Measuring Dissolved Methane in Aquatic Ecosystems Using An Optical Spectroscopy Gas Analyzer

1. Porewater sampling and analysis

1. Collect the samples using porewater dialysis samplers (peepers)¹¹. Deploy the peepers in relevant locations of the study site. In the demonstration study, 6 peepers were deployed on the two dominant vegetation patches of a freshwater marsh: three on a patch dominated by Sagitaria lancifolia and the other three on a patch co-dominated by S. lancifolia and Typha latifolia vegetation.
2. Collect samples 4x in June, September, October, and December. Collect samples at 10 different depths from the soil surface down to ~50 cm below it. The sampling followed the method described in^12,13.
   1. Fill sampling cells with 61 mL of deionized water ~20 days before sampling. Bubble deionized water with N₂ for 5 min in the lab to remove the oxygen before using it in the field.
   2. During sampling, withdraw 25 mL of water from the cell with a syringe connected to the cell's extraction line. Then, refill cells with fresh deionized water pre-bubbled with N₂ through a secondary cell recharge line. If concentration measurements are required right after collection, continue to step 2.1.
   3. Add the extracted water to 10 mL glass vials preserved with 0.2 mL of 0.1 M HCl, cap with septum, and refrigerate until analysis in the laboratory.
      NOTE: Users of this method are welcome to modify the collection depths, sampling schedule, and frequency based on specific study needs.

2. Greenhouse gas concentration measurement

1. Perform headspace equilibration of porewater samples in a syringe
   1. Create headspace gas samples using the equilibration technique¹⁴. Use a 30 mL syringe to draw 5 mL of water (V_pw) from the samples collected in the field and then add 15 mL of N₂ (Ultra High Purity 99.999%) to create the headspace (V_hs).
   2. Agitate the syringe vigorously and consistently for 5 min, manually or using a rocking shaker (if available). Inject 12 mL of headspace gas subsample into a 10 mL pre-evacuated vial used for gas concentration measurements with the optical analyzer in the following steps (Figure 1). Evacuate the vials with a peristaltic pump¹⁵ or manually pull the plunger of a 60 mL syringe on the closed vial and pump the air out 3x.
      NOTE: He can be used instead of N₂ to create the headspace. The procedure to add N₂ or He to the syringe depends on the specific adaptation or regulator of the actual tank available to users. This addition typically involves coupling the syringe to the tank using matching tubing or extraction with the syringe from a sampling port adapted to the tank. Depending on site conditions and associated logistics, users may use ambient air to create the headspace.
2. Create the injection chamber
   1. Manufacture an injection chamber that can accept a small volume of air (~1 – 5 mL) into a sealed volume of air that connects to the gas analyzer inflow and outflow to form a closed loop (Table of Materials). The injection chamber and the analyzer are the two main elements of the system (Figure 2).
   2. Modify the metallic lid of a 365 mL mason jar by drilling one 11 mm diameter hole to fit one septum as the injection port and two 7 mm diameter holes to insert stopcock valves that connect with the analyzer. Use epoxy glue to tighten the injection port and fittings and ensure chamber sealing.
3. Connect the injection chamber to the optical analyzer
   1. Connect the jar with the inlet and outlet ports of the optical analyzer using 5/32 inch inner diameter (I.D.) and ¼ inch outer diameter (O.D.) PFA plastic tubing and account for their added volume. Ensure the tubing follows the instrument manufacturer's recommendation and is clean and dry without condensation.
   2. Set the valves that connect the injection chamber to the instrument to open to create a closed-loop air circuit. Wait for gas concentrations to stabilize.
4. Inject a sample in the chamber
   1. When the concentration in the chamber and the signal in the analyzer have stabilized (i.e., standard deviation <0.03 ppm), inject 2 mL of a subsample from the vials containing the headspace sample created in step 2.1.2. Wait for concentrations in the analyzer to stabilize again before injecting the next subsample (Figure 3).
   2. Inject up to 20 subsamples consecutively or less if the concentration gets closer to 100 ppm (see⁸for effects on accuracy above this threshold). Analyze CH₄ check standards for every 5 samples and evaluate the difference with actual measurements using the relative standard deviation (RSD).
   3. When done with the set of stacked injections, unplug one of the lines connected to the instrument to reset the chamber to ambient pressure and avoid significant pressure and methane concentration build-up.
      NOTE: Based on multiple test runs, it was determined that the signal stabilized ~15 s after the injection, but different times may be required in response to other instrument recording specifications (Figure 3). The flow rate of the instrument used was ~1 L/min with a response time of 5 s and cavity pressure during the different analyses on different days ranging from 720 to 745 Torr.
5. Calculate CH₄ concentrations in the headspace
   1. For each subsample, calculate the concentration of the target GHG (e.g., CH₄, CO_2, N₂O) in the headspace (C_hs – µmol/mol) with equation 1:
           equation [1]
      Where V_hs is the volume of the headspace subsample.
   2. Calculate the concentrations before (C_bi – µmol mol^-1) and after (C_ai – µmol mol^-1) injections as the mean of 15 measurements (~15 s) before the injection and 15 after the stabilization of the signal post-injection. Calculate the volumes in mL, before (V_bi) and after (V_ai) injections with equations 2 and 3:
           [2]
      and
           [3]
      Where V_j (mL) is the added volume of the jar (365 mL) and hosing (125.4 mL), and V_i (mL) is the accumulated volume of the subsamples injected.
6. Calculate porewater concentration
   1. Calculate the partial pressure (P_i – MPa) of CO₂, CH_4, or N₂O in the headspace using the mole fraction of gas in the headspace and the atmospheric pressure (P) during the processing of the sample using equation 4:
           [4]
   2. Calculate the aqueous equivalent concentration of GHGs in porewater using a dilution equation (equation 5) with headspace and aqueous equivalent concentrations and volumes:
           [5]
      Where R is the universal gas constant (0.008314 L MPa mol⁻¹ K⁻¹), is the air temperature (K), V_hs is the volume of the headspace (mL), K_H^cp is Henry's volatility constant of CO₂, CH_4, or N₂O (2.96, 67.13, and 3.82 L MPa/mol, respectively)¹⁶, and V_pw the volume of the liquid subsample used to create the headspace (mL).
   3. Use recorded T and P during the measurements. The average T and P during the analysis of the demonstration project samples were 295.15 K and 0.101325 MPa, respectively.

3. Validation against standard chromatography measurements

1. Analyze different known CH₄ concentrations in 10 mL vials in a conventional gas chromatograph. Use three identical groups of 20 standards each. The lowest concentrations ranged from 5 to 100 ppm and increased at 5 ppm increments.
2. Determine CH₄ concentrations by flame ionization detection on a gas chromatograph. The chromatograph used a porous polymer absorbent packed column with He (25 mL/min) as the carrier gas. To run the measurements of the standards' concentrations, calibrate the chromatograph to a curve fit (r²) > 0.99.
3. After chromatography, proceed to determine CH₄ concentration in the same standard vials using the method described above, using an optical analyzer calibrated by the manufacturer (i.e., precision (300 s, 1σ): 0.3 ppb CH₄).
4. Assess the accuracy of the method by fitting a linear regression to the chromatography and optical analyzer results of the three standard groups. Assess the replicability of the method by comparing the slopes and intercepts of regressions performed within each group of standards. Evaluate the comparison using a Standard Least Square Model with an emphasis on Effect Leverage. Evaluate all regressions and comparisons at a significance level of 0.05.