Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream - PubMed (original) (raw)

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

Manabu Nishizawa et al. Appl Environ Microbiol. 2016.

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 (δ(15)NNO2- and δ(18)ONO2-, 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 δ(18)O value of nitrite produced from ammonia oxidation varied with the δ(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 δ(18)ONO2- 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.

Importance: Because 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.

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Figures

FIG 1

FIG 1

Schematic illustration of ammonia oxidation pathways in ammonia-oxidizing bacteria (A) and archaea (B) and associated isotope effects (based on reference 18). It is generally accepted that not ammonium but ammonia is the substrate for bacterial ammonia monooxygenase (AMO) (65), while the true substrate for archaeal AMO remains to be elucidated. Abbreviations: HAO, hydroxylamine dehydrogenase; NIR, nitrite reductase; NOR, nitric oxide reductase.

FIG 2

FIG 2

Prokaryotic 16S rRNA gene tag sequence compositions of the original microbial mat and enriched samples. In total, 19,901 and 14,973 sequences were identified in the original microbial mat (before incubation) and enriched samples (45 days after incubation). The “Ca. Nitrosocaldus” sp. within the phylum Thaumarchaeota is defined as the tag sequences exhibiting ≥97% sequence similarity to the clonal sequence obtained in this study (see also Fig. S3 and S4 in the supplemental material). Taxonomic groups presenting <1.5% of the total sequences were contained in in the other phyla.

FIG 3

FIG 3

Concentrations of inorganic nitrogen compounds during batch culture experiments. Identical colors indicate data obtained from the same batches.

FIG 4

FIG 4

Change in δ15NNO2− values during ammonia oxidation during batch culture experiments. The δ15NNO2− data on the plot were taken from the experimental periods when the ΣNH3 concentration was higher than 10 μM.

FIG 5

FIG 5

Time course behavior of the δ18ONO2− value during ammonia oxidation in batch culture experiments with water, of which the δ18OH2O values were 116‰ (A), 41‰ (B), and −8‰ (C). Error bars (1σ) were assigned to the δ18ONO2− values. Abbreviation: VSMOW, Vienna Standard Mean Ocean Water.

FIG 6

FIG 6

Correlation diagram between the δ18O values of water and nitrite produced at five sampling periods (0 to 195 min, 195 to 411 min, 411 to 574 min, 574 to 778 min, and 778 to 1,044 min) of batch culture experiments.

FIG 7

FIG 7

Time course behavior of the δ18ONO2− value in the abiotic oxygen isotope exchange experiments. Error bars (1σ) were assigned to the symbols.

FIG 8

FIG 8

Relationship between δ18ONO2−,pro and δ18ONO2−,eq expected in geothermal environments. Error bars (2σ) were assigned to the symbols.

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