole cells of Thermoanaerobacter kivui

Abstract

Background: In times of global warming there is an urgent need to replace fossil fuel-based energy vectors by less carbon dioxide (CO₂)-emitting alternatives. One attractive option is the use of molecular hydrogen (H₂) since its combustion emits water (H₂O) and not CO₂. Therefore, H₂ is regarded as a non-polluting fuel. The ways to produce H₂ can be diverse, but steam reformation of conventional fossil fuel sources is still the main producer of H₂ gas up to date. Biohydrogen production via microbes could be an alternative, environmentally friendly and renewable way of future H₂ production, especially when the flexible and inexpensive C1 compound formate is used as substrate.

Results: In this study, the versatile compound formate was used as substrate to drive H₂ production by whole cells of the thermophilic acetogenic bacterium Thermoanaerobacter kivui which harbors a highly active hydrogen-dependent CO₂ reductase (HDCR) to oxidize formate to H₂ and CO₂ and vice versa. Under optimized reaction conditions, T. kivui cells demonstrated the highest H₂ production rates (qH₂ = 685 mmol g^-1 h^-1) which were so far reported in the literature for wild-type organisms. Additionally, high yields (Y_(H2/formate)) of 0.86 mol mol^-1 and a hydrogen evolution rate (HER) of 999 mmol L^-1 h^-1 were observed. Finally, stirred-tank bioreactor experiments demonstrated the upscaling feasibility of the applied whole cell system and indicated the importance of pH control for the reaction of formate-driven H₂ production.

Conclusions: The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst for the oxidation of formate to H₂ (and CO₂). The existing genetic tool box of acetogenic bacteria bears further potential to optimize biohydrogen production in future and to contribute to a future sustainable formate/H₂ bio-economy.

Keywords: Acetogenic bacteria; Biohydrogen; Bioreactor; Dark fermentation; HER; Hydrogen-dependent CO2 reductase (HDCR); Optimization; Scale-up; qH2.

Fig. 1

H₂ production from formate by resting T. kivui cells in presence or absence of metabolic inhibitors. Resting cells (0.6 mg mL⁻¹) were added to preheated (60 °C) imidazole buffer (50 mM imidazole, 20 mM MgSO₄, 20 mM KCl, 2 mM DTE, 4 µM resazurin, pH 7) under a N₂ atmosphere. a The reaction was started by addition of 150 mM of sodium formate. H₂ (black squares) and formate concentrations (black triangles up) are plotted against time. b 30 µM DCCD, TCS or ETH2120 or 20 µL ethanol was added to the serum bottles 10 min before the reaction was started by adding 150 mM of sodium, potassium or ammonium formate as indicated. The specific hydrogen production rate was calculated based on the first 15 min after start of the reaction. All data points are mean ± SD, N = 2

Fig. 2

Influence of cell densities on volumetric and specific H₂ production rates of T. kivui. Resting cells (0.3–4 mg mL⁻¹) were added to preheated (60 °C) imidazole buffer (50 mM imidazole, 20 mM MgSO₄, 20 mM KCl, 2 mM DTE, 4 µM resazurin, pH 7) under a N₂ atmosphere. The reaction was started by addition of 300 mM of sodium formate. Specific H₂ production rate (open squares) and volumetric H₂ production rate (black squares) are plotted against the cell densities. The specific H₂ production rates were calculated based on the first 15 min after start of the reaction. All data points are mean ± SD, N = 2

Fig. 3

Formate- and cell density-dependent H₂ production at optimal pH and temperature. Resting cells were added to preheated (70 °C) imidazole buffer (50 mM imidazole, 20 mM MgSO₄, 20 mM KCl, 2 mM DTE, 4 µM resazurin, pH 7) under N₂ atmosphere. The reaction was started by adding sodium formate. Black squares, 0.3 mg mL⁻¹ and 300 mM sodium formate; black triangles down, 4 mg mL⁻¹ and 600 mM sodium formate. All data points are mean ± SD, N = 2

Fig. 4

Formate-driven H₂ production in a batch-operated stirred-tank bioreactor with pH control [Batch 1]. The bioreactor contained imidazole buffer (50 mM imidazole, 20 mM KCl, 2 mM DTE, pH 7.0) at a temperature of 60 °C. The stirrer speed was set at 400 rpm and a continuous gas flow rate of 50 mL min⁻¹ with 100% N₂ was applied. 600 mM of sodium formate was added prior to the start of the experiment. The reaction was started by transferring resting cells to a final total cell protein concentration of 0.6 mg mL⁻¹ into the bioreactor and the pH value was kept constant at 7.2 over the whole process by titration with 4 M H₃PO₄. a The formate consumption, acetate formation and pH curve are shown over time. b Gas evolution of H₂ and CO₂ during the experiment. c Initial formate oxidation and H₂ production kinetics of the first 2 h. d Optical density and total cell protein concentration in the bioreactor during the whole process time. Black triangles up, formate; black squares, acetate; black circles, pH value; black diamonds, H₂; black triangles down, CO₂; empty triangles up, total cell protein concentration; empty diamonds, optical density at 600 nm. All data points are mean ± SD, N = 2

Fig. 5

Formate-driven H₂ production in a batch-operated stirred-tank bioreactor without pH control [Batch 2]. The bioreactor contained imidazole buffer (50 mM imidazole, 20 mM KCl, 2 mM DTE, pH 7.0) at a temperature of 60 °C. The stirrer speed was set at 400 rpm and a continuous gas flow rate of 50 mL min⁻¹ with 100% N₂ was applied. 600 mM of sodium formate was added prior to the start of the experiment. The reaction was started by adding resting cells to a final total cell protein concentration of 0.6 mg mL⁻¹ into the bioreactor. The pH value was not controlled. a The formate consumption and pH are shown over time. b Gas evolution of H₂ and CO₂ during the experiment. c Optical density and total cell protein concentration in the bioreactor during the whole process time. Black triangles up, formate; black circles, pH value; black diamonds, H₂; black triangles down, CO₂; empty triangles up, total cell protein concentration; empty diamonds, optical density at 600 nm. All data points are mean ± SD, N = 2

Fig. 6

Model of the bioenergetics and biochemistry of acetogenesis from formate in T. kivui. HDCR, hydrogen-dependent CO₂ reductase; HydABC, electron-bifurcating hydrogenase; CODH/ACS, CO dehydrogenase/acetyl-CoA synthase; THF, tetrahydrofolate; HCO-THF, formyl-THF; HC-THF, methenyl-THF; H₂C-THF, methylene-THF; H₃C-THF, methyl-THF; Ech, energy-converting hydrogenase; Ech-MetFV, energy-converting hydrogenase complex coupled to methylene-THF reductase; CoFeSP, corrinoid-iron-sulfur-protein; Fd²⁻, reduced ferredoxin. The Ech-MetFV complex is hypothetical. The ion stoichiometries for the membrane proteins have not been determined experimentally. Adapted from Katsyv et al. [55]