Nitrification in Aquarium 1 (Lab Journal)

This is the detailed lab journal of the Nitrification in Aquarium 1 experiment. For a structured summary, please refer to the report of the experiment.

General comments

• The aquariophilic kits and pH strips we use are color-based indicators, and can be quite imprecise. The small variations of concentration can probably be considered un-significant.
• We could not measure ammonium content in the culture before August 7th (day 16), as we could not find the ammonium measuring kit in the aquariophilic shop, and had to order it.

Day 0 - 22/7/2015 - 20h

We started the NA1 experiment on this day.

Culture

We started the nitrifyers culture by mixing 2.5 l of pond water (including 1 l of water from the bottom of the pond + soil) with tap water, to reach a total amount of 40 l.

The pond from which pond water was collected was located away of any potential runoff of agricultural nitrate.

Bioreactor

Our culture was poured in a 60 L aquarium, in which a pump drips 380l/h of water through two diffusers.

Urine

The culture was fed with urine from a first batch collected from mainly one donor. The pH of urine was measured below 6.5, which indicates that urea was probably not hydrolysed to ammonium yet (pH would otherwise be much higher).

The culture was fed with 4.5 ml of this urine batch. This volume was calculated from an assumed nitrogen-concentration of 4k-5k mgN/l in urine, and a target concentration of 0,5 mgN/l in our culture, from our aquaponics protocol [1].

Day 1 - 23/7/2015 - 20h

Culture

We took out 45 ml of culture for analysis purposes.

The measured pH of the culture was 7.7.

We measured a nitrate concentration of 1 mgN/l, and anitrite concentration of 0.01 mgN/l. This indicates that the nitrification has probably already started, transforming urea from urine in small amounts of nitrate and nitrite.

Bioreactor

We approximately covered the aquarium, protecting our culture from light to avoid algae growth and nitrate consumption.

Urine

We fed the culture with 10 ml of urine, from a sample tube prepared from the main 5l batch. We try to feed the culture more, to see if potentially higher amounts of urea can induce more visible amounts of nitrate after nitrification.

The pH of urine was still below 6.5, indicating that urea hydrolosys has probably not taken place yet.

Day 5 - 27/7/2015 - 10h30

Culture

We took out 35 ml of culture for analysis purposes.

We measured a nitrate concentration of 2 (2-5) mgN/l, and a nitrite concentration of 0.02 mgN/. This a slight, although maybe unsignificant increase in the nitrification reaction.

Urine

We fed the culture with 10 ml of urine, from the same sample tube as the day before.

The pH of urine was measured at 7.1, maybe indicating a slight start of urea hydrolysis.

Day 6 - 28/7/2015 - 22h30

Culture

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was between 7,1 and 7,4.

We measured a nitrate concentration of 4 (3-5) mgN/l, and a nitrite concentration of 0,05 mgN/l. This a slight, although maybe unsignificant increase in the nitrification reaction.

Bioreactor

Aeration was reduced.

Urine

We fed the culture with 10 ml of urine, from the same sample tube as the day before.

The pH of urine was measured at 7.4, maybe indicating a continuation of the urea hydrolysis process ?

Day 7 - 29/7/2015 - 20h15

Culture

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was between 6.8 and 7.1. It is getting more acidic.

We measured a declining nitrate concentration of 1 mgN/l. Is this indicating that reduced aearation induces a consumption of nitrates by bacteria/algae ?

We measured a stable nitrite concentration of 0.05 mgN/l.

Bioreactor

Concerned by the fact that reduced aeration may have been responsible for nitrate consumption, aeration was raised back to its original level.

Urine

We fed the culture with 20 ml of urine, from the same sample tube as the day before. We try to feed the culture more, to see if potentially bigger amounts of urea can induce more visible amounts of nitrate after transformation.

The pH of urine was measured at 7.5, maybe indicating a continuation of the urea hydrolysis process ?

Day 8 - 30/7/2015 - 20h30

Culture

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was between 6.5 and 6.8. It is getting even more acidic.

We measured a concentration nitrate of 1 mgN/l, which is stable from the day before. Is nitrate being produced and eaten up at the same time ? Is the low pH inhibiting the nitrification process ?

We measured a slightly increasing nitrite concentration of 0.1 mgN/l, suggesting that at least the nitrite-producing nitrifyers are still slightly active.

Urine

We fed the culture with 50 ml of urine, directly from the big container. We try to feed the culture more, to see if potentially bigger amounts of urea can induce more visible amounts of nitrate after transformation.

The pH of urine was measured at 7.4, which is still low for urine with hydrolysed urea.

Day 9 - 31/7/2015 - 19h

Culture

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was 6.8, which is more or less stable since the last measurement.

We measured a slightly increasing nitrate concentration of 2 mgN/l, suggesting that nitrification may be at least slightly happening.

We measured a stable nitrite concentration of 0.1 mgN/l.

Bioreactor

We covered the aquarium more thoroughly, protecting our culture from light to avoid algae growth and nitrate consumption.

Urine

We fed the culture with 50 ml of urine, directly from the big container.

The pH of urine was measured at 7.4 (stable), which is still low for urine with hydrolysed-urea.

Day 10 - 1/8/2015 - 16h

Culture

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture slightly increased to 6.8-7.1.

We measured a stable concentration of 2 mgN/l, suggesting that nitrification may be at least slightly happening.

We measured a stable nitrite concentration of 0.1 mgN/l.

