"Biodiversity is dead, long live biodiversity" 10 month microbiome data from BRStv.

[taricha]

you're saying without his data we're not able to know when to utilize those modes? I can see how his data shows changes after employing those modes, but reefers are in it for the physical expression within the tank...the visual changes we see after usage.

Not at all.
My view is that the observable changes - as you say, the functional results are primary. The bacterial testing (hopefully) increases our understanding of how and why. (Some will care about that more than others.)

 

[metalle]

I'm scratching my head at this comment, but can't really find the right emoji to express it.

When I'm extracting clients DNA, amplifying specific genetic markers, attaching synthetic oligonucleotides, and quantifying the products for sequencing -- where exactly do you suggest I'm doing this? In my garage or something?

I agree it’s not the nicest way to express feedback. However there is something in the post that gives an opportunity to respond. I am sure there is a lot of research, proven methods, technology needed to perform testing your company does and I am
Personally thankful for what you are offering. Now comes the opportunity to share where you are as a company and how does the company plan to grow. How methods are validated or in plans to standardize. For example an non mandatory ISO standard like ISO 17025.
The indirect question of credibility was not answered and still benefits to be answered.
Again, I believe there is a great opportunity here.

 

[taricha]

For any of those interested, here is Eli giving about a 10 minute explanation of the process during one of his presentations.
PNWMAS meeting

 

[Lasse]

I have never thought of it that way. I think it is a slug fest between bacteria competing for real estate and in an unstable system, that real estate changes. You can add all the bacteria you want over time, but if the enviornment isn't conducive to that bacteria, or the 'apartment building' is full, that bacteria is not going to thrive or become dominant. Tanks with no or little competition, like dry rock starts, might be able to achieve better balance scores for bacteria sooner because there is less to fight for, they can just kinda move in.

I don't think that getting that balance to be better sooner in the BRS tests means that sterile set ups is superior methodology, but that is my thinking about diversity and balance.

I am not sure we actually care about diversity, we may care more about dominant strains.

I agree in this. Especially about a tendency for monoculture in older tanks

Aquabionetics, also told my reef at the time had less biodiversity than most tanks. And my tank was over 45 years old at the time.

Interesting - my tank get a similar result - different dominant strains though. However - I don't think it's strange if an aquarium goes towards less bacterial diversity over time. We have all learned that consistent care is important for stability. This tends to favor bacterial cultures that thrive under the exact living conditions that we create in our consistent care. Since we also do not manage the aquariums in the same way, dominant species will shift between different aquariums (with different owners). The example in a previous post where the same person submitted two different aquariums supports this. The result was that they were very similar with regard to bacteria. I´ll think we can assume that the aquariums was managed exactly the same way.

Maybe it is that biodiversity among the bacterial community in our aquariums is not the most important factor for a good looking (and working aquarium) Maybe it is that biodiversity among other animals and photosynthetic organisms is significantly more important and that there is a thought about predation in the set-up of the aquarium.

Good write up and follow up @taricha

Sincerely Lasse

 

[taricha]

Maybe it is that biodiversity among other animals and photosynthetic organisms is significantly more important and that there is a thought about predation in the set-up of the aquarium.

This lines up nicely with the ideas explored in the second half of the BRS experiment. Various photosynthetic uglies get added to all the tanks, and after determining how the tanks respond for a few weeks, copepods are added to each system.
The presence or absence of various individual nuisances and the Predators for those nuisances largely determines the progression of the tanks during that portion of the experiment.

 

[sixty_reefer]

I've meant to start this discussion thread for a while... so here we go.

Reef hobbyists have a lot of ideas about biodiversity and what it means in a reef system. A lot of these ideas (mine included) are reasoning from personal experience of myself and others, and how we think it ought to work. Mostly this is because the concepts are fuzzy and we just don't have much data - so reasoning from collective experience is the best we've got in these data-sparse areas.
But that's changing - there are a lot of concepts in this discussion that we now actually have pretty good data to ground ourselves in and think more concretely about - surrounding the concepts of biodiversity, maturity, balance etc. BRS did a series of 11 videos (full playlist) in 2022 "Biome Cycling" involving 12 side by side aquaria run in parallel for 10 months, with detailed observations and data. This was done at a scale and expense that the hobbyist can't duplicate and I think the results are of good quality, and the data is worth digesting even if I don't buy all his reasoning and conclusions. Given how much we think and talk about these ideas like the "ugly phase", biodiversity, maturity, balance etc - this data deserves way more discussion than it has received so far. It's a ton of data, observations, and reasoning to sift through. Way more than one post,
So I'll try to occasionally pull one big idea at a time that I think is well demonstrated by the data, and is worth talking about.

