In this anaerobic experiment we add nitrite in our cultures with the Nir transformed cells, in order to test how nitrite is converted to nitrous oxide.

  1. Make an overnight culture with the Nir transformed cells and then pour 100 ml in each of the 3 bioreactor vessels with 800 ml DM modified minimal medium.
  2. Set the bioreactor at 37oC.
  3. Add 0.8 ml of 0.916 M ammonium chloride solution in each of the three vessels.
  4. Add 8 ml of 50mM of nitrite stock solution to each of the three vessels.
  5. Take 100 mL as the first sample t=0 for both colorimetric analysis and nitrous oxide measurement, from each of the 3 vessels.
  6. After 10 hours take another 100 ml of sample to measure nitrous oxide and make the colorimetric analysis, from each of the 3 vessels.
  7. After taking the samples pour them in 200 ml sealed vessels with syringes in order to keep the conditions anaerobic.
  8. To finish gathering data, make the colorimetric measurements for nitrite and ammonium for each of the samples collected by using the protocols Nitrite colorimetric measurements and Ammonium colorimetric measurements, respectively.