DNA Testing for the Reef Tank - Ask me anything

[AquaBiomics]

Hi everyone,

I'm starting this thread to collect any questions from the community in one place, so I can make sure I answer them.

I run AquaBiomics, a DNA testing company focusing on the saltwater aquarium hobby. We use DNA sequencing to study the communities in saltwater aquariums, using the same methdos and databases developed by leading experts in the field of environmental microbiology.

DNA sequencing includes microbiome sequencing, which provides information on the kinds of microbes in your aquarium, and their levels. We also use eDNA sequencing to study the rest of the community, such as nuisance algae and the parasites that cause fish disease.

I know these subjects have been discussed here, but I usually miss the discussions. Between running a business, raising 3 kids, and operating a small farm, I don't find as much time as I'd like to stay on top of interesting discussions about reef tank DNA.

So I thought I'd make a thread and get these questions in one place. Who's got a question about DNA testing for the aquarium? If you ask a question in this thread, I'll make sure it gets answered.

Thanks!

 

[Randy Holmes-Farley]

Your reports sometimes make suggestions how one can change the bacterial types in a reef tank to look more like what you see in typical/average reef tanks. Not because of pathogens or known problems, but simply from deviating from average. Such changes including removing a UV, dosing amino acids, etc.

What benefit is there to an individual aquarium making these changes that are a consequence of the bacteria change, and what is the evidence that this benefit is attained if one makes these changes?

Some folks get the reports and do not seem to know what to do with them. Some changes are suggested, but it's not clear what they should expect to observe if those changes are made.

 

[AquaBiomics]

Your reports sometimes make suggestions how one can change the bacterial types in a reef tank to look more like what you see in typical/average reef tanks. Not because of pathogens or known problems, but simply from deviating from average. Such changes including removing a UV, dosing amino acids, etc.

What benefit is there to an individual aquarium making these changes that are a consequence of the bacteria change, and what is the evidence that this benefit is attained if one makes these changes?

Some folks get the reports and do not seem to know what to do with them. Some changes are suggested, but it's not clear what they should expect to observe if those changes are made.

Thanks, thats a nice clear question that I think others have also wondered about.

The answer is that I work hard to avoid making claims that arent well supported by data. Even if sometimes that may be what clients wish for.

We see it often reef keeping, right? People making claims about their favorite additive, without data to support those claims beyond their own anecdotal experience. Its important to me that we avoid that.

You will not find a claim in the report that "if you increase the levels of this bacterial family, your coral will grow faster". Because we lack the data to make such a claim. We, in terms of "we, the scientific community, don't know that yet".

This means that some bacterial types will have a lot of information attached to them. Nitrifying microbes. Pathogens. Bacteria whose functional association with corals or fish has been well established in peer reviewed studies. Other bacterial types will have much less information attached to them.

So naturally, our suggestions will similarly fall across a range of different strengths. The report may say in one case, "your sample has a known coral pathogen, here's a link to learn more about it" in big red text. While in another case our suggestions are more subtle. "This bacterial family is present at 10x higher levels in your tank than the usual reef tank - if you'd like to reduce it here are some evidence-based suggestions".

My approach is to try to make the data as useful as possible, while avoiding overstating what we really know.

We don't know everything about every bacterial group, but we also know a lot more than nothing about them. The reality is somewhere in between, and thats what leads to this inevitable challenge in communicating the results. I try to make this distinction clear in our reports but its not an easy task and I certainly can see room for improvement.

We all want to know more about these bacteria, and that takes experiments. The community is actively working in that direction. Just this week in collaboration with Reef Builders we've shipped out 27 kits for a big community science experiment on Carbon Dosing. One step at a time, we can use these data to answer the questions we all share about the roles of these microbes in our tanks.

 

[telegraham]

I'll share how I've used the service.

1. I've sampled several off-the-shelf reef hobby bacterial products, some of them multiple times, and as money allows, I continue sampling in an effort to give others visibility. One of those products was quite diverse. One of those products contains a potential coral pathogen, leading to manufacturer testing. That testing is ongoing.
2. I sampled my tank when it was young, sampled again several months later to watch the tank progress, chemically treated for cyano, took steps to recover potential damage to the microbiome, and have tested again. I'm looking forward to those results.
3. I've sampled a 100L cryptic container that's located in my garage and attached to my system to understand if that container trends differently than the tank. I'm also using those results to justify sharing of live sand/rock/media with those who wish to start a system with a seasoned microbiome.
4. As I continue to satisfy my interest in looking inside products that are offered to the hobby, I look forward to testing online coral vendors' shipments and I look forward to testing others' live sand/rubble/rock products.