Short summary of the overall exercise. (meet the tanks - Ep 3)
They started 12 tanks from different initial ingredients:
1) Dry sand and Rock - control
2) Dry sand and Rock + Coral frags/colonies
3) Dry rock, live sand in a bag
4) Synthetic live rock cured in seawater
5) Dry Rock and sand + live rock rubble cured in the dark, in dark sump
6) Live Rock and Sand directly from established tank
7) Dry rockand sand + 2 cups of established tank sand
8) Paper-wet indonesian live rock and dry sand
9) Gulf live rock shipped in water and dry sand
10) Dry rock and sand + Aquaforest reef mud
11) Dry rock and sand + 100% water from established system
12) Dry rock and sand + biobrick from established system

All tanks were given two clownfish and were kept dark for 4 weeks, then moderate "LPS lighting" from weeks 5-10, then high "SPS lighting" from weeks 11-15.
They tested each tank via aquabiomics at week 2, week 4, 10, and 15.
(What aquabiomics is and what was measured - Ep 4)


Idea 1: Biodiversity is dead, Balance is the new "biodiversity"
Aquabiomics gives two overall statistical measures/scores:
Biodiversity is a statistical measure of the bacterial families that make up more than 1% of the measured genetic material.
Balance is a measure of how much the present bacterial families look similar to those in established reef systems.

Here's the backwards thing that kills many ideas about biodiversity (like mine). In essentially every system that was started with any live material, The Biodiversity fell while the Balance rose. The biodiversity is initially higher especially in tanks started with a lot of live material like the live rock, but gradually falls. The Balance score - on the other hand, starts extremely low, but in every decent-looking tank climbs over time to more closely resemble typical reef tanks.
So early on, the high biodiversity likely represents a disturbed system, with many food sources of dead, disturbed and out of place organisms, and quick bacterial growth in response - but those early bacterial families look nothing like the eventual expected families that will make up a well-established tank.
Put another way, it is the death and loss of early bacterial diversity that helps shape the microbiome to look more like eventual reef systems. The Balance is a far better measure of this process of moving towards an established system than the biodiversity is, and perhaps the balance score isn't a terrible marker for biofilm maturity and system stability overall.
Ryan explains this idea of high biodiversity in tanks that look awful, and the balance being a better indicator here Ep 7 3:01-3:48

This data is in the videos Ep 5, 6, 7.









And here's Idea 2a and 2b in post 51

Idea 3 in post 73

here's a rundown of what aquabiomics has measured or concluded about many Frequently Asked Questions regarding hobby tank microbiomes in post 101

Idea 4 in post 110

Great work taricha, have you noticed that the order of the tanks change trough out the episodes?

Also have you noticed that every tank that had nuisance by week 15, that they were introduced into the display by the BRS team? Not sure if on purpose or accidental but every tank that was ugly by week 15 had an introduction of a contaminated organism in display that will compete with bacteria.

did you observe that balance and nuisance are directly connected in the first part of the episode with the best looking tanks having a higher balance compared to the ones with nuisance?

 

[taricha]

Great work taricha, have you noticed that the order of the tanks change trough out the episodes?

Thanks!
Yep. the tank order in "meet the tanks" was different than the order they gave the results in (more by logical groupings - comparing indo with gulf rock, and caribsea live sand and aquaforest live mud. etc.)

Also have you noticed that every tank that had nuisance by week 15, that they were introduced into the display by the BRS team? Not sure if on purpose or accidental but every tank that was ugly by week 15 had an introduction of a contaminated organism in display that will compete with bacteria.

Right:
Phase 1 was the microbiome part with all the aquabiomics tests and week 1-4 of darkness, week 5-10 of med light and week 11-15 of high light.
Phase 2 they add the uglies to all tanks.
Phase 3 they add pods to all tanks.
(frankly, I've spent most of my thinking on the phase 1 - the microbiome phase because it's the most complicated. The story is much more straightforward in phases 2 and 3 - really clear what's driving the nuisances.)

did you observe that balance and nuisance are directly connected in the first part of the episode with the best looking tanks having a higher balance compared to the ones with nuisance?

indeed!

