Enhanced growth of Acidovorax sp. strain 2AN during nitrate-dependent Fe(II) oxidation in batch and continuous-flow systems

Abstract

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO₃⁻, 8 to 9 mM Fe²⁺, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO₃⁻, 20 μM acetate, and 50 to 250 μM Fe²⁺, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.

Fig. 1.

(a and b) Transformations of NO₃⁻ and Fe²⁺ by Acidovorax sp. 2AN in batch reactors using different media. NO₃⁻ was present in all of the replicates at an initial concentration of 4.6 to 6 mM. When present, Fe²⁺ and acetate were present at initial concentrations of 8 to 8.4 mM and 1.5 to 1.7 mM, respectively. Medium O (organotrophic) contained only acetate as the electron donor, medium C (chemolithotrophic) contained only Fe²⁺ as the electron donor, and medium M (mixotrophic) contained both acetate and Fe²⁺. The data for heat-killed controls are presented in the supplemental material. (c) Comparative growth of strain 2AN in different media when sequentially transferred for 30 days. The data are presented as mean ± the standard deviations (n = 3). When not shown, error bars are smaller than the symbol size.

Fig. 2.

(a) Transformations of NO₃⁻, Fe²⁺, and acetate by Acidovorax sp. 2AN under mixotrophic (containing both Fe²⁺ and acetate) culture conditions. (b) Cell numbers. Nitrate was present in all of the replicates at an initial concentration of 5 mM. Fe²⁺ and acetate were present at initial concentrations of 8.3 and 1.6 mM, respectively. An additional 1.6 mM acetate was added on day 3. The data for heat-killed controls were similar to that shown in the supplemental material and is omitted for clarity. The data are presented as mean ± the standard deviations (n = 3). When not shown, error bars are smaller than the symbol size.

Fig. 3.

(a) Transformations of NO₃⁻ and Fe(II)-EDTA by Acidovorax sp. 2AN in batch cultures. Medium C contained only 5 mM Fe(II)-EDTA as the electron donor. Medium M contained both 5 mM Fe(II)-EDTA and 1.2 mM acetate. All media and controls contained 5 to 6 mM NO₃⁻. The data for heat-killed controls are presented in the supplemental material. The dotted and dashed lines, respectively, represent points when 7 mM nitrate and 4 mM Fe(II)-EDTA were re-added. (b) Consumption of acetate and accumulation of extracellular nitrite in the medium. The dashed lines represent the points when ca. 1 mM acetate was re-added. (c) Growth of strain 2AN in medium C and medium M. The data are presented as mean ± the standard deviations (n = 3). When not shown, error bars are smaller than the symbol size.

Fig. 4.

Comparisons of growth curves of Acidovorax sp. 2AN in organotrophic and mixotrophic media with various Fe²⁺ concentrations. Each panel represents a different set of experiments. The same inoculum was used in each set of experiments as described in the text. All media contained 100 μM NO₃⁻ and, except where noted in panel d, 20 μM acetate. The data are presented as mean ± the standard deviations for cell counts in 10 microscopic fields.

Fig. 5.

Relationship of initial growth rate of Acidovorax sp. 2AN to [Fe²⁺] used in the continuous-flow experiments. Mean growth rates were calculated using the cell count data from day 2 to day 4 depicted in Fig. 4. The dashed line represents the regression line with R² value of 0.966.

Fig. 6.

SEM images of Acidovorax sp. 2AN grown in different conditions. (a) 4-day-old cells grown on organotrophic medium O in batch culture. (b) A single cell with uniform Fe(III) oxyhydroxide encrustations after 2 h of incubation in mixotrophic medium M in batch culture. (c) Heavily encrusted cells and cell-mineral aggregates after a 4-day incubation in medium M in batch culture. (d) Cells from continuous-flow systems containing 250 μM Fe²⁺ after 16 days showing no cellular encrustation or cell-mineral aggregates. The images in panels a and b were made on gold-coated samples in high vacuum mode, and the images in panels c and d were made on uncoated samples in low vacuum mode.