TITLE: 15N2 label preparation (dissolved method)
AUTHORS: Molly Moynihan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for making pre-dissolved 15N2 label in sterile seawater</div></div>

[STEPS]
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The following protocol is based on the dissolved gas method described by Mohr et al. 2010 and Klawoon et al. 2015. This protocol makes 4x 500mL serum bottles of 15N2 labels enriched to ~90%. Enrichment of each serum bottle depends on the vacuum pressure used for degassing. The following calculations can be used to estimate degassing potential based on the vacuum pressure. [N2]seawater = 0.000398 mol/L = 398 mol/L at 24°C, 1 atm[N2] = TGP * XN2* kH [N2] is the concentration of N2 after reaching equilibrium at pressure p.  TGP is the total gas pressure, which is the difference between atmospheric pressure (patm) and vacuum pressure (pv). (TGP = patm - pv).  XN2 is the mole fraction of dinitrogen gas. XN2 = 0.7804kH is Henry's Law constant for nitrogen gas at 35ppt and 24°C. kH = 0.00051 mol/L atm  ABCD1pv (mbar)pv (atm)[N2] mol/LDegassing %22000.200.0003194442036000.590.0001623245949500.940.00002484594References: Klawonn, I., Lavik, G., Böning, P.,  Marchant, H., Dekaezemacker, J., Mohr, W., & Ploug, H. (2015).  Simple approach for the preparation of 15− 15N2-enriched water for  nitrogen fixation assessments: evaluation, application and  recommendations. Frontiers in microbiology, 6, 769.Mohr, W., Grosskopf, T., Wallace, D.  W., & LaRoche, J. (2010). Methodological underestimation of oceanic  nitrogen fixation rates. PloS one, 5(9), e12583.
ABCD1pv (mbar)pv (atm)[N2] mol/LDegassing %22000.200.0003194442036000.590.0001623245949500.940.00002484594
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Wash all glassware, tubing, rubber septa, etc. with 10% HCl and then rise 4x with Elga water (18.2MΩ).
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Filter sterilize 3L of seawater using 0.22um pore size filters. Only ~2.5L of this is needed to fill serum bottles but it is good to have some extra when siphoning (step 5).
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Shake serum bottles for a few minutes and then store horizontally, such that the gas bubble is not in contact with the septa. Store for 24 hours prior to use to increase dissolution (see Klawonn 2015 et al. Front. Microbiology)Note: Injecting 100mL of this water into 1L incubation chambers gives an enrichment of ~9.7 atom% 15N2
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Turn the serum bottle upside-down. Insert both the input and output needles/syringes into the septa. Slowly inject the gas label. Injecting upside prevents any gas from escaping through the outflow.
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When preparing gas syringe, flush any tubing/syringe being used 2x with 15N2 gas to remove any air.
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Using a needle and syringe, inject 15N2 gas in a ratio of 2ml/100mL into serum bottles (i.e. 11 mL for a 500mL serum bottle, as the headspace free volume is 550mL). A second needle and syringe should be used during injection as an outflow.
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Using the siphon, fill serum bottles headspace free and crimp seal. Depending on the rubber septa used, this step can be tricky and requires a bit of practice to not introduce an air bubble when inserting the septa.
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Using acid washed tubing, transfer degassed water from the vacuum flask to serum bottles by creating a siphon. Use a syringe to start the siphon.Using a siphon avoids pouring the water an potentially reintroducing gas into the water. Take care to keep the tubing ends at the bottom of both the flask and the serum bottle to avoid splashing.
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Using a vacuum flask, vacuum pump, magnetic stir plate and stir rod, degas 2L of water at ≥ 900 mbar for at least 20 minutes.
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After all the gas is injected, water accumulated in the outflow can be used to re-pressurize the serum bottle. Re-pressuring can increase dissolution (see Klawonn 2015 et al. Front. Microbiology) If there is any air in the outflow syringe, purge this air out prior to re-pressurizing so that only degassed water is being used to re-pressurize the bottle.
?. [Illustration of protocol]
Illustration by M. Moynihan