From watching the videos I tried to give each tank a subjective "uglies score" based on amount and apparent "health" of the nuisance growth at week 15. I rated each tank zero to 10, with zero being spotless and 10 being the most uglies.

You can see how the diversity doesn't reflect the eyeball assessment of uglies (not that it's meant to, but people wonder if it does). The balance on the other hand does seem to correlate decently with the visual assessment of uglies.

Additionally in terms of correlations - the systems that dropped most notably in balance percentile from weeks 10 to 15 also had an increase in uglies during that time period.

 

[sixty_reefer]

Right:
Phase 1 was the microbiome part with all the aquabiomics tests and week 1-4 of darkness, week 5-10 of med light and week 11-15 of high light.
Phase 2 they add the uglies to all tanks.
Phase 3 they add pods to all tanks.
(frankly, I've spent most of my thinking on the phase 1 - the microbiome phase because it's the most complicated. The story is much more straightforward in phases 2 and 3 - really clear what's driving the nuisances.)

I find phase 1 very self explanatory, and the nuisances introduced in phase one allowed for those variables.

If we look at the best 4 in phase 1 that was

tank 2 rock + dry sand

tank 3 dry rock + ocean direct sand

tank 6 dry rock + dry sand + (dark) cured rubble

tank 10 real reef rock (possibly dry cured as rock is very clean and coraline is painted) + dry sand

all the above 4 tanks done the best results in phase 1 due to not having no nuisances, although that changes in phase 2 and do really bad at keeping nuisance at bay meaning the only reason systems done relatively well in phase 1 is by not introducing photosynthetic nuisance in the systems that will be competing directly with the microbiology of the system.
it’s a shame there isn’t a balance for week 25 to illustrate that.

Edit:
If we evaluate all 12 methods in phase 1, the ones that failed at week 15 were kinda set to fail from day one.

Tank 1 contains live rock with photosynthetic

Tank 4 contains sand from a established system with photosynthetic

Tank 5 contains coral with live rubble that contains photosynthetic

Tank 7 established rock and sand containing photosynthetic

Tank 8 water from the brs that will have photosynthetic

Tank 9 wet gulf rock that contains photosynthetic

Tank 11 not sure we’re the mud comes from

Tank 12 marine pure block from a Refugium containing photosynthetic

I believe all tanks would of done well if rocks, sand, block that have a possibility to contain photosynthetic organisms were added to a dark sump like in tank 6 (dark cured rubble)

 

[taricha]

Idea 5a: There were a couple of headscratching results in the BRS microbiome tests. I'll only worry about the weird things were replicated across large numbers of tanks.

( @AquaBiomics thanks for popping in this thread, I really appreciate it. Would love it if you could share your thoughts / shoot down my speculation.)

There was a bacterial family that is usually a small component - just a bit player in typical tank community but was a major component in all 12 tanks at some point in the 2,4,10 and 15 week samples.
Methylophilaceae - light brown bar.

The light brown bar is in all 12 tanks and a major family in most of the 4 test periods in each of the 12 tanks. It was barely present or absent entirely from the samples of the 3 BRS established tanks that donated some material to the different tanks.

The aquabiomics info page on this family doesn't really tell anything that jumps out to me to explain this.
https://aquabiomics.com/info/prokaryotes/methylophilaceae

My best blind guess is that it's a family that might be driven by the nutrient in the food or fish waste?
And many of these tanks started nearly clean, the two clownfish and fish food might be a strong or dominating influence on the bacterial community for this phase.

A tiny bit of supporting evidence to my speculative idea is this paper:
Feeding rapidly alters microbiome composition and gene transcription in the clownfish gut

In the paper they had 120 small clownfish - 10 each in 12 tanks (10 gal) and looked at the biome in the food, the fish intestines, and the water.
Methylophilaceae was not found to be major in the food or intestines, but it was one of the dominant groups in the water.

"More than 90% of sequences in the water (across all replicates) were identified as belonging to the bacterial Families Flavobacteriaceae (Bacteroidetes), Methylophilaceae (Alphaproteobacteria), or Rhodobacteraceae (Alphaproteobacteria)."

