Direct injection method for ethylene and acetylene δ13C analyses by GC-C-IRMS
Following the direct injection approach of classical ISARA12 with a few modifications, ARA samples with high ethylene yield ( >500 ppmv) in 10% v/v acetylene were manually injected into a Thermo Scientific Trace GC Ultra-Isolink with a combustion reactor connected to a Thermo Scientific Delta V Plus isotope ratio mass spectrometer (GC-C-IRMS; Fig. 1a). Modifications include the replacement of silver ferrules in the GC oven with Valcon polymide (graphite reinforced polymer) ferrules to limit memory effects from acetylene. The combustion reactor was oxidized with pure oxygen for 1 hour before each run and brief (15 min) seed oxidations were performed between measurement batches (i.e., required every ~6-8 ethylene injections, ~4-6 acetylene injections) to regenerate reactor oxidation capacity and minimize drift in δ13C values. See Table S1a for additional instrument settings.
EPCon method
ARA samples with <500 ppmv ethylene were analyzed using an ethylene pre-concentration system fabricated in-house (EPCon, Fig. 1b). The EPCon is a fully automated on-line gas preparation system that uses a series of precisely timed valves, cryogenic traps, and a gas chromatograph (GC) to remove background components (particularly water and acetylene) and concentrate ethylene before this analyte is introduced into the GC-C-IRMS. The EPCon was developed through modification of a similar in-house system designed to measure nitrogen and oxygen isotopes in seawater and freshwater nitrate35-37 and optimized for measurement of low concentration ethylene δ13C. Differences from its direct predecessor35 include direct plumbing into the capillary GC-C of the commercial GC-C-IRMS system, eliminating the need for some of the original features (e.g., second GC column within fabricated system). Flow rates, pressures, valve and trap timings were adjusted to effectively separate ethylene and acetylene such that acetylene could be removed from the analyte stream, and ethylene could be cryogenically focused into a small volume prior to introduction into the GC-C-IRMS. See Supplementary Methods S1 and Table S1b-c for detailed instrument information and settings.
Chemical precipitation of headspace acetylene
For ISARA samples with < ~20 ppmv ethylene, complete GC separation of acetylene and ethylene within the EPCon system was unachievable due to extreme mass imbalance in analytes. Prior to EPCon δ13Cethylene analysis of these samples, we performed off-line acetylene removal from sample headspace by chemical precipitation of acetylene with silver nitrate in ammonia, producing a silver carbide salt34. Ammoniacal AgNO3 solution (0.5 g AgNO3 in 10 mL water) was added to each sample (0.5 mL AgNO3/10 mL headspace containing 10% v/v acetylene). Once the reaction was complete (~10 min), sample headspace was transferred to an autosampler vial for EPCon analysis, and the remaining carbide salt solution was neutralized (1 mL of 5 N HCl). Complete acetylene removal was verified by GC-FID. We estimated the influence of chemical precipitation of acetylene on the δ13Cethylene using control samples made with 2,000 ppm ethylene (from tank EY-4) with and without 10% v/v acetylene (n=3, Table 1). Given the highly reactive nature of the silver carbide salt product of precipitation when dry, acetylene precipitation needs to be handle with great care34 and was only performed as necessary (e.g., sample ethylene < 20 ppmv).
Quality controls and data processing
To ensure within-run repeatability and long-term reproducibility, we used commercially available ethylene and acetylene gas tanks as in-house tank standards (ethylene EY-4, EY-8, acetylene AY1, AY-4, Table 1) for drift correction and daily quality assurance checks. Quality control standards to test IRMS and EPCon performance were analyzed before each batch of sample runs. All δ13C measurements produced by the EPCon-GC-C-IRMS during long (~30 hour) runs were corrected for drift in instrumental response over time relative to the drift correction standard (EY-4) that was measured at uniform intervals throughout sample runs using linear interpolation between drift correction standards. A second standard (EY-8 or a separate batch of EY-4 standards) was used to independently validate the drift correction process. Data from direct injections were processed according to Zhang et al., 201612, and did not require drift correction due the frequent seed oxidation of the reactor. See Table S3 for daily QC checks and sample loading details and SI DataS1 for data processing calculations.