I'm aware of Andrew's impact studies, and I like what he's doing.

I'm aware of your information sharing here - https://www.reef2reef.com/threads/e...ies-in-my-tanks-updated-with-new-data.684209/

As an AquaBiomics customer, I'm very aware of the tested lots of live sand/rubble that you offer. I'm also aware that you might be one of the few, if the only provider, of tested live products that include the testing results.

@AquaBiomics ... I have two question. Why is it that the focus is always on "actionable?" How do you define actionable?

 

[dangles]

Hey Eli. Thanks for this thread. I'm eager to follow along!

First, I've only done the TankDNA test (not the microbiome test). But I really liked how in addition to the sample, the customer is asked a series of questions about their system to assess the overall health, and whether there are any current problems being experienced.
In addition to just "does this tank look typical, and how so?" I think this will give great insight into what a HEALTHY system looks like at different stages of its evolution, and what bacteria/pathogens are associated with which common (or uncommon) tank problems. I like this. Over time, I think this is the kind of information that will benefit the hobby in a lot of ways.

I don't have a "general" question, but I do have a question regarding a test I had done a few months ago.

I had done a TankDNA test to rule out fish parasites in my system after a couple of unexpected deaths (LOVE this particular service btw!).

I got a clean bill of health in regards to the usual suspects (yay!), but there was an odd finding in my system... an over-abundance of a fresh water ciliate: Hemiophrys macrostoma. No fish illnesses/deaths since the two I had prior to sending in the test.

Are there any steps I can take from here to find out more? I didn't find much using Google (not that the info wasn't there, it was just beyond my ability to parse out!). Is there other testing I can do - even just a followup test? Test my source water? Would those ciliates even make it through a 7-stage RODI system, and how do they survive in a marine environment?

Mine is an odd case I realize, but what do you suggest as next-steps - if any?

 

[AquaBiomics]

Hey Eli. Thanks for this thread. I'm eager to follow along!

First, I've only done the TankDNA test (not the microbiome test). But I really liked how in addition to the sample, the customer is asked a series of questions about their system to assess the overall health, and whether there are any current problems being experienced.
In addition to just "does this tank look typical, and how so?" I think this will give great insight into what a HEALTHY system looks like at different stages of its evolution, and what bacteria/pathogens are associated with which common (or uncommon) tank problems. I like this. Over time, I think this is the kind of information that will benefit the hobby in a lot of ways.

I don't have a "general" question, but I do have a question regarding a test I had done a few months ago.

I had done a TankDNA test to rule out fish parasites in my system after a couple of unexpected deaths (LOVE this particular service btw!).

I got a clean bill of health in regards to the usual suspects (yay!), but there was an odd finding in my system... an over-abundance of a fresh water ciliate: Hemiophrys macrostoma. No fish illnesses/deaths since the two I had prior to sending in the test.

Are there any steps I can take from here to find out more? I didn't find much using Google (not that the info wasn't there, it was just beyond my ability to parse out!). Is there other testing I can do - even just a followup test? Test my source water? Would those ciliates even make it through a 7-stage RODI system, and how do they survive in a marine environment?

Mine is an odd case I realize, but what do you suggest as next-steps - if any?

I remember your case - that was an unexpected result and I meant to write to you about it. Glad we get the chance to discuss it here.

60% of the sample, an unprecedented level compared with anything we've seen before.

Hemiophrys macrostoma is a freshwater ciliate - and as a result it rarely shows up in our samples. It appears to be at least commensal and maybe parasitic on carp. This ciliate has been renamed to Pseudoamphileptus macrostoma.

But what is it really, is it a freshwater ciliate that can actually survive saltwater? I wouldnt say its impossible but my immediate gut feeling is its probably a close relative adapted for saltwater.

Looking at the sequence I see that the best match (no ties) is H. macrostoma. Its 98% identical - not a perfect match but close enough to accept. Interestingly the next best match is very close (97.6%), and its an undescribed marine ciliate helpfully labeled Amphileptus sp. PHB08122401.

