Nitrate Concentration Conundrum

[Cadd]

Hey! I've got a project going but seem to be running into an interesting problem with nitrate concentration and I know there are probably people out there who can figure this out for me. I've been assessing nitrate concentrations after bacterial growth on the following base media modifications: +Nitrate, 1 mM Urea, 1 mM Urea + Nitrate, 10 mM Urea, 10 mM Urea + Nitrate, 1 mM Ammonium Chloride, 1 mM Ammonium Chloride + Nitrate, 10 mM Ammonium Chloride, and 10 mM Ammonium Chloride + Nitrate. Initial concentration of nitrate in all conditions supplemented with it should be at 5 g/L due to the potassium nitrate; meaning that 5 g/L should be the maximum amount of nitrate present unless more is being created by bacterial processes or (the part that I am unsure about) if certain chemicals are reacting to produce more nitrate(?). The final nitrate concentrations are yielding about 250 mg/L (ppm) at a dilution of 100 µL/20 mL for all the controls but this would translate to a starting concentration of 50 g/L of nitrate so I'm not sure where this may be going wrong. Media specifications, additions, and calculations are down below. Thanks!

Base media:
Ammonia-free Trace metals - 1 mL
Sodium Acetate - 3 g/L
Sodium Chloride - 0.6 g/L
Magnesium Sulfate Heptahydrate - 0.3 g/L
Dipotassium Phosphate - 1.0 g/L
Calcium Chloride Dihydrate - 0.05 g/L

+Nitrate:
Potassium Nitrate - 5 g/L

+Urea (1 or 10 mM):
1 mM - 0.06006 g/L
10 mM - 0.6006 g/L

+Ammonium Chloride (1 or 10 mM):
1 mM - 0.05349 g/L
10 mM - 0.5349 g/L

Calculation I'm getting:
Final nitrate concentration: 250 mg/L = .25 g/L
Dilution factor: (.1 mL)/(20 mL) = 0.005 = 5 x 10^-3
Initial nitrate concentration: (.25 g/L)/(0.005) = 50 g Nitrate
50 g obviously does not equal the 5 g/L that was actually added

[norman40]

Hi Cadd,

I don't think any of the chemicals you've listed will oxidize ammonia or urea to nitrate. So you may have a bacterial nitrification process that is increasing the nitrate concentration:

https://en.wikipedia.org/wiki/Nitrifying_bacteria

Another potential explanation might be some kind of experimental error with your nitrate concentration measurements. You might want to review your assay procedure for possible sources of error.

I hope this helps and good luck. Please post again if you have more questions.

A. Norman