Award: OCE-1946516

Nitrification is a step in the nitrogen cycle that links the most reduced form, ammonium, to the most oxidized form, nitrate.  Ammonium, which is released during the decomposition of organic matter, is oxidized in the first step of nitrification to nitrite.  The second step of nitrification is the oxidation of nitrite to nitrate by nitrite-oxidizing bacteria (NOB).  Both of these steps are thought to be obligately aerobic.  That is, the microbes that oxidize ammonium and nitrite require molecular oxygen for their respiration.  We observed many years ago that nitrite was apparently oxidized to nitrate in seawater that naturally contained no oxygen. This was highly controversial because all known NOB are obligate aerobes.  We continued to measure and report nitrite oxidation in the absence of oxygen in the severely oxygen depleted depth intervals of the oceanic oxygen minimum zones found offshore of Mexico and South America.  In light of this mounting evidence, others suggested potential explanations, including the use of terminal electron acceptors other than oxygen, undetected contamination with oxygen in the supposedly anoxic incubations, stress-induced reversible nitrate-reduction, etc.  We investigated as many of these as possible experimentally, including experiments carried out for the first time on our research cruise to the Eastern Tropical South Pacific in 2023.  Those experiments were designed to test the following hypothesis: Normal aerobic NOB were performing the apparently anoxic nitrite oxidation.  Their aerobic metabolism was supported by episodic intrusions of dissolved oxygen into the oxygen depleted layer.  In our experiment, nitrite oxidation rates were measured in three treatments:  No oxygen, a short spike of oxygen (to mimic an intrusion), continuous low oxygen.  Incubation bottles were plumbed continuously with gas mixtures and monitored with oxygen sensors to maintain treatment oxygen conditions.  The samples resulting from this experiment are still being analyzed but the results so far do not support the intrusion hypothesis.  In fact, exposing anoxic water to brief pulses of oxygen resulted in less nitrite oxidation than the same water that had not been exposed to oxygen at all.  As part of this project, we also characterized draft genomes derived from NOB living in the anoxic water. We found that the OMZ NOB are the dominant type of NOB in oxygen minimum zones.  And these OMZ NOB are distinct from the more common NOB that are quite abundant in oxygenated regions of the ocean.  The OMZ NOB are present in all the world’s oxygen minimum zones, but not in the rest of the ocean.  Thus, we conclude this project with a remaining mystery.  NOB that are clearly adapted to life without oxygen appear to be performing nitrite oxidation in the absence of oxygen using a mechanism that remains unknown. 

The fact that nitrite can be oxidized to nitrate in the absence of oxygen implies that fixed nitrogen can be retained in the system, even when oxygen is absent.  Usually when oxygen is depleted in the ocean, microbes begin to respire using nitrate as an alternative electron acceptor in the process called denitrification.  Denitrification leads to the production of nutrient (fixed) nitrogen as dinitrogen gas.  Denitrification is considered a loss of fixed nitrogen because most organisms cannot use dinitrogen gas as a nitrogenous nutrient source.  Nitrate, on the other hand, is a good nutrient source for most algae and many other microbes.  Nitrite is a pivot point between loss and retention of fixed nitrogen. Thus, this mysterious mechanism of apparently anaerobic nitrite oxidation acts as a previously unrecognized gate keeper for the retention of biologically available nitrogen.

The main event of this project was the research cruise to the Eastern Tropical South Pacific in November-December 2023.  In addition to the primary research group from Princeton University, we were joined by collaborators from MIT, Stanford University, University of South Carolina, University of Concepcion (Chile), University of Cadiz (Spain) and University of Basel (Switzerland).  Several graduate students and post docs participated in the cruise, and many undergraduates contributed to analysis of the samples back in the laboratory.  Thus this project contributed to training and education and provided novel experiences and inspiration for the next generations informed citizens and future ocean scientists.

 

Last Modified: 09/18/2025
Modified by: Bettie B Ward