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

[mmoner]

Idea 3: Almost none of the 12 tank starting schemes acheived average (>50 percentile) Diversity anytime during the test period weeks 2 to 15. Only one system even got close, and it took 15 weeks to get to that result. None of the others got over 34 percentile at any point, with most systems spending the whole 15 weeks around and below 10th percentile.

But in Balance score, more than half the systems achieved an average ( >50 percentile) balance score by the end of the 15 week period. A third (4/12) systems got there by the 2nd measurement at week 4.

This happened in systems both with strong initial material influences (Coral, Indo live rock) and some with no intentional seed material at all (dry rock/sand). These very different starting materials were able to achieve a balance that looked like a typical reef tank in this time frame.
If we say this criteria is a goal - to have our major bacterial components look like the rest of the reef hobby - then this goal is achievable in a fairly short time regardless of starting material. Quite an optimistic takeaway.

But if we say diversity matching a hobby tank is the goal - then this is a long slog - even with carefully chosen material, and no guarantees that the system gets there at all. Not so rosy a scenario.

One thing both views (diversity and balance) show is that there are quantifiable answers to a question that we often pose. Cycling takes a 1-2 weeks, but we feel like tank "maturity" takes much longer - on the scale of months. But we don't have good answers too what we think happens between 2 weeks and a few months. One of those answers is bacterial succession is clearly a much slower process than a couple of weeks.
This is a snapshot of all 4 microbiome tests over 15 weeks on all 12 tanks... No tanks are totally settled across any time frame within this 15 week period. Some are in more flux than others.

Thanks for graphs, can we see trendlines with each lighting period is highlighted? So afaik balanced tanks looks sleek and clean while diverse ones are much dirty with some nasty patches here and there.

A personal experience from one of my setups, i used brand new pack live sand with rocks from a newly broken down established tank and i had the nastiest stages ever for months (with 3ppm PO4 right from the start).

 

[Dan_P]

Not at all, really.
He's talked about the fact that reef tanks are such unstudied systems, that he's taken an agnostic approach to what a "good" tank is. So he simply throws out from the statistical average any coral aquaculture facilities (they are not really reef tanks), experimental setups, and any systems reported as bad or crashed, dead livestock etc.

Other than that, it's just a statistical average of hundreds of hobby systems. So the stacked color bar that says "typical" really is meant to represent typical - not "good" tanks.

For me, this clarification of the term “balance” seriously discounts its usefulness. And when you consider that the data behind “balance” suffers from the same flaw as does ICP data (no standard deviation), the “balance” term might actually be useless.

By the way, if you had a look at the data, I am pretty sure you could provide a more sophisticated analysis.

 

[Dan_P]

Idea 3: Almost none of the 12 tank starting schemes acheived average (>50 percentile) Diversity anytime during the test period weeks 2 to 15. Only one system even got close, and it took 15 weeks to get to that result. None of the others got over 34 percentile at any point, with most systems spending the whole 15 weeks around and below 10th percentile.

But in Balance score, more than half the systems achieved an average ( >50 percentile) balance score by the end of the 15 week period. A third (4/12) systems got there by the 2nd measurement at week 4.

This happened in systems both with strong initial material influences (Coral, Indo live rock) and some with no intentional seed material at all (dry rock/sand). These very different starting materials were able to achieve a balance that looked like a typical reef tank in this time frame.
If we say this criteria is a goal - to have our major bacterial components look like the rest of the reef hobby - then this goal is achievable in a fairly short time regardless of starting material. Quite an optimistic takeaway.

But if we say diversity matching a hobby tank is the goal - then this is a long slog - even with carefully chosen material, and no guarantees that the system gets there at all. Not so rosy a scenario.

One thing both views (diversity and balance) show is that there are quantifiable answers to a question that we often pose. Cycling takes a 1-2 weeks, but we feel like tank "maturity" takes much longer - on the scale of months. But we don't have good answers too what we think happens between 2 weeks and a few months. One of those answers is bacterial succession is clearly a much slower process than a couple of weeks.
This is a snapshot of all 4 microbiome tests over 15 weeks on all 12 tanks... No tanks are totally settled across any time frame within this 15 week period. Some are in more flux than others.

Here are some thoughts and questions. I really could not top your analysis. I found your analysis very useful.

I wonder what the thinking was not to get a week zero read out? Not having it is liking trying to figure out Game of Thrones coming in the middle of the third season. How much biodiversity is initially introduced by freshly prepared saltwater? What factors might have driven it down?

In the biodiversity trends, I see there may only be two slopes between week 10 and 15? Were these rate differences a result of similar bacteria growing or not growing, or just a lucky combination of dozens of families growing or not growing. I guess we would have to back out the trends for each bacteria family found at weeks 10 and 15 to see if certain players were creating these two slopes or it was just noise in the data.

The fluctuation of biodiversity might expected in bacteria populations and this fluctuation rate could be more interesting than the point in time “balance score”. In fact “balance score” might actually be a grand average across all tanks and all fluctuations, a very blunt metric.

Does the trend in biodiversity parallel the accumulation of organics or algae in these aquaria? If the aquaria were fed differently, would the trend in diversity change?

