CO Metabolism in the Thermophilic Acetogen Thermoanaerobacter kivui

Abstract

The thermophilic acetogenic bacterium Thermoanaerobacter kivui, previously described not to use carbon monoxide as a carbon and energy source, was adapted to grow on CO. This was achieved by using a preculture grown on H2 plus CO2 and by increasing the CO concentration in small, 10% increments.T. kivui was finally able to grow within a 100% CO atmosphere. Growth on CO was found in complex and mineral media, and vitamins were not required. Carbon monoxide consumption was accompanied by acetate and hydrogen production. Cells also grew on synthesis gas (syngas) with the simultaneous use of CO and H2 coupled to acetate production. CO oxidation in resting cells was coupled to hydrogen and acetate production and accompanied by the synthesis of ATP. A protonophore abolished ATP synthesis but stimulated H2 production, which is consistent with a chemiosmotic mechanism of ATP synthesis. Hydrogenase activity was highest in crude extracts of CO-grown cells, and carbon monoxide dehydrogenase (CODH) activity was highest in H2-plus-CO2- or CO-grown cells. The genome of T. kivui harbors two CODH gene clusters, and both CODH proteins were present in crude extracts, but one CODH was more prevalent in crude extracts from CO-grown cells.

FIG 1

Adaptation of T. kivui to growth on CO. (A) A culture adapted to growth on H₂ plus CO₂ served as the preculture for inoculation of fresh medium in the absence of CO (■) or in the presence of 10% CO (●) or 30% CO (○). (B) The culture cultivated on 10% CO from panel A served as the preculture to reinoculate fresh medium in the absence of CO (■) or in the presence of 10% CO (●) or 20% CO (○). (C) The 20% CO culture from panel B was used as the preculture for cultivation on 30% CO (□), which in turn served as the preculture for cultivation on 40% CO (△). The latter served as the preculture for growth on 50% CO (▼). N₂ plus CO₂ (80:20 [vol/vol]) was used as the makeup gas at a final pressure of 2 × 10⁵ Pa. All cultures were grown in complex medium, and growth was measured by monitoring the optical density at 600 nm. Shown are data from one representative experiment out of three independent replicates.

FIG 2

Growth of T. kivui adapted to CO. All cultures were grown in complex medium with increasing concentrations of CO (●, 20%; ■, 50%; ○, 70%; □, 90%; △, 100%) (A) or in mineral medium with 50% CO in the absence (▼) or presence (▲) of additional vitamins (B). N₂ plus CO₂ (80:20 [vol/vol]) was used as the makeup gas at a final pressure of 2 × 10⁵ Pa. Growth was measured by monitoring the optical density at 600 nm. Cultures adapted to growth on CO (at least 3 transfers at the given concentrations) served as precultures for this experiment. Shown are data from one representative experiment out of three independent replicates.

FIG 3

CO consumption, acetate production, and H₂ formation in cells growing on increasing concentrations of CO. The gas phase consisted of 30% CO (A), 50% CO (B), or 70% CO (C). N₂ plus CO₂ (80:20 [vol/vol]) was used as the makeup gas at a final pressure of 2 × 10⁵ Pa. All cultures were grown in complex medium, and growth was measured by monitoring the optical density (●) at 600 nm. The concentrations of CO (▼) and H₂ (◆) in the gas phase and of acetate in the liquid phase (○) were determined by gas chromatography. Shown are data from one representative experiment out of three independent replicates.

FIG 4

Simultaneous consumption of CO and H₂ in cells growing on syngas. The gas phase consisted of 30% CO, 50% H₂, 14% CO₂, and 6% N₂ (A); 50% H₂, 20% CO₂, and 30% N₂ (B); or 30% CO, 14% CO₂, and 56% N₂ (C) at a pressure of 2 × 10⁵ Pa. All cultures were grown in complex medium, and growth was measured by monitoring the optical density (●) at 600 nm. The concentrations of CO (▼) and H₂ (◆) in the gas phase and of acetate in the liquid phase (○) were determined by gas chromatography. Shown are data from one representative experiment out of three independent replicates.

FIG 5

CO consumption and acetate production in resting cells. T. kivui was grown on CO; cells were harvested, washed, and resuspended to a protein concentration of 1 mg/ml in 20 ml buffer (50 mM imidazole, 50 mM KHCO₃, 20 mM MgSO₄, 20 mM KCl, 20 mM NaCl, 4 mM DTE, 4 μM resazurin [pH 7.0]) in 120-ml serum bottles. The cell suspension experiments were carried out at 60°C in a shaking water bath. The gas phase contained 10% CO, and N₂ plus CO₂ (80:20 [vol/vol]) was used as the makeup gas at a final pressure of 2 × 10⁵ Pa. The concentrations of CO (■) in the gas phase and acetate (□) in the liquid phase were determined by gas chromatography. Shown are data from one representative experiment out of three independent replicates.

FIG 6

Influence of a protonophore on acetate, H₂, and ATP formation from CO in resting cells. T. kivui was grown on CO, cells were harvested, washed, and resuspended to a protein concentration of 1 mg/ml in 20 ml of buffer (50 mM imidazole, 50 mM KHCO₃, 20 mM MgSO₄, 20 mM KCl, 20 mM NaCl, 4 mM DTE, 4 μM resazurin [pH 7.0]) in 120-ml serum bottles. Experiments were carried out at 60°C in a shaking water bath. N₂ plus CO₂ (80:20 [vol/vol]) was used as the makeup gas at a pressure of 1 × 10⁵ Pa. Cells and the protonophore were preincubated for 10 min at 60°C. Vials were flushed with CO for 20 s. (A) The ATP content was measured by using the luciferin-luciferase assay with an assay mixture containing no protonophore (●) or 30 μM TCS (○). The arrow indicates the time point of CO flushing. (B) Hydrogen (squares) and acetate (circles) formation was measured by gas chromatography in an assay mixture in the absence of the protonophore (filled symbols) or in an assay mixture containing 30 μM TCS (hollow symbols). Shown are data from one representative experiment out of three independent replicates.

FIG 7

Detection of AcsA and CooS in crude extracts. Cells were grown on CO, H₂ plus CO₂, or glucose and harvested in the exponential growth phase. The cell extract was prepared, and 10 μg protein from each sample was separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The presence of AcsA (A) or CooS (B) was determined immunologically with antibodies raised against heterologously produced proteins.