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Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (¹³C/¹²C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. Here we describe a high sensitivity method to measure ethylene δ¹³C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoformAzotobacter vinelandiimutants and environmental sample assays rely on a common acetylene source for ethylene production. Application of the Low BNF activity ISARA (LISARA) method to low nitrogen-fixing activity soils, leaf litter, decayed wood, cryptogams, and termites indicates complementary BNF in most sample types, calling for additional studies of isoform-specific BNF.

 

Abstract. Oxygen minimum zones (OMZs), due to their large volumes of perennially deoxygenated waters, are critical regions for understanding how the interplay between anaerobic and aerobic nitrogen (N) cycling microbial pathways affects the marine N budget. Here, we present a suite of measurements of the most significant OMZ N cycling rates, which all involve nitrite (NO2-) as a product, reactant, or intermediate, in the eastern tropical North Pacific (ETNP) OMZ. These measurements and comparisons to data from previously published OMZ cruisespresent additional evidence that NO3- reduction is the predominant OMZ N flux, followed by NO2- oxidation back to NO3-. The combined rates of both of these N recycling processes were observed to be much greater (up to nearly 200 times) thanthe combined rates of the N loss processes of anammox and denitrification, especially in waters near the anoxic–oxic interface. We also showthat NO2- oxidation can occur when O2 is maintained near 1 nM by a continuous-purge system, NO2-oxidation and O2 measurements that further strengthen the case for truly anaerobic NO2- oxidation. We also evaluate thepossibility that NO2- dismutation provides the oxidative power for anaerobic NO2- oxidation. The partitioning ofN loss between anammox and denitrification differed widely from stoichiometric predictions of at most 29 % anammox; in fact,N loss rates at many depths were entirely due to anammox. Our new NO3- reduction, NO2- oxidation, dismutation, andN loss data shed light on many open questions in OMZ N cycling research, especially the possibility of truly anaerobicNO2- oxidation.

 

Abstract. Oxygen-deficient zones (ODZs) are major sites of net naturalnitrous oxide (N2O) production and emissions. In order to understandchanges in the magnitude of N2O production in response to globalchange, knowledge on the individual contributions of the major microbialpathways (nitrification and denitrification) to N2O production andtheir regulation is needed. In the ODZ in the coastal area off Peru, thesensitivity of N2O production to oxygen and organic matter wasinvestigated using 15N tracer experiments in combination with quantitative PCR (qPCR) andmicroarray analysis of total and active functional genes targeting archaeal amoAand nirS as marker genes for nitrification and denitrification, respectively.Denitrification was responsible for the highest N2O production with amean of 8.7 nmol L−1 d−1 but up to 118±27.8 nmol L−1 d−1 just below the oxic–anoxic interface. The highest N2O productionfrom ammonium oxidation (AO) of 0.16±0.003 nmol L−1 d−1occurred in the upper oxycline at O2 concentrations of 10–30 µmol L−1 which coincided with the highest archaeal amoA transcripts/genes.Hybrid N2O formation (i.e., N2O with one N atom from NH4+and the other from other substrates such as NO2-) was the dominantspecies, comprising 70 %–85 % of total produced N2O fromNH4+, regardless of the ammonium oxidation rate or O2concentrations. Oxygen responses of N2O production varied withsubstrate, but production and yields were generally highest below 10 µmol L−1 O2. Particulate organic matter additions increasedN2O production by denitrification up to 5-fold, suggesting increasedN2O production during times of high particulate organic matter export.High N2O yields of 2.1 % from AO were measured, but the overallcontribution by AO to N2O production was still an order of magnitudelower than that of denitrification. Hence, these findings show thatdenitrification is the most important N2O production process in low-oxygen conditions fueled by organic carbon supply, which implies a positivefeedback of the total oceanic N2O sources in response to increasingoceanic deoxygenation. 

Bulk sediment δ¹⁵N records from the eastern tropical Pacific (ETP) extending back to the last ice age most often show low glacial δ¹⁵N, then a deglacial δ¹⁵N maximum, followed by a gradual decline to a late Holocene δ¹⁵N that is typically higher than that of the Last Glacial Maximum (LGM). The lower δ¹⁵N of the LGM has been interpreted to reflect an ice age reduction in water column denitrification. We report foraminifera shell‐bound nitrogen isotope (FB‐δ¹⁵N) measurements for the two speciesNeogloboquadrina dutertreiandNeogloboquadrina incomptaover the last 35 ka in two sediment cores from the eastern equatorial Pacific (EEP), both of which have the typical LGM‐to‐Holocene increase in bulk sediment δ¹⁵N. FB‐δ¹⁵N contrasts with bulk sediment δ¹⁵N by not indicating a lower δ¹⁵N during the LGM. Instead, the FB‐δ¹⁵N records are dominated by a deglacial δ¹⁵N maximum, with comparable LGM and Holocene values. The lower LGM δ¹⁵N of the bulk sediment records may be an artifact, possibly related to greater exogenous N inputs and/or weaker sedimentary diagenesis during the LGM. The new data raise the possibility that the previously inferred glacial reduction in ETP water column denitrification was incorrect. A review of reconstructed ice age conditions and geochemical box model output provides mechanistic support for this possibility. However, equatorial ocean circulation and nitrate‐rich surface water overlying both core sites allow for other possible interpretations, calling for replication at non‐equatorial ETP sites.