Anaerobic soil slurries are made by mixing 10 g fresh weight of a sieved soil sample with 20 mL nutrient solution in 125 mL Wheaton bottles fitted with septa. Bottles are evacuated and purged with N2 three times to evacuate ambient air, vented to atmospheric pressure after the last N2 flush, then half of the bottles over-pressurized with addition of 16 mL C2H2to achieve a composition of 10% C2H2, and the other half with additional N2. Bottles are placed on a rotary shaker table at 125 rpm at room temperature and 3 mL gas samples collected 30, 60, and 90 minutes after C2H2 addition are transferred to evacuated storage vials.

Gas samples are analyzed for N2O concentration on a gas chromatograph (GC) in batches by statistical block and are calibrated against the corresponding 7-point standard curve. Two sets of standards are made. One set of standards are made with every batch by diluting a known concentration of a N2O standard gas in 90% N2 + 10% C2H2. The other set are made in 100% N2. Samples are analyzed within a month of collection and N2O concentrations are corrected for N2O in the aqueous phase using the Bunsen absorption coefficient (Groffman et al. 1999).

Equipment

• Vacuum/gas manifold system attached to N2 tank to evacuate and flush bottles and vials
• Rotary shaker table
• Gas chromatograph (e.g., Agilent 7890 equipped with a 63Ni electron capture detector (ECD))

Materials

• Wheaton bottles, 125 mL (http://wheaton.com/125-ml-btl-media-clr-type-i-grad-no-cap.html#1 VWR 16159-700)
• Butyl caps (http://wheaton.com/33-430-cap-phen-blk-opn-butyl-septa.html VWR 66012-658)
• Septa (http://wheaton.com/33-mm-septa-interlock-butyl-gry.html VWR 16199-969)
• Potassium nitrate (KNO3)
• Dextrose
• Timer
• Data sheets to record exact time points
• Syringes (1ml, 3ml, 10ml, and 20ml)
• Nanopure deionized water
• Calcium carbide (CaC2)
• Exetainer vials, 5.9 mL, with caps and septa
• Hypodermic needles, 22 gauge
• Chloramphenicol (if use – see note below)

Preparations Prior to Incubation Set-up

A. Nutrient media

1. Add 1 L of deionized water to a larger flask on a magnetic stir plate.
2. Add 1.44 g KNO3 (0.2 g N/L) and 500 mg Dextrose (0.5 g/L) to flask.
3. Mix for 1 hour with magnetic stir bar.
4. Bubble with N2 gas for 30 minutes prior to use to degas oxygen.

*Chloramphenicol is sometimes added to short-term incubations to inhibit protein synthesis. In the DEA, chloramphenicol addition would prevent denitrifying bacteria from synthesizing additional enzymes in response to optimal conditions (anoxic conditions, good supply of NO3- and DOC). Assays conducted with the antibiotic can prolong the linear range of N2O production and provide a more accurate measure of rates of denitrification. In soil microcosms, Smith and Tiedje (1979) used chloramphenicol in conjunction with the acetylene block method and identified two phases of N2O production (i.e., denitrification). In assays without chloramphenicol, there was a short-term, linear rate of denitrification, Phase 1, followed by an increased, often non-linear rate, Phase II. With chloramphenicol, the difference between Phase I and Phase II was slight or nonexistent. They concluded that Phase I represented actual field rates of denitrification whereas Phase II reflected the synthesis of denitrifying enzymes and microbial growth. Phase II thus represents the enzymatic potential for denitrification but is unlikely to reflect rates in situ (estimated in Phase I). Brooks et al. (1992) found that a chloramphenicol concentration of 0.9 mmol significantly inhibited N2O production, so assay concentrations should be well below this level. Additionally, chloramphenicol is also known to decrease the activity of existing enzymes (Pell et al. 1996). In our extensive testing with two-hour incubations using KBS soils, we found no effect of chloramphenicol on rates over those two hours, so we do not use it.

B. Gas sample vials

1. Fit an exetainer cap with a septum and screw tightly onto exetainer vial.
2. Insert needle from the vacuum/gas manifold system through the septum.
3. Insert a free needle through the septum to serve as an N2 vent.
4. Turn on N2 and gently flush vials for 1 minute.
5. Remove vent needle and allow vials to over-pressurize.
6. Turn the gas manifold valve to vent until no bubbles are in the bubbler system (usually 10-15 seconds); the bubbler prevents backflow of air into the vials.
7. Repeat steps three times.

C. Prepare C2H2

Note: C2H2 has a distinctive smell and must be prepared under a properly functioning hood or you might make yourself and people around you feel sick.
Note: Add water a tiny amount at a time to prevent the bottom of the bottle from blowing out due to pressure expansion as the CaC2 dissolves to make C2H2 gas.

