Acidification Enhances Hybrid N 2 O Production Associated with Aquatic Ammonia-Oxidizing Microorganisms

Ammonia-oxidizing microorganisms are an important source of the greenhouse gas nitrous oxide (N O) in aquatic environments. Identifying the impact of pH on N O production by ammonia oxidizers is key to understanding how aquatic greenhouse gas fluxes will respond to naturally occurring pH changes, as well as acidification driven by anthropogenic CO . We assessed N O production rates and formation mechanisms by communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in a lake and a marine environment, using incubation-based nitrogen (N) stable isotope tracer methods with N-labeled ammonium ( [Formula: see text]) and nitrite ( [Formula: see text]), and also measurements of the natural abundance N and O isotopic composition of dissolved N O. N O production during incubations of water from the shallow hypolimnion of Lake Lugano (Switzerland) was significantly higher when the pH was reduced from 7.54 (untreated pH) to 7.20 (reduced pH), while ammonia oxidation rates were similar between treatments. In all incubations, added [Formula: see text] was the source of most of the N incorporated into N O, suggesting that the main N O production pathway involved hydroxylamine (NH OH) and/or [Formula: see text] produced by ammonia oxidation during the incubation period. A small but significant amount of N derived from exogenous/added [Formula: see text] was also incorporated into N O, but only during the reduced-pH incubations. Mass spectra of this N O revealed that [Formula: see text] and [Formula: see text] each contributed N equally to N O by a "hybrid-N O" mechanism consistent with a reaction between NH OH and [Formula: see text], or compounds derived from these two molecules. Nitrifier denitrification was not an important source of N O. Isotopomeric N O analyses in Lake Lugano were consistent with incubation results, as N enrichment of the internal N vs. external N atoms produced site preferences (25.0-34.4‰) consistent with NH OH-dependent hybrid-N O production. Hybrid-N O formation was also observed during incubations of seawater from coastal Namibia with [Formula: see text] and [Formula: see text]. However, the site preference of dissolved N O here was low (4.9‰), indicating that another mechanism, not captured during the incubations, was important. Multiplex sequencing of 16S rRNA revealed distinct ammonia oxidizer communities: AOB dominated numerically in Lake Lugano, and AOA dominated in the seawater. Potential for hybrid N O formation exists among bo