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

[taricha]

I´m not sure that this is the best place to take sample from. If I do the test again - I will swab in the sand instead

I cultured up some of the bacteria released when I shook up my sand. When that was analyzed by aquabiomics, the results showed dominance of species very rarely seen in his testing database. (even though my normal aquarium sample was very typical.)

So if you sample in a non-standard way, you may expect to see rare and hard to interpret results.

 

[Lasse]

see rare and hard to interpret results

But you can´t expect very much bacteria (or bacteria-DNA) if you swab - as i did - in the return pipe. There is a biofilm but very thin. The rare result - maybe is from the pipes.

Sincerely Lasse

 

[Dan_P]

Here's my issue. It's a bit of a paradox.
1) I measure almost no nitrification in my water, and I easily measure nitrification in my sand.
2) Aquabiomics finds that the vast majority of Ammonia oxidizing microbe sequences in my sample are archaea with only a tiny slice being bacteria.
3) microbiology folks say that archaea are active mostly in the water...



I'm not sure what the resolution is to the paradox.
Could simply be that the notion about their activity in the water is incorrect and that our saltwater archaea are active on surfaces as much or more. (It sounds flip to say that maybe the people who study archaea are wrong, but from what I gather, they are mostly studied by picking up their genes and enzymes in the environment. rarely cultured.)
Or it could be that they are a tiny population of cells that sheds a lot of genetic sequences in the water where they are easy to collect (vs biofilm), or maybe the cells themselves have a tiny appetite for ammonia compared to a cell of ammonia oxidizing bacteria.

if it's either of the last two, then it goes to Dan's point below. The correlation between detected sequences and metabolism of ammonia would be pretty weak. Some of this ought to be answered in published papers, I think.


That study was done early. I wonder if it was before aquabiomics switched to adding a biofilm swab as a standard part of the test. "swab inside a dark contiinuously submerged pipe." Also, it's worth pointing out aquabiomics isn't measuring live cells, or even cells at all - just genetic sequences.

All of this gets at some of @MnFish1 skepticism as well. Maybe the test protocol just isn't a good way to measure your nitrifier population.

@Rick Mathew once said that trying to understand an aquarium through water testing is like trying to understand an automobile by studying tailpipe emissions. I am going to suggest that this idea may apply to DNA sampling. However, as with water testing, I like DNA sampling.

As soon as I become a bit more expert at growing biofilms, I want to use Aquabiomics to give me a read on biofilm inhabitants as I nudge biofilm development by varying conditions under which they mature.

 

[MnFish1]

Here's my issue. It's a bit of a paradox.
1) I measure almost no nitrification in my water, and I easily measure nitrification in my sand.
2) Aquabiomics finds that the vast majority of Ammonia oxidizing microbe sequences in my sample are archaea with only a tiny slice being bacteria.
3) microbiology folks say that archaea are active mostly in the water...



I'm not sure what the resolution is to the paradox.
Could simply be that the notion about their activity in the water is incorrect and that our saltwater archaea are active on surfaces as much or more. (It sounds flip to say that maybe the people who study archaea are wrong, but from what I gather, they are mostly studied by picking up their genes and enzymes in the environment. rarely cultured.)
Or it could be that they are a tiny population of cells that sheds a lot of genetic sequences in the water where they are easy to collect (vs biofilm), or maybe the cells themselves have a tiny appetite for ammonia compared to a cell of ammonia oxidizing bacteria.

if it's either of the last two, then it goes to Dan's point below. The correlation between detected sequences and metabolism of ammonia would be pretty weak. Some of this ought to be answered in published papers, I think.


That study was done early. I wonder if it was before aquabiomics switched to adding a biofilm swab as a standard part of the test. "swab inside a dark contiinuously submerged pipe." Also, it's worth pointing out aquabiomics isn't measuring live cells, or even cells at all - just genetic sequences.

All of this gets at some of @MnFish1 skepticism as well. Maybe the test protocol just isn't a good way to measure your nitrifier population.

I do not think you can draw these conclusions. Your comment about 'just because you're numbers are above the 50th percentile means most people's levels are below yours is true - BUT - it does not suggest/prove/or mean (IMHO) that this implies that other tanks are MEANINGFULLY lower than yours. My understanding is that archaea are also 'attached' - more as compared to free-swimming. As always its an interesting discussion. As I have posted, depending on where a sample is taken, can markedly change the diversity percentage, etc.

 

[MnFish1]

I cultured up some of the bacteria released when I shook up my sand. When that was analyzed by aquabiomics, the results showed dominance of species very rarely seen in his testing database. (even though my normal aquarium sample was very typical.)

So if you sample in a non-standard way, you may expect to see rare and hard to interpret results.

It would be my strong impression, that if you sampled one area of sand - and another one a foot away they numbers could we widely disparate. The problem (even with "standardized" sampling areas is that the inside of one tanks 'pipe' may have higher or lower flow, etc. - or for example one person is using carbon, another is dosing amino acids into their sump, etc etc etc. Without multiple samples from multiple areas (which In my tank showed extreme variability) - from one of the least diverse samples in the database to one of the most (based solely on a different location sampled). I believe the method is accurate - in that the 2 samples that I divided up and sent twice came out quite similar (i.e. little variation). I do not believe you can use this test accurately - except perhaps in an experimental situation - with controlled tanks.