So your mystery ciliate is
a) not a perfect match to the freshwater ciliate, but very close
b) almost as closely related to a different species isolated from saltwater
c) present at very high levels in your sample

Looking back at my notes I see that actually we re-tested your sample and found essentially the same results on the re-run, confirming it wasnt some weird PCR fluke. Its a real feature of your sample, whatever its name.

I think you've got a real population of a marine version of this ciliate. I don't see any reason to take immediate action - none of the papers I've found call this a parasite of concern for aquaculture. Purely out of curiosity I'd love to get a swab sample from one of your fish, to see if its commensal or free living. Any chance you can catch your fish easily? I'd be happy to send a swab!

 

[areefer01]

Looking back at my notes I see that actually we re-tested your sample and found essentially the same results on the re-run, confirming it wasnt some weird PCR fluke. Its a real feature of your sample, whatever its name.

I may be breaking the rules or flow of the thread but I wanted to call something out that you mentioned in your reply above. Bold and italic for reference.

Would it be a fair question to ask about spot checks, or key warning indicators, that flag samples for retesting?

Having used the service, and been part of a retest, it seems that you do a high level review checking for anomalies. I do not see this type of work being done with ICP. Not apple to apple I know but my point is that it seems you have some checks and balances in place.

 

[dangles]

I think you've got a real population of a marine version of this ciliate. I don't see any reason to take immediate action - none of the papers I've found call this a parasite of concern for aquaculture. Purely out of curiosity I'd love to get a swab sample from one of your fish, to see if its commensal or free living. Any chance you can catch your fish easily? I'd be happy to send a swab!

I can probably snatch one of them. I imagine sampling procedure will be included in the sample kit?

I’m curious if another water sample now would show the same proportions of the ciliate. My tank is still less than a year old so I’m sure the biome is still getting settled in.

Do you know if any other customers have used Tampa Bay Saltwater sand or rock? I used their sand. I wonder if it came from there or maybe from something at my LFS.

 

[AquaBiomics]

I may be breaking the rules or flow of the thread but I wanted to call something out that you mentioned in your reply above. Bold and italic for reference.

Would it be a fair question to ask about spot checks, or key warning indicators, that flag samples for retesting?

Having used the service, and been part of a retest, it seems that you do a high level review checking for anomalies. I do not see this type of work being done with ICP. Not apple to apple I know but my point is that it seems you have some checks and balances in place.

Absolutely right - I review each report personally and if they fail any of these checks I flag it for re-testing.

1. Coverage - we need to have a minimum number of DNA sequences to get a meaningful picture of the sample. Theres a lot of work in the lab to make sure everyone's sample gets enough sequences, but at the end of the run sometimes a sample doesnt have enough.

2. Sample composition - if the same is dominated too heavily by DNA from a single source, this obscures the presence of less abundant members of the community, making the test less sensitive. In sense this comes down to "the sample composition doesnt look normal", but this over-representation of a single type is what I'm really checking for.

In both cases we've produced a report and from a purely business perspective maybe I should leave it at that. Re-running certainly costs time and resources But as a scientist and a hobbyist myself, if that was my sample, I'd want to take another look.

So in practice thats what we do - if the data fail QC at the end for either of these reasons, we either re-run the sample or send a kit to take a new sample.

 

[BeanAnimal]

Thanks, thats a nice clear question that I think others have also wondered about.

The answer is that I work hard to avoid making claims that arent well supported by data. Even if sometimes that may be what clients wish for.

We see it often reef keeping, right? People making claims about their favorite additive, without data to support those claims beyond their own anecdotal experience. Its important to me that we avoid that.

You will not find a claim in the report that "if you increase the levels of this bacterial family, your coral will grow faster". Because we lack the data to make such a claim. We, in terms of "we, the scientific community, don't know that yet".

This means that some bacterial types will have a lot of information attached to them. Nitrifying microbes. Pathogens. Bacteria whose functional association with corals or fish has been well established in peer reviewed studies. Other bacterial types will have much less information attached to them.

So naturally, our suggestions will similarly fall across a range of different strengths. The report may say in one case, "your sample has a known coral pathogen, here's a link to learn more about it" in big red text. While in another case our suggestions are more subtle. "This bacterial family is present at 10x higher levels in your tank than the usual reef tank - if you'd like to reduce it here are some evidence-based suggestions".

