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Chemicals and reagents
Sodium chloride (S3014), potassium chloride (P9541), sodium phosphate dibasic (S3264), potassium phosphate monobasic (P9791), potassium bicarbonate (12602), ammonium chloride (A9434), ethylene diamine tetra acetic acid di-sodium salt (E6635), bovine serum albumin (BSA) (5482), absolute ethanol (100983), methanol (34860) were purchased from Sigma–Aldrich, St. Louis, MO, United States. CD 34, Sca 1, and CBA Flex Set which contains IL-3 (558346), IL-6 (558301), TNFα (558299), IFN γ (562233), G-CSF (560152), GM-CSF (558347), IL-1α (560157), IL-1β (560232), Mouse/Rat Soluble Protein Master Buffer Kit (558266) were procured from BD Biosciences, United States. May-Grunwalds Stain (S039) and Giemsa Stain Solution (TCL083) were procured from Hi-Media India.
1 L 1X PBS (8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, 0.25 g of potassium phosphate monobasic to 1 L at pH 7.4), 1 L 1X RBC lysis buffer (1 g of potassium bicarbonate, 8 g of ammonium chloride and 0.03 g of di-sodium EDTA), 1% BSA in PBS, 70% ethanol in PBS, and may-grunwald-giemsa stain mixed in 3:1 ratio were prepared in the laboratory.
Animals and groupings
Pathogen free Strain ‘A’ male mice weighing 25.0–30.0 gm were received from Institute of Nuclear Medicine and Allied Science (INMAS) experimental animal facility at the age of 9 weeks, which is equivalent to young adult humans. Six mice per cage were housed under 25 ± 3 °C temperature and relative humidity of 30–70% in 12 h light/dark cycle with standard food and water. The study design strictly adhered to the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of our institute, Institute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organization (DRDO), Delhi, India (Institute Animals Ethics Committee number: INM/IAEC/2019/01).
After 1 week of acclimatization under ambient conditions, mice were grouped for the survival assay according to localized gamma radiation dose i.e. 7, 9, 10, 12, 15, 17 and 19 Gy. Eighteen animals were assigned in each group for this assay.
For all other parameters mice were divided into two groups with six animals in each. Group 1, consisted of untreated or sham irradiated animals. Group 2 was exposed to 15 Gy localized irradiation. Mice were sacrificed at different time points (1, 2, 4, 7, 10, 15, 20, 25, and 30 days) post treatment. Hematopoietic stem cell marker CD 34 and Sca1 were measured on 1, 4, 7, and 10th day post-irradiation.
Irradiation
A Cobalt-60 teletherapy (Model: Bhabhatron-II, Panacea Medical Technologies Pvt Ltd, India) machine was used to irradiate the hinds of the three mice at a time (Fig. 1). The cobalt-60 beam was calibrated following IAEA TRS-398 protocol, with measurement done in water phantom (30 cm × 30 cm × 30 cm), for a field size 10 cm × 10 cm at source to surface distance (SSD) of 80 cm. A Farmer type, 0.6 cc volume, ionization chamber was utilised and the same was recently calibrated for absorbed dose to water (NDW) at Secondary Standard Dosimetry Laboratory (SSDL) of India. The measured absorbed dose rate (water, 10 cm × 10 cm, 80 SSD, Dmax) was 1.39 Gy/min.
Figure 1

Representative image of animal exposure with γ-rays in a field size of 2 cm × 35 cm.

