Hackuarium - User contributions [en]

Nitrification in Aquarium 1 (Report)

2017-03-07T13:08:22Z

Michka: /* Bioreactor */

This is the summarized report of the Nitrification in Aquarium 1 experiment. For more details, please refer to the [[Nitrification in Aquarium 1 (Lab Journal)|detailed 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== Bioreactor ====

Our culture was poured in a 60 L aquarium, in which a pump drips 380l/h of air 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 [[Nitrification in Aquarium 1 (Lab Journal)|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.
* if stronger aeration induces more evaporation.
* if feeding more urine to a more oxygenated container induces the production of larger amounts of nitrates.

The [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|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. [[Nitrification in Aquarium 3 (Report)|NA3]] will be fed with large amounts of urine. [[Nitrification in Aquarium 2 (Report)|NA2]] will be fed with small amounts of urine, to avoid nitrite pisoning of nitrifyers, and will thereby act as a backup for [[Nitrification in Aquarium 3 (Report)|NA3]].

Nitrification in Aquarium 3 (Report)

2016-11-03T13:30:58Z

Michka: /* Discussion */

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 ([[Nitrification in Aquarium 1 (Report)|NA1]]) we conducted. We started from the same culture as [[Nitrification in Aquarium 1 (Report)|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 [[Nitrification in Aquarium 1 (Report)|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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 [[Nitrification in Aquarium 2|NA2]]. However, some specific competing bacteria may have developed in [[Nitrification in Aquarium 3|NA3]]. Moreover, the pump in [[Nitrification in Aquarium 2|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 [[Nitrification in Aquarium 2 (Report)|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 ([http://wiki.hackuarium.ch/w/NA2_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|7.4 on day 6, nitrate concentration of 80 mgN/l]) and NA3 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|8.5 on day 6, nitrate concentration of 6 mgN/l]), although low pH conditions led to low nitrate conditions during NA1 ([http://wiki.hackuarium.ch/w/NA1_lab_journal#Day_17_-_8.2F8.2F2015_-_20h|7.4 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 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_1_-_13.2F08.2F2015_-_16h|see 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.

Nitrification in Aquarium 3 (Report)

2016-11-03T13:30:30Z

Michka: /* Results */

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 ([[Nitrification in Aquarium 1 (Report)|NA1]]) we conducted. We started from the same culture as [[Nitrification in Aquarium 1 (Report)|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 [[Nitrification in Aquarium 1 (Report)|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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 [[Nitrification in Aquarium 2|NA2]]. However, some specific competing bacteria may have developed in [[Nitrification in Aquarium 3|NA3]]. Moreover, the pump in [[Nitrification in Aquarium 2|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 [[Nitrification in Aquarium 2 (Report)|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 ([http://wiki.hackuarium.ch/w/NA2_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|7.4 on day 6, nitrate concentration of 80 mgN/l]) and NA3 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|8.5 on day 6, nitrate concentration of 6 mgN/l]), although low pH conditions led to low nitrate conditions during NA1 ([http://wiki.hackuarium.ch/w/NA1_lab_journal#Day_17_-_8.2F8.2F2015_-_20h|7.4 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 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_1_-_13.2F08.2F2015_-_16h|see 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 where 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.

Nitrification in Aquarium 3 (Report)

2016-11-03T13:25:07Z

Michka: /* Conclusion */

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 ([[Nitrification in Aquarium 1 (Report)|NA1]]) we conducted. We started from the same culture as [[Nitrification in Aquarium 1 (Report)|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 [[Nitrification in Aquarium 1 (Report)|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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 [[Nitrification in Aquarium 2|NA2]]. However, some specific competing bacteria may have developed in [[Nitrification in Aquarium 3|NA3]]. Moreover, the pump in [[Nitrification in Aquarium 2|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 [[Nitrification in Aquarium 2 (Report)|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 ([http://wiki.hackuarium.ch/w/NA2_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|7.4 on day 6, nitrate concentration of 80 mgN/l]) and NA3 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|8.5 on day 6, nitrate concentration of 6 mgN/l]), although low pH conditions led to low nitrate conditions during NA1 ([http://wiki.hackuarium.ch/w/NA1_lab_journal#Day_17_-_8.2F8.2F2015_-_20h|7.4 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 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_1_-_13.2F08.2F2015_-_16h|see 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 where 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.

Edible wall

2016-01-05T13:05:51Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we ([https://ch.linkedin.com/in/alexandraflorin/fr Alexandra Florin] and [http://fr.linkedin.com/pub/michka-m%C3%A9lo/43/877/169 Michka Mélo]) work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” (NA) experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

[[Nitrification in Aquarium 2 (Report)|NA2]] led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why [[Nitrification in Aquarium 2 (Report)|NA2]] was a success while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-05T13:04:08Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we (Alexandra Florin and Michka Mélo) work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” (NA) experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

[[Nitrification in Aquarium 2 (Report)|NA2]] led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why [[Nitrification in Aquarium 2 (Report)|NA2]] was a success while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Nitrification in Aquarium 2 (Lab Journal)

2016-01-04T10:27:49Z

Michka:

This is the detailed lab journal of the Nitrification in Aquarium 2 experiment. For a structured summary, please refer to the [[Nitrification in Aquarium 2 (Report)|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 be considered as insignificant.

== Day 0 - 12/08/2015 - 22h ==

We started the NA2 experiment on this day.

