Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

A. woodii grows on 1,2-PD and converts it to propanol and propionate. A. woodii was grown in 50 ml medium in 150-ml serum bottles with 15 mM 1,2-PD. Growth was followed by measuring the OD at 600 nm (circles). Concentrations of 1,2-PD (squares), propanol (inverted triangles), propionaldehyde (triangles), and propionate (diamonds) were determined by gas chromatography. Acetate was determined enzymatically but was not formed during the experiment. All data points are means ± SEM; n = 3 independent experiments.

Conversion of 1,2-PD by resting cells. A. woodii was grown on 1,2-PD, harvested, washed twice, and resuspended to a final protein concentration of 1 mg ml⁻¹ in 10 ml buffer in 150-ml serum bottles under N₂ atmosphere. Concentrations of 1,2-PD (squares), propanol (inverted triangles), propionaldehyde (triangles), and propionate (diamonds) were determined by gas chromatography. Acetate was determined enzymatically but was not formed during the experiment. All data points are means ± SEM; n = 3 independent experiments.

Cell extract (CFE) of A. woodii catalyzes propionaldehyde-dependent NAD reduction and NADH oxidation. The cell extract was prepared from cells grown on 1,2-PD as the substrate. (A) CFE catalyzed propionaldehyde and CoA-dependent reduction of NAD⁺. After the addition of 10 μl cell extract (protein concentration of 8 mg ml⁻¹), CoA (250 μM), NAD⁺ (2 mM), and propionaldehyde (20 mM) were added and the reduction of NAD⁺ was followed at 340 nm. (B) CFE also catalyzed propionaldehyde-specific oxidation of NADH. After the addition of NADH (500 μM), 20 μl cell extract (protein concentration of 6 mg ml⁻¹) and propionaldehyde (20 mM) were added and the oxidation of NADH was followed at 340 nm.

Cellular levels of PduB in cells grown on different substrates. A. woodii was grown on the substrates as indicated and harvested in the late exponential growth phase. Cell extracts were separated on a 12% SDS-PAGE gel. The presence of PduB was determined immunologically with antibodies raised against heterologously produced PduB.

Enzymatic activities of key enzymes of the WLP and 1,2-PD metabolism in cells grown on 1,2-PD, fructose, or H₂ and CO₂. (A) A. woodii was grown on the substrates as indicated, and hydrogenase (H₂ase), formate dehydrogenase (FDH), carbon monoxide dehydrogenase (CODH), and Fd:NAD oxidoreductase as key enzymes for the WLP were measured in the cell extract. (B) Propanol dehydrogenase and propionaldehyde dehydrogenase were measured in the cell extract as key enzymes of the 1,2-PD metabolism. The specific activities in cell extract from cells grown on fructose were set to 1, and the other activities are relative to this value. Actual activities in cell extract from fructose-grown cells were 62 ± 2 U mg⁻¹ (H₂ase), 2 ± 0.1 U mg⁻¹ (FDH), 3.4 ± 0.2 U mg⁻¹ (CODH), 0.15 ± 0.01 U mg⁻¹ (Fd:NAD oxidoreductase), 7 ± 2 mU mg⁻¹ (propanol dehydrogenase), and 0.64 ± 0.12 U mg⁻¹ (propionaldehyde dehydrogenase).

Electron microscopic images of A. woodii grown on 1,2-PD (A) or lactate (B). Cells were grown on the respective substrate to the late exponential growth phase. Cells grown on 1,2-PD showed structures with sizes around 100 to 200 nm, resembling microcompartments (m), and much larger structures that could be storage compartments (s).