Full size image

The localized bone marrow irradiation was done using single Posteroanterior (PA) beam with 2 cm × 35 cm field size at 80 cm SSD opened through secondary collimators of the machine. The 5 mm thick acrylic re-strainers were used to immobilize each mouse which also provides the build-up thickness for the irradiation. The dose prescription point was the surface of the hinds i.e. 5 mm below the surface of the re-strainer. The whole irradiation platform was placed on 5 cm thick acrylic slab to provide sufficient back scatter and all the air gaps between the three mice were filled with tissue equivalent gel bolus or wet cotton to compensate for missing side scatter. The prescription doses were absorbed dose to water and not corrected for any tissue heterogeneity.
The absolute and relative dosimetry of collimated field (2 cm × 35 cm) were performed using pin point (0.04 cc volume) ionization chamber and EBT3 (ISP, Wayne, USA) Gafchromic dosimetry films in solid water phantom. The response of the pin point chamber and EBT3 films was calibrated against the SSDL calibrated ionization chamber in solid water phantom at the depth of 5 cm and field size 10 cm × 10 cm in the same beam quality (i.e. cobalt-60). The film scanning was performed using Epson 10000XL flatbed scanner as per the manufacturers scanning protocol. The beam profiles were obtained from the scanned films and the full width at half maximum (FWHM) in transverse direction was 1.78 cm for the opening of 2.0 cm. The dose uniformity was better than 4% in central 80% of the field. The absolute dose rate (dose to water, dmax, 2 cm × 35 cm, SSD 80 cm) measured at the centre of the field was 1 Gy/min. The overall uncertainty in dose calculations is 6.5% with enhancement or coverage factor (k = 2) at 95% confidence. The main body of the mice was covered with 15 mm lead to prevent any scatter radiation reaching at other body parts of the animal. The surface dose reaching at the shielded body parts of the mice was measured using 0.4 cc ionisation chamber. The measured mean surface dose was 0.024 ± 0.014% of the open field dose. This dose is negligible and considered to be acceptable.
Survival assay in mice
For survival assay, 18 mice from each experimental group i.e. 7, 9, 10, 12, 15, 17 and 19 Gy, were irradiated at a fixed dose rate of 1 Gy/min. Groups were inspected daily for their morbidity and mortality status for a total duration of 30 days. All the surviving mice were euthanized humanely using intra-peritoneal injection (i.p.) with dexmedetomidine (0.3 mg/kg) in combination with ketamine (190 mg/kg) at the completion of the observation period.
Validation of hematopoietic syndrome model
The model of hematopoietic syndrome was validated with localized radiation of 15 Gy as this was the maximum dose at which all the animals survived as observed in the survival results. Mice were euthanized using intra-peritoneal injection (i.p.) with dexmedetomidine (0.3 mg/kg) in combination with ketamine (190 mg/kg) at 1, 2, 4, 7, 10, 15, 20, 25, and 30th day time points. Further analysis such as organ weight, cell counting, and serum cytokines measurement assays were conducted on various organs such as femur bone, spleen, thymus, lungs, kidneys (L), kidneys (R), liver and heart and blood at above mentioned time points. Hematopoietic stem cell markers (CD 34 and Sca 1) were measured in bone marrow cells of mice at 1, 4, 7, and 10th day time points.
Body and organ weights
The control and 15 Gy locally irradiated group animals were sacrificed at 1, 4, 7, 10, 15, 20, 25, and 30th day time points. The organs (spleen, thymus, lungs, kidneys (L), kidneys (R), liver and heart) were dissected out and weighed individually. Relative organ weight was calculated as the ratio between organ weight and body weight.
Haematology
The blood samples were collected from untreated and 15 Gy localized radiation group at 1, 4, 7, 10, 15, 20, 25, and 30th day time points in EDTA vial by cardiac puncture. 20 µl of collected blood from each experimental animal was taken for haematology and analysed using Nihon Kohden Celltac α, Tokyo, Japan, a fully automatic 3 part haematology analyzer.
Bone marrow cell counting
Total bone marrow cells were flushed out at 1, 4, 7, 10, 15, 20, 25 and 30th day points in 1 ml PBS from both femurs of the untreated mice and 15 Gy localized radiation group. The PBS containing cells was centrifuged at 2000 rpm for 8 min and the supernatant was discarded. The cells were washed with 1 ml PBS and the cell pellet was re-suspended in 1 ml of PBS. The re-suspended sample was diluted as required. The cells were then counted using improved Neubauer chamber under Olympus BX-63 microscope.
Bone marrow smears study
Bone marrow cells collected at 1, 4, 7, 10, 15, 20, 25, and 30th day post-irradiation from different treatment groups were centrifuged at 2000 rpm for 8 min and re-suspended in 50 μl of Fetal Bovine Serum (FBS). A small drop of cells suspension was dropped on a clean microscopic slide and then smeared thinly over an area of 2–3 cm by pulling another glass slide held at a 45° angle. Cells were fixed with methanol and stained with may-grunwald-giemsa. Slides were then analysed using Olympus BX-63 microscope.
Splenocytes and thymocytes counts
The organs excised at 1, 4, 7, 10, 15, 20, 25, and 30th day time-points were cleaned in chilled PBS. Using sterile-chilled frosted slides both the thymus and spleen were minced into a cell suspension and which was further centrifuged at 2000 rpm for 8 min. After centrifugation the supernatant was discarded. The RBCs were lysed using RBC lysis buffer and the cells were washed with 1 ml PBS. The cells were then counted using improved Neubauer chamber under Olympus BX-63 microscope.
Measurement of hematopoietic stem cell marker CD 34 and Sca1
Bone marrow cells were isolated from both the femurs from various treatment groups at different time points. Cell were washed with chilled PBS and fixed with 70% chilled ethanol and stored overnight at – 20 °C. Following day, five million cells were blocked with 1% BSA-PBS and stained with CD 34 and Sca 1 cell-surface markers for 20 min at room temperature. 10,000 cells from each sample were analyzed using FACS LSR II (BD Biosciences, San Jose, CA) and results were visualized by the BD FACSDiva V7 software (BD Biosciences, San Jose, CA).
Cytokines expression changes
Mice blood samples were collected in BD serum separation tube at different time-points post treatment (1, 4, 7, 10, 15, 25, and 30 days) via. cardiac puncture from different treatment groups. Samples were incubated at room temperature for 2 h and after incubation centrifuged at 6000g for 15 min. Serum was separated and stored at − 80 °C until analysis. The Levels of IL-3, IL-6, TNFα, IFN γ, G-CSF, GM-CSF, IL-1α,and IL-1β cytokines in mice serum were determined by using respective BD Cytometric Bead Array (CBA) Flex Set (BD Biosciences, USA) on dual laser flow analyzer (LSR-II, Becton Dickinson Biosciences, USA) according to manufacturer’s instructions. Results were analysed using FCAP Array V3 software (BD Biosciences, San Jose, CA).
Statistical analysis
All values obtained in our results are represented as mean ± SD of three independent replicates. The 30 day survival data was plotted using Kaplan–Meier analysis. The difference between the experimental groups was evaluated by one-way analysis of variance, with Newman–Keuls multiple comparison test (V, 8.01; GraphPad Prism, San Diego, CA, USA). The comparisons were made among the untreated and 15 Gy locally irradiated groups for all experimental parameters except the survival study. A value of p  More

