Microaerobic Conditions Are Required for Magnetite Formation Within Aquaspirillum magnetotacticum

The amount of magnetite (Fe304) within mag­ netosomes of the microaerophilic bacterium AquQspirillum magnetotacticum varies with oxygen and nitrogen supply. The development of optical methods for directly measuring cell magnetism in culture samples has enabled us to quantitate bacterial Fe304 yields. We measured final cell yields. average cell magnetic moments, and magnetosome Yields of growing cells. Cultures were grown with N03-, NH4+, or both, in sealed, unshaken vials with initial headspace P02 values ranging from 0 (trace) to 21 kPa. More than 50% of cells had detectable magnetosomes only when grown in the range of 0.5-5.0 kPa Oz. Optimum cell magnetism (and Fe304 formation) occurred under mi­ croaerobic conditions (initial headspace POz of 0.5-1 kPa) re­ gardless of the N source. At optimal conditions for Fe304 formation, denitrifying cultures produced more of this mineral than those growing with Oz as the sole terminal electron ac­ ceptor. This suggests that competition for Oz exists between processes involving respiratory electron disposal and FC304 formation. Oxygen may also be required for Fe304 formation by other species of magnetotactic bacteria. Bacterial Fe304 appears to persist in sediments after death and lysis of cells. The presence of bacterial Fe304 in the fossil and paleomagnetic records may be of use as a retrospective indicator of sedimentation that has occurred in microaerobic waters. Introduction Magnetotactic bacteria contain intracellular, enveloped, mag­ netic particles or "magnetosomes" (Fig. 1). The magnetosomes of strains that have been studied consist of Fe304, the iron oxide magnetite (Frankel et aI., 1983; Towe and Moench, 1981). Aquaspirillum magnetotacticum strain MS-l, the most thorFig. 1. Transmission electron micrograph of negatively stained cells of A. magnetotacticum. Cells contain chains of magnetosomes which are often bisected by division planes. Bar = I fLm. oughly studied magnetic bacterium, is an obligate micro­ aerophile. Cells denitrify microaerobically and concomitantly consume Oz. However, unlike most other denitrifiers, they do not grow under strictly anaerobic conditions, even with N03­ present (Blakemore et al., 1979; Bazylinski and Blakemore, 1983a). Rates of growth and intracellular Fe304 formation (Blakemore et al., 1979), denitrification (Bazylinski and Blake­ more, 1983a), and nitrogen fixation (Bazylinski and Blakemore, 1983b) by this organism are noticeably depressed when the ini­ tial Oz tension in the culture headspace is greater than 6 kPa. Cultures growing with Oz as the terminal electron acceptor (e.g., with NH4+ in lieu of N03as the sole N source) contain fewer magnetic cells than those which are denitrifying. However, the effect of Oz and N source on Fe304 formation by cells has not previously been quantitated due to lack of a suitable method to measure cell magnetism. We have previously estimated cell magnetism in liquid cul­ tures by microscopically noting the extent to which suspended cells align with an applied magnetic field, or by qualitative esti­ mates of their differential light scattering when cultures are held in a continuously rotating magnetic field (e.g., over a laboratory magnetic stirrer). The cells exhibit optical birefringence, how­ ever, and methods have been developed to quantitate cell mag­ netic moments (values of j..L) from measurements of magnetic field-dependent birefringence (Rosenblatt et al., 1982). We undertook the present study to clarify the relationship between available Oz and cell magnetism in cultures grown with N03and/or NH4+ as the sole N source. Our findings are rele­ vant to the biogeochemistry of iron, and, if representative of other species, indicate that bacteria produce Fe304 oniy with Oz available but under microaerobic conditions. Materials and Methods A. magnetotacticum was cultured in a chemically defined medium (Blakemore et al., 1979) containing tartaric acid in lieu of succinic as a carbon source, and either 2 mM NaN03, (NH4hS04, or both N sources, each" at 1 mM. Cells were in­ oculated into 155-ml sealed serum vials, each containing 55 ml of culture medium under a gas atmosphere of known composition. Cultures were prepared in triplicate using seven different initial headspace gas compositions: 0.0 (trace) 0.5, 1.0,2.5,5.0, 10, and 21% (vol/vol) of O2 in N2• It is important to note that the culture system used lacked extensive redox buffering. Medium contain­ ing resazurin was colorless prior to inoculation but was faintly pink just afterward. Thus, trace amounts of O2 added with the inoculum were sufficient to allow some cell growth. No sample in this study was strictly anaerobic prior to incubation. In confirmation of earlier published findings, A. magnetotacticum, when tested under stringent conditions of anoxia, failed to grow even with N03 present. Cultures were each inoculated to 1.3 x 10 cells/ml and incubated at 30°C without shaking. At the end of growth the following were evaluated: 1. cells/ml (by means of direct microscopic counts) 2. magnetosomes per cell (by means of direct transmission electron microscopic examination; magnetosomes within 100 cells from each culture were counted) 3. average cell magnetic moment (by measuring magnetic field-dependent culture birefringence) The apparatus used to measure magnetic field-dependent cul­ ture birefringence has been described (Rosenblatt et aI., 1982). Bacteria in 5 ml of culture fluid were fixed with a drop of 10% glutaraldehyde. Fixed cells in suspension were placed in a 3-ml cuvette (I-cm path length) situated within a Helmholtz coil pair that was used to vary the magnetic field applied to the sample. This entire assembly was placed within a Mumetal canister to cancel the ambient laboratory magnetic field on the cells. The optic axis of the sample was perpendicular to the applied mag­ netic field. Increases in the field strength over the range 0.1-25.0 Oe produced a corresponding increase in measured birefring­ ence. Only magnetic cells contributed to the measured effect. The data were analyzed to yield the average value and distribu­ tion of Jl. for those cells.