Analytical method validation
For each measurement method (i.e., direct injection, EPCon, and chemical precipitation + EPCon), we determined the sensitivity, limit of quantification, linearity range, intraday repeatability, and within lab reproducibility (as defined in Carter and Barwick, 201138) by repeated analysis of the main in-house ethylene tank standard (EY-4) under various conditions (Table 1). Sensitivity was determined by linear regression of the IRMS response mass 44 signal (area in Vs) relative to the amount of ethylene C loaded (in nmols C). Linearity range was defined by the lowest and highest quantities of ethylene carbon that could be directly injected into the GC or loaded into the EPCon autosampler to obtain a mass 44 peak amplitude of 1-6 V (typical conservative analytical range). Samples were loaded with a goal of ~2 V for the mass 44 signal. Repeatability (i.e., intraday variability) was estimated as the average of the standard deviations for each day over 26 days for the EPCon, and 6 days for direct injection. Within lab reproducibility was calculated using the standard deviation of average δ13C measurements for each day over 26 days for the EPCon and 6 days for direct injection.
The limit of quantification (LOQ) was determined based on the minimum ethylene concentration (in ppmv) that could be measured using each method. The technical LOQ applies to ethylene standards and samples with no acetylene, and is bounded by the minimum accepted peak amplitude (1 V for mass 44) and the maximum loading volumes for each method (direct injection, 1 mL as constrained by injector and GC column loading; EPCon and chemical precipitation methods, 20 mL as constrained by autosampler vial volume). The methodological LOQ for samples with a 10% matrix, set by the maximum loading volume that avoids overloading the system with acetylene, is 0.5 mL for acetylene and 1.5 mL for EPCon. The methodological LOQ when Chemical precipitation was used is ~2 ppmv, the lowest sample concentration before the background ethylene concentration in acetylene generated from calcium carbide is greater than ethylene from sample acetylene reduction.
Bacterial cultures
Azotobacter vinelandii mutants39-41 utilizing only Mo-nitrogenase (‘Mo-Nase’ mutant, strain CA1.70) or only V-nitrogenase (‘V-Nase’ mutant, strain CA11.70) for nitrogen fixation were grown aerobically at 30oC in a modified Burks medium12,42 with 100 nM NaMoO4 (strain CA1.70) or 1 mM NaVO3 (strain CA11.70). Exponential phase cells (OD620nm ~0.3-0.8)were sampled to initiate acetylene reduction assays. See Methods S2 for details.
Environmental samples
Natural surface samples (mosses, cyanolichens, leaf litter, topsoil, decaying wood) and wood-feeding termites with low BNF activity were assessed for complementary nitrogenase activity. Natural surface samples were collected from forested sites in central New Jersey (Institute of Advance Studies, Stony Ford Reserve, Pine Barrens, Watershed Institute) and New Hampshire (Mount Moosilauke) from 2019-2021. At each site, triplicates of each sample type were collected from 1 or more stations (10 m x 10 m per station separated by 500-1000 m). Samples, stored at room temperature, were assessed by ARAs within 5 days of collection. Wood-feeding termites (genus Zootermopsis) were obtained from Ward Scientific (https://www.wardsci.com) and maintained within controlled laboratory habitats for 2-16 days prior to ARA. See Methods S3 and Table S3 for details.
Acetylene Reduction Assays
Acetylene reduction assays43(ARAs) were performed on Azotobacter cultures and environmental samples using 10% v/v acetylene generated from calcium carbide. Headspace ethylene concentration was monitored by GC-FID. See Methods S2, S3 and Table S3 for ARA details.
Azotobacter ARAs were conducted at 30o C, 200-250 rpm shaking in 25-240 mL serum bottles sealed with 20 mm blue butyl stoppers, containing 90% v/v air and 10% v/v acetylene headspace. Headspace with ~100-2000 ppmv ethylene from MoNase mutant ARAs and ~50-200 ppmv ethylene from VNase mutant ARAs was saved in evacuated 10 mL serum vials sealed with 20 mM butyl stoppers for isotopic analyses (ethylene standards EY-Mo-1 and EY-V-1, Table 1, Fig. 2).
Field sample ARAs were conducted in 100-500 ml glass canning jars (Mason, Ball) with metal lids fitted with 20 mm blue butyl stoppers (leaf litter, soil, and wood, Table S3); in 30 mL glass vials with screw caps fitted with PTFE/silicone septa (moss, lichens, soil, Table S3); or in 15 mL serum vials sealed with 20 mm butyl stoppers (termites, Table S3). Control incubations (no acetylene added) were performed with leaf litter, soil, decaying wood (Mt. Moosilauke, Pine Barrens), and termite samples to assess natural ethylene production independent of acetylene reduction. At the end of ARAs (~2-300 hrs depending on sample type, Table S3), headspace (< 3mL) was subsampled to measure ethylene concentration by GC-FID, with the remaining headspace transferred to evacuated sealed vials (10 mL) for later isotopic analysis.
