Nitrogen Cycle and Ammonia-Oxidizing Archaea Study Notes

AOA are one of the most abundant groups of microbes in the world’s oceans.
They play a crucial role in the nitrogen cycle, particularly in the oxidation of ammonia to nitrite where oxygen is required.
Despite their dependence on oxygen, AOA can thrive in oxygen-depleted environments, indicating unique metabolic adaptations.

AOA oxidize ammonia (NH3) to nitrite (NO2−) as per the reaction:
$NH3 + 1.5O2 → NO2− + H2O + H^+$
This process typically requires oxygen, making the presence of AOA in low or anoxic conditions puzzling.

The study utilized trace luminescence oxygen sensors to measure oxygen levels in the presence of Nitrosopumilus maritimus.
Following oxygen depletion, N. maritimus was observed to produce dinitrogen (N2) and a small amount of oxygen, challenging previous notions.

Dark non-photosynthetic oxygen production is rarely observed in nature, known pathways include:
1. Chlorite dismutation during perchlorate and chlorate respiration.
  $ClO2− → Cl− + O2$
2. Detoxification of reactive oxygen species (e.g., H2O2 dismutation).
  $H2O2 → H2 + O2$
3. Nitric oxide (NO) dismutation:
  $2NO2− → 2NO → N2 + O2$
The production pathway in N. maritimus is distinct from these known pathways, involving NO dismutation but producing oxygen and nitrous oxide (N2O).

N. maritimus was cultured aerobically then subjected to conditions of low oxygen (< 5 mM).
Following the consumption of oxygen down to limits of detection (1 nM), oxygen concentration demonstrated an unexpected increase over time.
Multiple controls were employed to eliminate possibilities of abiotic oxygen production:
- No oxygen buildup was seen in abiotic controls or dead cell cultures (killed with mercuric chloride).
- Similar results were observed across various incubation methods, ruling out contamination.
- Oxygen microelectrodes corroborated observations made by optodes.

Oxygen accumulation was linked to ammonia oxidation, leading to reduced oxygen production when cyanide (0.5 mM) was added, indicating dependency.
- Cyanide reduced ammonia oxidation and significantly increased oxygen production (from 14 ± 2 to 65 ± 12 nmol liter⁻¹ hour⁻¹).

Experiments using 15N-labeled ammonium (15N-NH4+) confirmed ongoing ammonia oxidation under oxygen production, with rates indicated:
- 46 nM/hour (Experiment I)
- 39 nM/hour (Experiment II)
Oxygen production rates were calculated to be between 60 and 69 nM/hour, indicating most oxygen was immediately consumed rather than accumulating.
The average cell density during these experiments was about 1.3 ± 0.53 × 10⁷ cells ml⁻¹, resulting in an ammonia oxidation rate per cell between 3-3.5 attomoles cell⁻¹ hour⁻¹.

The production of dinitrogen (N2) was predominantly traced back to the reduction of nitrite as opposed to ammonium.
Source of N2 identified as originating from nitrite reduction, with isotopic analysis supporting the hypothesis that no immediate conversion from ammonium to N2 was detected.
Nitrous oxide (N2O) emerged as an intermediate, suggesting that two-step reactions involving NO and N2O could account for the observed production rates of nitrogen species.

The proposed metabolic pathway indicates:
- Nitrite is converted to nitric oxide (NO) which then dismutates to produce N2 and O2.
- The oxygen produced is consumed for ongoing ammonia oxidation to nitrite, while N2 production is decoupled from oxygen production in specific scenarios.
- The imbalance in production rates suggests other intermediates might exist between NO and the final products (N2, O2).
- N2O is highlighted as a potential critical intermediate in the oxygen production pathway.

The discovery of an oxygen-producing pathway in AOA like N. maritimus can explain their presence in oxygen-depleted marine environments and may require a reevaluation of nitrogen cycling in these contexts.
AOA's ability to produce oxygen from nitrite could significantly affect biogeochemical cycles in the marine ecosystem, particularly in relation to nitrogen fixation and cycling rates.

Researchers suggest that this newly identified oxygen and nitrogen production pathway in N. maritimus contributes to a broader understanding of microbial ecology in anoxic conditions, and challenges prior conceptions of nitrogen cycle dynamics in low-oxygen environments.