Procedure

The anaerobic experiment is performed in a sealed bottle, where two polarographic electrodes that will measure nitric oxide and nitrous oxide are carefully attached to the lid. The electrodes are connected to a sensor to measure the different concentrations. The bottle is placed to a magnetic stirrer on 270 rpm and 37^oC.

There is one control flask, as well, containing only DM medium which will be spiked with NO₂^- (as will be done for the experimental flasks).

Additionally, we will measure the response of only anaerobic E. coli biomass without any NO₂^- added, and the same for anaerobic P. aeruginosa. This will be done in the experimental flasks prior to spiking with NO₂^-.

There will be 2 experimental flasks:

1. E. coli, to which spikes of NO₂^- will be added.
2. P. aeruginosa, to which spikes of NO₂^- will be added.

In addition to the continuous gas measurements, we take samples to measure the concentration of ammonia, nitrite and nitrate at the beginning and end of each spike.

The response time of the cells to spikes of NO₂^- is expected to be on the order of minutes, and we will run the experiment until the solution has reached saturation with NO₂^-.

EQUIPMENT NEEDED

• 1 400 mL bottle with a lid that the electrodes can be fastened air tightly to
• 1 magnetic stirrer
• 1 NO probe for NO measuring
• 1 N₂O probe for NO₂O measuring
• Syringes with long needles and small needles
• Syringe filters with pore size 0.2μm
• Stopwatch
• Modified DM Minimal Medium (Appendix 6)
• E. coli overnight culture
• P. aeruginosa overnight culture
• Flat bottom centrifuge tubes
• 2mL Eppendorf tubes
• 10mL, 1mL, 200μL pipettes with tips.
• MilliQ water

EXPERIMENTAL PROCEDURE

First,

1. Prepare DM minimal medium with ammonium chloride as a nitrogen source. Add 0.745 g NH₄Cl to 1L of prepared DM medium.
2. Start overnight cultures.
3. Prepare test solutions for ammonium, nitrite and nitrate kits.
4. Label eppendorf tubes and test tubes for colorimetric samples.
5. Grow E. coli top10 overnight/ P. aeruginosa in 10mL of DM medium + NH₄Cl prepared in step 1 at 37^oC.
6. Take 4mL of E. coli/P. aeruginosa overnight culture and add to 200mL fresh DM medium + NH₄Cl.
7. Grow the cells at 37^oC in 200 RPM until OD=0.35 (about 3 hours; about 5 hours for P. aeruginosa).
8. Pellet down the 200mL culture, 3000g for 4 min (Cool down the centrifuge if it is needed for 30 min at 4^oC).
9. Wash with 5 ml cold Modified DM minimal medium and centrifuge again.
10. Pour off the supernatant and resuspend the cell pellet in 200mL Modified DM Minimal medium in a centrifuge tube and pour samples together if they were made in more than one tube.
11. Measure OD of the 200 mL cell suspension and add Modified DM minimal medium until OD=0.3 (note the exact value).
12. Pure the 200 mL of the OD=0.3 suspension to a bottle. This is the experimental flask.
13. For the control: Remove one aliquot of 200mL of DM medium (without the added NH₄Cl).
14. Make the anaerobic experiment by saturating with pure N₂ following the method for injecting N₂ (Appendix 1), for 5 min the nitrite stock solution and 10 min the cell suspensions and control.
15. Start the experiment by adjusting the NO and N₂O electrodes in one flask at a time.
16. Put the flask on the magnetic stirrer and start with 270 rpm at 37^oC.
17. Remove 2 mL as the first sample t=0 colorimetric analysis.
18. Add 0.5 mL of 50mM nitrite stock solution to 100 ml of the cell suspension.
19. Watch the concentrations of NO and N₂O, and continue adding nitrite when they are not changing (about 10 min).
20. Repeat steps 18-20 until the solution exceeds the sensitivity of the sensor (1mM).
21. Make the colorimetric measurements for nitrite, nitrate and ammonium for each of the samples collected by using the Ammonium measurement in order to finish gathering data.