Acetylene Block

Contact: Kristin Searcy

1. Fill 250-mL serum bottles with 40 mL of DEA solution.
2. DEA solution
   1. Add 200 mL of distilled water to a 1 L volumetric flask.
   2. Dissolve in flask:
      1. 0.1442 g potassium nitrate (KNO₃)
      2. 1.8 g dextrose
      3. 0.225 chloramphenicol
   3. Dilute solution 1 L
   4. Store in brown bottle in refrigerator (N.B. Solution spoils in 3-5 days)
3. Transfer bacteria sample to serum bottle.
   1. For biofilm samples, I remove the bacteria from the attached surface into a PBS solution through cycles of water bath sonication (one minute), ice (one minute), and vortexing (30 seconds), and then transfer the bacteria-laden PBS to the DEA solution.
   2. For sediment samples, I transfer 30 g of sediment to the DEA solution.
   3. Make sure to either measure the dry weight or perform direct counts on your samples depending on how you want to report your results.
4. Purge each sample in the serum bottle with nitrogen gas for 5 minutes in order in create an anaerobic environment. (Shake sample occasionally during purging to release any trapped oxygen.)
5. Inject 10 mL of acetylene gas (C₂H₂) into the headspace of the serum bottle. (This inhibits the transformation of N₂O to N₂.)
6. Use gas chromatography to measure the concentration of nitrous oxide in the headspace of the samples following acetylene injection. I use a ³Ni electron capture detector and a stainless steel Porapak Q (80/100 mesh) column. Flush the column with N₂ as carrier gas at 15 mL/min.  The column and detector should be operated at 60°C and 320°C, respectively. 
7. Make a standard curve by injecting known volumes of nitrous oxide into serum bottles filled with nitrogen gas, and allow the mixture a few minutes to equilibrate. Inject ~20 μL of the standard mixture into the GC and take a reading. (Vary the amount of nitrous oxide used in the standard mixtures, but keep constant the volume of gas that is injected into the GC when making the standard curve.)
8. Following the injection of the acetylene into the sample bottles, take headspace samples every half-hour, and directly inject the sample into the GC to measure the concentration of nitrous oxide over time.