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

Nano sapiens

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Good point about activity from Biospira and not much from rock. If the typical live rock is like your “lazy sand”, the hobby might be overestimating the capacity of live rock to oxidize ammonia though it would be a excellent source of bacteria as Aquabiomics has demonstrated.

It would be interesting to know the ammonia oxidation rate for your live rock before adding anything. There might be a simple way to roughly estimate the volume and surface area so we could compare its capacity to your sand.

I recollect an experiment done some time ago comparing the nitrification/denitrification capacity of live rock vs. live sand (just can't find it on the Internet). In this experiment, live sand was found to be much more effective than live rock.

The experiment I linked to a post or two above shows:

"The results demonstrated that LR mainly removed ammonium (NH4⁺) from the water with a mean efficiency of 0.141 mg/(kg·h), while the removal of nitrate (NO3–) was not significant. Bacterial diversity analysis showed that ammonia-oxidizing bacteria (AOB) were the most common bacteria on LR, which accounted for 0.5%–1.4% of the total bacterial population, followed by denitrifying bacteria, which accounted for 0.2% of the total population, and the ammonia-oxidizing archaea (AOA) were the least common type (<0.01%). The low abundance of denitrifying bacteria may be responsible for the poor nitrate (NO3–) removal of LR"

My take away from this is that while live rock is not very effective at direct nitrification/denitrification, it actually is beneficial since it does process ammonium , thus resulting in less nitrite/nitrate being produced.

One additional feature of live rock not often discussed is the surface community of periphyton/biofilms. These communities are very effective at capturing/sequestering POM (particulate organic matter), which helps to keep a system oligotrophic and the water clear. The surface biofilms also contribute to denitrification as indicated in the Reefs.com article.

"The results of this study stand in stark contrast to the prevailing wisdom, finding that “live rock” had no noticeable effect on nitrate levels during the 96-hour trial. And, somewhat counterintuitively, it was also shown that the denitrifying bacteria are up to 15 times more abundant on the surface of “live rock” than within it."
 
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Aqua Man

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Not sure what livestock you are thinking that could push the nitrifier population way down.
Actually wondering about the opposite. It’s said that some tanks loose bacteria diversity. So not surprising that Battle coral has a good bacteria report. Or a local LFS that has high turn over of frags from around the globe.
leads me to believe the majority of the activity I saw in the first two weeks is from the Biospira bottle and maybe not the rock.
Mind blow!! So adding bottle bac may not be such a bad idea after all!!
 

Dan_P

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Thanks! Great find.

I want to point out the first sentence in the first link ,

“The bane of most aquarists is the slow buildup of organic wastes in the form of nitrate.”

The article writer has things mixed up.

Anyway very interesting abstract (second link). I requested the full paper.
 
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taricha

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Neat experiment! Curious why the bio Spira was added? While low quality, the rock was live. Any testing done for nitrate?
follow up on this...
..in reading through an aquabiomics article I was a little taken aback at how slow the nitrification response was from "maricultured live rock".

Here's what I was looking at....
no2-and-no3.jpeg

"Figure 9. Production of nitrite and nitrate by microbial communities in each tank after while ammonia levels were maintained at approximately 1 ppm. Each symbol represents the average of duplicate tanks." Article: Establishing a Healthy Microbiome in a New Aquarium Using Live Rock

So compare that to what I saw in my cycling tanks. Three ammonia doses (~1.5ppm each) over 6 days were cleared by day 8.
Nitrite spiked and is almost cleared by day 12 as you can see here.
NO2 processing.png

The tanks will all have cleared Nitrite by day 13. Or at least it'll be low enough I can start to measure NO3.

Compared to Aquabiomics data above where Nitrite didn't start climbing (indicating rapid ammonia oxidation) until after day 10 for any of the rock. Also the "bad" live rock still had a nitrite spike at day 40, and the "good" rock didn't start to bring down the nitrite spike until day 30.
This is why I think I'm mostly measuring early activity from the biospira rather than the low quality rock.

