Does my decade old sand bed actually nitrify? Who eats Ammonia in our tanks?

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taricha

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I agree with you - wading through the data about archaea is difficult - and unless someone who is an expert - that has read/synthesized all of the review articles on it, you will get conflicting results (just like you are with aquabiomics).

on the archaea,
aquabiomics has said that hobbywide most of the detected ammonia oxidizing organisms (or at least sequences) are from archaea. I think I'll dig up posted aquabiomics reports to see if that's generally the case, or if I'm misinterpreting this.

Now - one important caveat about what I said above - its only true if you're counting by species. Most of the nitrifying types are in the biofilm.

But in fact by far the most abundant ammonia-oxidizer in saltwater aquariums are not Bacteria, but rather Archaea. In this case they are free-living Archaea that live in the water column. So the often repeated statement that nitrifiers don't live in the water is not strictly true. In fact, in terms of cell numbers, thats probably where most of them are. But many types (especially nitrite-oxidizing bacteria) are much more abundant in the biofilm.

If hobbywide, everybody has sand that nitrifies somewhat, but nobody has water that nitrifies much at all....
then either
1) the idea that the archaea are mostly doing their work in the water is wrong, and their activity is surface associated for us in the reef hobby.
or 2) archaea are much better at putting a bunch of genes in the water than they are at oxidizing ammonia in the water
or 3) aquabiomics protocol samples water well and biofilm poorly, skewing the results and making water-borne microbes look much more numerous than surface ones.
or 4) the premise is wrong, and lots of systems in the hobby have significant nitrification in the water alone, just not mine or @Dan_P (anybody ever actually observe this?)

@Brew12 has read a ton more cycling posts than most.
I wonder if he's seen any hobbyists find significant nitrification from tank water alone - no substrate?
 
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Dan_P

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on the archaea,
aquabiomics has said that hobbywide most of the detected ammonia oxidizing organisms (or at least sequences) are from archaea. I think I'll dig up posted aquabiomics reports to see if that's generally the case, or if I'm misinterpreting this.



If hobbywide, everybody has sand that nitrifies somewhat, but nobody has water that nitrifies much at all....
then either
1) the idea that the archaea are mostly doing their work in the water is wrong, and their activity is surface associated for us in the reef hobby.
or 2) archaea are much better at putting a bunch of genes in the water than they are at oxidizing ammonia in the water
or 3) aquabiomics protocol samples water well and biofilm poorly, skewing the results and making water-borne microbes look much more numerous than surface ones.
or 4) the premise is wrong, and lots of systems in the hobby have significant nitrification in the water alone, just not mine or @Dan_P (anybody ever actually observe this?)

@Brew12 has read a ton more cycling posts than most.
I wonder if he's seen any hobbyists find significant nitrification from tank water alone - no substrate?

I assume for now there is no a major flaw in the DNA snippet counting process nor the “number of DNA snippets is correlated with population size” assumption. I do wonder whether Aquabiomics ever validated their collection methodology to show that a water sample or water sample plus a biofilm sample is a good representation of the aquarium‘s microbiome. If not, we might be dealing with a red herring (Your #’s 2 and 3).
 

MnFish1

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Agreed on replicates. My plan is to continue using algicide and export to essentially rid my system of green algae. Then after that has been in place for a couple of weeks I'll retest. Then I'll shift to less flake food and more frozen food (keeping total protein input roughly the same) - to shift the carb/protein ratio and retest after a few weeks of that.
I've measured already that the chemistry shows changes with ammonia uptake and oxidation by killing and removing algae, and I expect that would continue.
This will allow me to see a) how much the measured rates of ammonia oxidation by sand and uptake by the system overall is effected by these system changes, and b) if or how well those changes are reflected in the aquabiomics data.
I would also be certain that algaecide does not affect bacterial populations
 

MnFish1

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I assume for now there is no a major flaw in the DNA snippet counting process nor the “number of DNA snippets is correlated with population size” assumption. I do wonder whether Aquabiomics ever validated their collection methodology to show that a water sample or water sample plus a biofilm sample is a good representation of the aquarium‘s microbiome. If not, we might be dealing with a red herring (Your #’s 2 and 3).
I got wildly different results from the same tank - from 'Most diverse ever (I believe above the 95th percentile) and least diverse ever (<5 percent) - depending on the area sampled.
 

MnFish1

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I assume for now there is no a major flaw in the DNA snippet counting process nor the “number of DNA snippets is correlated with population size” assumption. I do wonder whether Aquabiomics ever validated their collection methodology to show that a water sample or water sample plus a biofilm sample is a good representation of the aquarium‘s microbiome. If not, we might be dealing with a red herring (Your #’s 2 and 3).
Why? Just curious?
 
