Nitrifying Bacteria. Where Are You?

[Dan_P]

I feel pretty confident that if you had fed 2ppm ammonia under the same conditions you would have seen a big scale-up.

You called this one correctly.

I used the bare box and the box with sand to observe what happens when I ramp up the initial ammonia concentration. I measured the total NH3 and nitrite concentrations at 24 hours.

Results. Data plotted below. Error bars for replicate aquaria are 2 standard deviations.

Activity increases rapidly. A second dose increased activity a little (the second data point on the plot for each ammonia concentration above 0.5 ppm). One dose is still enough in fish-less cycling. The surface area effect seems to have disappeared at higher ammonia concentrations. The only explanation is that there is no surface area effect with my experimental conditions. Nitrite consumption was about half that of ammonia consumption. The last experiment used two levels of PO4, 0.2 and 1.0 ppm. This had no effect on consumption rates.

My very last experiment is to monitor ammonia consumption activity as the BioSpira biofilm sits in aquarium water in the dark, fed either NH3 or the amino acid glutamine.

 

[MnFish1]

You called this one correctly.

I used the bare box and the box with sand to observe what happens when I ramp up the initial ammonia concentration. I measured the total NH3 and nitrite concentrations at 24 hours.

Results. Data plotted below. Error bars for replicate aquaria are 2 standard deviations.

Activity increases rapidly. A second dose increased activity a little (the second data point on the plot for each ammonia concentration above 0.5 ppm). One dose is still enough in fish-less cycling. The surface area effect seems to have disappeared at higher ammonia concentrations. The only explanation is that there is no surface area effect with my experimental conditions. Nitrite consumption was about half that of ammonia consumption. The last experiment used two levels of PO4, 0.2 and 1.0 ppm. This had no effect on consumption rates.

My very last experiment is to monitor ammonia consumption activity as the BioSpira biofilm sits in aquarium water in the dark, fed either NH3 or the amino acid glutamine.

Perfect - this makes sense!!!!

 

[MnFish1]

You called this one correctly.

I used the bare box and the box with sand to observe what happens when I ramp up the initial ammonia concentration. I measured the total NH3 and nitrite concentrations at 24 hours.

Results. Data plotted below. Error bars for replicate aquaria are 2 standard deviations.

Activity increases rapidly. A second dose increased activity a little (the second data point on the plot for each ammonia concentration above 0.5 ppm). One dose is still enough in fish-less cycling. The surface area effect seems to have disappeared at higher ammonia concentrations. The only explanation is that there is no surface area effect with my experimental conditions. Nitrite consumption was about half that of ammonia consumption. The last experiment used two levels of PO4, 0.2 and 1.0 ppm. This had no effect on consumption rates.

My very last experiment is to monitor ammonia consumption activity as the BioSpira biofilm sits in aquarium water in the dark, fed either NH3 or the amino acid glutamine.

PS - it would be interesting if you tried it with 0 ppm PO4 - unless biospira has PO4 in it. Also replicating with Fritz or another bacteria brand.

 

[Dan_P]

PS - it would be interesting if you tried it with 0 ppm PO4 - unless biospira has PO4 in it. Also replicating with Fritz or another bacteria brand.

Those would all be extremely interesting things to investigate. I will be switching gears though and moving equipment over to support my study of algal biofilms. I do have one more BioSpira experiment to report on. Results in a couple weeks.

 

[taricha]

Activity increases rapidly. A second dose increased activity a little (the second data point on the plot for each ammonia concentration above 0.5 ppm).

I love a confusing experimental result as much as the next guy, but it's also nice to be reassured that our biofilms inhabit the same universe.


Still thinking through the implications that a bunch of replicates say 0.5ppm ammonia dosing didn't scale up nitrification ability (from a default biospira dose), but 2ppm ammonia does.
Can't wait to see nitrifying films grown on amino acid.

 

[Dan_P]

Still thinking through the implications that a bunch of replicates say 0.5ppm ammonia dosing didn't scale up nitrification ability (from a default biospira dose), but 2ppm ammonia does.

