Nitrification in Aquarium 3 (Report)

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This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the detailed lab journal of this experiment.

Introduction

As stated on the main project page, our third “Nitrification in Aquarium” experiment (NA3) 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.

This second experiment is a variation of the first one (NA1) we conducted. We started from the same culture as NA1, but reduced the reaction volume to 15 l (instead of 40 l for NA1), while keeping the same aeration rate, to have a better oxygenation of the system. This NA3 experiment will be fed with large amounts of urine everyday, approximately reaching the target 1/30 volume/volume urine dilution we plan to use to feed the hydroponics wall. We hope to observe the production of larger amounts of nitrate, or to definitely rule out the hypothesis stating that we might be bringing too low amounts of nitrogen to the system.

Material & Methods

Experimental setup

Culture

We started the culture for NA3 on August 12th, 2015, from 15 l of the mix from NA1.

Recalling the main concentrations for NA1 culture at 7 pm :

  • pH = 7.4
  • Ammonium concentration = 1.5 mgN/l
  • Nitrate concentration = 20 mgN/l
  • Nitrite concentration = 0.8 mgN/l

However, we measured different values when measuring in our new “bioreactor”

  • pH = 7.2
  • Ammonium concentration = 3 mgN/l
  • Nitrate concentration = 10 mgN/l
  • Nitrite concentration = 0.7 mgN/l

(We took out 35 ml of culture for analysis purposes.)

On day 1 (Augsut 13th, 2015), we reinforced the culture with nitrifying bacteria from a stock culture bought at the aquariophilic shop.

This difference in theses measurements most probably comes from the inaccuracy of our analysis kits.

We fed the culture with 0.5 l of urine, from the urine batch started on August 12th.

A strong smell of urine is observed after feeding.

Bioreactor

This culture was poured in a 20 L opaque bucket , in which a pump (comparable to the one used for NA1) was dripping air through two diffusers. The bucket was covered with a piece of cardboard cut in a pizza box, to avoid light to get in the bioreactor.

Concentrations measurement

We used aquariophilic measurement kits for ammonium [2], nitrite [3] & nitrate [4] from JBL. A manual on how to use the kit for concentration monitoring is included in each kit.

When concentrations were high, we sometimes used preliminary urine dilutions to get a better precision in our measurements.

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

The experiment was terminated on August 21st, 2015, after 9 days.

  • Nitrate and ammonium concentrations display quite strong variations, which are difficult to explain.
  • The nitrate concentration stays in a quite low range, even though the hypothetic amounts of nitrogen brought by urine is quite high.
  • The addition of nitrifyers from the aquariophilic culture did not help.

Discussion

The high variations in the result are at least partly due to the inaccurracy of the measuring kits.

We may have not fed enough urine to the culture, thereby inducing low nitrate consumption. However, we added up to a hundred 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 very 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 (NA1, 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. We also reduced the reaction volume from 40 l to 15 l while keeping the same reaction rate, and it did not help. 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. Finally, similar oxygenation conditions led to a quite good nitrate concentration (160 mgN/l) in NA2. However, some specific competing bacteria may have developed in NA3. Moreover, the pump in NA2 being slightly more powerful, we cannot totally rule out the fact that lack of oxygen may be a reason for low nitrate concentration.

Nitrifyers I and/or II may be inhibited by lack of oxygen (indepedently of the presence of competing bacteria). It seems unlikely, as oxygenation in the reactor is quite strong, but should maybe still be tested, for the reasons stated in the previous paragraph.

Nitrifyers I and/or II may be inhibited by light conditions. It is however unlikely, as the opaque bucket 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. This last reason seams unlikely too, as similar conditions did not inhibit nitrification in NA2.

Nitrifyers I and/or II may be inhibited by low pH conditions. This seems very unlikely, as pH was quite high, especially by the end of the experiment.

It is likely that nitrogen may be evaporating in the form of ammonia, after conversion of urea to ammonium. The sometimes strong urine smell observed around the bioreactor is reinforcing this hypothesis. More urine fed may mean more ammonia evaporation. Maybe ammonia evaporates at a higher rate than the ammonium processing rate by nitrifyers.

It also remains likely that urea hydrolysis may be a slower process than what we thought initially, happening within days and not within hours. Optimal light, pH and oxygenation conditions for a quick urea hydrolysis should be investigated. For instance, we can observe a quite significant difference in pH conditions between NA2 (on day 6, nitrate concentration of 80 mgN/l) and NA3 (on day 6, nitrate concentration of 6 mgN/l), although low pH conditions led to low nitrate conditions during NA1 (on day 17, nitrate concentration of 8 mgN/l). Urea hydrolysis however does not seem sensitive to light conditions, as it happens both in dark piping systems and translucid Falcon tubes. Neither does it seem to be sensitive to oxygen, as it happens in aerated piping system as well as in airtight Falcon tubes.

We should also point out that adding up nitrifyers from the aquariophilic shop stock culture did not give a boost to the nitrate concentration, even though we added ten times the recommended dose on the bottle (lab journal NA3, day 1, August 13th, 2015). Unfortunately, we could not measure the concentration of nitrifying bacteria present in the culture, this would have helped us to understand if the bacteria we added were killed or inhibited by inappropriate culture conditions (low ammonium concentration because of ammonia evaporation, lack of oxygen because of other competing bacteria, lack of physical support to host the nitrifyers…), or if the nitrate they produced was consumed by competing bacteria.

Conclusion

The most probable explanation for the low nitrate concentration at this stage of investigations seems to be a mismatch between the dynamics of the processes at stake in the reactor. The ability of nitrifyers to process ammonium may be the limiting factor, inducing unprocessed ammonium to evaporate due to the strong aeration rate. The urine smell observed around the bioreactor reinforces the hypothesis of ammonium losses by evaporation. The nitrifyers culture may be unable to develop strongly because of lack of physical support. Urea hydrolysis, maybe being negatively impacted by culture conditions, may also impact this overall dynamics.

Bringing large amounts of nitrogen through large amounts of urine does not seem to help nitrification to happen, and nitrate concentration to rise. This invalidates the hypothesis of the amount of nitrogen brought to the culture as limiting factor.