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Experimental site
Seawater samples were collected from two stations (Fig. S1a, offshore OMZ station PS2 and coastal OMZ station PS3) in the ETNP in March and April 2018 on board R/V Sally Ride (Cruise ID: SR 1805). NO2− kinetics and O2 effects experiments (≤1 day incubation) were performed at the oxycline, the oxic/anoxic interface (top) of the ODZ and the core of the ODZ at each station (Fig. S1b and Table S1). Long incubations (6–7 days) were performed at the core of the ODZ at both stations to investigate nitrogen reaction rates at very low O2 concentrations.
Sampling and incubation experiments for NO2− kinetics and O2 effects
Twelve 30-L Niskin bottles on a rosette with a conductivity–temperature–depth (CTD) profiler were used to collect seawater while recording in situ O2 concentration, temperature, pressure, salinity, and chlorophyll fluorescence. O2 concentration at selected ODZ depths was measured by STOX sensor (on the CTD profiler) with detection limit of 10 nM [42]. NO2− concentrations were measured by standard spectrophotometric methods onboard. O2 and NO2− concentrations were used to select sampling depths. NO3− and NO2− concentrations in incubation samples were measured on a chemiluminescence NO/NOx Analyzer (Teledyne API, San Diego, CA, USA) in the lab.
Seawater was collected from Niskin bottles into 60 mL air-tight serum bottles after overflowing three times in order to minimize O2 contamination. Serum bottles were sealed with rubber septa and aluminum seals ensuring the absence of bubbles inside bottles. Septa were deoxygenated in anaerobic chambers with three cycles of vacuum/helium flushing over a period of 1 month prior to the cruise. A helium headspace was created for each sample collected from anoxic depths (the top and the core of the ODZ), and then samples were flushed with helium for at least 15 min to remove O2 that might have been introduced during sampling.
To determine a single NO2− oxidation rate, a set of five serum bottles amended with 15NO2− tracer was incubated on board at 12 °C in the dark. Incubations were terminated in time series (one bottle at day zero (T0), two at 0.5 day and two at 1 day) by adding 0.2 mL of saturated ZnCl2. The T0 bottles served as killed controls for both tracer contamination and abiotic reactions. The observed temporal changes in isotopic signals that occurred in the live samples over a few days in the time courses would not be detected if abiotic reactions were taking place during the >3 months that elapsed before all samples were measured on the mass spec.
In the lab, NO2− was removed from samples using sulfamic acid, and NO3− in serum bottles was converted into N2O using the denitrifier method [43, 44]. Both concentration and isotopic composition of N2O were measured on a mass spectrometer (Delta Vplus, Thermo Fisher Scientific, Waltham, MA, USA) for calculating nitrite oxidation rate from the linear regression of the five nitrate concentrations at the three time points as previously described [3]. NO2− kinetics of NO2− oxidation were determined by measuring NO2− oxidation rates under different 15NO2− tracer concentrations (0.5–13.8 μM). For responses of NO2− oxidation to O2, different amounts (0, 0.2, 0.5, 1, 2, and 5 mL) of O2 saturated seawater collected from the same Niskin bottle were added into serum bottles to achieve different O2 concentrations. O2 concentrations in serum bottles were monitored by optical oxygen sensors with a detection limit of 62.5 nM (PyroScience GmbH, Aachen, Germany).
Half-saturation constant
Half-saturation constant (Km) is the nitrite concentration at which nitrite oxidation rate (V) equals half of the potential maximum rate (Vm). The curve fitting tool in MATLAB_R2015a was used to fit the Michaelis–Menten model (Eq. (2)) to determine Km.

