Geobacter species have previously been considered strict anaerobes (4, 18, 19). However, when organic electron donors are introduced into previously oxic subsurface sediments in order to stimulate anaerobic dissimilatory metal reduction, Geobacter species rapidly become the predominant Fe(III)-reducing microorganisms in a diversity of subsurface sediments (2, 13, 29). Thus, it appears that there is a reservoir of Geobacter species in oxic aquifers.

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 H₂SO₄ 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. ​(Fig.2A).2A). 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. ​(Fig.2A)2A) 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. ​(Fig.2).2). 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.

The finding that G. sulfurreducens can survive exposure to atmospheric oxygen and readily grows at low oxygen concentrations has important implications for the growth and survival of Geobacter species in subsurface environments. In water-saturated soils and aquatic sediments, Fe(III) is likely to be most abundant near the oxic-anoxic interface, where the oxidation of Fe(II) can regenerate Fe(III) produced from microbial Fe(III) reduction (30). Thus, oxygen intrusions into the zone of Fe(III) reduction may be common, especially in sediments with substantial bioturbation or other physical disturbances. In shallow aquifers, an environment where Fe(III) reduction is often an important terminal electron-accepting process, periods of high surface recharge can result in the introduction of dissolved oxygen into previously anoxic zones (31). Microorganisms carrying out anaerobic respiration in the interior anoxic zone of soil aggregates (24, 25, 26, 27) in otherwise oxic soils are also likely to face periodic oxygen exposure. The ability to tolerate exposure to high levels of oxygen and to grow at low oxygen levels may permit G. sulfurreducens to thrive in these environments. It is well documented that once oxygen is consumed in subsurface environments, Geobacter species can rapidly become the predominant Fe(III)-reducing microorganisms (2, 13, 29).