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Dan_P

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Agreed on replicates. My plan is to continue using algicide and export to essentially rid my system of green algae. Then after that has been in place for a couple of weeks I'll retest. Then I'll shift to less flake food and more frozen food (keeping total protein input roughly the same) - to shift the carb/protein ratio and retest after a few weeks of that.
I've measured already that the chemistry shows changes with ammonia uptake and oxidation by killing and removing algae, and I expect that would continue.
This will allow me to see a) how much the measured rates of ammonia oxidation by sand and uptake by the system overall is effected by these system changes, and b) if or how well those changes are reflected in the aquabiomics data.
You could turn off the lights and you would find out very quickly what all the photosynthetic organisms are doing. One day might be enough to get a hint without killing everything. I don’t know how much nitrogen uptake goes on in the dark so it is not a perfect experiment.
 

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Why would you assume that there is not a sampling variation in different areas of different reef tanks? I.e. that the issues seen are related to samples (and different tank conditions) - as compared to diversity, nitrification, etc
I do indeed feel that sampling location in space and time could be important. I am contemplating spending money to find out whether three samples taken in a row from the same place provide the same information or same place 6 hours apart. This is all in the planning phase. I need a more detailed rationale for selecting sampling locations.
 

MnFish1

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I do indeed feel that sampling location in space and time could be important. I am contemplating spending money to find out whether three samples taken in a row from the same place provide the same information or same place 6 hours apart. This is all in the planning phase. I need a more detailed rationale for selecting sampling locations.
I did this experiment. 2 samples (that I sent) from the same place, and time - were identical. I also thought about doing it at different times of the day but did not. But - samples from the rock itself were markedly different than the inside of the return tube, etc
 
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Nano sapiens

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...or same place 6 hours apart.

If you (or anyone else) decides to do this, I'd suggest a sample taken during the middle of the reef aquarium's light period and also one from the middle of the tank's dark period (both taken from the same locations). Reason being, sampling on the natural reef showed marked variation in the microbial community during a diel cycle:

https://www.nature.com/articles/s41467-019-09419-z

My comments from a previous post directed at Microbiomics:

"This research deals specifically with the microbial communities on four of the Southern Line Islands (some of the most pristine coral reef communities in the world). Not only the composition of the microbial community is determined (some interesting microbial differences from our reef systems stand out), but it also answers the question of how does the microbial community differ in a 24 hr period? Turns out, the difference is 'day and night', literally! The daytime community makeup is quite different from the one at night. In fact, the difference is more pronounced from day to night in one specific spot than from one island to another island."
 

Dan_P

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I did this experiment. 2 samples (that I sent) from the same place, and time - were identical. I also thought about doing it at different times of the day but did not. But - samples from the rock itself were markedly different than the inside of the return tube, etc
I have been growing biofilms and found differences between these films ability to consume ammonia depending on whether they grow on glass or aragonite. Under the microscope, there are visual differences between the glass surface near the bottom of the slide compared to the top of the slde (these microscope slides are heldin a vertical position). As a result of these observations, I expect surface samples to be highly variable.
 

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@Brew12 has read a ton more cycling posts than most.
I wonder if he's seen any hobbyists find significant nitrification from tank water alone - no substrate?
I'm not sure I can add too much to this discussion. I have seen documented ammonia spikes from removing media in HOB filters in QT tanks. The implication I would take from this is that in an immature system the water alone offers little nitrification.
In more mature systems I've seen ammonia spikes when large amounts of media/rock are removed. I've assumed this was because of removing the surface where AOB's were concentrated.
I've also seen documented ammonia spikes when large amounts of algae were removed from a system.

Not scientific by any means, but in a mature reef system I would feel that coral/algae consume the most ammonia, followed by bacteria on surfaces, followed by water biota.

Just my quick thoughts, not very scientific.
 

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Brew that removal of surface area + increased ammonia in QT test was directly proven on seneye by Jon M in his seneye works thread, agreed it was a noted spike with lowering of surface area, and a direct regain of steady state within minutes when it was added back. api would've shown .5 the whole time but that digital meter let us see a change from thousandths ppm to hundredths, upon removal. very precise rise n fall.
 
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taricha

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You could turn off the lights and you would find out very quickly what all the photosynthetic organisms are doing. One day might be enough to get a hint without killing everything. I don’t know how much nitrogen uptake goes on in the dark so it is not a perfect experiment.
Unfortunately, algae can eat a ton of ammonia even in the dark, hence why I tested nitrification rate by pulling surface sand samples.
See example below (Surge ammonium uptake in macroalgae from a coral atoll)
Screen Shot 2021-08-03 at 11.03.56 AM.png




I would also be certain that algaecide does not affect bacterial populations
That is a question I was curious about as well.
Here's a relevant part of my aquabiomics report, and comment from Aquabiomics email.