$${{V}} ,=, {{V}}_{mathrm{m}} ,times,left[ {{mathrm{NO}}_2^-} right]{mathrm{/}}left( {left[ {{mathrm{NO}}_2^-} right] ,+, {{K}}_{mathrm{m}}} right).$$
( 2)

Long incubation experiments
Longer (≥6 days) incubations were performed in 12-mL exetainers. Seawater from the core of the ODZ (250 m at station PS2, 160 m at station PS3) was sampled into 320 mL ground glass-stoppered bottles, which were immediately transferred into a N2 flushed glove bag. 15NO2− was added into these bottles to reach final concentrations of 7.24 and 8.01 µM at stations PS2 and PS3, respectively. 15NO2− labeled seawater was aliquoted into exetainers and capped within the glove bag. The septa had been stored under helium for at least 1 month. Exetainers were purged with helium for 5 min. Every 12 h, microbial activity in triplicate exetainers was terminated by adding 0.05 mL of 50% w/v ZnCl2. In the lab, N2 produced in exetainers was measured on a mass spectrometer (Europa Scientific 20–20, Crewe, UK) [45]. Denitrification and anammox rates were computed from linear regression of N2 produced at three time points. 15NO3− was measured (as described above) in the same exetainers, and NO2– oxidation rates were computed from linear regression of NO3− produced at three time points. O2 was monitored throughout the incubations in parallel exetainer vials using LUMOS sensors [12, 13]. Each O2 production or consumption rate was determined by linear regression of O2 concentrations at 32 time points. One sensing spot, glued inside an exetainer vial, allowed the optode to measure O2 concentrations every 5 min from the outside. Detection limit and resolution of LUMOS sensors was ≈1 nM.
DNA sampling, extraction, sequencing, metagenomics, and metatranscriptomics analysis
Particulate DNA samples were collected by filtration onto 0.22 µm Sterivex filters from the ODZ core samples (250 m at station PS2, 160 m at station PS3). DNA was extracted using the modified plant tissue protocol (All Prep DNA/RNA Mini Kit, Qiagen, Valencia, CA, USA), and subjected to paired-end sequencing on an Illumina MiSeq to generate over 10 million read pairs for each sample by the Genomics Core Facility of Lewis-Sigler Institute for Integrative Genomics at Princeton University. Quality control of raw reads was performed by BBDuk (DOE Joint Genome Institute, Walnut Creek, CA, USA), and assembled into contigs using metaSPAdes v3.12.0 [46] with specified options (-k 21, 33, 55, 77, 99, 127 -m 500). Metagenome-assembled genomes (MAGs) were constructed using MetaBAT v2.12.1 [47] with default (sensitive) mode and contigs longer than 1500 bp. The quality of MAGs was determined by checkM [48]. Taxonomy of MAGs was predicted by GTDB-tk [49]. ETNP PS2 MAG-11 from the ODZ core at station PS2 and ETNP PS3 MAG-54 from the ODZ core at station PS3 were identified as Nitrospina-like NOB. The taxonomy of these two NOB MAGs (ETNP PS2 MAG-11 and ETNP PS3 MAG-54) was further confirmed by comparing them with known OMZ NOB MAGs (MAG-1 and MAG-2) [8] using ANI. ANI between MAGs was assessed using enveomics [50]. MAG-2 [8], ETNP PS2 MAG-11 and ETNP PS3 MAG-54 belong to the same species (threshold for species: ANI ≥95%) based on their ANI values (Table S3). Considering the low completeness of MAG-11 and the high contamination of MAG-54 constructed from the two new metagenomes here (Table S4), we decided to use MAG-2 as the representative for this NOB species.
We estimated the relative abundance and the transcriptional activity of the two known OMZ NOB species in different oceanic regions (including the two ETNP stations in this study) by mapping metagenomes and metatranscriptomes from this and other studies to MAG-1 and MAG-2. The relative abundance of MAG-1 or MAG-2 at stations PS2 and PS3 in the ETNP was calculated as RPKG (the number of metagenomic reads obtained in this study mapped to a MAG per MAG length (kb) per genome equivalents) [15]. Genome equivalents were estimated using MicrobeCensus v1.1.1 [15]. The transcriptional activity of MAG-1 or MAG-2 in ETNP and ETSP OMZs was assessed by mapping published metatranscriptomic reads from the ETNP (PRJNA263621) [51] and the ETSP (SRA023632.1) [52] to MAG-1 and MAG-2. The relative abundance of RNA in Fig. S4 was calculated as the number of metatranscriptomic reads mapped to a MAG divided by the number of total reads. Mapping was performed by Bowtie2 [53] using “very-sensitive” mode, and only reads with a mapping quality above 20 were included as mapped reads.