Background ethylene correction
Due to low BNF activity, δ13Cethylene was corrected for isotopic influence of background ethylene (~2 ppmv) carried over into ARAs by 10% v/v source acetylene (Methods S4, Eqn. S1). Background correction was required for ARA samples producing <20 ppmv ethylene; no information on nitrogenase could be derived from samples producing <5 ppmv ethylene due isotopic influence of background ethylene. For ARAs with ethylene >5000 ppmv (i.e., 5% of acetylene concentration), δ13Cethylene was also corrected for Rayleigh fractionation44 (Methods S4).
Direct δ13Cethylene and 13εAR scaling methods to quantify complementary Nase contribution
One of three methods (Fig. 2) was used to quantify the contribution of complementary nitrogenase to acetylene reduction (as %VNase or %FeNase) in ARAs using δ13Cethylene and δ13Cacetylene. The particular method used was dependent on whether precise measurements of source δ13Cacetylene were achievable, given sample availability and technical difficulties in chromatography and combustion. EPCon-GC-IRMS was used to measure δ13Cethylene. See Methods S5, Table S4, and Data S1 for calculation details.
Method 1- The direct scaling approach (Fig. 2), which circumvents the need to measure δ13Cacetylene, was used to calculate complementary nitrogenase contribution when the same source of acetylene stock was used in environmental sample ARAs as a set of calibration ARAs performed with MoNase and VNase strains of Azotobacter vinelandii. Measured δ13Cethylene in environmental sample ARAs is converted to %VNase using endmember δ13Cethylene values (e.g., ethylene scaling standards, Table 1, Fig. 2) diagnostic of 0% and 100% VNase activity generated, respectively, by MoNase and VNase Azotobacter calibration ARAs (Eqn. S3). See Methods S2 for details on setup and analyses of Azotobacter ARAs.
When source acetylene stock used in sample ARAs was not processed in Azotobacter calibration ARAs, we quantified complementary Nase contribution using classical ISARA approaches12 (Fig. 2, methods 2 and 3), which require knowledge of sample δ13Cacetylene and δ13Cethylene to account for isotopic variation in different acetylene stocks in calculations of 13εAR (= δ13Cacetylene – δ13Cethylene).
Method 2- The δ13Cacetylene of different acetylene stocks used in sample and Azotobacter ARAs, measured with the direct injection method, was used with sample δ13Cethylene to calculate 13εAR, followed by calibration to the %VNase scale using 13εV and13εMo of Azotobacter and other diazotrophs, Rhodopseudomonas palustris and Anabaena variabilis (Fig. 2b, Methods S5, Table S4, Data S1; calculation modified from Zhang et al., 201612).
Method 3- When precise measurement of δ13Cacetylene by direct injection for the specific stock of acetylene within an ARA was unachievable, we used the mean and standard deviation of δ13Cacetylene for seven different batches of acetylene generated from calcium carbide over the past four years (δ13Cacetylene = 14.9 ± 0.9 ‰, n=8; Fig. S1; Eqn. S5) in 13εAR calculations. %VNase was calculated using 13εV and 13εMo values from Azotobacter and other diazotrophs as in method 2 (Fig. 2c, Methods S5, Table S4, Data S1).
Unstable growth of the A. vinelandii Fe-only Nase strain (RP1.1110) precluded calculations of %FeNase based on Azotobacter. Calculations %FeNase (Fig. 2c, Methods S5, Table S4, Data S1) used EPCon derived 13εFe = 5.2 ± 0.7‰ (s.d.) from Rhodopseudomonas palustris using only FeNase12 in ARAs . Because εFe < εV < εMo12, significant Fe-Nase activity can lead to %VNase values > 100% (i.e., 100% FeNase is equivalent to ~140% VNase; Table S4). Estimated uncertainty on the % FeNase scale is at most ~20%.
Complementary Nase contributions to N2 fixation and isoform adjusted total N2 fixation rates can be calculated using %VNase or %FeNase to AR (see above) and R ratios specifying the rate of AR to N2 fixation for each nitrogenase (RMo= 4 , RV and Fe= 2) 12.