Now that I understand “balance score” more clearly (Thank you) I think that I have a better understanding of why the balance score data is a mess. The “balance score” is arbitrarily tracking a subset of bacteria growth and producing an almost uninterpretable jumble of numbers. For the BRS data, it could make more sense trending the individual families and looking at what that might be informing us about the aquarium microbiome development relative to its set up. Plotting parameters like biodiversity and “balance score” masks potentially useful information and trends. I understand fully the daunting task of looking at all the data rather than a derived parameter, but I propose that the parameters are of dubious worth for our purposes. We need to start from scratch.

 

[Hats_]

they used the eDNA system at my internship before. they were all over the moon by the technology and the results. basic way that it works is that you supply the system with markers that can bind to certain scraps of dna that animals release into the environment (think mucous and feces for example) then when those are found in the sample they attach and can be measured. The process is extremely expensive and to conserve the dna and prevent the sample from changing my internships froze the samples after collection, samples can be kept like this for years to be measured later. One problem they noticed is determining abundance of certain strains or animals is very difficult since not all animals leave the same amount of environmental dna (take for example the slimecoat of a wrasse compared to other fish).

So from what i can see is that AB can have a lot of inaccuracies because of three issues:
- Not having all the markers for all the possible strains that can exist in seawater
- Not being able to accurately predict abundance yet due to there not being a lot known about how much eDNA certain strains of bacteria leave behind.
- samples can morph since they are not being shipped frozen AFAIK

just my 2 cents, being a bit more familiar with the technique

 

[taricha]

I wonder what the thinking was not to get a week zero read out?

Remember that the bacterial balance and bar chart of who's in what relative prevanceis based on the genetic material in the water. The film swab is for more sensitivity toward the nitrifying community and more counts for diversity. (because people's sampling of water is roughly comparable - but how people swab a dark pipe is not)
So week zero, you'd just be sampling new saltwater in all the tanks except the one that got 100% reef tank water. It would just be amplified noise.

In the biodiversity trends, I see there may only be two slopes between week 10 and 15? Were these rate differences a result of similar bacteria growing or not growing, or just a lucky combination of dozens of families growing or not growing.

rather than look at the slope of percentile which may not be super-meaningful, look at the slopes of the raw counts (quoted below). You could still argue there are low and high groups, but there's no obvious (to me) connection between the tank types and hi/low slopes - nor to specific groups increasing or decreasing.

First chart is the Diversity scores (the raw number of types detected.)

Notice how the diversity drops across the board during the early dark phase, while the reverse happens in the early dark phase to the tanks' balance scores below.

These are the Balance scores - a.k.a similarity to core microbiome of Reef Tanks ( again raw scores, not percentiles)

The “balance score” is arbitrarily tracking a subset of bacteria growth and producing an almost uninterpretable jumble of numbers. For the BRS data, it could make more sense trending the individual families and looking at what that might be informing us about the aquarium microbiome development relative to its set up. Plotting parameters like biodiversity and “balance score” masks potentially useful information and trends. I understand fully the daunting task of looking at all the data rather than a derived parameter, but I propose that the parameters are of dubious worth for our purposes. We need to start from scratch.

Well, not really arbitrary - it's tracking the most dominant groups across the hobby. But correct that the connection to function in a system is entirely unknown and a good connection may not exist.

But yes, I intend to drill down below the topline numbers, I just like to do the dumb way first to see how far that gets me.
(I'm not optimistic that trends of the more granular data will make even as much sense as the toplines do.)

So from what i can see is that AB can have a lot of inaccuracies because of three issues:
- Not having all the markers for all the possible strains that can exist in seawater
- Not being able to accurately predict abundance yet due to there not being a lot known about how much eDNA certain strains of bacteria leave behind.
- samples can morph since they are not being shipped frozen AFAIK

1) as far as I can tell, AB can group all of the detected bacteria into families. Species-level or even genus - no.
2) Right. Eli says the bacteria are better behaved than the eukaryotes at contributing DNA to the water. Small Inverts will sometimes spread enormous amounts of genetic material into the water and dominate the eukaryotic DNA distribution (snails and sponges are the common examples he gave).
But still, for the reason you mentioned - the abundance data is really only sensible for comparisons from one system to another or the same system over time. Can't really say that a larger bar for rhodobacters vs vibrios means for sure that there's more cells or biomass of one vs the other.
3) the samples are preserved on the user end at time of sampling, so that issue is avoided.

 

[livinlifeinBKK]

Saw the videos a while back and while I did find them interesting i recall not being really convinced for a few reasons as i recall...I'd have to rewatch the videos to remember why though

 

[Hats_]

2) Right. Eli says the bacteria are better behaved than the eukaryotes at contributing DNA to the water. Small Inverts will sometimes spread enormous amounts of genetic material into the water and dominate the eukaryotic DNA distribution (snails and sponges are the common examples he gave).
But still, for the reason you mentioned - the abundance data is really only sensible for comparisons from one system to another or the same system over time. Can't really say that a larger bar for rhodobacters vs vibrios means for sure that there's more cells or biomass of one vs the other.
3) the samples are preserved on the user end at time of sampling, so that issue is avoided.