My approach is to try to make the data as useful as possible, while avoiding overstating what we really know.

We don't know everything about every bacterial group, but we also know a lot more than nothing about them. The reality is somewhere in between, and thats what leads to this inevitable challenge in communicating the results. I try to make this distinction clear in our reports but its not an easy task and I certainly can see room for improvement.

We all want to know more about these bacteria, and that takes experiments. The community is actively working in that direction. Just this week in collaboration with Reef Builders we've shipped out 27 kits for a big community science experiment on Carbon Dosing. One step at a time, we can use these data to answer the questions we all share about the roles of these microbes in our tanks.

In the very kindest manner - That is a whole lot of words that actually say very little other than you know more about some bacteria than others. I can't speak for Randy (whatsoever) but I am no clearer on his question (or those of my own) after reading that.

I get that we can test for things, but what is the actual purpose if we don't have proven ways to alter them. To that end, what do we alter them toward? Known successful systems, NSW conditions on a reef? Etc. The science itself is extremely interesting, but I am having trouble with the application.

 

[Dan_P]

Hi everyone,

I'm starting this thread to collect any questions from the community in one place, so I can make sure I answer them.

I run AquaBiomics, a DNA testing company focusing on the saltwater aquarium hobby. We use DNA sequencing to study the communities in saltwater aquariums, using the same methdos and databases developed by leading experts in the field of environmental microbiology.

DNA sequencing includes microbiome sequencing, which provides information on the kinds of microbes in your aquarium, and their levels. We also use eDNA sequencing to study the rest of the community, such as nuisance algae and the parasites that cause fish disease.

I know these subjects have been discussed here, but I usually miss the discussions. Between running a business, raising 3 kids, and operating a small farm, I don't find as much time as I'd like to stay on top of interesting discussions about reef tank DNA.

So I thought I'd make a thread and get these questions in one place. Who's got a question about DNA testing for the aquarium? If you ask a question in this thread, I'll make sure it gets answered.

Thanks!

Thanks this quite nice.

With your current capabilities, is it possible to look at functioning genes rather than obtaining the names of bacteria? Questions to answer might be is my system denitrifying nitrate, fixing nitrogen, reducing nitrate to ammonia, are there many active virulence genes? Full disclosure, I’ve been reading.

 

[AquaBiomics]

In the very kindest manner - That is a whole lot of words that actually say very little other than you know more about some bacteria than others. I can't speak for Randy (whatsoever) but I am no clearer on his question (or those of my own) after reading that.

Sorry if that was unclear, I will try to rephrase. As a recovering academic I have a tendency to use a lot of words.

Randy asked about benefits for the aquarium. What benefits do the various bacterial groups have for the aquarium?

My answer is that we know a lot about some bacterial grounds, so we can make clear inferences about benefits for the aquarium. Nitrifying microbes - we know their function, and so we know this is a beneficial group the aquarium. Coral pathogens - we know something about their association with corals, so we know this is a harmful group to have in the aquarium. Other groups we know very much less about, so we can't make any clear inferences about benefits for the aquarium.

To get beyond inferences we need experiments, and those efforts are ongoing by the community.

That was my answer in a nutshell. Any clearer?

--

I get that we can test for thing, but what is the actual purpose if we don't have proven ways to alter them and to that end, what do we alter them toward? Known successful systems, NSW conditions on a reef? Etc.

There are lots of ways to alter these communities, the list is ever growing. We've been discussing this for a while now, although I confess that I've been too busy to post much in the last couple years.

• Increase diversity - add live sand or mud
• Increase nitrifying communities - add live sand or mud
• Increase/decrease Pelagibacteraceae - turn off/on the UV sterilizer
• Decrease Rhodobacteraceae - oxolinic acid
• Decrease Arcobacter - low doses of Ciprofloxacin

Thats just off the top of my head, based on hobbyist experiments. If we turn to the scientific literature there's a much wider range - I've incorporated those suggestions into the reports. For example if a tank has excess levels of Flavobacteria, research shows this group is promoted (in coral reef communities) by addition of algal nutrients. So the client may consider reducing algal inputs into their tank to alter this.

The bacteria that make up the aquarium community cannot be bought in a bottle, so it isnt as easy as adjusting trace elements based on ICP. I doubt it ever will be, but there are plenty of other ways to adjust the community. And every new experiment our clients run can add a new option to the list. A new dial we can turn to adjust the community.