=== Culture ===

We started the culture for NA2 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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

=== Bioreactor ===

This culture was poured in a 20 L bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

== Day 1 - 13/08/2015 - 16h ==

=== Culture ===

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a stable ammonium concentration of 3 mgN/l.

We measured a stable nitrate concentration of 10 mgN/l.

We measured a stable nitrite concentration of 0.8 mgN/l.

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.2 (stable), urea hydroloysis has probably not happened yet.

== Day 2 - 14/08/2015 - 12h30 ==

=== Culture ===

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a slightly increasing ammonium concentration of 5 mgN/l.

We measured a sloghtly decreasing nitrate concentration of 6 mgN/l.

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

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4 (slightly increasing), urea hydroloysis has probably not happened yet.

== 20h30 ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

We think that ammonia may be evaporating, and that the amount of ammonia evaporated may be increased by increased aeration. We therefore decided to measure ammonium concentration again a few hours after feeding the system with urine.

In eight hours, we observed a slight decrease in ammonium concentration to 3 mgN/l (-2 mgN/l). This may indicate an evaporation of ammonia, but the measurement error margins being quite large, we cannot be certain.

=== Urine ===

We fed the culture with 40 ml of urine, from the batch started on August 12th.

== Day 5 - 17/08/2015 - 19h ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

We measured a slightly increasing ammonium concentration of 5 mgN/l.

We measured a drastically increasing nitrate concentration of 80 mgN/l.

We measured an increasing nitrite concentration of 10 mgN/l.

=== Urine ===

We fed the culture with 65 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4 (stable), urea hydroloysis has probably not happened yet.

== Day 6 - 18/08/2015 - 21h ==

=== Culture ===

We took out 25 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a stable ammonium concentration of 5 mgN/l.

We measured a stable nitrate concentration of 80 mgN/l.

We measured a stable nitrite concentration of 10 mgN/l.

=== Urine ===

No urine was fed to the culture, as the nitrite level stayed high.

== Day 7 - 19/08/2015 - 15h ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

We measured a stable ammonium concentration of 5 mgN/l.

We measured a drastically increasing nitrate concentration of 160 mgN/l.

We measured a slightly decreasing nitrite concentration of 9 mgN/l.

The level of the culture surface got about 3 cm lower overnight.

=== Urine ===

As the culture seems to consume ammonium, we fed the culture with 65 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.7 (slightly increasing), urea hydroloysis may be happening.

== Day 9 - 21/08/2015 - 15h30 ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The measured pH of the culture was 7.1 (stable).

We measured a slightly decreasing ammonium concentration of 4.5 mgN/l.

We measured a stable nitrate concentration of 160 mgN/l.

We measured an increasing nitrite concentration of 16 mgN/l.

We ended the experiment at that stage.

Nitrification in Aquarium 3 (Lab Journal)

2016-01-04T10:27:37Z

Michka:

This is the detailed lab journal of the Nitrification in Aquarium 3 experiment. For a structured summary, please refer to the [[Nitrification in Aquarium 3 (Report)|report]] of the experiment.

== Day 0 - 12/08/2015 - 22h ==

We started the NA3 experiment on this day.

=== Culture ===

We started the culture for NA3 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.)

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

=== Bioreactor ===

This culture was poured in a 20 L bucket , in which a pump (comparable to the one used for NA1) was dripping air through two diffusers.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

A strong smell of urine stays after feeding.

== Day 1 - 13/08/2015 - 16h ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (slightly increasing).

We measured an increasing ammonium concentration of 8 mgN/l.

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

We measured a stable nitrite concentration of 0.8 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

We can still smell urine after feeding the culture.

== 19h30 ==

We added 15 ml of an aquariophilic concentrated nitrifyers culture (10x recommended dose), in order to see if launching a thriving culture helps to increase nitrate concentration.

== Day 2 - 14/08/2015 - 12h30 ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

The measured pH of the culture was 7.5 (slightly increasing).

We measured a decreasing ammonium concentration of 2 mgN/l.

We measured an increasing nitrate concentration of 20 mgN/l.

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

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

== 19h30 ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

The measured pH of the culture was 7.8 (slightly increasing).

We think that ammonia may be evaporating because of aeration. We therefore decided to measure ammonium concentration again a few hours after feeding the system with urine.

We observed an ammonium concentration of 10 mgN/l. This is quite high, but not so high compared to the fact that we fed the culture with 500 ml of urine (supposed concentration of 4k-5k mgN/l) only seven hours earlier. This may indicate an evaporation of ammonia

== Day 5 - 17/08/2015 - 19h ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

The measured pH of the culture was 8.4 (increasing).

We measured an increasing ammonium concentration of 30-60 mgN/l. This may indicate a slow urea hydrolysis, slightly converting urea into ammonium.

We measured an imprecise nitrate concentration, in the range of 2-20 mgN/l.

=== Urine ===

The increase in ammonium concentration suggests that nitrogen may be accumulating in the culture in the form of urea, which would be hydrolysed only slowly. To avoid an excessively high concentration of ammonium, leading to a potentially toxic concentration of nitrite, no urine was fed to the culture today.

The pH of urine was measured at 7.4, urea hydroloysis has probably not happened yet.

No urine smell detected.

== Day 6 - 18/08/2015 - 21h ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

The measured pH of the culture was 8.5 (increasing).

We measured a drastically decreasing ammonium concentration of 0.5 mgN/l.

We measured a nitrate concentration of 6 mgN/l.

We measured a slightly increasing nitrite concentration of 2 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4-7.7, urea hydroloysis may be starting.

Low urine smell detected.

