We did a variety of experiments to characterize the behaviour of transformed and untransformed E. coli in the presence of ammonium and nitrite.

• Toxicity of Nitrite, Nitrate and Ammonium to E. coli
• Verify nitrite stability in anaerobic, untransformed E. coli

In order to verify and characterize our transformants, we performed the following experiments:

• Measure production of N₂O from Nitrite NO₂^- anaerobically
• Measure consumption of Ammonium in Mutant 1 transformants compared to native E. coli

Finally, in order to see if the long term behaviour of our Nir transformant, we grew it in a bioreactor.

Toxicity of Nitrite, Nitrate and Ammonium to E. coli

Determine whether the ions nitrite, nitrate and ammonium are toxic to E. coli, and at what concentration. This experiment will use the Biolector to grow an E. coli strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth. We found that at high concentrations ammonium (>=500mM) and nitrate (>=25mM) were toxic to E. coli. However, these concentrations are much higher than we would expect to find in wastewater, so we do not expect these ions to be toxic to our cells.

Toxicity Experiment

Nitrite stability in anaerobic E. coli

To ensure that any nitrite we produce from Mutant 1 will not be consumed by any of the pathways in native E. coli, we grew E. coli anaerobically in the presence of varying concentrations of nitrite and measured the amount of ammonia and nitrite present. We found that a pathway to convert nitrite to ammonium is active, and therefore our Nir pathway will need to outcompete this.

Native anaerobic stability of nitrite in E. coli

Anaerobic production of nitrous oxide from nitrite in E. coli

Grow our transformed Mutant 2 anaerobically in the presence of varying concentrations of nitrite; measure the amount of nitrous oxide produced. Controls are untransformed E. coli and P.aeroginosa.

Experiment 2

Measure consumption of Ammonium in Mutant 1 transformants compared to native E. coli

Grow AMO and HAO transformants aerobically in the presence of ammonia; measure concentration of ammonia.

Experiment 4

Growth of Nir transformant in Bioreactor

Bioreactor