It would be interesting to know the ammonia oxidation rate for your live rock before adding anything. There might be a simple way to roughly estimate the volume and surface area so we could compare its capacity to your sand.
oops. That would've been nice to get the tank's ammonia processing rate before biospira got added.

Maybe Petco will still have a rock or two in a few days.
 
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taricha

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So an update that's more interesting than I expected.
I was curious if I could mostly cut out the ammonia uptake by algae and re-measure the total ammonia consumption of the tank (3.8 & 3.2 ppm Total Ammonia Nitrogen/day) and the ammonia oxidation of the sand (0.1-0.2 ppm TAN/day).

I manually removed a bunch of algae from rocks and all algae from the sump, I did a couple of doses of algacide (algaefix marine), and sucked up dead algae as I noticed it.

When I re-checked the sand ammonia consumption rate, it's basically doubled.
Post_algacide sand rate.png


(Right graph, still provably Nitrification - sorry using less accurate NO3 test)

Here is is for comparison with my previous data. The data from my sandbed is in black. the the stars are what I measured now after removing a bunch of algae over the last month, compared to the circles and x's originally.
Sand rate comparison.png


So does just removing the majority of algae cause nitrifiers to take up a more substantial role? There's some complications here. I removed algae but I also killed a lot and dead material falling to the sandbed and decomposing is feeding ammonia to the sand. So it's not only removing competition - they also got an additional ammonia input.

But it's interesting to watch, the algae is not all gone - I'll purge the rest of it and see if the sand capacity continues to climb. That would imply the corals were not the major players suppressing the sand nitrification in my system.
 
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Dan_P

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So an update that's more interesting than I expected.
I was curious if I could mostly cut out the ammonia uptake by algae and re-measure the total ammonia consumption of the tank (3.8 & 3.2 ppm Total Ammonia Nitrogen/day) and the ammonia oxidation of the sand (0.1-0.2 ppm TAN/day).

I manually removed a bunch of algae from rocks and all algae from the sump, I did a couple of doses of algacide (algaefix marine), and sucked up dead algae as I noticed it.

When I re-checked the sand ammonia consumption rate, it's basically doubled.
Post_algacide sand rate.png


(Right graph, still provably Nitrification - sorry using less accurate NO3 test)

Here is is for comparison with my previous data. The data from my sandbed is in black. the the stars are what I measured now after removing a bunch of algae over the last month, compared to the circles and x's originally.
Sand rate comparison.png


So does just removing the majority of algae cause nitrifiers to take up a more substantial role? There's some complications here. I removed algae but I also killed a lot and dead material falling to the sandbed and decomposing is feeding ammonia to the sand. So it's not only removing competition - they also got an additional ammonia input.

But it's interesting to watch, the algae is not all gone - I'll purge the rest of it and see if the sand capacity continues to climb. That would imply the corals were not the major players suppressing the sand nitrification in my system.
Could you tell if the nitrate production increased? Any nitrite showing up? Did PO4 level increase with loss of algae? On a different topic, did cyanobacteria appear as algae died?
 
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taricha

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Could you tell if the nitrate production increased? Any nitrite showing up? Did PO4 level increase with loss of algae? On a different topic, did cyanobacteria appear as algae died?
The system overall is still running solid zeros on NO2/NO3.
PO4 may have gone up - haven't checked - but it was never much below 0.10ppm, so no P limitation there.
Red-brown slime of cyano (with a few dino cells) grew on the stressed/dying/dead algae. So there's clearly nutrient release associated with killing off the algae.
 