== Day 7 - 19/08/2015 - 15h ==

=== Culture ===

We took out 25 ml of culture for analysis purposes. We also took out 1 l of culture to compensate the volume of urine added, and avoid overflow from our 20 l bioreactor.

The measured pH of the culture was 8.5 (stable).

We measured a drastically increasing ammonium concentration of 50 mgN/l.

We measured an increasing nitrate concentration of 20 mgN/l.

We measured a stable nitrite concentration of 2 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.7, urea hydroloysis may be starting.

Low urine smell detected.

== Day 9 - 21/08/2015 - 15h30 ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The pH of the culture reached its highest point at 8.9.

The ammonium concentration remains high, around 50-150 mgN/l.

Nitrate concentration decreased to 8 mgN/l.

Nitrite concentration decreased to 1 mgN/l.

The decrease of nitrate and nitrite concentration may be due to the removal of culture and addition of urine in the context of a quite unstable microbial culture.

=== Urine ===

The culture was not fed with urine, as the ammonium concentration is still quite high.

The pH of urine was measured at 8.5, urea hydroloysis has probably happened.

Low urine smell detected.

The experiment was terminated that night.

Nitrification in Aquarium 2 (Lab Journal)

2016-01-04T10:27:17Z

Michka:

Nitrification in Aquarium 1 (Lab Journal)

2016-01-04T10:26:49Z

Michka:

This is the detailed lab journal of the Nitrification in Aquarium 1 experiment. For a structured summary, please refer to the [[Nitrification in Aquarium 1 (Report)|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 [http://aquaponie.net/demarrage-cycler-aquaponie].

== 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.

Nitrification in Aquarium 3 (Report)

2016-01-04T10:24:50Z

Michka: /* Discussion */

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 ([[Nitrification in Aquarium 1 (Report)|NA1]]) we conducted. We started from the same culture as [[Nitrification in Aquarium 1 (Report)|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 [[Nitrification in Aquarium 1 (Report)|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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 [[Nitrification in Aquarium 2|NA2]]. However, some specific competing bacteria may have developed in [[Nitrification in Aquarium 3|NA3]]. Moreover, the pump in [[Nitrification in Aquarium 2|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 [[Nitrification in Aquarium 2 (Report)|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 ([http://wiki.hackuarium.ch/w/NA2_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|7.4 on day 6, nitrate concentration of 80 mgN/l]) and NA3 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|8.5 on day 6, nitrate concentration of 6 mgN/l]), although low pH conditions led to low nitrate conditions during NA1 ([http://wiki.hackuarium.ch/w/NA1_lab_journal#Day_17_-_8.2F8.2F2015_-_20h|7.4 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 ([http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_1_-_13.2F08.2F2015_-_16h|see 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 where 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 process 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.

Nitrification in Aquarium 3 (Report)

2016-01-04T10:22:39Z

Michka:

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 ([[Nitrification in Aquarium 1 (Report)|NA1]]) we conducted. We started from the same culture as [[Nitrification in Aquarium 1 (Report)|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 [[Nitrification in Aquarium 1 (Report)|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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 [[Nitrification in Aquarium 2 (Report)|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 ([[http://wiki.hackuarium.ch/w/NA2_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|7.4 on day 6, nitrate concentration of 80 mgN/l]]) and NA3 ([[http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_6_-_18.2F08.2F2015_-_21h|8.5 on day 6, nitrate concentration of 6 mgN/l]]), although low pH conditions led to low nitrate conditions during NA1 ([[http://wiki.hackuarium.ch/w/NA1_lab_journal#Day_17_-_8.2F8.2F2015_-_20h|7.4 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 ([[http://wiki.hackuarium.ch/w/NA3_lab_journal#Day_1_-_13.2F08.2F2015_-_16h|see 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 where 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 process 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.

Nitrification in Aquarium 2 (Report)

2016-01-04T10:16:04Z

Michka: /* Discussion & Conclusion */

This is the summarized report of the Nitrification in Aquarium 2 experiment. For more details, please refer to the [[Nitrification in Aquarium 2 (Lab Journal)|detailed lab journal]] of this experiment.

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 NA2 experiment will be fed with relatively low amounts of urine everyday, to make sure that we do not produce large amounts of nitrite which might poison nitrifyers I & II. It is thereby intended as a backup for NA3.

== Material & Methods ==

=== Experimental setup ===

==== Culture ====

We started the culture for NA2 on August 12th, 2015, from 15 l of the mix from NA1. The bucket was covered with a piece of cardboard cut in a pizza box, to avoid light to get in the bioreactor.

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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

==== Bioreactor ====

This culture was poured in a 20 L opaque bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Concentrations measurement ===

We used aquariophilic measurement kits for ammonium [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 ==

We terminated the experiment on August 21st, 2015, after 9 days.

We obtained a quite satisfying nitrate concentration (160 mgN/l) at the end of the experiment. This concentration is in the range of those used in aquaponics/hydroponics to feed plants.

== Discussion & Conclusion ==

Comparing [[Nitrification in Aquarium 1 (Report)|NA1]], NA2 and [[Nitrification in Aquarium 3 (Report)|NA3]], it is difficult to understand why N2 led to a satisfying ammonium concentration while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] did not.

The most probable explanation seems to be a good coordination between processes at stake, which failed to happen in the previous experiments. Potential contamination by competing bacteria in [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]], which would have failed to develop in NA2 may also be an explanation.

The rise to a nitrate concentration of 80 mgN/l, and then to 160 mgN/l did not follow any specific change in the conditions, neither in the urine fed to the system nor in the culture pH, light or aeration conditions.