 

[Dan_P]

My best blind guess is that it's a family that might be driven by the nutrient in the food or fish waste?
And many of these tanks started nearly clean, the two clownfish and fish food might be a strong or dominating influence on the bacterial community for this phase.

Did you get a sense of how carefully BRS avoided bacteria contamination during set up and cross contamination between tanks during the experiment by the technicians?

 

[taricha]

I believe all tanks would of done well if rocks, sand, block that have a possibility to contain photosynthetic organisms were added to a dark sump like in tank 6 (dark cured rubble)

Yes, if we set aside the whole discussion of microbiome and just ask about the Uglies in Phase 1, then introduction of direct lighted material into the lighted portion of the tank is probably the cleanest explanation.

 

[sixty_reefer]

Yes, if we set aside the whole discussion of microbiome and just ask about the Uglies in Phase 1, then introduction of direct lighted material into the lighted portion of the tank is probably the cleanest explanation.

I agree, the issue here is that some of this photosynthetic organisms can influence greatly on the bacterial diversity, for example if we look at tank 9 (wet gulf rock) that end up at week 15 with a fairly high diversity wile full of ugly nuisance, this system got over-rune with diatoms that are one of the larger producers of methanol in the ocean.
turning the lights on seems to be a key point for heterotrophic bacteria numbers, we can’t only believe that Doc is coming from foods as the system must of been fed since the fish where introduced.
In addition many other photosynthetic organisms do release organic carbon into the water column that will also affect bacterial shifts ex. Cyanobacteria, macro and micro algae’s etc…

 

[taricha]

Did you get a sense of how carefully BRS avoided bacteria contamination during set up and cross contamination between tanks during the experiment by the technicians?

From messing around with culturing bacteria and seeing what's actually required to keep a sample sterile, hobby tank activities are lightyears away from sterile technique that would prevent contamination. The BRS experiment was no exception to that.

Having said that, I'm not that concerned about accidental contamination as a big complication. The reason that doesn't bother me is that even intentional addition of large amounts of material often did not actually transfer major families that were in the parent systems of that material. Or rather, transferring those families over didn't make them an important part of the test tank biome after the transfer. I used vibrio as an example of this effect here.

Idea 4:
Just because you add a bacterial type, doesn't mean it will become resident in the tank. Surprisingly, even if you add a bacterial type that is common to reef tanks, and you run reef tank conditions, doesn't ensure that it'll become a detectable part of the community.

As an example, take the bacterial family Vibrionaceae: In the BRS data, all of the established source tanks had significant amounts of this family, which is closely associated with animal and especially coral surfaces. It can be at very high levels in coral-packed systems.

But note the almost total absence of Vibrionaceae (hot pink bar on the bottom) by week 15 in all 12 treatment tanks.

In each treatment in this chart, the rightmost bar is week 15.
Only the Gulf Rock has a tiny detectable amount of this family.
All the established BRS tanks had it, and many of the 12 experimental tanks had material pulled from those 3 tanks: rubble from dark sump, sand, rock & sand, 100% water, biobrick, and even corals.

So even if you know you are adding a certain type of bacteria, and even if you know it usually reaches significant levels in reef tanks, it's still not a slam dunk that it will become significant in any modest time frame.
It's apparently more complicated than that to add and meet all the requirements for a certain family to become a significant resident in a system.

(I suspect the coral added tank would have shown notable Vibrionaceae eventually, but apparently "eventually" is even longer than 15 weeks.
Recurring theme, some of these processes look way slower than the stuff we normally think of in starting a tank. )

All the mature tanks had large vibrio populations, but even intentional transfer of that material did not lead to detectable vibrio in any of the 12 test tanks. So I don't think accidental contamination is that large of an issue.

To put it another way, I know there was contamination, but the test tanks ended up with very different bacterial family outcomes anyway, so the contamination must not have been all that important. (Could be wrong, but makes sense in my head)

 

[Lasse]

Methylophilaceae was not found to be major in the food or intestines, but it was one of the dominant groups in the water.

"More than 90% of sequences in the water (across all replicates) were identified as belonging to the bacterial Families Flavobacteriaceae (Bacteroidetes), Methylophilaceae (Alphaproteobacteria), or Rhodobacteraceae (Alphaproteobacteria)."