 

[AquaBiomics]

Thanks this quite nice.

With your current capabilities, is it possible to look at functioning genes rather than obtaining the names of bacteria? Questions to answer might be is my system denitrifying nitrate, fixing nitrogen, reducing nitrate to ammonia, are there many active virulence genes? Full disclosure, I’ve been reading.

Hi Dan,

No, this is definitely a limitation of the "barcoding" approach - we don't see genes. So from the data we're currently generating, the best we could do would be to look up which genera or species have each gene, and make inferences on that basis. As you're aware, there are limitations to this.

Gene based questions like you're bringing up are better addressed with gene based primers. There are lots of these developed, its easy to adapt our workflow to new primer pairs. So far we've run 16s, 18s, A chloroplast marker, an HSP marker specific for Mycobacteria, and an HSP marker specific for Vibrio.. all with this same workflow, sequenced together. So I'm optimistic about the ability to add another target.

The limitation of course is that each would be gene specific - so we could dig up some amoA primers to profile ammonia oxidation genes, for example. But we'd need a different set of primers for nitrite oxidation, etc.

Another approach entirely would be metagenome sequencing, but that gets expensive and would be a different workflow, not easily compatible with what I'm doing.

 

[Dan_P]

Hi Dan,

No, this is definitely a limitation of the "barcoding" approach - we don't see genes. So from the data we're currently generating, the best we could do would be to look up which genera or species have each gene, and make inferences on that basis. As you're aware, there are limitations to this.

Gene based questions like you're bringing up are better addressed with gene based primers. There are lots of these developed, its easy to adapt our workflow to new primer pairs. So far we've run 16s, 18s, A chloroplast marker, an HSP marker specific for Mycobacteria, and an HSP marker specific for Vibrio.. all with this same workflow, sequenced together. So I'm optimistic about the ability to add another target.

The limitation of course is that each would be gene specific - so we could dig up some amoA primers to profile ammonia oxidation genes, for example. But we'd need a different set of primers for nitrite oxidation, etc.

Another approach entirely would be metagenome sequencing, but that gets expensive and would be a different workflow, not easily compatible with what I'm doing.

Thanks for the explanation. I appreciate having this idea put into perspective.

 

[taricha]

Thanks for your time and expertise Eli. I'd argue it took the hobby a decade to get a feel for what ICP does well and where it is limited. Forgive me for trying to "fast forward" a few years and get a similar picture for this analytical tool.

Questions

(I feel like this has been answered, but I don't know and can't find it. fine to point to where you've answered it before. )
1) the user samples water and biofilm separately and receives a combined community composition. How are those two samples combined to generate the community composition snapshot? Or is community composition from the water, and the biofilm only adds more detected strains for biodiversity and other measures in the report?

2) I've heard some people in the hobby who work with eDNA sequencing suggest that 10k is a really low number of reads. (papers like this for context, where 60k is typical, and 100k to 1mill is done when desired.)
As an analytical technique for the hobby, what is still done very well at 10k reads? and what is a limitation of that number of reads that would be better handled by the far higher number of reads that are done in papers like the above? In other words, what are we giving up for the presumably lower expense of 10k reads vs say 100k?

3) Maybe relatedly, what pathogens/diseases do you find are well-measured by the service, and what seems to be harder to catch on the reports?

4) What is the fixative? I had some older test kits that I hadn't used and when I went to use them, the fixative had evaporated (even in double-sealed tubes!). In a pinch, after googling a bit, I used 99.5% ethanol as a substitute and threw the kit in the freezer after sampling. Acceptable, or don't bother sending?

5) Can you point to some resources that have done sequencing that might look similar-ish to your reports but done on natural reefs? I know they will be profoundly different, but it would be of interest nonetheless.




The next questions aren't exactly about your service, but are questions that you might be able to answer off the top of your head, but would be very difficult for us to answer otherwise. I'm asking on the chance that you can answer them easily - punt if they are too time consuming.

6) echoing one of Dan's questions. Do you find Nitrogen-fixers in reef tanks?

7) people want to say bacteria = coral food. But at one point recently the answer was: we have no idea which bacteria "SPS" corals actually eat, and the likely candidates aren't known to be culturable.
Has that answer changed in the last few years?