Further investigation should be performed to understand better the dynamics at stake.

Nitrification in Aquarium 2 (Report)

2016-01-04T10:15:07Z

Michka: /* Discussion & Conclusion */

This is the summarized report of the Nitrification in Aquarium 2 experiment. For more details, please refer to the [[Nitrification in Aquarium 2 (Lab Journal)|detailed lab journal]] of this experiment.

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 NA2 experiment will be fed with relatively low amounts of urine everyday, to make sure that we do not produce large amounts of nitrite which might poison nitrifyers I & II. It is thereby intended as a backup for NA3.

== Material & Methods ==

=== Experimental setup ===

==== Culture ====

We started the culture for NA2 on August 12th, 2015, from 15 l of the mix from NA1. The bucket was covered with a piece of cardboard cut in a pizza box, to avoid light to get in the bioreactor.

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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

==== Bioreactor ====

This culture was poured in a 20 L opaque bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Concentrations measurement ===

We used aquariophilic measurement kits for ammonium [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 ==

We terminated the experiment on August 21st, 2015, after 9 days.

We obtained a quite satisfying nitrate concentration (160 mgN/l) at the end of the experiment. This concentration is in the range of those used in aquaponics/hydroponics to feed plants.

== Discussion & Conclusion ==

Comparing [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]], it is difficult to understand why [[Nitrification in Aquarium 2 (Report)|NA2]] led to a satisfying ammonium concentration while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] did not.

The most probable explanation seems to be a good coordination between processes at stake, which failed to happen in the previous experiments. Potential contamination by competing bacteria in NA1 and NA3, which would have failed to develop in NA2 may also be an explanation.

The rise to a nitrate concentration of 80 mgN/l, and then to 160 mgN/l did not follow any specific change in the conditions, neither in the urine fed to the system nor in the culture pH, light or aeration conditions.

Further investigation should be performed to understand better the dynamics at stake.

Nitrification in Aquarium 1 (Report)

2016-01-04T10:14:00Z

Michka: /* Conclusion */

This is the summarized report of the Nitrification in Aquarium 1 experiment. For more details, please refer to the [[Nitrification in Aquarium 1 (Lab Journal)|detailed 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 [[Nitrification in Aquarium 1 (Lab Journal)|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.
* if stronger aeration induces more evaporation.
* if feeding more urine to a more oxygenated container induces the production of larger amounts of nitrates.

The [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|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. [[Nitrification in Aquarium 3 (Report)|NA3]] will be fed with large amounts of urine. [[Nitrification in Aquarium 2 (Report)|NA2]] will be fed with small amounts of urine, to avoid nitrite pisoning of nitrifyers, and will thereby act as a backup for [[Nitrification in Aquarium 3 (Report)|NA3]].

Nitrification in Aquarium 1 (Report)

2016-01-04T10:12:04Z

Michka: /* Results */

This is the summarized report of the Nitrification in Aquarium 1 experiment. For more details, please refer to the [[Nitrification in Aquarium 1 (Lab Journal)|detailed 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 [[Nitrification in Aquarium 1 (Lab Journal)|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.

Nitrification in Aquarium 1 (Report)

2016-01-04T10:11:31Z

Michka: /* Results */

This is the summarized report of the Nitrification in Aquarium 1 experiment. For more details, please refer to the [[Nitrification in Aquarium 1 (Lab Journal)|detailed 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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.

Nitrification in Aquarium 3 (Report)

2016-01-04T10:10:58Z

Michka:

This is the summarized report of the Nitrification in Aquarium 3 experiment. For more details, please refer to the [[Nitrification in Aquarium 3 (Lab Journal)|detailed lab journal]] of this experiment.

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 (7.4 on day 6, nitrate concentration of 80 mgN/l) and NA3 (8.5 on day 6, nitrate concentration of 6 mgN/l), although low pH conditions led to low nitrate conditions during NA1 (7.4 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 (see 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 where 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 process 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.

Nitrification in Aquarium 2 (Report)

2016-01-04T10:10:38Z

Michka:

This is the summarized report of the Nitrification in Aquarium 2 experiment. For more details, please refer to the [[Nitrification in Aquarium 2 (Lab Journal)|detailed lab journal]] of this experiment.

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 NA2 experiment will be fed with relatively low amounts of urine everyday, to make sure that we do not produce large amounts of nitrite which might poison nitrifyers I & II. It is thereby intended as a backup for NA3.

== Material & Methods ==

=== Experimental setup ===

==== Culture ====

We started the culture for NA2 on August 12th, 2015, from 15 l of the mix from NA1. The bucket was covered with a piece of cardboard cut in a pizza box, to avoid light to get in the bioreactor.

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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

==== Bioreactor ====

This culture was poured in a 20 L opaque bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Concentrations measurement ===

We used aquariophilic measurement kits for ammonium [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 ==

We terminated the experiment on August 21st, 2015, after 9 days.

We obtained a quite satisfying nitrate concentration (160 mgN/l) at the end of the experiment. This concentration is in the range of those used in aquaponics/hydroponics to feed plants.

== Discussion & Conclusion ==

Comparing NA1, NA2 and NA3, it is difficult to understand why NA2 led to a satisfying ammonium concentration while NA1 and NA3 did not.

The most probable explanation seems to be a good coordination between processes at stake, which failed to happen in the previous experiments. Potential contamination by competing bacteria in NA1 and NA3, which would have failed to develop in NA2 may also be an explanation.