Screen Shot 2021-08-03 at 11.27.05 AM.png

Screen Shot 2021-08-03 at 11.27.24 AM.png


Screen Shot 2021-08-03 at 11.06.56 AM.png


"...the microbiome test revealed a community with diversity on the low side of normal, but not an extreme value. The composition of this community on the other hand was textbook normal, except for the near absence of Alteromonadaceae. This is a group that blooms in nutrient addition (especially carbohydrates), so I suspect this may just reflect lower nutrients than many tanks. "

I take this together to mean that the algicide (algaefix marine) that I did a few rounds of - and then several weeks of none prior to the aquabiomics test - did not substantially harm the bacteria.
Did it shift populations? who knows? I'm certain that lots of dying algae is a factor that would shift populations, as all food input changes would. I manually removed whatever I could.



I got wildly different results from the same tank - from 'Most diverse ever (I believe above the 95th percentile) and least diverse ever (<5 percent) - depending on the area sampled.
I'll need to look at what your low percentile diversity test was, but your "most diverse ever" was when you swirled the water around over the surfaces and lifted suspended material into the water sample.
Rather than surprising, I found it very reassuring that when your sample included many suspended particles from surfaces, that the bacteria found were a far more diverse collection than undisturbed tank water.
Your "water sample", in effect sampled many locations at once.
 
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I have seen documented ammonia spikes from removing media in HOB filters in QT tanks. The implication I would take from this is that in an immature system the water alone offers little nitrification.
In more mature systems I've seen ammonia spikes when large amounts of media/rock are removed. I've assumed this was because of removing the surface where AOB's were concentrated.
I've also seen documented ammonia spikes when large amounts of algae were removed from a system.

Not scientific by any means, but in a mature reef system I would feel that coral/algae consume the most ammonia, followed by bacteria on surfaces, followed by water biota.

Awesome. I knew you'd have a good way to think of this. By subtraction systems that pulled surfaces or algae can see ammonia spikes, implying these systems have water that can't keep up with the ammonia processing rate for the tank.
 
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Man, I missed that this archaea question had kind of been answered - in at least @Nano sapiens case.

An important question for our discussions about where various microbes live in the tank.

Its not a part of the standard output but I did a little digging. Since Ralph has shared his results I'll use his results as an example to answer the question.

Ammonia oxidizers:
Nitrosomonadaceae -- both, 3.8-fold higher in biofilm
Nitrososphaeraceae -- only detected in biofilm
Cenarchaeaceae -- both, 5.1-fold higher in biofilm

Nitrite oxidizers:
Nitrospiraceae -- both, 2.6-fold higher in biofilm

This matches my general impression across multiple samples, although again this tank has relatively high levels of nitrifying microbes overall compared to the range I've tested. In some tanks neither group is detected. In many tanks, ammonia oxidizers are low and nitrite oxidizers below detection. I believe what is happening is the two groups vary together across different tanks, with nitrite oxidizers always at lower levels.

The archaea were also the dominant (by number) ammonia oxidizers for his system, as they were in mine.

And they were found heavily skewed to biofilm and not the water.
 
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taricha

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I did this experiment. 2 samples (that I sent) from the same place, and time - were identical. I also thought about doing it at different times of the day but did not. But - samples from the rock itself were markedly different than the inside of the return tube, etc
I can't re-find the results of your experiments with varying the sampling method. got a link? or is that info gone now and only in our heads?

removal of surface area + increased ammonia in QT test was directly proven on seneye by Jon M in his seneye works thread, agreed it was a noted spike with lowering of surface area, and a direct regain of steady state within minutes when it was added back.
any chance you could link to a post or link the thread and tell post number where to look?
 

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I'll need to look at what your low percentile diversity test was, but your "most diverse ever" was when you swirled the water around over the surfaces and lifted suspended material into the water sample.
Rather than surprising, I found it very reassuring that when your sample included many suspended particles from surfaces, that the bacteria found were a far more diverse collection than undisturbed tank water.
Your "water sample", in effect sampled many locations at once.
Right - but that was the point. Who cares whether the return tube has a less diverse population than 99 percent of tanks (for example) - if the entire tank contains a diverse population. This is why where (and how) the sample is taken is so important.

For example - in a tank with 'low flow' across the rocks, one might expect a lower diversity - than a tank with 'high flow' accross the rocks (based on my results) - but that is not entered into the equation. There are numerous other uncontrolled variables though in the test itself, location is).

BTW - my 'diverse sample' was me swirling my hand across my rocks - to create a flow - there was not a huge amount of sediment floating around the tank. I will say - it was a 'high flow' as compared to a swirl.

None of my comments are directed at Aquabiomics testing itself - because controlling for all of these variables would require a supercomputer and perhaps millions of samples. I'm just saying - perhaps some of the conclusions you're making may not be justified - given some of the limitations of the test.
I can't re-find the results of your experiments with varying the sampling method. got a link? or is that info gone now and only in our heads?


any chance you could link to a post or link the thread and tell post number where to look?
I am on vacation - I can get the results of the tests from Aquabiomics website - but - I sent them in as blinded samples - so I can't really tell you. I will try to find out some more information
 
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