 

[taricha]

BUT - it does not suggest/prove/or mean (IMHO) that this implies that other tanks are MEANINGFULLY lower than yours.

Sorry. I'm slow. I think I finally appreciate your argument a little more. If the large variations are due to sample to sample inconsistency, then me finding higher-than-average Nitrifier sequences could just mean it's a "lucky" sample and my actual nitrification population is low - in agreement with the chemical tests.
I'm more inclined to believe that the large sample to sample discrepancies in detected Nitrafiers across the hobby reflect the actual large differences in how those systems process ammonia.

But that's tricky. I think it will be very hard to say which is the larger source of variation in the aquabiomics data set. I need to re-read the differences in the samples that you sent in. I've forgotten some details.

 

[Dan_P]

Sorry. I'm slow. I think I finally appreciate your argument a little more. If the large variations are due to sample to sample inconsistency, then me finding higher-than-average Nitrifier sequences could just mean it's a "lucky" sample and my actual nitrification population is low - in agreement with the chemical tests.
I'm more inclined to believe that the large sample to sample discrepancies in detected Nitrafiers across the hobby reflect the actual large differences in how those systems process ammonia.

But that's tricky. I think it will be very hard to say which is the larger source of variation in the aquabiomics data set. I need to re-read the differences in the samples that you sent in. I've forgotten some details.

Good discussion here on variation. When variation in ICP results is understood a bit better, exploring Aquabiomics test results variation could be useful.

 

[MnFish1]

Sorry. I'm slow. I think I finally appreciate your argument a little more. If the large variations are due to sample to sample inconsistency, then me finding higher-than-average Nitrifier sequences could just mean it's a "lucky" sample and my actual nitrification population is low - in agreement with the chemical tests.
I'm more inclined to believe that the large sample to sample discrepancies in detected Nitrafiers across the hobby reflect the actual large differences in how those systems process ammonia.

But that's tricky. I think it will be very hard to say which is the larger source of variation in the aquabiomics data set. I need to re-read the differences in the samples that you sent in. I've forgotten some details.

I don't think you're slow - I don't think I was clearly making my point - maybe this picture will help (why I said it is important to know the 'curve' that you're looking at. Pretend you're looking at the picture below -Concentrate on the green and the red curves. Lets say each curve represents the numbers of 1 type of bacteria in 100 reef tanks today. Lets say you're at 51 percent (near the peak/mean/median). On the green curve - 'normal' would be a much larger range than 'normal' would be on the red curve (with 'normal' being defined as 1 or 2 standard deviations above/below the mean. (edit - ignore the actual numbers - obviously there can't be 'negative' numbers of bacteria. Pretend the mean of the green and red curves are both '2'

 

[Randy Holmes-Farley]

Good discussion here on variation. When variation in ICP results is understood a bit better, exploring Aquabiomics test results variation could be useful.

"Random" variation is a big issue in science. Normally, "random" just means we do not yet know what is causing the variability.

My company is trying to prove a 2-3 fold change in something that is inherently somewhat noisy, and it takes many experiments and/or a very large number of test subjects to convincingly demonstrate that effect.

 

[Belgian Anthias]

Never heard that - always heard that it is in a biofilm

Sincerely Lasse

In the ocean Archaea are most active in so called " Oxygen Minimum Zones", in deeper water layers. Archae are mainly active in zones where the oxygen availability becomes insufficient for AOB, ref: MB CMF De Haes. The end product, nitrite, then may be used for ANAMMOX as there will be little competition with NOB in OMZ. ANAMMOX bacteria use an anaerobic pathway. ref: MB CMF De Haes 2020
In an aquarium also OMZ are created but on a different scale. In an aquarium Archaea are mainly active part of biofilms in bio-filters, where the biofilms are less disturbed.

 

[Lasse]

In the ocean Archaea are most active in so called " Oxygen Minimum Zones", in deeper water layers. Archae are mainly active in zones where the oxygen availability becomes insufficient for AOB, ref: MB CMF De Haes. The end product, nitrite, then may be used for ANAMMOX as there will be little competition with NOB in OMZ. ANAMMOX bacteria use an anaerobic pathway. ref: MB CMF De Haes 2020
In an aquarium also OMZ are created but on a different scale. In an aquarium Archaea are mainly active part of biofilms in bio-filters, where the biofilms are less disturbed.

But do not they need a substrate - can they be free swimming?

Sincerely Lasse

 

[Rick Mathew]

"Random" variation is a big issue in science. Normally, "random" just means we do not yet know what is causing the variability.

My company is trying to prove a 2-3 fold change in something that is inherently somewhat noisy, and it takes many experiments and/or a very large number of test subjects to convincingly demonstrate that effect.