The rise to a nitrate concentration of 80 mgN/l, and then to 160 mgN/l did not follow any specific change in the conditions, neither in the urine fed to the system nor in the culture pH, light or aeration conditions.

Further investigation should be performed to understand better the dynamics at stake.

Nitrification in Aquarium 1 (Report)

2016-01-04T10:10:07Z

Michka:

This is the summarized report of the Nitrification in Aquarium 1 experiment. For more details, please refer to the [[Nitrification in Aquarium 1 (Lab Journal)|detailed 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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.

Nitrification in Aquarium 1 (Report)

2016-01-04T10:09:18Z

Michka:

This is the summarized report of the Nitrification in Aquarium 1 experiment, for more details, please refer to the [[Nitrification in Aquarium 1 (Lab journal)|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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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.

Edible wall

2016-01-04T10:07:27Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” (NA) experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

[[Nitrification in Aquarium 2 (Report)|NA2]] led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why [[Nitrification in Aquarium 2 (Report)|NA2]] was a success while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T10:06:58Z

Michka: /* Conclusion */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

[[Nitrification in Aquarium 2 (Report)|NA2]] led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why [[Nitrification in Aquarium 2 (Report)|NA2]] was a success while [[Nitrification in Aquarium 1 (Report)|NA1]] and [[Nitrification in Aquarium 3 (Report)|NA3]] failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T10:06:00Z

Michka: /* Conclusion */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

[Nitrification in Aquarium 2 (Report)|NA2] led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. [Nitrification in Aquarium 1 (Report)|NA1] and [Nitrification in Aquarium 3 (Report)|NA3] failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why [Nitrification in Aquarium 2 (Report)|NA2] was a success while [Nitrification in Aquarium 1 (Report)|NA1] and [Nitrification in Aquarium 3 (Report)|NA3] failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T10:03:42Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living [https://en.wikipedia.org/wiki/Green_wall green walls] from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T09:59:18Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow [https://en.wikipedia.org/wiki/Nitrifying_bacteria nitrifying bacteria] (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in [https://en.wikipedia.org/wiki/Aquaponics aquaponics], where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T09:58:00Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in aquaponics, where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T09:57:41Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in aquaponics, where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium (NA)” experiments ([[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] & [[Nitrification in Aquarium 3 (Report)|NA3]]) will therefore be adaptations 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 [[http://aquaponie.net/demarrage-cycler-aquaponie/ this protocol]].

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T09:55:38Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in aquaponics, where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments ([[Nitrification in Aquarium 1 (report) NA1]], NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2016-01-04T09:55:00Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are very important in aquaponics, where we grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2015-12-28T12:13:56Z

Michka:

== Introduction ==

At [http://est2e.com/ ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are present in aquaponic systems. Such systems allow to grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2015-12-28T12:13:42Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ | ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are present in aquaponic systems. Such systems allow to grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2015-12-28T12:13:32Z

Michka: /* Introduction */

== Introduction ==

At [http://est2e.com/ |ESTEE], we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are present in aquaponic systems. Such systems allow to grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2015-12-28T12:12:41Z

Michka: /* Experiments */

== Introduction ==

At ESTEE, we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are present in aquaponic systems. Such systems allow to grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], [[Nitrification in Aquarium 2 (Report)|NA2]] and [[Nitrification in Aquarium 3 (Report)|NA3]] include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for [[Nitrification in Aquarium 1 (Lab Journal)|NA1]], [[Nitrification in Aquarium 2 (Lab Journal)|NA2]] and [[Nitrification in Aquarium 3 (Lab Journal)|NA3]] include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

Edible wall

2015-12-28T12:11:28Z

Michka: /* Experiments */

== Introduction ==

At ESTEE, we work on a project aiming at feeding living green walls from urine to produce food. We will first aim at feeding hydroponics green walls with urine as a nutrients source. We will then identify which edible and/or useful plants are suitable to grow in such an environment.

Our very first step will be to grow nitrifying bacteria (or nitrifyers) to convert urine into nitrate. Such nitrifying bacteria are present in aquaponic systems. Such systems allow to grow fish and plants in the same system, the waste from the fish providing nutrients to plants for their growth. However, fish waste need (at least partly) processing by bacteria to be edible for plants. Such bacteria include nitrifyers, which will transform ammonium from fish waste into nitrate, edible by plants.

Our first “Nitrification in Aquarium” experiments (NA1, NA2 & NA3) will therefore be adaptations 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.

== From urine to nitrate : how does it work ? ==

Urea is hydrolysed into ammonium (NH4+) by bacteria containing an enzyme called urease.
Ammonium is transformed into nitrite (NO2-) by a first class of nitrifyers, which we will call nitrifyers I.
Nitrite is transformed into nitrate (NO3) by a second class of nitrifyers, which we will call nitrifyers II.
The transformation of urine into nitrate is called nitrification.

=== Urea hydrolysis ===

Urease-containing bacteria are ubiquitous, and urea hydrolysis happens naturally in any traditional toilet system.

When urine is hydrolysed into ammonium, its pH gets quite high (around 8.5). Ammonium is part of an acido-alkaline with ammonia (NH3), which pKa value is 9. Ammonia is a gas. If stored in an open container, ammonia will fly out until pH of hydrolyzed urine reaches 9, causing nitrogen losses and reduced efficiency in converting urine to nitrate. Urine should therefore be stored in a tightly sealed container.

Hydrolysing urine is also a way to sterilize it, as high pH will kill pathogens.

