Science of Nitrification

[Sun.singh1991]

For as long as I’ve been keeping fish I wanted to know what was going on in our self contained ecosystems. Early on in my fish keep journey I discovered that it was actually bacteria that allowed us to keep such beautiful aquariums alive. This is what inspired me to go to school for a B.S degree in Chemistry and Biology. I eventually found myself asking questions about what really was going on in our tanks down to the molecular level. Resources online typically point you to a basic understating of the nitrogen cycle, pH, and temperature requirements but rarely do they explain how all these factors affect each other on the micro scale. 

In this post I will explain the processes that keep our fish alive in greater detail. I will also try to keep things as easy to understand as I possibly can. This is not a scientific journal as I intend for this entry to be accessible  to anyone regardless of educational or experience level. The journey to understanding our aquariums starts with the nitrogen cycle. Nitrogen is introduced into our aquariums by fish food; as proteins are broken down the fish release ammonia(NH3) as a waste product. Ammonia is toxic to fish because it’s buildup makes it more difficult for fish to excrete it into the surrounding water, causing metabolic shutdown and eventually organ and brain damage in fish. Now total ammonia concentration is measured as both ammonia(NH3) and the ionized form ammonium(NH4+). The relationship between these two forms is dependent on pH; at a higher pH  the ratio of ammonia is much higher than ammonium. And conversely at lower pH the ratio of ammonium is higher.  Ammonium  is completely non toxic to fish whereas ammonia is very toxic. Therefore a fundamental relationship exists between pH and ammonia toxicity. pH is a  measurement of hydrogen ions (H+) in water, at lower pH more H+ is available to bind with ammonia(NH3) to form ammonium(NH4+). This is why tanks that run higher pH levels can be more dangerous to have a ammonia spike in. 
 

This is where the bacteria that keep our fish alive come in. Autotrophic Nitrifying bacteria  oxidize ammonia in a two step process that consumes large amounts of dissolved oxygen and carbonate, also known as alkalinity, from the water. Alkalinity is commonly known as dKH in testing. Bicarbonate and carbonate are essential to our bacteria colonies because they are the inorganic carbon sources that provide energy for them to grow and multiply. Alkalinity is also the buffering system that prevents large changes of pH in our aquariums. In the first step of nitrification a genus of bacteria known as Nitrosomonas take ammonia(NH3), oxygen, and carbonate to oxidize NH3 to Nitrite (NO2). Nitrite is also very deadly to fish because it binds with hemoglobin, which prevents fish from getting oxygen to their organs and can cause death. In addition to NO2 this step also produces H+ ions that increase the acidity of the water over time. In a second step a genus of bacteria known as Nitrobacter take the nitrite produced in the first step and further oxidize it to nitrate(NO3). Acidity in the form of H+ ions is also produced in this step. Overall this two step process requires approximately 4.6 milligrams of dissolved oxygen and 7.1 milligrams  of alkalinity  to oxidize one milligram of ammonia into one milligram of nitrate . This reaction is therefore dependent heavily on the supply of oxygen and alkalinity in the water. Temperature also plays a key role in this reaction as the bacteria are able to grow and multiply best at temperatures between 77 and 86 degrees Fahrenheit. 

Nitrate(NO3) is generally considered safe for fish at concentrations up to 80 ppm. However, it should be noted that the EPA requires municipal water plants to maintain Nitrates at less than 10 ppm. Therefore it can be concluded that nitrates in higher concentrations do have negative implications for fish. At this point in the nitrogen cycle the Nirates can be removed in from the system in a few ways. A water change can immediately lower nitrates to acceptable levels, depending on the percentage of water changed. Another option is the use of plants to uptake nitrates, it should be noted that this solution is dependent on the amount of type of plants used in the aquarium. Faster growing plants will uptake more nitrates than slower growing ones. A third solution is a process called denitrification in which Nitrates are reduced to nitrogen gas which is gassed off from the water rapidly. Denitrification is performed by groups of heterotrophs that require anoxic conditions.  

 

 

 

[TheSwissAquarist]

It's great seeing someone really dive in deep into the subject! 

The prospect is quite daunting!

[Guppysnail]

This is a very nice write up to help folks understand the process a bit better. Thank you for sharing. 