Found a presentation on nitrifying bacteria that contained a plot showing that the ammonia removal rate of a nitrification filter was correlated to the concentration of ammonia. At higher levels the curve flattens.

 

[Lasse]

Can't wait to see nitrifying films grown on amino acid

Why should it not? Amino acid will be breakdown to NH3/NH4 rather rapidly by other bacteria

Sincerely Lasse

 

[taricha]

@Dan_P I was thinking about the 0.5ppm vs 2ppm ammonia result in the context of fishless vs fish-in cycling. If you are doing fish-in cycling, and you started with a dose of biospira, you might never reach ammonia levels that would actually spur growth of the nitrifiers in the films. So those that start with an ammonia dose and the same bacteria ought to be expected to have a higher number of (biospira) nitrifiers post-cycle.
Not that one is better than the other, just that the populations are going to be different for fishless vs fish-in cycling.

Why should it not? Amino acid will be breakdown to NH3/NH4 rather rapidly by other bacteria

yep. it ought to work just fine, just that Dan and I haven't done it before.
We have looked a bunch at feeding heterotrophs amino acids and feeding nitrifiers ammonia. Everything seems pretty well behaved. But haven't fed a nitrifying biofilm organics that would require the nitrifiers to wait in line behind the heterotrophs to get a shot at the ammonia.
There ought to be room for all. Genetic testing of nitrifying films finds that the nitrifiers may only be a few % of the population. So room for all and happy neighbors probably.

 

[J1a]

@Dan_P I was thinking about the 0.5ppm vs 2ppm ammonia result in the context of fishless vs fish-in cycling. If you are doing fish-in cycling, and you started with a dose of biospira, you might never reach ammonia levels that would actually spur growth of the nitrifiers in the films. So those that start with an ammonia dose and the same bacteria ought to be expected to have a higher number of (biospira) nitrifiers post-cycle.
Not that one is better than the other, just that the populations are going to be different for fishless vs fish-in cycling.



yep. it ought to work just fine, just that Dan and I haven't done it before.
We have looked a bunch at feeding heterotrophs amino acids and feeding nitrifiers ammonia. Everything seems pretty well behaved. But haven't fed a nitrifying biofilm organics that would require the nitrifiers to wait in line behind the heterotrophs to get a shot at the ammonia.
There ought to be room for all. Genetic testing of nitrifying films finds that the nitrifiers may only be a few % of the population. So room for all and happy neighbors probably.

These are some interesting conclusions. Perhaps some extension questions:

1. To what extend does nitrifiers compete with corals for ammonium? If this is significant, perhaps lower the amount of nitrifier through removal of media can allow more ammonium to be take up for corals.

2.in the event of cohabitation of heterotroph and AOB, there a competition for oxygen. Could gas exchange be a limiting factor?

 

[Lasse]

These are some interesting conclusions. Perhaps some extension questions:

1. To what extend does nitrifiers compete with corals for ammonium? If this is significant, perhaps lower the amount of nitrifier through removal of media can allow more ammonium to be take up for corals.

IMO -this competition is often governed by chance and the distance between production and consumption;. However if the corals take the majority of the NH3/NH4 produced - ir means a decline of the population of nitrifiers - but if the nitrifiers use of ammonium are dominated - the corals get their second chance from the NO3 produced by the nitrifier

2.in the event of cohabitation of heterotroph and AOB, there a competition for oxygen. Could gas exchange be a limiting factor?

Yes - lack of gas exchange and a low O2 pressure will limit the depth in the biofilm there nitrifier can work. However the limited factor for the heterotrophs is the availability of organic Carbon - DOC (dissolved Organic Carbon mostly.

Sincerely Lasse

 

[Dan_P]

@Dan_P I was thinking about the 0.5ppm vs 2ppm ammonia result in the context of fishless vs fish-in cycling. If you are doing fish-in cycling, and you started with a dose of biospira, you might never reach ammonia levels that would actually spur growth of the nitrifiers in the films. So those that start with an ammonia dose and the same bacteria ought to be expected to have a higher number of (biospira) nitrifiers post-cycle.
Not that one is better than the other, just that the populations are going to be different for fishless vs fish-in cycling.