High pH also induces precipitation and slight degradation of minerals.

=== Nitrifying bacteria ===

Both nitrifyers I & II are sensitive to nitrite (NO2), which is toxic for them and inhibits the nitrification reaction. We therefore have to feed the nitrifyer culture with small amounts of urine, because a too large amount at once may lead to a high production of nitrite by nitrifyers I, and lead to poisoning of both nitrifyers I & II.

Nitrifyers I have a tendency to make the culture more acidic, and nitrifyers II are inhibited by low pH conditions (below 6). Higher pH conditions (around 8) are therefore more favorable for nitrification [http://aquaponie.net/demarrage-cycler-aquaponie].

Nitrifyers are sensitive to light [http://aquaponie.net/demarrage-cycler-aquaponie].

=== Urea, ammonium & evaporation ===

Urea is not volatile.
Ammonium (in the form of ammonia) is volatile.

== Experiments ==

So far, we performed three experiments, all having a dedicated report page and detailed lab journal page.

Reports for [[Nitrification in Aquarium 1 (Report)|NA1]], NA2 and NA3 include introduction, material and methods, main results, discussion and conclusions.

Detailed lab journal for NA1, NA2 and NA3 include day-by-day report of measurements and actions taken.

A summary of the measurements in the form of an Excel sheet can be found here.

== Conclusion ==

NA2 led to a nitrate concentration of 160 mgN/l, which is in the range of the concentration used in hydroponics to feed plants. NA1 and NA3 failed to reach a nitrate concentration above 20 mgN/l.

It is yet unclear why NA2 was a success while NA1 and NA3 failed, further investigation needs to be performed to have a better understanding of the dynamics at stake.

== Next steps ==

To simplify the complexity of the investigation, urea hydrolysis should be performed before feeding urine to our system. Its dynamics should be observed by frequent pH measurement, to observe if it happens within days or within hours.

Ammonium content in urine should be measured before being fed to the bioreactor.

Ammonium/nitrite/nitrate concentrations in the bioreactor should be monitored as frequently as possible after feeding, to have a better understanding of the possible evaporation issue. A second experiment may compare the potential evaporation rate in different aeration rate & bioreactor volume combinations, to test if increased aeration induces increased evaporation.

A protocol should be designed to try to discriminate between failed nitrification, nitrate consumption, or even possibly ammonium consumption by competing bacteria.

The need for physical support to host nitrifyers should also be investigated and tested.

During experiment & experimental set up design, it should be kept in mind that:
* It is wiser to protect the bioreactor from light
* Low aeration did not seem to help
* Feeding very large quantities of urine to the system did not seem to help
* Performing urea hydrolysis before feeding urine to the system will strongly help investigation

== Open questions ==

If you have any elements which would help us solve these open questions, you are most welcome to edit this section. Please add references when possible.

* Is nitrifying bacteria growth inhibited by lack of physical support (f.i. clay beads) to host them ?
* What are the appropriate pH, light & oxygen conditions for a quick urea hydrolysis by urease-contaning bacteria ?
* Which bacteria may compete with nitrifyers for ammonium in the same culture conditions ?

NA1 report

2015-12-28T12:10:41Z

Michka: Michka moved page NA1 report to Nitrification in Aquarium 1 (Report)

#REDIRECT [[Nitrification in Aquarium 1 (Report)]]

Nitrification in Aquarium 1 (Report)

2015-12-28T12:10:41Z

Michka: Michka moved page NA1 report to Nitrification in Aquarium 1 (Report)

Report of Nitrification in Aquarium 1

== 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

==== 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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.

NA2 report

2015-12-28T12:10:26Z

Michka: Michka moved page NA2 report to Nitrification in Aquarium 2 (Report)

#REDIRECT [[Nitrification in Aquarium 2 (Report)]]

Nitrification in Aquarium 2 (Report)

2015-12-28T12:10:26Z

Michka: Michka moved page NA2 report to Nitrification in Aquarium 2 (Report)

Report of Nitrification in Aquarium 2

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 NA2 experiment will be fed with relatively low amounts of urine everyday, to make sure that we do not produce large amounts of nitrite which might poison nitrifyers I & II. It is thereby intended as a backup for NA3.

== Material & Methods ==

=== Experimental setup ===

==== Culture ====

We started the culture for NA2 on August 12th, 2015, from 15 l of the mix from NA1. The bucket was covered with a piece of cardboard cut in a pizza box, to avoid light to get in the bioreactor.

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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

==== Bioreactor ====

This culture was poured in a 20 L opaque bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Concentrations measurement ===

We used aquariophilic measurement kits for ammonium [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 ==

We terminated the experiment on August 21st, 2015, after 9 days.

We obtained a quite satisfying nitrate concentration (160 mgN/l) at the end of the experiment. This concentration is in the range of those used in aquaponics/hydroponics to feed plants.

== Discussion & Conclusion ==

Comparing NA1, NA2 and NA3, it is difficult to understand why NA2 led to a satisfying ammonium concentration while NA1 and NA3 did not.

The most probable explanation seems to be a good coordination between processes at stake, which failed to happen in the previous experiments. Potential contamination by competing bacteria in NA1 and NA3, which would have failed to develop in NA2 may also be an explanation.

The rise to a nitrate concentration of 80 mgN/l, and then to 160 mgN/l did not follow any specific change in the conditions, neither in the urine fed to the system nor in the culture pH, light or aeration conditions.

Further investigation should be performed to understand better the dynamics at stake.

