Description: We investigated the microbial and abiotic N2O cycle in the water column of iron-rich, meromictic Lake La Cruz, Spain, during two sampling campaigns in March 2015 and March 2017. At the deepest point of the lake, we used a profiling in situ analyzer equipped with several probes and optodes to detect physicochemical parameters. In addition, we collected water column samples via an in situ pump in order to analyze concentrations of N, S, and Fe species as well as isotope characteristics of several N species. In 2017, we used a Niskin bottle to take water samples from 8.0 and 14.5 m depth for two types of incubation experiments. In the first set of experiments, we added 15N-labeled substrates, and in some incubations Fe2+, to filtered and unfiltered lake water, and analyzed the produced N2O, N2, and NH4+. In the other experiment, we determined the N and O isotope effects of NO2- and N2O during chemodenitrification (reaction of NO2- and Fe2+) in anoxic and sterile lake water from 14.5 m depth.

Description: Upwelling systems are significant sources of atmospheric nitrous oxide (N₂O). The Benguela Upwelling System is one of the most productive regions worldwide and a temporally variable source of N₂O. Strong O₂ depletions above the shelf are favoring periodically OMZ formations. We aimed to assess underlying N₂O production and consumption processes on different temporal and spatial scales during austral winter in the Benguela Upwelling System, when O₂-deficiency in the water column is relatively low. The fieldwork took place during the cruise M157 (August 4th – September 16th 2019) onboard the R/V METEOR. This expedition included four close-coastal regions around Walvis Bay at 23°S, which presented the lowest O₂ concentrations near the seafloor and thus may provide hotspots of N₂O production. Seawater was collected in 10 L free-flow bottles by using a rosette system equipped with conductivity-temperature-depth (CTD) sensors (SBE 911plus, Seabird-electronics, USA). Incubation experiments were performed using stable isotope ¹⁵N-tracers. Seawater samples for ¹⁵N-tracer incubations and natural abundance N₂O analysis were collected from 10 L free-flow bottles and filled bubble-free into 125 mL serum bottles. The samples for natural abundance N₂O analysis were immediately fixed with saturated HgCl₂ and stored in the dark. To perform the incubation, we added ¹⁵N-labeled NO₂-, NO₃⁻ and NH₄⁺ to estimate the in-situ N₂O production rates and associated reactions. To determine a single rate, the bottles were sacrificed after tracer addition, and within the time interval of 12 h, 24 h and 48 h by adding HgCl₂. Rates were calculated based on a linear regression over time. Total N₂O and natural abundance isotopologues of N₂O were analyzed by using an isotope ratio mass spectrometer (IRMS, Delta V Plus, Thermo Scientific). NO₂- production was additionally analyzed by transforming ¹⁵NO₂- to ¹⁵N₂O following the azide method after McIlvin & Altabet (2005) and the nitrogen isotope ratio of N₂O was measured by an IRMS. N₂ production was determined via an IRMS (Flash-EA-ConfloIV-DELTA V Advanced, Thermo Scientific) by injecting headspace from exetainers. The N₂O yield per nitrite produced and the N₂O yield during denitrification was calculated. Samples for natural abundance N₂O was sampled and measured in triplicates and is shown as an average with standard deviation (SD). In order to estimate the contribution of different N₂O producing pathways by major biological processes and the extent of N₂O reduction to N₂, the dual-isotope mapping approach was applied to natural abundance isotopologues of N₂O, which uses the relative position of background-subtracted N₂O samples in a δ¹⁵Nˢᴾ-N₂O vs. δ¹⁸O-N₂O diagram (Yu et al., 2020; Lewicka-Szczebak et al., 2020).

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.