[Sun.singh1991]

On 11/4/2022 at 1:30 AM, TheSwissAquarist said:

It's great seeing someone really dive in deep into the subject! 

The prospect is quite daunting!

Thank you!

[Luciferkrist]

Nice, concise, and not too esoteric to understand. So now I kind of understand why adding a bubbler or airstone to a new tank helps is cycle faster! It adds more dissolved O2 into the water for the bacteria to neutralize the ammonia!

The one thing about all I've been learning and re-learning that I always seem to never find is exactly what bacteria we are all depending on! Like are there specific 'species' of bacteria that we are looking for? Half of us here can give the scientific names of the plants or fish in our tanks ( I admit, I'm not good at this part), but I never hear about the specific single-celled helpers. Is there was a way to encourage those particular types to colonize instead of just waiting for a colony to form in our tanks by luck/time?

[Sun.singh1991]

On 11/5/2022 at 7:27 AM, Luciferkrist said:

Nice, concise, and not too esoteric to understand. So now I kind of understand why adding a bubbler or airstone to a new tank helps is cycle faster! It adds more dissolved O2 into the water for the bacteria to neutralize the ammonia!

The one thing about all I've been learning and re-learning that I always seem to never find is exactly what bacteria we are all depending on! Like are there specific 'species' of bacteria that we are looking for? Half of us here can give the scientific names of the plants or fish in our tanks ( I admit, I'm not good at this part), but I never hear about the specific single-celled helpers. Is there was a way to encourage those particular types to colonize instead of just waiting for a colony to form in our tanks by luck/time?

The specific species are probably the same as the ones used in our wastewater systems. 
https://genome.jgi.doe.gov/portal/niteu/niteu.home.html 

As for how to grow the colonies faster the best way is to maintain temperatures around 78-82 degrees, have lots of oxygenation, maintain a PH between 7-8, and provide a source of ammonia to the tank. 

[modified lung]

On 11/5/2022 at 8:57 AM, Sun.singh1991 said:

The specific species are probably the same as the ones used in our wastewater systems. 

That's actually been found not to be the case. In aquariums and aquaculture systems we mostly find ammonia-oxidizing archaea and nitrite-oxidizing bacteria from the genus Nitrospira because they do better in the low nitrogen concentrations. There's even some "comammox" species of Nitrospira that can oxidize both ammonia and nitrite.

There's over 400 species of nitrifiers most of which have a specific environmental niche. So that fastest way to grow your population is stable parameters. But the more stable the environment, the less your population will be able to handle change. So there's a trade-off between speed and robustness.

 

[Sun.singh1991]

On 11/5/2022 at 5:11 PM, modified lung said:

That's actually been found not to be the case. In aquariums and aquaculture systems we mostly find ammonia-oxidizing archaea and nitrite-oxidizing bacteria from the genus Nitrospira because they do better in the low nitrogen concentrations. There's even some "comammox" species of Nitrospira that can oxidize both ammonia and nitrite.

There's over 400 species of nitrifiers most of which have a specific environmental niche. So that fastest way to grow your population is stable parameters. But the more stable the environment, the less your population will be able to handle change. So there's a trade-off between speed and robustness.

 

That’s interesting, I’ve always believed it was the same species.  That’s good to know. Have you found any specific products that can help? Such as Fritzyme or Seachem Stability. 

[modified lung]

On 11/5/2022 at 6:20 PM, Sun.singh1991 said:

That’s interesting, I’ve always believed it was the same species.  That’s good to know. Have you found any specific products that can help? Such as Fritzyme or Seachem Stability. 

😁 That's what everyone assumed until someone decided to actually look.

I've only used one bacteria product from Pentair because my employer at the time kept buying it. The label said it contained Nitrosonomas and Nitrobacter. It didn't work at all. I actually think it might have made things worse.

If I were to try anything, it would be Dr Tim's. He was that guy who actually looked at what's was in an aquarium. Otherwise, I would only consider using something that says it has Nitrospira on the label. I hear a lot of positive reviews about Fritz too.