After these measurement, we added 50g of Na2CO3 to the culture and strongly mixed it (manually). Adding this chemical aims at strongly increasing the pH. Knowing that the nitrifyers operate better in high pH conditions, we decided to see if increasing drastically the pH of the culture would increase the amount of nitrate produced.

Urine

After some nitrogen mass balance calculations, we realized that the total amount of nitrogen added to the culture in urea/ammonium form was way more important than what we could find in nitrite/nitrate form. Being afraid that ammonium (which we cannot measure yet) might be accumulating in the culture, we did not feed the culture with urine today.

Half an hour later, at 16h30

Culture

The pH of the culture has reached 7.9-8.0. The addition of Na2CO3 reached its goal.

Day 12 - 3/8/2015 - 19h

Culture

We took out 30 ml of culture for analysis purposes.

The pH of the culture has reached 8.7-8.8, which is quite high.

We measured a slightly decreasing nitrate concentration of 1 mgN/l.

We measured a stable nitrite concentration of 0.1 mgN/l.

Given the fact that nitrification was not improved during the last two days of culture at high pH, we decided to revert back to initial conditions. We therefore kept 1/5 (8l) of the total volume of the culture (40l), and mixed it with tap water to reach 40l. We also kept the bottom sludge.

Urine

As

• not much nitrite or nitrate was produced,
• we still cannot measure ammonium concentration,

we did not feed the culture with urine today.

Day 13 - 4/8/2015 - 13h30

Culture

We took out 30 ml of culture for analysis purposes.

The pH of the culture was brought back to 7.2.

We measured a slightly increasing nitrate concentration of 4 mgN/l.

We measured a slightly decreasing nitrite concentration of 0.05 mgN/l.

Urine

As

• not much nitrite or nitrate was produced,
• we still cannot measure ammonium concentration,

we did not feed the culture with urine today.

Day 14 - 5/8/2015 - 21h

Culture

We took out 30 ml of culture for analysis purposes.

The pH of the culture is slightly decreasing to 7.1.

We measured a stable nitrate concentration of 4 mgN/l.

We measured a slightly increasing nitrite concentration of 0.1 mgN/l.

Urine

As

• not much nitrite or nitrate was produced,
• we still cannot measure ammonium concentration,

we did not feed the culture with urine today.

Day 16 - 7/8/2015 - 19h

Culture

We took out 40 ml of culture for analysis purposes.

The pH of the culture is slightly decreasing to 7.1.

We measured a very low ammonium concentration of 0.3 mgN/l.

We measured a slightly increasing nitrate concentration of 7 (5-10) mgN/l.

We measured a stable nitrite concentration of 0.1 mgN/l.

Urine

We fed the culture with 25 ml of urine, directly from the big container.

The pH of urine was measured at 7.8, which is still lower than usual for urine with hydrolysed-urea.

While taking today’s 25 ml of urine from the container, we contaminated the urine stock (on purpose) by using a pipette which was dipped in our culture. We thereby hope to bring urease-containing bacteria from the culture in the urine stock, and accelerate urea hydrolysis.

Day 17 - 8/8/2015 - 20h

Culture

We took out 35 ml of culture for analysis purposes.

The pH of the culture is slightly increasing to 7.4.

We measured a slightly increasing, but still very low ammonium concentration of 0.5 mgN/l.

We measured a slightly increasing nitrate concentration of 8 mgN/l.

We measured a slightly increasing nitrite concentration of 0.5 mgN/l.

Urine

We fed the culture with 23 ml of urine, directly from the big container.

The pH of urine was measured at 8.5, which is in the normal range for urine with hydrolysed-urea.

36 Day 19 - 10/8/2015 - 18h

Culture

We took out 15 ml of culture for analysis purposes.

The pH of the culture is slightly increasing to 7.7.

We measured a slightly increasing, but still low ammonium concentration of 1 mgN/l.

We measured a slightly increasing nitrate concentration of 9 (8-10) mgN/l.

We measured a slightly increasing nitrite concentration of 0.7 mgN/l.

Urine

We fed the culture with 50 ml from a new batch of urine. We try to feed the culture with more urine, to see if potentially bigger amounts of urea can induce more visible amounts of nitrate after transformation.

The pH of urine was measured at 6.5, which is again showing that urea hydroloysis probably has not happened yet.

Day 20 - 11/8/2015 - 19h

Culture

We took out 35 ml of culture for analysis purposes.

The pH of the culture is slightly decreasing to 7.4.

We measured a slightly decreasing ammonium concentration of 0.6 mgN/l.

We measured a slightly increasing nitrate concentration of 9 (8-10) mgN/l.

We measured a slightly increasing nitrite concentration of 0.6 mgN/l.

Bioreactor

When we arrived for measuring, the tubes connecting the pump to the diffusers were disconnected, the culture was therefore not oxygenated for at least a few hours, maybe even 25. We hope that the culture was not killed...

Urine

We fed the culture with 50 ml from a new urine batch.

The pH of urine slightly increased to 6.8, urea hydroloysis probably has not happened yet.

Day 21 - 12/8/2015 - 19h

Culture

We took out 35 ml of culture for analysis purposes.

The pH of the culture is stable to 7.4.

We measured a slightly increasing ammonium concentration of 1.5 mgN/l.

We measured an increasing nitrate concentration of 20 mgN/l, which is a good sign that the culture is still alive, even after yesterday’s aeration accident.

We measured a slightly increasing nitrite concentration of 1 mgN/l.

We ended up the NA1 experiment here, and started the NA2 and NA3 experiments on the same night.