Calibration of NO and N₂O electrodes/probes

• Cleaning the probes

1. Prepare 96% ethanol solution
2. Prepare 0.01M HCl solution
3. Put both probes in ethanol solution for 1 minute
4. Put both probes in HCl solution for 1 minute
5. Rinse with demineralized water

• Calibration

1. Make standard solution NO

i. Make 200 ml solution 1 M NaOH ii. Make it anoxic by bubbling with N₂ for 5 minutes iii. Make MQ water anoxic iv. Bubble 5% NO solution through NaOH solution v. Convey towards the water flask for 20 minutes

1. Make standard solution of N₂O

i. Make solution anoxic by bubbling 5 minutes with N₂ ii. Flush with N₂O for 20 minutes.

• Procedure

1. Pure 200 ml of DM minimal medium in the bottle
2. Adjust the temperature at around 37^oC by using a magnetic stirrer
3. Adjust both of the probes in the lid of the bottle
4. Connect the electrodes with the microsensors
5. Make the solution anoxic by bubbling with N2 for 10 minutes
6. Start by calibrating N₂O sensor (the adding points are the concentration of N₂O in mg/L)

i. Add the first point “0” on the sensor ii. Spike 0.5 ml of N₂O in 200 ml and add point “60.35” iii. Spike 0.5 ml of N₂O and add point “120.7” iv. Spike 0.5 ml of N₂O and add point “181.06” v. Spike 0.5 ml of N₂O and add point “2241.41” vi. Spike 1 ml of N₂O and add point “362.11” vii. Spike 1 ml of N₂O and add point “482.81” viii. Then apply the calibration for the sensor

7. Continue by calibrating NO sensor (the adding points are the concentration of NO in mg/L) i. Add the first point “0” on the sensor ii. Spike 1 ml of NO in 200 ml and add point “0.47” iii. Spike 1 ml of NO and add point “0.94” iv. Spike 1 ml of NO and add point “1.4” v. Spike 1 ml of NO and add point “1.86” vi. Spike 1 ml of NO and add point “2.31” viii. Then apply the calibration for the sensor