Exogenous nitrate attenuates nitrite toxicity to anaerobic ammonium oxidizing (anammox) bacteria

Anaerobic ammonium oxidizing bacteria (anammox) can be severely inhibited by one of its main substrates, nitrite (NO₂^-). At present, there is limited information on the processes by which anammox bacteria are able to tolerate toxic NO₂^-. Intracellular consumption or electrochemically driven (transmembrane proton motive force) NO₂^- export are considered the main mechanisms of NO₂^- detoxification. In this work, we evaluated the potential of exogenous nitrate (NO₃^-) on relieving NO₂^- toxicity, putatively facilitated by NarK, a NO₃^-/NO₂^- transporter encoded in the anammox genome. The relative contribution of NO₃^- to NO₂^- detoxification was found to be pH dependent. Exposure of anammox cells to NO₂^- in absence of their electron donating substrate, ammonium (NH₄⁺), causes NO₂^- stress. At pH 6.7 and 7.0, the activity of NO₂^- stressed cells was respectively 0 and 27% of the non-stressed control activity (NO₂^- and NH₄⁺ fed simultaneously). Exogenous NO₃^- addition caused the recovery to 42% and 80% of the control activity at pH 6.7 and 7.0, respectively. The recovery of the activity of NO₂^- stressed cells improved with increasing NO₃^- concentration, the maximum recovery being achieved at 0.85 mM. The NO₃^- pre-incubation time is less significant at pH 7.0 than at pH 6.7 due to a more severe NO₂^- toxicity at lower pH. Additionally, NO₃^- caused almost complete attenuation of NO₂^- toxicity in cells exposed to the proton gradient disruptor carbonyl cyanide m-chlorophenyl hydrazone at pH 7.5, providing evidence that the NO₃^- attenuation is independent of the proton motive force. The absence of a measurable NO₃^- consumption (or NO₃^- dependent N₂ production) during the batch tests leaves NO₃^- dependent active transport of NO₂^- as the only plausible explanation for the relief of NO₂^- inhibition. We suggest that anammox cells can use a secondary transport system facilitated by exogenous NO₃^- to alleviate NO₂^- toxicity.