In order to explore the possibility of the presence of other NOB in the ODZ core at stations PS2 and PS3, we also estimated the relative abundance of other (putative) NOB using their marker gene, nxrB (nitrite oxidoreductase). First, we downloaded previously identified (putative) nxrB sequences from the ETSP OMZ [8]. Then, we color coded the genes in Fig. 4 based on previously defined clusters in a phylogenetic tree (see Fig. 3 in [8]): the Nitrospina cluster (blue) contains nxrB grouped with cultured marine NOB, Nitrospina gracilis. Based on BLASTp search results, amino acid sequence identities between all the OMZ nxrB in this cluster and that of Nitrospina gracilis were 96.71%, 96.71%, and 96.87% for NODE_69234, NODE_114897, and NODE_102582, respectively. Only this cluster is associated with known NOB, and the nitrite oxidation capacity of all the other genes associated with nxrB needs to be confirmed. The anammox cluster (red) contains anammox nxrB sequences. The putative cluster (black) contains nxrB grouped with microbes in which nitrite oxidation capacity has not been proven. This putative cluster fell between known NOB and anammox, and was implied to represent unidentified NOB [54]. The last cluster is called unknown nxrB (gray) because neither their phylogeny nor function can be determined based on their distant relationship with known NOB or anammox nxrB. Finally, we mapped the ETNP metagenomic reads obtained in this study to each nxrB gene. Relative abundance of nxrB genes was also expressed as RPKG: relative abundance of nxrB gene = (number of mapped reads to a certain nxrB gene)/(length of this nxrB in kb)/(genome equivalents).
To explore the potential metabolisms of NOB in anoxic ODZ cores, we searched for chlorite dismutase and NO dismutase (nod) genes in the NOB MAGs. First, protein-coding sequences in the two new NOB MAGs obtained here (ETNP PS2 MAG-11 and ETNP PS3 MAG-54) were predicted by Prodigal v2.6.3 [55]. The protein-coding sequences were annotated by the best BLASTp hits against the nr protein database. DNA sequences encoding Cld were identified in both ETNP NOB MAGs. Predicted Cld amino acid sequences encoded by ETNP MAGs were too short (120 aa) to be compared to Cld of Candidatus Nitrospira defluvii, but they only had one mismatch with Cld amino acid sequences of MAG-2 from the ETSP OMZ based on MUSCLE alignment using MEGA 7 software (Fig. S5b). Thus, we looked for the arginine173, the Cld activity marker, in longer Cld sequences of MAG-1 and MAG-2 by aligning their Cld with the Cld of Candidatus Nitrospira defluvii using MUSCLE in MEGA 7 software. Since nod was not found in the ETNP MAGs via annotation and was not reported in MAG-1 and MAG-2 in the previous study [8], gene search (i.e., searching nod against NOB MAGs) was performed using Hidden Markov Models by HMMER3 [56]. Reference sequences of the search included a nod sequence retrieved from Candidatus Methylomirabilis oxyfera [32] genome (accession numbers FP565575.1), and three environmental nod sequences (accession numbers: KX364450.1, KX364454.1, and KU933965.1). Search queries were the two ETNP MAGs and two ETSP MAGs in Table S3.
Estimation of NO2− oxidation through disproportionation using an inverse isotope model
We simulated the distribution of NO2− oxidation rates via disproportionation using a 1-D inverse isotope model [37] in the anoxic ODZ core from two stations in the ETSP OMZ (Fig. S6), where the complete suite of isotope and rate data have been previously published (Table S7). Briefly, the net biochemical production or consumption rate of each nitrogen compound (R14Ammonium, R14Nitrite, R14Nitrate, R15Nitrite, and R15Nitrate) was balanced by vertical diffusion and advection at steady state, and then five equations were used to balance measured 14NH4+, 14NO2−, 15NO2−, 14NO3−, and 15NO3− concentrations (Eqs. (3–7)). The exclusion of horizontal processes was justified [37] since the model was not run all the way to the surface, and using a constant vertical advection term does not violate continuity of the model. F is the rate of each nitrogen cycle process represented in this model (Fig. 5a, b). We modified the previous model by replacing canonical NO2− oxidation with NO2− disproportionation and using the recently determined stoichiometry of nitrate production by anammox from 0.3 to 0.16 [24]. Since OMZ NOB genomes encode nitrite oxidoreductase (catalyzes NO2−→NO3−) and nitrite reductase (catalyzes NO2−→NO), we assumed that the fractionation factor of nitrate production from nitrite by disproportionation is the same as by canonical nitrite oxidation (αDis = αNxr) based on the close clustering between OMZ NOB nxr and aerobic NOB (Nitrospina gracilis) nxr [8], and the fractionation factor of N2 produced by disproportionation is the same as by denitrification (αDisN2 = αNir). Rates of NO2− disproportionation (FDis), NO3− reduction (FNar), denitrification (FNir), and anammox (FAmx) were solved from the equations by the nonnegative least squares optimization routine (lsqnonneg) in MATLAB_R2015a as described previously [37].

