Nitrifying Bacteria. Where Are You?

[Lasse]

What puzzled me though (slightly tangential) is that fishless cycle does not call for addition of phosphate sources. Any thoughts?

IMO - it should but I still think this method just crash all others - humble as I am :D:D:D

Sincerely Lasse

 

[Dan_P]

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I would suppose having po4 slightly in excess can exclude it as the limiting reagent. It will also unlikely to interfere with ammonium oxidation. Make sense.

What puzzled me though (slightly tangential) is that fishless cycle does not call for addition of phosphate sources. Any thoughts?

I seem to recall that bottled bacteria medium contains enough PO4 that a recommended dose is likely enough to meet the needs of the bacteria. @taricha will have some thoughts on this from his research on bottled bacteria.

 

[MnFish1]

I seem to recall that bottled bacteria medium contains enough PO4 that a recommended dose is likely enough to meet the needs of the bacteria. @taricha will have some thoughts on this from his research on bottled bacteria.

I think you're right.

 

[Dan_P]

IMO - it should but I still think this method just crash all others - humble as I am :D:D:D

Sincerely Lasse

I love this article. Anyone follow these directions?

 

[MnFish1]

I love this article. Anyone follow these directions?

Yes - I think one of the problems with experiments using test tubes - is its hard to mimic the actual 'stuff' going on in our tanks.

 

[taricha]

What puzzled me though (slightly tangential) is that fishless cycle does not call for addition of phosphate sources. Any thoughts?

I seem to recall that bottled bacteria medium contains enough PO4 that a recommended dose is likely enough to meet the needs of the bacteria. @taricha will have some thoughts on this from his research on bottled bacteria.

True, there was some in Waste Away - just enough for a full dose to be +0.03 PO4 in a system. But not really significant for heterotrophs. Compared to Dan's nitrifying experiments, when I grew heterotrophs, they'd slurp down the PO4. In some cases, it looked like the amount of available PO4 in the fish food or whatever I fed them was the limiter for the maximum culture population size. By comparison, Dan's nitrifiers just didn't consume much or need much.
I take his experiments where 0.00 (hanna) and 0.20 PO4 levels were very similar in terms of nitrification rate (slight slow down in low PO4) to be more relevant data. Seems pretty hard to Phosphate - limit nitrification.

 

[J1a]

Seems pretty hard to Phosphate - limit nitrification.

So wouldn't this suggest there is no significant increase in bacteria population?

 

[taricha]

So wouldn't this suggest there is no significant increase in bacteria population?

I think it's more that you can nitrify a ton of ammonia with a small number of true nitrifiers. So it takes absurdly small amounts of PO4 to keep them happy and growing. You might even be able to double or quadruple the nitrifiers in an initial dose of biospira with barely detectable PO4.
You'll need way more cells (and probably more P) to handle ammonia if you were talking about heterotrophs.

 

[Dan_P]

I think it's more that you can nitrify a ton of ammonia with a small number of true nitrifiers. So it takes absurdly small amounts of PO4 to keep them happy and growing. You might even be able to double or quadruple the nitrifiers in an initial dose of biospira with barely detectable PO4.
You'll need way more cells (and probably more P) to handle ammonia if you were talking about heterotrophs.

Good points. Nitrifyers use ammonia oxidation as an energy source and grow slowly. This means nitrogen uptake with a small biomass production. This leads me to the unanswerable question, when I see an increase in ammonia oxidation, is the nitrifyer population growing or just breathing a bit faster?

