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

Abstract

Ammonia 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 the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (␦ 15 N NO2؊ and ␦ 18 O NO2؊ , respectively) are geochemical tracers for evaluating the sources and the in situ rate 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 thermophilic Thaumarchaeota populations composed almost entirely of "Candidatus Nitrosocaldus." The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The ␦ 18 O value of nitrite produced from ammonia oxidation varied with the ␦ 18 O 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 thermophilic Thaumarchaeota could be estimated using ␦ 18 O NO2؊ 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 thermophilic Thaumarchaeota and 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.

Ammonia-Oxidizing Archaea (AOA) Play with Ammonia-Oxidizing Bacteria (AOB) in Nitrogen Removal from Wastewater

“… Schematic illustration of ammonia oxidation pathways in ammonia-oxidizing bacteria (a) and archaea (b). The figure is reproduced from Kozlowski et al and Nishizawa et al [ 66 , 67 ]. Abbreviations: HAO, hydroxylamine dehydrogenase; NIR, nitrite reductase; NOR, nitric oxide reductase.…”

Candidatus Nitrosocaldus cavascurensis, an Ammonia Oxidizing, Extremely Thermophilic Archaeon with a Highly Mobile Genome

“…With the help of molecular marker genes, diverse ammonia oxidizing thaumarchaea were found in hot terrestrial and marine environments ( Reigstad et al, 2008 ; Zhang et al, 2008 ; Jiang et al, 2010 ; Cole et al, 2013 ). Furthermore, in situ nitrification activities up to 84°C were measured using isotopic techniques in AOA-containing habitats in Iceland ( Reigstad et al, 2008 ), in Yellowstone National park ( Dodsworth et al, 2011 ), in a Japanese geothermal water stream ( Nishizawa et al, 2016 ), and in enrichment cultures from Tengchong Geothermal Field in China ( Li et al, 2015 ). In addition, an enrichment of an ammonia oxidizing thaumarchaeon, Ca.…”

Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle

“…Uncultivated thermophiles detected in a high-temperature environment potentially play significant roles in the biogeochemical nitrogen cycle, as indicated by their metabolic potential ( 4 – 6 , 27 ). In order to obtain a better understanding of the mechanisms by which as well as what thermophilic microbial populations contribute to the nitrogen cycle in the natural environment, we collected a microbial mat sample in an ammonia-rich geothermal groundwater stream in Japan and performed continuous cultivation at 65°C and 70°C using flow-through bioreactors with the mat sample as the inoculum as previously reported ( 25 ). At the original sampling site, discharged groundwater has constantly shown a high temperature (69–72°C), circumneutral pH (6.1–6.4), low salinity (0.1%), and high concentrations of NH 4 + (>100 μM) and Fe 2+ (>70 μM) ( 14 , 17 , 24 , 33 ).…”

“…Furthermore, the oxidation of ammonia to nitrite by “ Nitrosocaldus ” spp. at 70°C was demonstrated using bioreactors for more than 2 years ( 25 ). These findings have suggested that the nitrogen cycle, such as nitrification, is driven by these uncultivated microorganisms in this high-temperature environment.…”