1. Add a few grams of CaC2 to an uncapped Wheaton bottle. If the CaC2 is powdered, add enough to cover the bottom of the vial. If the CaC2 is granulated, add 15-20 pea-sized granules.
2. Cap the Wheaton bottle with screw-on cap with septum.
3. Evacuate the bottle, creating a vacuum.
4. Inject ~1 mL deionized water to the CaC2 pellets and gently agitate.
5. Use a 50-60 mL syringe with a 25 guage needle to carefully remove C2H2 from the bottle. The bottle will be under pressure so use your thumb to put pressure on syringe plunger so it does not shoot out like a champagne cork. If there is no positive pressure, then add more water to create more gas (assuming CaC2 is not fully reacted). Do not remove so much gas that there is negative pressure in the vial.
6. Store C2H2 in bottles unless using directly from the CaC2 vials.
7. Use C2H2 to make a 90% N2 + 10% C2H2 mixture to replenish DEA incubations after sampling points.
8. Continue using the CaC2 bottles until pressure has been reduced. Wait until pressure is minimal before adding another 1 mL of water to make more C2H2. The CaC2 is exhausted when it no longer fizzes on adding water.

D. Soil preparation

1. Collect soil samples to 20 cm with a soil corer.
2. Sieve fresh soil samples with a 4 mm sieve.
3. Set some soil aside for moisture determination.

Incubations

A. Set-up

1. Add 10 to 25 g to of fresh soil to each Wheaton bottle using a scoopula. Tests may be needed to determine soil mass and gas sampling intervals for other soils or conditions.
2. Add 20 mL nutrient media to each bottle using an automated pipette.
3. Screw-on the cap with septum.
4. Flush bottles with N2 three times using the same procedure as for exetainer preparation, leaving 1 atm of N2 in each bottle. Gently swirl bottles during each evacuation to allow for maximum removal of porewater N2O and O2. Do not shake: shaking can clog vent needles and disperse soil on the sides of bottles, away from NO3- and C resources.
5. Add 16 mL C2H2 to half of the bottles and 16 mL N2 to the other half of the bottles.
6. Put bottles immediately on rotary shaker table at 125 rpm at room temperature.

B. Gas sampling

1. After 30 minutes of incubation, insert the needle of a 3 mL syringe into the bottle’s septum and pump the syringe 3 times to mix the headspace. Then fill the syringe, remove the needle from the bottle, and inject sample gas into an appropriately labeled Exetainer to create an overpressure in the Exetainer (prior to sampling the Exetainer will be at 1 atm if prepared as described above). **Note: The headspace gas is lighter than air. If you do not use a gas tight syringe keep the syringe pointing downward while and after withdrawing the needle from the incubation bottle.
2. Record time of sampling each jar.
3. Repeat steps after 60 and 90 minutes of incubation time. 120 minutes is optional.

C. Gas analysis

Samples are analyzed for N2O with an Agilent 7890 Gas Chromatograph using the protocol detailed at https://lter.kbs.msu.edu/protocols/159 but there are two sets of standards. In addition to the normal set of standards, another set with gas standards prepared from 90% N2 + 10% C2H2 mixture instead of N2 to account for the additional effects of C2H2 on the electron capture detector (ECD) (since C2H2 is a quench gas).

Calculations

1. Adjust N2O concentrations determined for gas samples for N2O dissolved in media using the Bunsen coefficient:

M=Cg x (Vg +Vl )
where:
M = total amount of N2O in water and gas phase
Cg = concentration of N2O in the gas phase (headspace) as determined from the gas chromatograph, in μg N / L
Vg = volume of gas phase at 1 atm (125 mL bottle volume + 16 mL C2H2 or N2)
Vl = volume of liquid phase (20 mL)
 = Bunsen absorption coefficient at assay temperature (1.06 at 05 °C; 0.882 at 10 °C; 0.743 at 15 °C; 0.632 at 20 °C; 0.544 at 25 °C; 0.472 at 30 °C)





2. Calculate the denitrification rate (DR) by regressing the amount of N2O in each bottle against time. For assays with two sampling time points rather than three, the equation below can be used:

DR = [(C2*H)- (C1*H)]/ (D*T)
where:
DR = Denitrification rate (μg N∙〖kg soil〗^(-1)∙h^(-1))
C1 = N2O concentration at time point 1 (μg N_2 O-N/L headspace)
C2 = N2O concentration at time point 2 (μg N_2 O-N/L headspace)
H = flask head space (L) at 1 atm (do your own calculation based on your soil, media, and sample size)
T = time interval (h)
D = soil dry weight (kg)





3. Rates of N2O production are evaluated for linearity. If the rate is non-linear or otherwise poorly fits the time course series, discard the rate due to sampling error. With additional time course sampling, a non-linear fit that takes exponential growth into consideration might be used (Pell et al. 1996).

References:

Groffman, P. M., E. A. Holland, D. D. Myrold, G. P. Robertson, and X. Zou. 1999. Denitrification. Pages 272-288 in G. P. Robertson, D. C. Coleman, C. S. Bledsoe, and P. Sollins, editors. Standard Soil Methods for Long-term Ecological Research. Oxford University Press, New York, New York, USA.
Brooks, P. J., R. L. Smith, and D. L. Macalady. 1992. Inhibition of existing denitrification enzyme activity by chloromaphenicol. Appl. Envrion. Microbiol 58:1746-1753.
Pell, M., B. Stenberg, J. Stemstron, and L. Torstensson. 1996. Potential denitrification activity assay in soil — with or without chloramphenicol. Soil Biology and Biochemistry 28:393-398.
Smith, M. S. and J. M. Tiedje. 1979. Phases of denitrification following oxygen depletion in soil. Soil Biology and Biochemistry 11:262-267.