 

[MnFish1]

Good points. Nitrifyers use ammonia oxidation as an energy source and grow slowly. This means nitrogen uptake with a small biomass production. This leads me to the unanswerable question, when I see an increase in ammonia oxidation, is the nitrifyer population growing or just breathing a bit faster?

and is it actual 'nitriiers' or 'heterotrophs'. Heterotrophs far more likely. IMHO

 

[Lasse]

When experimenting this way is in an early stages - in spite of to much P or not - as long as you not dose organic carbon - the heterotrophs will not grow. However - After a while when biofilm is too dense to allowing oxygen to penetrate deeper (hence not surviving autotrophs) the dead autothrops create the organic carbon and heterotrophs can grow. But in the start - nope - in this type of experiment at least

Sincerely Lasse

 

[J1a]

When experimenting this way is in an early stages - in spite of to much P or not - as long as you not dose organic carbon - the heterotrophs will not grow. However - After a while when biofilm is too dense to allowing oxygen to penetrate deeper (hence not surviving autotrophs) the dead autothrops create the organic carbon and heterotrophs can grow. But in the start - nope - in this type of experiment at least

Sincerely Lasse

The autotrophe -> heterotrophe development make sense. If autotroph population were to increase, they will still need to take up phosphate from the environment, yes?

So if we remove all phosphate from the water sample, as well as the biospira itself, we can perhaps know if the mechanism is by autotrophic growth, or just whatever existing autotroph doing the heavy lifting.

 

[Lasse]

So if we remove all phosphate from the water sample, as well as the biospira itself, we can perhaps know if the mechanism is by autotrophic growth, or just whatever existing autotroph doing the heavy lifting.

You can do a reversed experiment. In water with PO4 and NH3/NH4 - have enough of alkalinity (above 2-3 dkH) and nitrospira - just measure the ammonia and total N in the water over time. analyse the N species

The same set up but adding a lot of DOC to the water and add heterotrophs - do the same - measure the ammonia and total N in the water over time. analyse the N species.

what i think you will seer is that in the first experiment - NH3/NH4 will disappear (as specie) rather fast but total nitogen in the water column will be near the same. In experiment 2 - NH3/NH4 will be - as a species - much longer time in the water column but total dissolved inorganic N will be lesser

Chose your path - bring down NH3/NH4 as soon as possible or bring down total inorganic N

Sincerely Lasse

 

[J1a]

what i think you will seer is that in the first experiment - NH3/NH4 will disappear (as specie) rather fast but total nitogen in the water column will be near the same. In experiment 2 - NH3/NH4 will be - as a species - much longer time in the water column but total dissolved inorganic N will be lesser

This sounds right.

Let's see what other insights will arise from further experiments.

 

[MnFish1]

@Lasse - perhaps I'm hallucinating - but it is (was) my impression that various pseudomonas sp. can merely use ammonia without much - if any carbon. I have tried looking it up - but can't find it completely - but was curious about your thoughts.

 

[taricha]

This leads me to the unanswerable question, when I see an increase in ammonia oxidation, is the nitrifyer population growing or just breathing a bit faster?

Here's one that looks fairly persuasive to me...
This was sand that was cycled with biospira, ramped the capacity way up, then left it "curing" (circulating in the dark without input) for a couple of months, the capacity for nitrification dropped considerably but did not disappear.
Then I pulled some of the sand, added it to tank water spiked with ~1ppm total ammonia-N.

The green is the ammonia, but look at the blue - NO2-N. It's remarkable how cleanly you can fit it to an exponential for ~3 days.
The exponential function for that fit is y = 0.0486*e^(.824*t) - 0.048
That rate constant implies a nitrite doubling time of 0.841 days = 20.2hrs.
It stops fitting at 3+ days because then NO2->NO3 becomes significant, and by day 3.5 there's significantly lower ammonia concentration left to process.

Note that the ammonia decrease is much less clean - no evidence of exponential population growth, because there are a bunch of ways to consume ammonia. The ammonia oxidizers weren't the only path. But they were the only path to NO2 production and that capacity expanded in exactly the relationship you would expect for population growth.

If you just want to see pretty exponential curves, start with a tiny population and track nitrite until the NO3 producers catch up.

 

[MnFish1]

Curious - what Is the reason for using biospira - as compared to others

 

[J1a]

Here's one that looks fairly persuasive to me...
This was sand that was cycled with biospira, ramped the capacity way up, then left it "curing" (circulating in the dark without input) for a couple of months, the capacity for nitrification dropped considerably but did not disappear.
Then I pulled some of the sand, added it to tank water spiked with ~1ppm total ammonia-N.

