Determining concentration of nitrogen compounds

Experiment 1a: Verify nitrite stability in anaerobic, untransformed E. coli

Characterization of the behavior of E. coli wild type while growing in the presence of nitrite in an anaerobic reaction chamber by measuring the conversion of nitrite to ammonium.

In this anaerobic experiment three different concentrations of nitrite are added to a medium where a native strain of E. coli is growing, in order to test the rate at which nitrite is converted to ammonium. The hypothesis is that nitrite will be converted into ammonium. We want to determine how readily, and at what concentrations this happens. The anaerobic experiments will be performed in a sealed flask. The concentrations of NH₄⁺ and NO₂^- will be measured every 20 minutes and the experiment will last for 3 hours. The literature states that 10mmol of NO₂^- is converted into NH₄⁺ per mg cell biomass per hour. There will be also a magnetic stirrer on 250 rpm. Each sample with different concentrations of nitrite will be measured separately, as well as the controls. There will be three controls. One will be with only anaerobic biomass to check the behavior of cells without nitrite. A second one will be anaerobic abiotic with nitrite and medium to check that there is not any conversion of nitrite in the medium. A third anaerobic with killed biomass to confirm that reaction is catalyzed only by microbial activity.

EQUIPMENT NEEDED

• 6 bottle flasks of 100 ml with lids
• 1 magnetic stirrer
• Syringes with long needles and small needles
• Syringe filters with pore size 0.2μm
• Stopwatch.
• Ammonium Bicarbonate (NH₄)HCO₃ puriss., meets analytical specification of Ph.Eur., BP, E 503, 99-101% (Sigma-Aldrich)
• Sodium nitrite – NaNO₂ , puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99% (Sigma-Aldrich)
• Modified DM minimal medium
• E. coli overnight culture
• LB-broth medium
• Flat bottom centrifuge tubes
• 50 mM nitrite stock solution (MW NaNO₂ =69 g/mol; 345 mg in 100 mL water)
• 2mL Eppendorf tubes
• Colorimetric test kits for ammonium and nitrite.
• Rack for the samples
• Glass tubes for the colorimetric tests
• 10mL, 1mL, 200μL pipettes with tips.
• Single use plastic cuvettes.
• MilliQ water.

All preparations with cells will be done on ice so that the cells do not grow.

EXPERIMENTAL PROCEDURE

1. Grow E. coli top10 overnight in 10mL of LB medium at 37◦C.
2. Take 5mL of overnight culture and add to 500mL fresh LB medium.
3. Grow the cells at 37^oC in 210 RPM until OD=0.35 (about 2 hours).
4. Cool down the centrifuge for 30 min at 4^oC.
5. Pellet down the 500mL culture, 3000g for 4 min at 4^oC.
6. Discard supernatants and wash each aliquot with 10-20 ml cold Modified DM minimal medium and centrifuge again.
7. Pour off the supernatant once again and resuspend the cell pellets in 200mL Modified DM Minimal medium.
8. Measure OD of the 200 mL cell suspension and add cold Modified DM minimal medium until OD=0.3 (note the exact value). The final volume should be around 520-570 mL.
9. Killed biomass control: Add 100 mL of biomass in a flask, label accordingly and autoclave it.
10. 3 nitrite experiments plus control: Pour 100 mL of the OD=0.3 suspension into each flask and keep on ice. Label the flasks accordingly (nitrite1, nitrite 2, nitrite 3, no nitrite).
11. Abiotic control: Add 100 mL of DM medium to a flask and label accordingly.
12. Make experiments anaerobic by sparging N₂ in the solution by following the method for injecting N₂ (Appendix 1), for 3 min the nitrite stock solution and 5 min the cell suspensions and controls. Seal all bottles with rubber stoppers and aluminium caps.
13. Add 0.5 mL of 50mM nitrite stock solution to 99.5 ml of the cell suspension in flask 1. Then the nitrite concentration is about 0.25mM.
14. Add 1 mL of of 50mM nitrite stock solution to 99 ml of the cell suspension in a flask 2 and adjust the electrode. Then the nitrite concentration is about 0.5mM.
15. Add 2 mL of 50mM nitrite stock solution to 98 ml of the cell suspension in a flask 3. Then the nitrite concentration is about 1mM.
16. In the abiotic anaerobic control add 1 mL of nitrite stock.
17. Add 1mL of nitrite stock solution the anaerobic control with killed biomass.
18. Remove two 2.5mL samples as the t=0 samples from each of the reaction chambers.
19. Remove an initial 2mL sample to measure the initial Nitrate concentration.
20. Put the flask on the magnetic stirrer and start with 250 rpm at 37^oC.
21. Every 20 min for 3 hours take two samples 2 ml for the colorimetric assay from each of the experiments.
22. Inject the sample using filter of pore size 0.2μm and fill into a 2mL Eppendorf tube.
23. Repeat the steps 22-23 for each flask.
24. After the end of the batches, take a final sample for colorimetric assay and measure OD+cell mass.
25. Take a final sample to measure the ending Nitrate concentration.
26. Make the colorimetric measurements for nitrite and ammonium by using the protocols Nitrite colorimetric measurements and Ammonium colorimetric measurements, respectively.