Food and intestine sample probably also contain bacteria bounded into a substrate - the water sample probably only contain bacteria that does not need a substrate or are less attached to the substrate. IMO - its nearly the same as to say that pear juice contain more pear than apple does

To put it another way, I know there was contamination, but the test tanks ended up with very different bacterial family outcomes anyway, so the contamination must not have been all that important. (Could be wrong, but makes sense in my head)

Or you can also say that´s probably the way of how you manage the tank that is the most important vector in the longer run. The food you use, the animals you have, the additive you use, which primary producer you have and so on If so - will rise a lot of questions about probiotics

Sincerely Lasse

 

[sixty_reefer]

Maybe a dumb question (but also the most important question), does anyone knows how the nitrogen cycle have been established in preparation to perform this experiments? I can’t find that information anywhere on the episodes and knowing the method could be helpful to interpret some data that may be influenced by the method used, only information I can find was that they were trying to deviate from bottled bacteria.

 

[taricha]

No cycling was done. All except dry rock/ sand control had material that would have been capable of processing ammonia. The control probably accumulated small amount of ammonia until lights-on after 4 weeks.

 

[sixty_reefer]

No cycling was done. All except dry rock/ sand control had material that would have been capable of processing ammonia. The control probably accumulated small amount of ammonia until lights-on after 4 weeks.

I just re watched all episodes as you really got me interested in the subject.

Are we sure about this? Randy explicit says that this is biome testing that differs from the nitrogen cycle (he’s words) they are testing for better ways to introduce diversity past the nitrogen cycle meaning that all tanks were put through a nitrogen cycle prior to the biome testing (making it safe for fish), that in my opinion will influence the initial biome depending on the method used.
No one in the right mind would have added two fish to this 12 experiments from day zero without ensuring that the nitrogen cycle is in place. Can you imagine the amount of grief they would have got into if that was the case and allow the fish to experiment an ammonia spike?

episode 3 is clear about this.
this series can be very misleading to many aquarists due to to many important factors have been left out, can you imagine someone miss interpreting this experiment and sets up a tank with 2 cups of cycled sand and adds two fish straight away?
same for all 12 tanks adding two fish in day zero without a established nitrogen cycle prior, would most likely kill the fish if additional bottled bacteria is not used.

 

[Dan_P]

From messing around with culturing bacteria and seeing what's actually required to keep a sample sterile, hobby tank activities are lightyears away from sterile technique that would prevent contamination. The BRS experiment was no exception to that.

Having said that, I'm not that concerned about accidental contamination as a big complication. The reason that doesn't bother me is that even intentional addition of large amounts of material often did not actually transfer major families that were in the parent systems of that material. Or rather, transferring those families over didn't make them an important part of the test tank biome after the transfer. I used vibrio as an example of this effect here.




All the mature tanks had large vibrio populations, but even intentional transfer of that material did not lead to detectable vibrio in any of the 12 test tanks. So I don't think accidental contamination is that large of an issue.

To put it another way, I know there was contamination, but the test tanks ended up with very different bacterial family outcomes anyway, so the contamination must not have been all that important. (Could be wrong, but makes sense in my head)

Nicely explained. Thanks.

 

[taricha]

Are we sure about this?

Nope, not sure. It's just my interpretation.

They "decided to avoid the biome in a bottle products", and referred to the dry rock/sand control as having "no obvious sources of biome seeding".

 

[sixty_reefer]

Nope, not sure. It's just my interpretation.

They "decided to avoid the biome in a bottle products", and referred to the dry rock/sand control as having "no obvious sources of biome seeding".

I find that statement from Randy confusing, as he states that they decided to avoid biome in a bottle, that was the statement that got me pondering, if they not using bottled bacteria what are they using to complete the nitrogen cycle that once we start looking into it becames obvious that all systems were put through a nitrogen cycle previously to the testing starting.

my interpretation so far is that they are not considering the nitrogen cycle as part of the tanks biome as it’s mainly done with nitrifying bacteria that shows on a separate eDNA sheet.
the biome that they referred during the experiment is mainly heterotrophic bacteria, zooplankton and other organisms that can only be introduced via donor colonies.
The question that raises eyebrows is what product was used to complete the nitrogen cycle as some products can introduce more than just autotrophic bacteria as you know, in a previous post you made a observation that all systems had a family of bacteria in common throughout the experiment (Methylophilaceae), I can’t stop but wonder if some of this species were introduced with the nitrogen cycle and in a way affect the final result.