The green is the ammonia, but look at the blue - NO2-N. It's remarkable how cleanly you can fit it to an exponential for ~3 days.
The exponential function for that fit is y = 0.0486*e^(.824*t) - 0.048
That rate constant implies a nitrite doubling time of 0.841 days = 20.2hrs.
It stops fitting at 3+ days because then NO2->NO3 becomes significant, and by day 3.5 there's significantly lower ammonia concentration left to process.

Note that the ammonia decrease is much less clean - no evidence of exponential population growth, because there are a bunch of ways to consume ammonia. The ammonia oxidizers weren't the only path. But they were the only path to NO2 production and that capacity expanded in exactly the relationship you would expect for population growth.

If you just want to see pretty exponential curves, start with a tiny population and track nitrite until the NO3 producers catch up.

Interesting data, I would like to draw some ideas from it.

If we take the nitrite graph as a proxy measurement of nitrosipira population, the lag phase is curiously absent. This would suggest that the population of nitrifyer not only survived, but also hardly disrupted in the process of curing and transfer. The nitrifyers added to the test tank is immediately nitrifying and reproducing.

So the follow up question about the nitrifyer in the transferred sand is: does the nitrifyer population dies back during curing (less live bacteria, but whatever lives are active) or does the bacteria population merely becomes dormant.

Based on the exponential fit and the apparent lack of lag phase, I lean towards the former pathway.

A side note, maybe we can take the derivative of the nitrate graph to be a better proxy of the nitrifyer population.

 

[Azedenkae]

@Lasse - perhaps I'm hallucinating - but it is (was) my impression that various pseudomonas sp. can merely use ammonia without much - if any carbon. I have tried looking it up - but can't find it completely - but was curious about your thoughts.

Some species can indeed utilize a lot of ammonia (ammonium) in heterotrophic nitrification, true. Iirc it's also linked to their capability to perform aerobic denitrification.

Here's one study: https://www.sciencedirect.com/science/article/abs/pii/S0960852411010868

I can't remember exactly what all the studies concluded, or even what the mechanism is, because I kinda came to the conclusion that yeah nah, autotrophic nitrification is still preferable.

Hence why I am so for the ammonia-dosing method of fishless cycling. The autotrophic nitrifiers grown here can seem to obtain not only phosphorous, but other elements quite easily, so it does not seem to be an issue. I presume there's enough present through whatever means (trapped in sand, in rocks perhaps, etc.) for them to utilize for growth.

 

[Dan_P]

Here's one that looks fairly persuasive to me...
This was sand that was cycled with biospira, ramped the capacity way up, then left it "curing" (circulating in the dark without input) for a couple of months, the capacity for nitrification dropped considerably but did not disappear.
Then I pulled some of the sand, added it to tank water spiked with ~1ppm total ammonia-N.

The green is the ammonia, but look at the blue - NO2-N. It's remarkable how cleanly you can fit it to an exponential for ~3 days.
The exponential function for that fit is y = 0.0486*e^(.824*t) - 0.048
That rate constant implies a nitrite doubling time of 0.841 days = 20.2hrs.
It stops fitting at 3+ days because then NO2->NO3 becomes significant, and by day 3.5 there's significantly lower ammonia concentration left to process.

Note that the ammonia decrease is much less clean - no evidence of exponential population growth, because there are a bunch of ways to consume ammonia. The ammonia oxidizers weren't the only path. But they were the only path to NO2 production and that capacity expanded in exactly the relationship you would expect for population growth.

If you just want to see pretty exponential curves, start with a tiny population and track nitrite until the NO3 producers catch up.

Nice demonstration of the staying power of AOB’s. Low death rate I guess.

There is one bit of information missing in my brain. What does the ammonia consumption look like for a resting population of AOB’s suddenly give ammonia? I read they can in some instances ramp up quickly (no pictures though) and in other instances they spend time turning on protein synthesis, sitting around not taking up ammonia. I am sorry now that I did not read more deeply.