Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria

Abstract

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with Nitrosomonas europaea suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with N. europaea and the ammonia oxidizer G5-7, a close relative of Nitrosomonas oligotropha belonging to Nitrosomonas cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of N. europaea. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete N. europaea at growth-limiting substrate concentrations of about 10 micro M ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However, N. europaea displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation, N. europaea became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The Nitrosomonas cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of N. europaea, belonging to Nitrosomonas cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of N. europaea to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.

FIG. 1.

Bacterium-specific DGGE of PCR products of DNA from cultures of N. europaea plus N. winogradskyi and of G5-7 mixed at different ratios. Cells had been grown in separate ammonium-limited chemostats at dilution rates of 0.014 h⁻¹ and were mixed within 6 h after sampling from the effluents of the continuous cultures. DNA was isolated from 10 ml of the mixture. PCR was done with 100 ng of DNA in a 50-μl PCR mixture. The labeled bands (N. europaea, N. winogradskyi, and G5-7) were excised and sequenced. The other bands (not labeled) belong to the heterotrophic bacteria in the enrichment culture. The marker consists of PCR products from Lactobacillus lactis, Escherichia coli, and Micrococcus luteus (from top to bottom).

FIG. 2.

Ammonium dynamics of the continuous cultures of the ammonia oxidizer G5-7 and of N. europaea plus N. winogradskyi before mixing of both cultures for the growth experiment.

FIG. 3.

Ammonia oxidizer G5-7 in coculture with N. europaea and N. winogradskyi in an ammonium-limited chemostat at a dilution rate of 0.014 h⁻¹. At the start of the experiment, 300 ml of the coculture of N. europaea and N. winogradskyi was inoculated into 700 ml of the culture of the ammonia oxidizer G5-7. (Upper panel) Ammonium dynamics. (Lower panel) Population dynamics indicated by DGGE of PCR products obtained with bacterium-specific primers. The marker consists of PCR products from L. lactis, E. coli, and M. luteus (from top to bottom).

FIG. 4.

N. europaea and N. winogradskyi in the coculture with ammonia oxidizer G5-7 in an ammonium-limited chemostat at a dilution rate of 0.014 h⁻¹. At the start of the experiment, 300 ml of the enrichment culture G5-7 was inoculated into 700 ml of the coculture of N. europaea and N. winogradskyi. (Upper panel) Ammonium dynamics. (Lower panel) Population dynamics indicated by DGGE of PCR products obtained with bacterium-specific primers. The marker consists of PCR products from L. lactis, E. coli, and M. luteus (from top to bottom).

FIG. 5.

Nitrite and nitrite plus nitrate production by N. europaea and the ammonia oxidizer G5-7 present in the enrichment culture after different periods of starvation (mean and standard deviation for four replicates). (A) Nitrite production by N. europaea over more than 200 h. (B) Nitrite production by N. europaea during the first 12 h. (C) Nitrite plus nitrate production by the enrichment culture containing the ammonia oxidizer G5-7 over more than 200 h. (D) Nitrite production by the ammonia oxidizer G5-7 during the first 12 h.