Rick Mathew

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Key points:
  • My system is capable of removing ammonia quickly: ~4ppm/day total ammonia-Nitrogen (TAN)
  • The sand processes ammonia slowly: ~0.1-0.2ppm/day TAN meaning the other 95+% happens somewhere else
  • The tank water itself also has detectable ammonia processing: ~0.05ppm/day TAN [Tank water itself not doing anything: see update in post 48]
  • In the sand and the water, the processing is provably Nitrification
  • A Recommended Minimum Dose of Biospira consumes ammonia faster straight out of the bottle than my 10 year old sand
  • I don't know if this is good or bad


The classic idea of nitrification is that ammonia is oxidized to nitrite and nitrate by bacteria in surfaces like sand, rock, and filters. But ammonia is also the preferred N source for photosynthetic organisms. Everything from coral to single cell algae. Additionally, heterotrophic bacteria can use whatever organic carbon sources are available to consume ammonia as well. So Just because ammonia disappears doesn't mean that it went through the ammonia -> NO2 -> NO3 "cycle", it might've just been consumed and become biomass instead.

There's a somewhat contrarian theory that in some tanks, ammonia mostly goes to algae and coral and very little gets nitrified at all.
See this article for that idea: #1 WHAT IF I TOLD YOU... Ammonia is causing your algae problems?
Also AquaBiomics finds some systems that have lots of nitrifiers and some that have barely any Ammonia Oxidizing Bacteria (AOB) and undetectably low Nitrite Oxidizing Bacteria (NOB). Article: The Microbial Community in a Professional Coral Aquaculture System



Anyway: Some things made me think I have a system that would fit this - processing ammonia in non "cycling" ways, so I went looking for data to measure my ammonia eaters. This answered a lot of Q's for me and raised a few more.

As a baseline comparison, here's the tank response to a one-time dose of ammonia during the day period, just a few measurement to get the general size and scale of the rates my system processes ammonia. (pH was 8.0 - this level of ammonia is well below EPA 1-hr level of 2.9 TAN @ pH 8)

Ammonia Tank Rate.png


The tank consumed ammonia at 0.161ppm/hr Total Ammonia Nitrogen (TAN). This works out to 3.9ppm/day.

So how much of this actually happens in the sand?

I dosed tank water to just under 0.5ppm TAN and split it into 100mL samples. Four samples got a series of different amounts of sand 0%,1%, 2% or 5% (a.k.a. 1, 2, or 5mL in 100mL sample) of white sand from the top of the sandbed where water flows across it pretty well.
Two other samples got1% sand + newly opened bottled starter products:
1% sand + BioSpira (recommended minimum dose)
1% sand + One and Only (5x recommended minimum dose)
They were placed in the dark on an orbital shaker at 70 rpm to keep the water moving and tested over 2.5 days.

Ammonia Rates .png


The series of samples with varying amounts of sand are in shades of blue, the 1% sand + biospira (red) acted more like the 5% sand. The 1% sand + One and Only (green) acted basically like just 1% sand. The trendlines were used to estimate the consumption rates.
Surprisingly, even no sand at all - just 100mL of water clearly consumed ammonia. [not repeatable: see post 48 update]

Here's visualizing the rate of ammonia consumption (slopes from previous graph) vs the amount of sand there was.
Amm Rate vs Sand.png


There is probably diminishing returns for higher levels of sand since at 5% the sand is piling up and a smaller portion is in contact with the moving water. BioSpira +1% sand clearly effective above what the 1% sand in the sample could do, while One and Only had no detectable effect. What proportion of sand is most applicable to my tank? My tank water is 40cm deep, and likely only the top 1cm of sand (or less) is reasonably in contact with moving water above, so 2% or less seems a good ballpark approximation to how my sandbed behaves.
I therefore estimate my tank sand can eat somewhere in the range of 0.10 to 0.15ppm/day TAN, or only <4% of my overall system consumption of 3.9ppm/day, meaning only 4% of that maximum daily ammonia consumption could be done in the sand+water.
Speaking of water, this graph makes it really clear that my tank water processes ammonia. In the 1% sand samples, the water may be doing half the work. Crazy! (Ammonia Oxidizing Archaea??)
[edit: Not repeatable. See post 48 for better data]

Okay one last thing. I haven't actually shown this ammonia was oxidized to nitrite/nitrate. It might have just been consumed by heterotrophs or algae in the sand. So let's look for NO2 / NO3.
Nitrite is not worth plotting a graph: Biospira produced 0.35ppm NO2, all other samples produced zero NO2. So maybe it was oxidized to NO3.

