Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream

ABSTRACTAmmonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in thein situquantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ15NNO2−and δ18ONO2−, respectively) are geochemical tracers for evaluating the sources and thein siturate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilicThaumarchaeotapopulations composed almost entirely of “CandidatusNitrosocaldus.” The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ18O value of nitrite produced from ammonia oxidation varied with the δ18O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilicThaumarchaeotacould be estimated using δ18ONO2−in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments.IMPORTANCEBecause ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilicThaumarchaeotaand elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

Isotopic Signature of N2O Produced by Marine Ammonia-Oxidizing Archaea

The ocean is an important global source of nitrous oxide (N2O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N2O, but the isotopic signatures of N2O produced by these processes are not consistent with the marine contribution to the global N2O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N2O. Natural-abundance stable isotope measurements indicate that the produced N2O had bulk δ15N and δ18O values higher than observed for ammonia-oxidizing bacteria but similar to the δ15N and δ18O values attributed to the oceanic N2O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N2O source.