Oxygen tolerance and detoxification mechanisms of highly enriched planktonic anaerobic ammonium-oxidizing (anammox) bacteria

Abstract

Oxygen is a key regulatory factor of anaerobic ammonium oxidation (anammox). Although the inhibitory effect of oxygen is evident, a wide range of oxygen sensitivities of anammox bacteria have been reported so far, which makes it difficult to model the marine nitrogen loss and design anammox-based technologies. Here, oxygen tolerance and detoxification mechanisms of four genera of anammox bacteria; one marine species ("Ca. Scalindua sp.") and four freshwater anammox species ("Ca. Brocadia sinica", "Ca. Brocadia sapporoensis", "Ca. Jettenia caeni", and "Ca. Kuenenia stuttgartiensis") were determined and then related to the activities of anti-oxidative enzymes. Highly enriched planktonic anammox cells were exposed to various levels of oxygen, and oxygen inhibition kinetics (50% inhibitory concentration (IC₅₀) and upper O₂ limits (DO_max) of anammox activity) were quantitatively determined. A marine anammox species, "Ca. Scalindua sp.", exhibited much higher oxygen tolerance capability (IC₅₀ = 18.0 µM and DO_max = 51.6 µM) than freshwater species (IC₅₀ = 2.7-4.2 µM and DO_max = 10.9-26.6 µM). The upper DO limit of "Ca. Scalindua sp." was much higher than the values reported so far (~20 µM). Furthermore, the oxygen inhibition was reversible even after exposed to ambient air for 12-24 h. The comparative genome analysis confirmed that all anammox species commonly possess the genes considered to function for reduction of O₂, superoxide anion (O₂^•-), and H₂O₂. However, the superoxide reductase (Sor)-peroxidase dependent detoxification system alone may not be sufficient for cell survival under microaerobic conditions. Despite the fact that anaerobes normally possess no or little superoxide dismutase (Sod) or catalase (Cat), only Scalindua exhibited high Sod activity of 22.6 ± 1.9 U/mg-protein with moderate Cat activity of 1.6 ± 0.7 U/mg-protein, which was consistent with the genome sequence analysis. This Sod-Cat dependent detoxification system could be responsible for the higher O₂ tolerance of Scalindua than other freshwater anammox species lacking the Sod activity.