In order to determine whether G. sulfurreducens
is able to grow with oxygen as the sole electron acceptor, G. sulfurreducens
was grown in a modification of the previously described anoxic, bicarbonate-buffered medium (7
) with excess acetate (15 mM) as the electron donor and limiting fumarate (20 mM) as the electron acceptor. Cysteine and resazurin were omitted from the medium. The atmosphere overlying the medium was He-CO2
(80:20). Cultures were grown in 1.1-liter bottles in a manner similar to the method described by Dilling and Cypionka (9
), with 200 ml of medium at 30°C with shaking at 45 rpm. Acetate and fumarate concentrations were measured with a liquid chromatograph (model SPD-10A; Shimadzu, Kyoto, Japan) equipped with a 100- by 7.8-mm Aminex Fast Acid column (Bio-Rad, Hercules, Calif.) with an 8 mM H2
mobile phase at a flow rate of 0.5 ml/min. Oxygen concentration in the headspace was measured with a Hewlett-Packard series HP6890 gas chromatograph equipped with a thermal conductivity detector (Agilent Technologies, Inc., Albany, N.Y.) and a Carboxen 1010 PLOT capillary column (Supelco, Bellefonte, Pa.). The temperatures of the detector, injector, and column were 230, 230, and 40°C, respectively. Cell number was determined with acridine orange staining and epifluorescence microscopy, as previously described (20
). When a 2% inoculum of mid-log-phase cells grown in anoxic acetate-fumarate medium was added to fresh anoxic medium, the cells consumed acetate and grew to an absorbance of ca. 0.3 within 2 days (Fig. ). At this point, all of the fumarate in the medium had been consumed, and no further growth was observed unless the medium was amended with additional fumarate (data not shown). When oxygen was added to the headspace at an initial concentration of 5 or 10%, G. sulfurreducens
grew, as was indicated by an increase in absorbance (Fig. ) and a net increase of 1.93 × 108
and 2.82 × 108
cells/ml, respectively. Cell growth was accompanied by the concurrent consumption of acetate and oxygen (Fig. ). If the concentration of oxygen was increased to 15 or 20%, oxygen consumption stopped within a day and cell growth was less than that with 5% oxygen, suggesting that aerobic growth could not be sustained at these higher oxygen levels.
FIG. 2. Oxygen-dependent growth of G. sulfurreducens. (A) Growth (open symbols) and acetate consumption (filled symbols) following the introduction of 5% (○), 10% (□), 15% (), or 20% () oxygen into the headspace of cultures pregrown (more ...)
In order to determine if sustained growth on acetate and oxygen was possible, cells were initially grown on acetate with 5 mM fumarate as the electron acceptor; this was followed by oxygen addition after fumarate was depleted. Growth on acetate and oxygen continued for 10 days with multiple additions of 5% oxygen and 15 mM acetate (Fig. ). When 10% oxygen was added, growth stopped after the second addition. However, growth could be sustained after an initial addition of 10% oxygen if 5% oxygen was used for subsequent additions (data not shown).
FIG. 3. Sustained growth of G. sulfurreducens on oxygen. (A) Growth (open symbols) and acetate consumption (filled symbols) following the introduction of 5% oxygen into the headspace of cultures pregrown on 5 mM fumarate; (B) oxygen consumption by G. sulfurreducens (more ...)
Growth on oxygen required that the cells be pregrown on fumarate. There was no growth when either anaerobically grown cells or cells that had been grown with 5 or 10% oxygen were inoculated directly into fresh medium with 5%, 10%, or atmospheric oxygen, even when the medium was supplemented with organic acids that were present in the fumarate-grown cultures, such as succinate and malate.
The stoichiometry of oxygen to acetate consumption was 2.4 ± 0.8 (mean ± standard deviation from four replicate experiments, as determined from the results shown in Fig. and ) for the cultures grown with an initial oxygen concentration of 5%, and there was no growth on oxygen in acetate-depleted cultures. These results suggest that acetate was oxidized to carbon dioxide, with oxygen serving as the sole electron acceptor according to the reaction CH3COOH + 2O2 → 2CO2 + 2H2O. In comparison, the stoichiometry of fumarate-to-acetate consumption for the initial growth on fumarate was 3.8 ± 0.6, which also corresponded well with the predicted reaction, where acetate is oxidized to CO2, with fumarate serving as the sole electron acceptor: 4COOHCHCHCOOH + CH3COOH + 2H2O → 4COOHCH2CH2COOH + 2CO2.The levels of growth (measured as the increase in cell number per millimole of acetate consumed) were similar in the presence of oxygen and in the presence of fumarate. Growth on fumarate alone yielded 5.7 × 107 cells per mmol of acetate, while growth on 5 and 10% oxygen yielded 5.9 × 107 and 6.3 × 107 cells per mmol of acetate, respectively.