$$R_{14Ammonium} ,=, 0.11,times {,}^{14}F_{Nir} ,+, 0.07,times {,}^{14}F_{Nar} ,- {,}^{14}F_{Amx},$$
( 3)

$$R_{14Nitrite} = – ,^{14}F_{Nir} ,+, {,}^{14}F_{Nar} ,-, (1 ,+, c) ,times,{,}^{14}F_{Amx} \ -, left( {5/3} right) ,times,{,}^{14}F_{Dis},$$
( 4)

$$R_{14Nitrate} ,=, – ^{14}F_{Nar} ,+, c ,times {,}^{14}F_{Amx} ,+, {,}^{14}F_{Dis},$$
( 5)

$$R_{15Nitrite} = -!! {,}^{14}F_{Nir}/alpha_{Nir} ,times,left( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right) \ +, {,}^{14}F_{Nar}/alpha_{Nar},times,left( {left[ {{,}^{15}{rm{NO}}_{3}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{3}^{-}} right]} right)\ -, {,}^{14}F_{Amx}/alpha_{Amx},times,left( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right) \ -, c times !{,}^{14}F_{Amx}/alpha_{NxrAmx},times,left( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right)\ -, {,}^{14}F_{Dis}/alpha_{Dis}timesleft( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right) \ -, left( {2/3} right) !times ! {,}^{14}F_{Dis}/alpha_{DisN2} !times left(! {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ !{{,}^{14}{rm{NO}}_{2}^{-}} right]} !right),$$
(6)

$$R_{15Nitrate} = -!! {,}^{14}F_{Nar}/alpha_{Nar} ,times,left( {left[ {{,}^{15}{rm{NO}}_{3}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{3}^{-}} right]} right) \ +, c times !{,}^{14}F_{Amx}/alpha_{NxrAmx},times,left( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right)\ +, ^{14}F_{Dis}/alpha_{Dis},times,left( {left[ {{,}^{15}{rm{NO}}_{2}^{-}} right]/left[ {{,}^{14}{rm{NO}}_{2}^{-}} right]} right).$$
(7) More

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