You are absolutely correct Sir...It has been my experience that it is not only science that must deal with randomness but many things in life

Rick

 

[taricha]

I'm still wading through google scholar on the archaea location and activity question.

It looks messy - like we'd either have to try to apply conflicting conclusions from studies on freshwater aquaria, or marine commercial aquaculture biofilters, or the open ocean, etc. and that seems dicey either way. One is nopt marine, the others are much higher nutrients and or much lower than aquaria.

All the hobby data I'm aware of leans in favor of benthic nitrification. Whatever oxidizes ammonia, it's on surfaces almost entirely. If archaea are primarily in water, it seems their metabolism may be tiny and irrelevant.

@Lasse if I remember, aquabiomics found your ammonia oxidizers were archaea (detected no AOB).

If you took tank water and spiked it with ~0.5ppm total ammonia and slow bubbled it in the dark for a couple of days, would it nitrify?
In my system, sand would deplete that (and show NO3 increase) in ~2 days, but water wouldn't move it more than 0.1ppm in that time.

 

[MnFish1]

I'm still wading through google scholar on the archaea location and activity question.

It looks messy - like we'd either have to try to apply conflicting conclusions from studies on freshwater aquaria, or marine commercial aquaculture biofilters, or the open ocean, etc. and that seems dicey either way. One is nopt marine, the others are much higher nutrients and or much lower than aquaria.

All the hobby data I'm aware of leans in favor of benthic nitrification. Whatever oxidizes ammonia, it's on surfaces almost entirely. If archaea are primarily in water, it seems their metabolism may be tiny and irrelevant.

@Lasse if I remember, aquabiomics found your ammonia oxidizers were archaea (detected no AOB).

If you took tank water and spiked it with ~0.5ppm total ammonia and slow bubbled it in the dark for a couple of days, would it nitrify?
In my system, sand would deplete that (and show NO3 increase) in ~2 days, but water wouldn't move it more than 0.1ppm in that time.

IMHO, the most likely scenario here is that the Aquabiomics data is suspect. I would suggest perhaps repeating the experiment - using more controls - and replications? 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).

There is also an extreme difference between comparing a water treatment facility open ocean and a reef tank. (I do not think there is good data for reef tanks - that I've seen). But here is a quote:

"Solving previously unexplained N-cycling processes in soils and oceans
The open ocean has extremely low concentrations of dissolved ammonium. Measurements revealed substantial discrepancies between ammonia oxidation rates observed in situ and those of cultivated AOB, with the half-saturation constant for activity in the open ocean orders of magnitude lower. However, the cultivation of marine AOA possessing extremely high specific affinities for ammonia, together with molecular surveys revealing a numerical dominance of N. maritimus-like archaea, demonstrated that AOA are primarily responsible for ammonia oxidation in the open ocean."

 

[Lasse]

but water wouldn't move it more than 0.1ppm in that time.

Run a test with filtrated water - 1-4 µm just because take away particles as substrates. In deep nearly anaerobic water - there is particles even in the ocean.

Sincerely Lasse

 

[MnFish1]

Run a test with filtrated water - 1-4 µm just because take away particles as substrates. In deep nearly anaerobic water - there is particles even in the ocean.

Sincerely Lasse

Question - I fully believe what I think you're saying - i.e. that particulates in the water allow bacteria to colonize them (even though we can't see them). That said - why would this not occur in our tanks as well? Skimming? etc etc?

 

[Lasse]

Question - I fully believe what I think you're saying - i.e. that particulates in the water allow bacteria to colonize them (even though we can't see them). That said - why would this not occur in our tanks as well? Skimming? etc etc?

Yes but the particle aggregate goes up in the skimmer cup and get anaerobic. And one important thing - if the particles are to "much" organic - the nitrification bacteria can´t concur with heterotrophs, It demand more inorganic particles. In the classic active sludge treatment you are concerned of the age of the sludge (often anaerobic sludge that is reused) For BOD removal not older than 2-4 days - for nitrification not younger than 5 days (normally) In SBR reactors they first run the sludge as BOD removal, When BOD is low - nitrification starts and finally they run the whole thing anaerobic for denitrification.

Sincerely Lasse

 

[MnFish1]

Yes but the particle aggregate goes up in the skimmer cup and get anaerobic. And one important thing - if the particles are to "much" organic - the nitrification bacteria can´t concur with heterotrophs, It demand more inorganic particles. In the classic active sludge treatment you are concerned of the age of the sludge (often anaerobic sludge that is reused) For BOD removal not older than 2-4 days - for nitrification not younger than 5 days (normally) In SBR reactors they first run the sludge as BOD removal, When BOD is low - nitrification starts and finally they run the whole thing anaerobic for denitrification.

Sincerely Lasse

Thanks!!

 

[Lasse]

This is the reason why I do not advocate for water change when nitrification have stuck

Sincerely Lasse

 

[taricha]

IMHO, the most likely scenario here is that the Aquabiomics data is suspect. I would suggest perhaps repeating the experiment - using more controls - and replications?

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.