Isolation of Ammonium-Oxidizing Iron Reducers from a Wetland Soil–Derived Enrichment Culture

Begin with a wetland soil-derived enrichment culture containing bacterial communities.

The culture exhibits Feammox activity, ammonium oxidation coupled to ferric iron reduction, indicating the enrichment of a bacterium capable of this metabolic process.

Serially dilute the culture using media.

Streak the diluted samples onto test agar plates supplemented with ferric iron and ammonium.

Use control plates that lack either ferric iron or ammonium, or contain ferric iron with organic carbon instead of ammonium, to rule out iron reduction through non-Feammox pathways.

Incubate all plates in an anaerobic glove bag containing a nitrogen-carbon dioxide mixture.

Bacteria that oxidize ammonium while reducing ferric to ferrous iron form gray colonies only on the test plate.

This confirms the presence of Feammox bacteria in the enrichment culture.

Other bacteria that use Feammox byproducts on the test plate or organic carbon on the control plate do not form gray colonies.

Isolate the gray colonies and subculture them for further analysis.