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USGS Scientists Measure New Bacterial Nitrogen Removal Process in Groundwater

USGS scientists prepare a tracer solution in a gas impermeable bladder
U.S. Geological Survey (USGS) scientists prepare a tracer solution to conduct a natural gradient tracer test to measure anammox activity in groundwater. The tracer solution is prepared by pumping groundwater into a gas impermeable bladder to maintain the in situ dissolved oxygen concentration and then mixing in the tracer chemicals. The bladder is contained in an ice-cooled water pool to maintain the groundwater temperature until the solution is pumped back into the aquifer. Photo Credit: Deborah Repert, USGS.

For the first time, U.S. Geological Survey (USGS), Virginia Institute of Marine Science, and the University of Connecticut scientists have detected active anammox bacteria in groundwater.

Anaerobic (without oxygen) ammonium oxidation, or anammox, is a process carried out by naturally occurring bacteria that can simultaneously remove ammonium and reduce nitrite to produce harmless nitrogen gas.

Scientists have known about anammox bacteria, but its presence in groundwater has not been confirmed until this study. In the past, denitrification (the process by which nitrate [NO3-] is reduced to form nitrites, other nitrogen oxides, and eventually nitrogen gas) was thought to be the only major process in groundwater that transformed nitrogen compounds to nitrogen gas. The scientists determined that anammox bacteria were active in the subsurface in a variety of geochemical conditions. The scientists then demonstrated that anammox activity in groundwater can rival that of bacteria responsible for denitrification, with the added benefit that anammox simultaneously removes ammonia, and that water-resource managers may need to consider it when assessing nitrogen contamination in groundwater.

Field Experiment

A diagram of the natural gradient tracer test used to detect anammox in groundwater
A diagram of the natural gradient tracer test used to detect anammox in groundwater. The tracer solution was injected in the well on the left, and wells on the right were used to monitor the subsurface plume caused by the tracer injection. Analytical testing for the isotopic ratios of nitrogen gas (30N2 or 29N2) helped determine rates for the denitrification of nitrate (NO3-) to nitrogen gas (N2) and for anammox of ammonium (NH4+) to N2. From Smith and others, 2015.

The scientists conducted a natural gradient tracer test at the Toxic Substances Hydrology Program's Cape Cod Research Site. The tracer test consisted of injecting a solution of chemical tracers into the subsurface and monitoring the groundwater chemistry and tracer concentrations downstream from the injection well (see diagram). The tracer solution consisted of isotopically labeled nitrite (15NO2, not radioactive) and bromide (Br). The 15NO2 allowed the scientists to determine rates of denitrification and anammox by measuring changes in the isotopic composition of the reaction products. Bromide was used to monitor the location of the injected solution in the subsurface. Advanced molecular techniques were used to detect genes from anammox bacteria (16S ribosomal ribonucleic acid genes [16S rRNA]) and the transcriptional products of those genes (16S messenger RNA [16S mRNA]).

The Results

The scientists determined that anammox was active in the subsurface in a variety of geochemical conditions, even where groundwater ammonium concentrations were low. Many of the geochemical conditions are commonly present in the groundwater and indicate that anammox can be an important process in many types of groundwater conditions. The report documented the competition between anammox and denitrification for nitrogen oxides and explored the effect of altered organic carbon concentrations on that competition.

Environmental Health Implications

Nitrogen contamination of groundwater used for drinking water is a public-health concern. Ingestion of nitrate by infants can interfere with oxygen-carrying capacity of blood in infants, leading to blue skin color known as methemoglobinemia or "blue baby disorder." The results from this study provide information needed to understand processes that remove nitrate from groundwater. Denitrification is usually considered by scientists to be the main mechanism for removal, but the confirmation of anammox in groundwater indicates that the biogeochemical processes affecting the fate and transport of nitrogen in groundwater are more complex than was previously thought. This could be especially true where groundwater has been affected by wastewater with excess nutrients.

The USGS Toxic Substances Hydrology and Hydrologic Research and Development Programs funded this study along with grants from the National Science FoundationEarth Sciences (EAR) Grants 1024662, 1329284, and 1024900.


Smith, R.L., Böhlke, J.K., Song, B., and Tobias, C.R., 2015, Role of anaerobic ammonium oxidation (anammox) in nitrogen removal from a freshwater aquifer: Environmental Science and Technology, v. 49, no. 20, p. 12169–12177, doi:10.1021/acs.est.5b02488.

More Information

This article was featured as an article in the USGS GeoHealth Newsletter, Vol. 13, No. 1, 2016

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