- Fill 250-mL serum bottles with 40 mL of DEA solution.
- DEA solution
- Add 200 mL of distilled water to a 1 L volumetric flask.
- Dissolve in flask:
- 0.1442 g potassium nitrate (KNO3)
- 1.8 g dextrose
- 0.225 chloramphenicol
- Dilute solution 1 L
- Store in brown bottle in refrigerator (N.B. Solution spoils in 3-5 days)
- Transfer bacteria sample to serum bottle.
- 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.
- For sediment samples, I transfer 30 g of sediment to the DEA solution.
- Make sure to either measure the dry weight or perform direct counts on your samples depending on how you want to report your results.
- 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.)
- Inject 10 mL of acetylene gas (C2H2) into the headspace of the serum bottle. (This inhibits the transformation of N2O to N2.)
- 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.
- 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 mL 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.)
- 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.
Manage space
Manage content
Integrations