Nitrification in Aquarium 1 (Report)

This is the summarized report of the Nitrification in Aquarium 1 experiment, for more details, please refer to the lab journal of this experiment.

Introduction

As stated on the main project page, our first “Nitrification in Aquarium” experiment (NA1) is an adaptation of the first “cycling” step of aquaponics. We will start from an aquarium, an air pump and drippers and some pond water. We will feed the system with urine everyday, as they do with ammonium in this protocol [1]. This is supposed to lead to the build up of a strong culture of nitrifyers, which will convert urine into nitrate.

Material & Methods

Experimental setup

Culture

We started the nitrifyers culture on July 22nd, 2015, 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.

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 [2].

Bioreactor

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

Collecting and storing urine

We stored urine directly after collection, in airtight clean containers, thereby avoiding losses of ammonia.

Our urine container may have been too clean, as urea hydrolysis did not happen before active inoculation with pond water.

Concentrations measurement

We used aquariophilic measurement kits for ammonium [3], nitrite [4] & nitrate [5] from JBL. A manual on how to use the kit for concentration monitoring is included in each kit.

We also used pH-measuring paper strips. Instructions are also included when you buy them.

All these color-based indicators were not very precise, inducing quite strong uncertainty in our measurements.

Results

We terminated the experiment on August 12th, 2015, after 21 days.

The main results of this first experiment are : - The amount of nitrate (and nitrite) stays lower than the amount of nitrogen fed in the form of urea/ammonium. - Reducing aeration led to (maybe unsignificant) nitrate consumption. - Increasing the pH of the culture above 8 for 2 days did not seem to enhance the nitrification reaction.

For detailed results, see the NA1 lab journal.

Discussion

Many hypothesis may expain why nitrate concentration does not rise.

We may have not fed enough urine to the culture, thereby inducing low nitrate consumption. However, we added up to ten times the amount of urine than what we should have done according to our calculations and supposed urine nitrogen concentration (4k-5k mgN/l). This did not significantly affect the amount of nitrate we produced. The lack of nitrogen fed to the culture is therefore unlikely.

Urease-containing bacteria may not be present in the culture. However, such bacteria are known to be uquitous. Moreover, when we contaminated our first urine batch with some of our culture (on day 16), urine pH raised drastically, suggesting that hydrolysis happened as a consequence of this contamination, and that urease-containing bacteria are therefore present in the culture.

Oxygen may be consumed by competing bacteria. It seems unlikely, as oxygenation in the reactor is quite strong (380l of air/h). Morevover, this happens mainly when there is a high organic content in the culture, which is not the case here, the volume of the bottom sludge being very low in proportion to the total reaction volume.

Nitrifyers I and/or II may be inhibited by lack of oxygen. It seems unlikely, as oxygenation in the reactor is quite strong (380l of air/h), but should maybe still be tested.

Nitrifyers I and/or II may be inhibited by light conditions. It is however unlikely, as the aquarium was covered with cardboard, thereby avoiding light to enter in the aquarium. Unless the sensitivity of nitrifyers to light is so high that the daily opening for measurement harms them.

Nitrifyers I and/or II may be inhibited by low pH conditions. It seems however unlikely, as 48 hours (day 10-12) at high pH did not significantly affect the nitrate concentration. Unless high pH should have been maintained for a longer period.

It is likely that nitrogen may be evaporating in the form of ammonia, after conversion of urea to ammonium.

It is likely that urea hydrolysis may be a slower process than what we though initially, happening within days and not within hours.

Conclusion

- It seems wiser to leave aeration to the maximum rate to avoid nitrate consumption, even though it may lead to increase in ammonia losses by evaporation. - It seems wiser to cover the aquarium to avoid algae growth and subsequent nitrate consumption. - Increasing the amount of urine fed (at the magnitude at which we did it) does not seem to help. - Increasing the pH of the solution does not seem to help significantly either.

We start two experiments in smaller containers, to test : - if stronger oxygenation helps. - i fstronger aeration induces more evaporation. - if feeding more urine to a more oxygenated container induces the production of larger amounts of nitrates.

The NA2 & NA3 experiment will be conducted in smaller 15 l nitrifyer cultures. Each will be oxygenated with a pump comparable to the one feeding the 40 l culture of NA1. NA3 will be fed with large amounts of urine. NA2 will be fed with small amounts of urine, to avoid nitrite pisoning of nitrifyers, and will thereby act as a backup for NA3.