So lets take a look at NO3.

NO3 produced.png


Wow that looks a lot like the Ammonia consumption data in the previous graph (except there's more NO3 than expected in the One and Only). So let's see how tightly they match up. (The NO3 measurement in Biospira sample was corrected for the Nitrite interference using @Dan_P work here)

Ammonia to NO2_3.png


This plot compares the amount of Total Ammonia - Nitrogen consumed over the 2.5 days (on the left) to the NO2 + NO3 - nitrogen accumulated at the end (on the right)
First, the sand-only samples (in blue) match up very well. Secondly, the bottle products (solid red and green lines) showed way too much NO3 for the amount of Ammonia they consumed, so I checked for NO3 in the bottles - they are nitrifiers after all. Indeed, recommended min dose of Biospira adds 2.8ppm NO3, and 5x Rec'd min dose of O&O adds 2.3ppm NO3. After correcting for the NO3 that came in the bottles, the ammonia consumption and NO2 & NO3 production line up very well (red and green dashed lines). So clearly the ammonia consumption that happens in the sand and water really is nitrification, oh, and the bottle of O&O really was dead - guess it got frozen at some point. (BTW the slight increase from ammonia-N consumption to NO3-N production in these samples is probably from some organics in the sand being remineralized during the 2.5 day experiment. Note the effect increases with increasing sand level.)

So here's the takeaway.
I can measure the consumption of ammonia by my sand (and water!) and show that it's nitrification. And it's really small - maybe only 4% of the rate of the ammonia consumption in my system overall. The bulk of ammonia in my system is probably eaten by algae and coral.
I think my tank may be an extreme case. My 70 gal system has probably a kg of mixed coral and several hundred grams of algae, algae grows and exports quickly. It's had a history of sometimes large carbon dosing, but nothing in the past few months. Either my tank or my sump is always lighted. The rates of ammonia consumption capable by coral and algae when lighted is huge. So it makes sense that nitrification looks like a bit player in my system, my tank always runs zero detectable NO3, and any that is dosed disappears quickly. I plan to send a sample to Aquabiomics tomorrow to see if the genetic approach tells the same story.
But I don't think I have the only tank like this! I think the hobby might have quite a few systems where the traditional nitrogen cycle is essentially a non-factor. Is this good? Bad? I don't know. But it's clear from the effect of biospira, that this would be easy to change if someone wanted to.
Awesome work!
 
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taricha

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So aquabiomics data for my tank incoming. :)
Will my tiny ammonia processing rates from the sand show up as low testable nitrifiers?
 
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NItrogen is cycled constantly. Heterotrophs retrieve their carbon source by braking down organics. After the job is done and all carbon is retrieved, most of that carbon has been used for providing the energy needed and is released as CO2, which means less as 50% can be used for heterotrophic growth using ammonia as a nitrogensource. A fish will release about 85% of total nitrogen .
This means most of the buildingmaterials are not used up while braking down the organics. In a biofilm ammonia left over is transformed into harmless nitrate as fast as possible, not by growth but by respiration, by autotrophs not needing organic carbon. Ammonia and nitrite is toxic for most bacteria and archaea when able to build up. This means most nitrogen left over is transformed into nitrate as fast as possible. In a normal world this nitrate is then used by slower growing autotrophic organisms, and Photo-autotrophs producing protein, storing the previously released CO2 and the surplus of building materials back into organic material.
This way the balance between producers and reducers can be maintained.
Organisms release ammonia constantly when using nitrate, transforming nitrate into usable ammonia-nitrogen, releasing what is not consumed.
Most bacteria only can use nitrate as a nitrogen source if ammonium is not available, most photo-autotrophs are able to use both the same time. A biofilm releasing ammonia in the environment instead of nitrate? I prefer nitrate.
 