[Sun.singh1991]

On 11/5/2022 at 7:27 AM, Luciferkrist said:

Nice, concise, and not too esoteric to understand. So now I kind of understand why adding a bubbler or airstone to a new tank helps is cycle faster! It adds more dissolved O2 into the water for the bacteria to neutralize the ammonia!

The one thing about all I've been learning and re-learning that I always seem to never find is exactly what bacteria we are all depending on! Like are there specific 'species' of bacteria that we are looking for? Half of us here can give the scientific names of the plants or fish in our tanks ( I admit, I'm not good at this part), but I never hear about the specific single-celled helpers. Is there was a way to encourage those particular types to colonize instead of just waiting for a colony to form in our tanks by luck/time?

So after doing some research it would seem I was incorrect. Recent studies have shown that it is actually Ammonia-oxidizing archaea(AOA) in the phylum Nitrososphaerota that oxidize ammonia in most of our aquariums. 
 

[TheSwissAquarist]

On 11/18/2022 at 10:18 AM, Sun.singh1991 said:

So after doing some research it would seem I was incorrect. Recent studies have shown that it is actually Ammonia-oxidizing archaea(AOA) in the phylum Nitrososphaerota that oxidize ammonia in most of our aquariums. 
 

Have you tested that theory??

[gardenman]

In terms of products to add, the best is anything from one of your already cycled tanks. Those bacteria will already be adjusted to your water type (and water isn't just water, it all varies) and will cycle your tank the quickest. I used to think fish stores could make a lot of money selling pre-cycled filter media, but if the bacteria didn't like the new water they went to, they could all die and become more of a problem than a help. The bacteria in your established tanks have evolved to thrive in your water. They are optimized for your conditions. Move those thriving, healthy bacterial colonies to a different water source and they could crash. The biomes in our aquariums are likely all unique in some manner and what works in one could fail miserably in another. I suspect the biomes evolve over time as conditions in the tanks change also. The colonies you had when the tank first cycles may not be similar to ones you have five years down the road. 

[Sun.singh1991]

On 11/18/2022 at 4:34 AM, TheSwissAquarist said:

Have you tested that theory??

Unfortunately I don’t have the necessary equipment to test for specific bacteria strains at home. 

[TheSwissAquarist]

On 11/18/2022 at 4:13 PM, Sun.singh1991 said:

Unfortunately I don’t have the necessary equipment to test for specific bacteria strains at home. 

I expect you’d need a whole laboratory for that 😂!

[Fish Folk]

Nice write up! I too have always read that Nitrosomonas and Nitrobacter are the crucial colonizers to complete the Ammonia > Nitrite > Nitrate cycle. Bottom line, the principle of "one colony that converts Ammonia to Nitrite / another colony that converts Nitrite to Nitrate" still seems sound, even if it turns out that it was Nitrospira all along. I would love to see some scholarly abstracts on aquarium biology that demonstrate Lab work on this topic. Here is a non-scholarly one. 

Excellent points on oxygen, temperature, and pH! I am puzzled about how bacteria in bottles is able to stay alive without oxygen once it sits on a shelf for long periods of time. Could that be another thread to pull on?? 

[Guppysnail]

On 11/18/2022 at 11:15 AM, Fish Folk said:

I am puzzled about how bacteria in bottles is able to stay alive without oxygen once it sits on a shelf for long periods of time.

You know I’m not a scientist so grain of salt here. My understanding is they go into a sort of anabiosis/cryptobiosis the way brine shrimp eggs are. They only become active in the presence of oxygen and possibly ammonia or something like that. 

[Sun.singh1991]

On 11/18/2022 at 10:15 AM, Fish Folk said:

Nice write up! I too have always read that Nitrosomonas and Nitrobacter are the crucial colonizers to complete the Ammonia > Nitrite > Nitrate cycle. Bottom line, the principle of "one colony that converts Ammonia to Nitrite / another colony that converts Nitrite to Nitrate" still seems sound, even if it turns out that it was Nitrospira all along. I would love to see some scholarly abstracts on aquarium biology that demonstrate Lab work on this topic. Here is a non-scholarly one. 

Excellent points on oxygen, temperature, and pH! I am puzzled about how bacteria in bottles is able to stay alive without oxygen once it sits on a shelf for long periods of time. Could that be another thread to pull on?? 