NA1 lab journal

2015-12-28T12:10:02Z

Michka: Michka moved page NA1 lab journal to Nitrification in Aquarium 1 (Lab Journal)

#REDIRECT [[Nitrification in Aquarium 1 (Lab Journal)]]

Nitrification in Aquarium 1 (Lab Journal)

2015-12-28T12:10:02Z

Michka: Michka moved page NA1 lab journal to Nitrification in Aquarium 1 (Lab Journal)

Lab Journal of Nitrification in Aquarium 1

== 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 [http://aquaponie.net/demarrage-cycler-aquaponie].

== 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.

NA2 lab journal

2015-12-28T12:09:33Z

Michka: Michka moved page NA2 lab journal to Nitrification in Aquarium 2 (Lab Journal)

#REDIRECT [[Nitrification in Aquarium 2 (Lab Journal)]]

Nitrification in Aquarium 2 (Lab Journal)

2015-12-28T12:09:33Z

Michka: Michka moved page NA2 lab journal to Nitrification in Aquarium 2 (Lab Journal)

Lab Journal of NA2

== 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 be considered as insignificant.

== Day 0 - 12/08/2015 - 22h ==

We started the NA2 experiment on this day.

=== Culture ===

We started the culture for NA2 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 = 7.4 (stable)
* Ammonium concentration = 3 mgN/l
* Nitrate concentration = 10 mgN/l
* Nitrite concentration = 0.8 mgN/l

(We took out 30 ml of culture for analysis purposes.)

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

=== Bioreactor ===

This culture was poured in a 20 L bucket , in which a pump (the one used for NA1) was dripping 380l/h of air through two diffusers.

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine slightly increased to 7.2, urea hydroloysis has probably not happened yet.

== Day 1 - 13/08/2015 - 16h ==

=== Culture ===

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a stable ammonium concentration of 3 mgN/l.

We measured a stable nitrate concentration of 10 mgN/l.

We measured a stable nitrite concentration of 0.8 mgN/l.

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.2 (stable), urea hydroloysis has probably not happened yet.

== Day 2 - 14/08/2015 - 12h30 ==

=== Culture ===

We took out 35 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a slightly increasing ammonium concentration of 5 mgN/l.

We measured a sloghtly decreasing nitrate concentration of 6 mgN/l.

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

=== Urine ===

We fed the culture with 25 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4 (slightly increasing), urea hydroloysis has probably not happened yet.

== 20h30 ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

We think that ammonia may be evaporating, and that the amount of ammonia evaporated may be increased by increased aeration. We therefore decided to measure ammonium concentration again a few hours after feeding the system with urine.

In eight hours, we observed a slight decrease in ammonium concentration to 3 mgN/l (-2 mgN/l). This may indicate an evaporation of ammonia, but the measurement error margins being quite large, we cannot be certain.

=== Urine ===

We fed the culture with 40 ml of urine, from the batch started on August 12th.

== Day 5 - 17/08/2015 - 19h ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

We measured a slightly increasing ammonium concentration of 5 mgN/l.

We measured a drastically increasing nitrate concentration of 80 mgN/l.

We measured an increasing nitrite concentration of 10 mgN/l.

=== Urine ===

We fed the culture with 65 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4 (stable), urea hydroloysis has probably not happened yet.

== Day 6 - 18/08/2015 - 21h ==

=== Culture ===

We took out 25 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (stable).

We measured a stable ammonium concentration of 5 mgN/l.

We measured a stable nitrate concentration of 80 mgN/l.

We measured a stable nitrite concentration of 10 mgN/l.

=== Urine ===

No urine was fed to the culture, as the nitrite level stayed high.

== Day 7 - 19/08/2015 - 15h ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

We measured a stable ammonium concentration of 5 mgN/l.

We measured a drastically increasing nitrate concentration of 160 mgN/l.

We measured a slightly decreasing nitrite concentration of 9 mgN/l.

The level of the culture surface got about 3 cm lower overnight.

=== Urine ===

As the culture seems to consume ammonium, we fed the culture with 65 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.7 (slightly increasing), urea hydroloysis may be happening.

== Day 9 - 21/08/2015 - 15h30 ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The measured pH of the culture was 7.1 (stable).

We measured a slightly decreasing ammonium concentration of 4.5 mgN/l.

We measured a stable nitrate concentration of 160 mgN/l.

We measured an increasing nitrite concentration of 16 mgN/l.

We ended the experiment at that stage.

NA3 lab journal

2015-12-28T12:09:12Z

Michka: Michka moved page NA3 lab journal to Nitrification in Aquarium 3 (Lab Journal)

#REDIRECT [[Nitrification in Aquarium 3 (Lab Journal)]]

Nitrification in Aquarium 3 (Lab Journal)

2015-12-28T12:09:12Z

Michka: Michka moved page NA3 lab journal to Nitrification in Aquarium 3 (Lab Journal)

Lab Journal of "Nitrification in Aquarium" 3

== Day 0 - 12/08/2015 - 22h ==

We started the NA3 experiment on this day.

=== Culture ===

We started the culture for NA3 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.)

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

=== Bioreactor ===

This culture was poured in a 20 L bucket , in which a pump (comparable to the one used for NA1) was dripping air through two diffusers.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

A strong smell of urine stays after feeding.

== Day 1 - 13/08/2015 - 16h ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The measured pH of the culture was 7.4 (slightly increasing).

We measured an increasing ammonium concentration of 8 mgN/l.