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Belgian Anthias

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Nitrifiers can be outcompeted for ammonia by coral, algae, and heterotrophs.
so...
No Light, no coral, no algae.
Start with ammonia dosing, not ghost feeding or a shrimp. Organics in the food will get the heterotrophs going, and the nitrifiers would only get ammonia as leftovers.
If you use a bottle starter, be sure it's true chemoautotroph nitrifiers. Some are actually "heterotrophic nitrifiers" that require Organic carbon. Adding ammonia-only, not a shrimp or fish food in the beginning will help distinguish.
Will they? Nitrifiers are mainly active in a biofilm although there are a lot of free living bacteria, also heterotrophs, which are able to nitrify ammonium. They grow very slow compared to heterotrophs but as nitrification is respiration, they are able to compete for ammonium with other organisms using ammonia for growth. In marine aquaria biofilters most nitrification is done by Archaea, in combination with bacteria. Archaea are very active in low oxygen zones within the biofilm.

chemoautotroph nitrifiers. in a bottle? How they will retrieve the energy needed to survive?
True nitrifying bacteria are considered to belong to the NITROBACTERACEAE families. These bacteria are strictly aerobic, gram-negative, chemolithic autotrophs. They need oxygen, usually use inorganic (carbonless) compounds as their energy source and require carbon dioxide (CO2) for their source of carbon. In the case of the Nitrobacteraceae these energy sources are derived from the chemical conversion of ammonia to nitrite, or, nitrite to nitrate.
They do NOT form endospores!

They are part of the divers bacterial community needed to form a nitrifying biofilm. A nitrifying biofilm can not be delivered in a bottle!

The Parable of the Bacteria in a Bottle

 

Lasse

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I couldn't help myself so I set up three 5 gal tanks...

3Tanks.jpg


Each tank got Instant Ocean water, 500mL Caribsea aragonite dry sand, and low quality "live" rock from Petco. (The rocks had all been kept in a pile in the same tank. I imagine they are pretty similar). Each tank got the minimum recommended dose of Biospira, and were left to circulate with the lights off.

Tank 1 is left to "cure", and gets no feeding except the few tenths ppm ammonia in I.O. and whatever organics comes from the rock.

Tank 2 and 3 were traditionally cycled using a schedule based on Dr. Tim's fishless cycling. Day 1, 3 and 6 the tanks got the same 1 to 1.5ppm ammonia drops.
Day 8 ammonia was zero in all 3 tanks.

Day 9 I added ammonia to each tank to test the consumption rates...

Cycling vs Curing.png


Tank 1 that was just left to "cure" did great. Data in blue, it consumed about 0.5ppm TAN/day.
Tanks 2 & 3 that were actually cycled were so fast they depleted my first dose and I had to re-dose ammonia to get a measure on the consumption rate.
Data in Red and Green, they are both showing consumption of around ~1.7ppm TAN per day.

In case anyone else wanted to visualize how much the nitrification ability is expanded during a normal cycling routine. Tanks ended the 9 day cycle with 3 to 4x the nitrification rate of a tank that was given the same bacteria but not fed.
This result did not surprise me at all. it is in line with all my experiences. In the two "cycled" aquarium - you have build up a larger standing stock of ammonia oxidation organisms (AOO) (probably mostly archaea). If the food supply reduces - they go dormant. They will be activated fast if new food will be present (food - read NH3/NH4 in this case). In the first aquarium - the "standing stock of AOO was to low to consume the new load of NH3/NH4. If you repeat the experiment a couple of times (i.e. add NH3/NH4 every day) - I´m sure the differences will disappear.