Does it matter that a nitrification biofilm is started with 0.5 or 2.0 ppm? I feel the answer is yes if you are starting up a fish farm, but a reef aquarium, maybe not.

If one had some free lab time, one could design some experiments to demonstrate how the two behave in response to ammonia impulses

 

[Dan_P]

1. To what extend does nitrifiers compete with corals for ammonium? If this is significant, perhaps lower the amount of nitrifier through removal of media can allow more ammonium to be take up for corals.

Now you got me thinking weird thoughts, like, if you are going to raise coral or start a reef aquarium, why bother starting a nitrifying biofilm first? If you grow seaweed you don’t need nitrifying bacteria. Why do you need nitrification for coral? Let them be the ammonia consumers.

 

[Lasse]

If you grow seaweed you don’t need nitrifying bacteria

But you will have a lot of them as biofilm on the algae, space, oxygen in excess (during daytime), no competition and so on. And closer to the source of NH3/NH4. In fact - there is examples of experimental treatment plants that use Elodea as a substrate for nitrification,

Sincerely Lasse

 

[Dan_P]

I love a confusing experimental result as much as the next guy, but it's also nice to be reassured that our biofilms inhabit the same universe.


Still thinking through the implications that a bunch of replicates say 0.5ppm ammonia dosing didn't scale up nitrification ability (from a default biospira dose), but 2ppm ammonia does.
Can't wait to see nitrifying films grown on amino acid.

Here is the final installment. I cultured the aragonite rock slices that were covered with a BioSpira biofilm under three conditions in the dark on a orbital shaker for 20 days before assessing their activity (see photo below). Medium was changed about every four days.

Each treatment had roughly the same rock slice surface area. The “IO+NH3” experiment was Instant Ocean with a total ammonia content of 0.5 ppm, “TW+NH3” was aquarium water and ammonia and the final treatment “TW+Gln” was aquarium water dosed with glutamine to the same amount of nitrogen as 0.5 ppm NH3.

The nitrification capacity assessment was run in fresh Instant Ocean dosed with PO4 and 0.5 ppm total ammonia in clean boxes. As you can see below (y-axis ppm N, X-axis hours) all three treatments ran approximately the same. The nitrate trend was calculated. The orange dot is the measured nitrate after nitrite was gone. The fourth plot (blue line IO+NH3, orange line TW+NH3, grey line TW+Gln) compares the nitrite concentration profile where you can see differences but there might not be anything noteworthy to learn other than “replicates are needed in biology experiments to figure out if anything happened”

The rock slices cultured in aquarium water and fed glutamine might be a potential analytical device for detecting and measuring organic nitrogen in a water sample. The combined heterotrophic-autotrophic population converted glutamine to nitrate almost quantitatively. I haven’t tried other nitrogen sources. My aquarium water is pretty much depleted of N except for NO3 according to a recent NDOC analysis.

 

[taricha]

Those curves are really pretty, and the NO3-N agreement is fantastic.

I don't understand the blue measured(?) ammonia in the TW+Gln run.
Did the system just decompose all the Gln to ammonia immediately? or is it calculated, or are you doing something fancy to measure glutamine as it's consumed?

 

[Dan_P]

Those curves are really pretty, and the NO3-N agreement is fantastic.

I don't understand the blue measured(?) ammonia in the TW+Gln run.
Did the system just decompose all the Gln to ammonia immediately? or is it calculated, or are you doing something fancy to measure glutamine as it's consumed?

The ”TW+Gln” refers to the condition under which the BioSpira covered rock slices were cultured. The nitrification capacity assessment medium was IO dosed with ammonium chloride to a total ammonia content of 0.5 ppm?

It would have been worth a separate post if I managed to measure the glutamine concentration. Some day.

Those nitrate measurements were made with the modified API nitrate kit and the Hanna HR Marine NO3 Checker.

 

[taricha]

The ”TW+Gln” refers to the condition under which the BioSpira covered rock slices were cultured.

I knew I should have read it one more time.

Your modified API nitrate result is tighter agreement with theoretical nitrification than when I've done it with the Hannah LR NO3 test. Impressive!