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

We measured a stable nitrite concentration of 0.8 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

We can still smell urine after feeding the culture.

== 19h30 ==

We added 15 ml of an aquariophilic concentrated nitrifyers culture (10x recommended dose), in order to see if launching a thriving culture helps to increase nitrate concentration.

== Day 2 - 14/08/2015 - 12h30 ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

The measured pH of the culture was 7.5 (slightly increasing).

We measured a decreasing ammonium concentration of 2 mgN/l.

We measured an increasing nitrate concentration of 20 mgN/l.

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

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

== 19h30 ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

The measured pH of the culture was 7.8 (slightly increasing).

We think that ammonia may be evaporating because of aeration. We therefore decided to measure ammonium concentration again a few hours after feeding the system with urine.

We observed an ammonium concentration of 10 mgN/l. This is quite high, but not so high compared to the fact that we fed the culture with 500 ml of urine (supposed concentration of 4k-5k mgN/l) only seven hours earlier. This may indicate an evaporation of ammonia

== Day 5 - 17/08/2015 - 19h ==

=== Culture ===

We took out 70 ml of culture for analysis purposes.

The measured pH of the culture was 8.4 (increasing).

We measured an increasing ammonium concentration of 30-60 mgN/l. This may indicate a slow urea hydrolysis, slightly converting urea into ammonium.

We measured an imprecise nitrate concentration, in the range of 2-20 mgN/l.

=== Urine ===

The increase in ammonium concentration suggests that nitrogen may be accumulating in the culture in the form of urea, which would be hydrolysed only slowly. To avoid an excessively high concentration of ammonium, leading to a potentially toxic concentration of nitrite, no urine was fed to the culture today.

The pH of urine was measured at 7.4, urea hydroloysis has probably not happened yet.

No urine smell detected.

== Day 6 - 18/08/2015 - 21h ==

=== Culture ===

We took out 20 ml of culture for analysis purposes.

The measured pH of the culture was 8.5 (increasing).

We measured a drastically decreasing ammonium concentration of 0.5 mgN/l.

We measured a nitrate concentration of 6 mgN/l.

We measured a slightly increasing nitrite concentration of 2 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.4-7.7, urea hydroloysis may be starting.

Low urine smell detected.

== Day 7 - 19/08/2015 - 15h ==

=== Culture ===

We took out 25 ml of culture for analysis purposes. We also took out 1 l of culture to compensate the volume of urine added, and avoid overflow from our 20 l bioreactor.

The measured pH of the culture was 8.5 (stable).

We measured a drastically increasing ammonium concentration of 50 mgN/l.

We measured an increasing nitrate concentration of 20 mgN/l.

We measured a stable nitrite concentration of 2 mgN/l.

=== Urine ===

We fed the culture with 500 ml of urine, from the batch started on August 12th.

The pH of urine was measured at 7.7, urea hydroloysis may be starting.

Low urine smell detected.

== Day 9 - 21/08/2015 - 15h30 ==

=== Culture ===

We took out 30 ml of culture for analysis purposes.

The pH of the culture reached its highest point at 8.9.

The ammonium concentration remains high, around 50-150 mgN/l.

Nitrate concentration decreased to 8 mgN/l.

Nitrite concentration decreased to 1 mgN/l.

The decrease of nitrate and nitrite concentration may be due to the removal of culture and addition of urine in the context of a quite unstable microbial culture.

=== Urine ===

The culture was not fed with urine, as the ammonium concentration is still quite high.

The pH of urine was measured at 8.5, urea hydroloysis has probably happened.

Low urine smell detected.

The experiment was terminated that night.

Nitrification in Aquarium 3 (Report)

2015-12-28T12:08:41Z

Michka:

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 (7.4 on day 6, nitrate concentration of 80 mgN/l) and NA3 (8.5 on day 6, nitrate concentration of 6 mgN/l), although low pH conditions led to low nitrate conditions during NA1 (7.4 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 (see 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 where 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 process 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.

NA3 report

2015-12-28T12:08:28Z

Michka: Michka moved page NA3 report to Nitrification in Aquarium 3 (Report)

#REDIRECT [[Nitrification in Aquarium 3 (Report)]]

Nitrification in Aquarium 3 (Report)

2015-12-28T12:08:27Z

Michka: Michka moved page NA3 report to Nitrification in Aquarium 3 (Report)

Report of Nitrification in Aquarium 3

== Introduction ==

As stated on the main project page, our second “Nitrification in Aquarium” experiment (NA2) 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 [http://aquaponie.net/demarrage-cycler-aquaponie]. 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 [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2438], nitrite [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2439] & nitrate [http://www.jbl.de/?lang=fr&mod=products&func=detail&id=2440] 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 aquriophilic 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 (7.4 on day 6, nitrate concentration of 80 mgN/l) and NA3 (8.5 on day 6, nitrate concentration of 6 mgN/l), although low pH conditions led to low nitrate conditions during NA1 (7.4 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 (see 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 where 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 process 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.

Nitrification in Aquarium 3 (Lab Journal)

2015-12-28T12:04:34Z

Michka: Created page with "Lab Journal of "Nitrification in Aquarium" 3 == Day 0 - 12/08/2015 - 22h == We started the NA3 experiment on this day. === Culture === We started the culture for NA3 from..."

Nitrification in Aquarium 2 (Lab Journal)

2015-12-28T12:01:38Z

Michka: Created page with "Lab Journal of NA2 == General comments == The aquariophilic kits and pH strips we use are color-based indicators, and can be quite imprecise. The small variations of concent..."