 

[Randy Holmes-Farley]

Thanks, thats a nice clear question that I think others have also wondered about.

The answer is that I work hard to avoid making claims that arent well supported by data. Even if sometimes that may be what clients wish for.

We see it often reef keeping, right? People making claims about their favorite additive, without data to support those claims beyond their own anecdotal experience. Its important to me that we avoid that.

You will not find a claim in the report that "if you increase the levels of this bacterial family, your coral will grow faster". Because we lack the data to make such a claim. We, in terms of "we, the scientific community, don't know that yet".

This means that some bacterial types will have a lot of information attached to them. Nitrifying microbes. Pathogens. Bacteria whose functional association with corals or fish has been well established in peer reviewed studies. Other bacterial types will have much less information attached to them.

So naturally, our suggestions will similarly fall across a range of different strengths. The report may say in one case, "your sample has a known coral pathogen, here's a link to learn more about it" in big red text. While in another case our suggestions are more subtle. "This bacterial family is present at 10x higher levels in your tank than the usual reef tank - if you'd like to reduce it here are some evidence-based suggestions".

My approach is to try to make the data as useful as possible, while avoiding overstating what we really know.

We don't know everything about every bacterial group, but we also know a lot more than nothing about them. The reality is somewhere in between, and thats what leads to this inevitable challenge in communicating the results. I try to make this distinction clear in our reports but its not an easy task and I certainly can see room for improvement.

We all want to know more about these bacteria, and that takes experiments. The community is actively working in that direction. Just this week in collaboration with Reef Builders we've shipped out 27 kits for a big community science experiment on Carbon Dosing. One step at a time, we can use these data to answer the questions we all share about the roles of these microbes in our tanks.

Thank you.

 

[Doctorgori]

I get that we can test for things, but what is the actual purpose if we don't have proven ways to alter them. To that end, what do we alter them toward? Known successful systems, NSW conditions on a reef? Etc. The science itself is extremely interesting, but I am having trouble with the application

Only quoting as I was going to say similar… I can see this being useful if we knew more (as a whole) …I paid for a few ICP test and while I could clearly see deviations from NSW, there wasn’t a clear cut problem/solution for a chemical neophyte like myself…
I can see opportunities for both snake oil and advancement here

Nitrifying microbes - we know their function, and so we know this is a beneficial group the aquarium.

This might be coming into question in light of a few recent threads…

Coral pathogens - we know something about their association with corals, so we know this is a harmful group to have in the aquarium.

STN/RTN ….I can see the application here but this issue has lots of room for discovery …
Have you any knowledge se in this regard, esp for BJD , torch loss, et et

 

[Randy Holmes-Farley]

AquaBiomics said:
Nitrifying microbes - we know their function, and so we know this is a beneficial group the aquarium.

This might be coming into question in light of a few recent threads…

As I noted in the other thread, that is where opinions come in with respect to what bacteria are and are not desirable, and may vary from aquarium to aquarium or hobbyist to hobbyist.

 

[Dan_P]

7) people want to say bacteria = coral food. But at one point recently the answer was: we have no idea which bacteria "SPS" corals actually eat, and the likely candidates aren't known to be culturable.
Has that answer changed in the last few years?

I picture bacteria sticking to coral mucus and the mixture being ingested. How would a coral select which bacteria to eat? Or is it which bacteria can a coral digest?

 

[livinlifeinBKK]

What is the microbiome of a healthy reef composed of or characterized by precisely? Can you link any publication or offer a reference that defines what a "healthy" microbiome looks like on a natural reef? I could easily be mistaken, but according to peer-reviewed studies, some published as recently as 2024, microbiomes supporting coral growth and health are not only species-specific, but show high plasticity. This coupled with the fact that the microbiome can quickly and easily shift due to a change in abiotic and/or biotic factors, there is a general lack of knowledge of what constitutes a healthy microbiome (Voolstra et al., 2024).

It seems that you define a healthy microbiome only by comparing samples to those of tanks considered healthy. Is that correct?

Also, I watched a Youtube video of you speaking a while back and believe I recall you mentioning that populations of bacteria compete which generally leads to less diversity and certain bacteria becoming far more abundant than others due to this competition. If I'm not mistaken, how would rubble rock help spread diversity in an already established tank, particularly if it's large? Wouldn't the small populations of different bacteria be quickly outcompeted I ask this because you sell rock rubble.