Some products like Seachem Stability actually contain the microbes in their spore forms, in favorable conditions these spores will become active. 

[LionelKirkham]

On 11/4/2022 at 9:54 AM, Sun.singh1991 said:

For as long as I’ve been keeping fish I wanted to know what was going on in our self contained ecosystems. Early on in my fish keep journey I discovered that it was actually bacteria that allowed us to keep such beautiful aquariums alive. This is what inspired me to go to school for a B.S degree in Chemistry and Biology. I eventually found myself asking questions about what really was going on in our tanks down to the molecular level. Resources online typically point you to a basic understating of the nitrogen cycle, pH, and temperature requirements but rarely do they explain how all these factors affect each other on the micro scale. 

In this post I will explain the processes that keep our fish alive in greater detail. I will also try to keep things as easy to understand as I possibly can. This is not a scientific journal as I intend for this entry to be accessible  to anyone regardless of educational or experience level. The journey to understanding our aquariums starts with the nitrogen cycle. Nitrogen is introduced into our aquariums by fish food; as proteins are broken down the fish release ammonia(NH3) as a waste product. Ammonia is toxic to fish because it’s buildup makes it more difficult for fish to excrete it into the surrounding water, causing metabolic shutdown and eventually organ and brain damage in fish. Now total ammonia concentration is measured as both ammonia(NH3) and the ionized form ammonium(NH4+). The relationship between these two forms is dependent on pH; at a higher pH  the ratio of ammonia is much higher than ammonium. And conversely at lower pH the ratio of ammonium is higher.  Ammonium  is completely non toxic to fish whereas ammonia is very toxic. Therefore a fundamental relationship exists between pH and ammonia toxicity. pH is a  measurement of hydrogen ions (H+) in water, at lower pH more H+ is available to bind with ammonia(NH3) to form ammonium(NH4+). This is why tanks that run higher pH levels can be more dangerous to have a ammonia spike in. 
 

This is where the bacteria that keep our fish alive come in. Autotrophic Nitrifying bacteria  oxidize ammonia in a two step process that consumes large amounts of dissolved oxygen and carbonate, also known as alkalinity, from the water. Alkalinity is commonly known as dKH in testing. Bicarbonate and carbonate are essential to our bacteria colonies because they are the inorganic carbon sources that provide energy for them to grow and multiply. Alkalinity is also the buffering system that prevents large changes of pH in our aquariums. In the first step of nitrification a genus of bacteria known as Nitrosomonas take ammonia(NH3), oxygen, and carbonate to oxidize NH3 to Nitrite (NO2). Nitrite is also very deadly to fish because it binds with hemoglobin, which prevents fish from getting oxygen to their organs and can cause death. In addition to NO2 this step also produces H+ ions that increase the acidity of the water over time. In a second step a genus of bacteria known as Nitrobacter take the nitrite produced in the first step and further oxidize it to nitrate(NO3). Acidity in the form of H+ ions is also produced in this step. Overall this two step process requires approximately 4.6 milligrams of dissolved oxygen and 7.1 milligrams  of alkalinity  to oxidize one milligram of ammonia into one milligram of nitrate . This reaction is therefore dependent heavily on the supply of oxygen and alkalinity in the water. Temperature also plays a key role in this reaction as the bacteria are able to grow and multiply best at temperatures between 77 and 86 degrees Fahrenheit. 

Nitrate(NO3) is generally considered safe for fish at concentrations up to 80 ppm. However, it should be noted that the EPA requires municipal water plants to maintain Nitrates at less than 10 ppm. Therefore it can be concluded that nitrates in higher concentrations do have negative implications for fish. At this point in the nitrogen cycle the Nirates can be removed in from the system in a few ways. A water change can immediately lower nitrates to acceptable levels, depending on the percentage of water changed. Another option is the use of plants to uptake nitrates, it should be noted that this solution is dependent on the amount of type of plants used in the aquarium. Faster growing plants will uptake more nitrates than slower growing ones. A third solution is a process called denitrification in which Nitrates are reduced to nitrogen gas which is gassed off from the water rapidly. Denitrification is performed by groups of heterotrophs that require anoxic conditions.  

 

 

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Edited November 29, 2022 by LionelKirkham