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  • 1
    Publication Date: 2020-02-06
    Description: Ammonia-oxidizing microorganisms are an important source of the greenhouse gas nitrous oxide (N2O) in aquatic environments. Identifying the impact of pH on N2O 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 CO2. We assessed N2O 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 15N-labeled ammonium (15NH4+) and nitrite (15NO2-), and also measurements of the natural abundance N and O isotopic composition of dissolved N2O. N2O 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 NH4+ was the source of most of the N incorporated into N2O, suggesting that the main N2O production pathway involved hydroxylamine (NH2OH) and/or NO2- produced by ammonia oxidation during the incubation period. A small but significant amount of N derived from exogenous/added 15NO2- was also incorporated into N2O, but only during the reduced-pH incubations. Mass spectra of this N2O revealed that NH4+ and 15NO2- each contributed N equally to N2O by a "hybrid-N2O" mechanism consistent with a reaction between NH2OH and NO2-, or compounds derived from these two molecules. Nitrifier denitrification was not an important source of N2O. Isotopomeric N2O analyses in Lake Lugano were consistent with incubation results, as 15N enrichment of the internal N vs. external N atoms produced site preferences (25.0-34.4%) consistent with NH2OH-dependent hybrid-N2O production. Hybrid-N2O formation was also observed during incubations of seawater from coastal Namibia with 15NH4+ and NO2-. However, the site preference of dissolved N2O 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 N2O formation exists among both communities, and at least in AOB-dominated environments, acidification may accelerate this mechanism.
    Type: Article , PeerReviewed
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  • 2
    Publication Date: 2023-03-08
    Description: © The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Visser, A.-N., Wankel, S., Frey, C., Kappler, A., & Lehmann, M. Unchanged nitrate and nitrite isotope fractionation during heterotrophic and Fe(II)-mixotrophic denitrification suggest a non-enzymatic link between denitrification and Fe(II) oxidation. Frontiers in Microbiology, 13, (2022): 927475, https://doi.org/10.3389/fmicb.2022.927475.
    Description: Natural-abundance measurements of nitrate and nitrite (NOx) isotope ratios (δ15N and δ18O) can be a valuable tool to study the biogeochemical fate of NOx species in the environment. A prerequisite for using NOx isotopes in this regard is an understanding of the mechanistic details of isotope fractionation (15ε, 18ε) associated with the biotic and abiotic NOx transformation processes involved (e.g., denitrification). However, possible impacts on isotope fractionation resulting from changing growth conditions during denitrification, different carbon substrates, or simply the presence of compounds that may be involved in NOx reduction as co-substrates [e.g., Fe(II)] remain uncertain. Here we investigated whether the type of organic substrate, i.e., short-chained organic acids, and the presence/absence of Fe(II) (mixotrophic vs. heterotrophic growth conditions) affect N and O isotope fractionation dynamics during nitrate (NO3–) and nitrite (NO2–) reduction in laboratory experiments with three strains of putative nitrate-dependent Fe(II)-oxidizing bacteria and one canonical denitrifier. Our results revealed that 15ε and 18ε values obtained for heterotrophic (15ε-NO3–: 17.6 ± 2.8‰, 18ε-NO3–:18.1 ± 2.5‰; 15ε-NO2–: 14.4 ± 3.2‰) vs. mixotrophic (15ε-NO3–: 20.2 ± 1.4‰, 18ε-NO3–: 19.5 ± 1.5‰; 15ε-NO2–: 16.1 ± 1.4‰) growth conditions are very similar and fall within the range previously reported for classical heterotrophic denitrification. Moreover, availability of different short-chain organic acids (succinate vs. acetate), while slightly affecting the NOx reduction dynamics, did not produce distinct differences in N and O isotope effects. N isotope fractionation in abiotic controls, although exhibiting fluctuating results, even expressed transient inverse isotope dynamics (15ε-NO2–: –12.4 ± 1.3 ‰). These findings imply that neither the mechanisms ordaining cellular uptake of short-chain organic acids nor the presence of Fe(II) seem to systematically impact the overall N and O isotope effect during NOx reduction. The similar isotope effects detected during mixotrophic and heterotrophic NOx reduction, as well as the results obtained from the abiotic controls, may not only imply that the enzymatic control of NOx reduction in putative NDFeOx bacteria is decoupled from Fe(II) oxidation, but also that Fe(II) oxidation is indirectly driven by biologically (i.e., via organic compounds) or abiotically (catalysis via reactive surfaces) mediated processes co-occurring during heterotrophic denitrification.
    Description: This study was supported by the German Research Foundation (DFG)-funded RTG 1708 “Molecular Principles of Bacterial Survival Strategies.” Work performed under the supervision of ML was supported by the University of Basel funds.
    Keywords: Denitrification ; Nitrate/nitrite isotopes ; Iron oxidation ; Isotope fractionation ; Carbon substrate
    Repository Name: Woods Hole Open Access Server
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