Sincerely Lasse
 

brandon429

why did you put a reef in that
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Lasse I have this question: twice on file now, linkable, friends testing out cycling have just completely cycled dry surfaces by merely adding saltwater and waiting. All food acquisition was natural


one completed in sixty days


the other, in 30


so how can aoo go dormant without food, those systems just up cycled when a cycling chart said they would, no feed input given by the reefer. Both systems were rocks and not just sand, higher surface area to amplify water presentation to the sparse new bacterial load, so not just sand but these cycles are self-feeding even when we withhold I’m certain. Also to consider: Dr Reef did a 14 month wait test on a -sealed- water system with high surface area and it could *not* pass oxidation testing, by excluding the natural daily inputs even after a year wait.


open topped = can’t be starved it seems
 

Lasse

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if you do not understand this - its your problem. For something to get dormant - it must have been there in enough numbers before the starvation. If you just add dry rocks that´s not have been cycled - there is not enough of AOO - and the few that is dormant take a time to develop - in your examples 60 and 30 days. But the fact that it works after 30 or 60 days shows that in these case - it must have been a few dormant organism when the experiment started. And if you have the same load during 1 year (nearly nothing) you can´t expect a huge amount of AOO in the system. I do not know how long time they can stay dormant - but in Taricha´s experiment and in my own experiments (days and weeks) they will have full effect after a starvation if you have had a high load before. It means - if you have a rate of 1 ppm daily - they will manage that dose after at least a week starvation but it will not manage 2 pmm directly - but if you have it to consume 2 ppm - they will manage 2 ppm after a starvation period of at least 1 week IME.

sincerely Lasse
 
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Lasse

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They are part of the divers bacterial community needed to form a nitrifying biofilm. A nitrifying biofilm can not be delivered in a bottle!
Still it works both in waste water treatments and aquarium if it is the right organisms

Sincerely Lasse
 

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why did you put a reef in that
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I disagree because we have also tracked out fallow systems that ran a strong bioload, removed for three months, put fish back and no gap in filtration turnover. I remember in sand removal threads prior that you said removing sandbeds will affect nitrification negatively, but this thread didn’t find that, and neither did our fifty page sand rinse removal thread. it found the polar opposite- nitrification overall for the tank unaffected and a few tests were confirmed on seneye as well. I didn’t find any of the aoo insults you’ve mentioned in our large post collections.

I realize we don’t get to read your experiments in links, we just get the overall summary but it’s odd that we don’t see it in large scale aquarium testing.
 
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Lasse

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fallow systems that ran a strong bioload, removed for three months, put fish back and no gap in filtration turnover.
Exactly what I say - Once a system is used of a load - it will start up nearly in the same effectivines again. AOO and NOO (Nitrite Oxidation Organisms) have gone dormant in the period between.

Sincerely Lasse
 

brandon429

why did you put a reef in that
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You are I are taking away polar opposite inferences from these things being posted online. If everyone agreed it wouldn’t be any fun tho


can you post something that we can objectively evaluate? Such as, using aquarium supplies, make a bucket reef study on some aspect of filtration, cycling or ammonia control and post that. How your tests benchmarked before stressing the system in some way, how they revealed the stress too. What they showed after the stress was alleviated… That will save us having to relay summaries for the data. I myself don’t make experiments… I watch and collect them from others who are using aquarium gear etc


the reason you and I never agree is because we keep finding completely opposite outcomes from the prediction when using aquarium specific testing and simple arrangements. The pages of biochemistry you are typing has never lined up with any seneye study on this site, though the formulas make sense we just don’t see the consequence portion in context—the loss, weakening or stressing of bacteria in wet media is not happening when someone takes time to make a simple paint bucket reef tank for example

when we see seneye studies on the site (our best chance at reading ammonia action clearly as reported by the masses) the arrangements always involve copious amounts of live rock. We have a surface area excess in every test…in my opinion this is the very aspect missing in any situation where ammonia-oxidizing organisms are shown weak or dormant or unable.
we test in a non reef setting, then state that applies to reef tanks.
 
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Dan_P

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So aquabiomics data for my tank incoming. :)
Will my tiny ammonia processing rates from the sand show up as low testable nitrifiers?
Do we know how a low amount of nitrifiers in a Aquabiomics test correlates to the measured ammonia oxidation rate of the aquarium?

There is always the unknown rate of detection by the method. Then there is the question of would the same absolute number of nitrifiers in an aquarium process ammonia at the same rate if they were 10% or 50% of the community?
 
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