I agree with you there, comparing one tank with itself over time will be able to shed some light on some things. I still think that since this technology is in very early stages and that it may not be as accurate as we hope. At least it is just an insight and people are not changing vital things in their tanks according to results from here so i dont see any harm in the service they provide.

I generally dont agree with the 3rd point you make here though. DNA degrades relatively quickly and people are not freezing it before sending it to AB, i dont expect it to arrive at their doorstep the next day so i do think that this can most certainly change the composition of the sample (also very weather dependable). What do you mean by 'preserved on the users end at time of sampling'? just to clarify

 

[taricha]

What do you mean by 'preserved on the users end at time of sampling'? just to clarify

The user takes a sample of water, filters it, then applies the fixative to the filter before shipping the filter back. Same thing for the biofilm swab - fixative is applied by the user at time of sampling, then swab is shipped back.
https://aquabiomics.com/sampling-instructions

 

[Hats_]

The user takes a sample of water, filters it, then applies the fixative to the filter before shipping the filter back. Same thing for the biofilm swab - fixative is applied by the user at time of sampling, then swab is shipped back.
https://aquabiomics.com/sampling-instructions

aaahh, i didnt know that, i couldnt find the instructions on how it works on their website, it just said "mail the sample" thanks for clarifying that!

 

[taricha]

Let me expand a little more on Balance Score , Diversity and visual judging of the systems.
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.

12 - biobrick
9 - gulf wet rock
5 - Coral
and 7 - tank rock/sand
...all had notable increases in nuisance growth from weeks 10-15, which can help to explain one way a system that has better "balance" score can regress on that metric during that time frame.

 

[livinlifeinBKK]

There just seen to be many more variables at play than they took into account (maybe they weren't sure how to take into account some of the variables, idk). Just my personal experience but I've always used live ocean rock without experiencing the "uglies" not just to the extent that their experiment showed but practically no "uglies" whatsoever. On the other hand, I see post after post here from people with very bad "uglies" and it seems like a large portion of these hobbyists went the dry rock/sand route. It really seems like it's a good bit more complex than portrayed in their videos and results.

 

[Hats_]

There just seen to be many more variables at play than they took into account (maybe they weren't sure how to take into account some of the variables, idk). Just my personal experience but I've always used live ocean rock without experiencing the "uglies" not just to the extent that their experiment showed but practically no "uglies" whatsoever. On the other hand, I see post after post here from people with very bad "uglies" and it seems like a large portion of these hobbyists went the dry rock/sand route. It really seems like it's a good bit more complex than portrayed in their videos and results.

i agree, i started with all dry rock but caribsea livesand, had no uglies whatsoever, only thing i had was diatoms that went away after just a couple of days and then coralline took its place. and my light were on full strength day one

I feel like there are way more variables in play than BRS' test shows

 

[taricha]

I've always used live ocean rock without experiencing the "uglies" not just to the extent that their experiment showed but practically no "uglies" whatsoever. On the other hand, I see post after post here from people with very bad "uglies" and it seems like a large portion of these hobbyists went the dry rock/sand route.

i agree, i started with all dry rock but caribsea livesand, had no uglies whatsoever, only thing i had was diatoms that went away after just a couple of days and then coralline took its place. and my light were on full strength day one

I feel like there are way more variables in play than BRS' test shows

Actually, everything I've talked about so far in this thread is just from phase 1 of the BRS test (because that's where the microbiome testing is done).
In phase 2 they add intentional uglies to all tanks and it comes out more like y'alls experience. The "livest" materials generally handle that the best, with dry rock/sand struggling to cope with new nuisance.

 

[livinlifeinBKK]

Actually, everything I've talked about so far in this thread is just from phase 1 of the BRS test (because that's where the microbiome testing is done).
In phase 2 they add intentional uglies to all tanks and it comes out more like y'alls experience. The "livest" materials generally handle that the best, with dry rock/sand struggling to cope with new nuisance.

Oh i see...I joined the thread not long ago so haven't had time to read everything here yet because it's a fairly long thread at this point...just saw the title and like i said, i actually did watch the series so i had the impression you were giving your assessment of the full series

 

[Aqua Man]

In phase 2 they add intentional uglies to all tanks and it comes out more like y'alls experience. The "livest" materials generally handle that the best, with dry rock/sand struggling to cope with new nuisance.

Data is great and I appreciate you putting it together. I found the phase 2 even more interesting, so I apologize for jumping too far ahead in your thread earlier!!

Maybe bacteria does not play as big a role as thought? Data is not my strong point. Seems that all the tanks eventually balanced out?

As Garth mentioned about food, did they say in the video what the food input was? Was there any mention of the nutrient levels? N/P?

 

[livinlifeinBKK]

As i recall, they didn't do anything to address the die off from the live rock they used which would obviously feed algae like crazy...this struck me as somewhat odd since most people would change the water if it were their tank which would greatly reduce algal growth... doesn't it seem like the results might not accurately represent a typical scenario for this tank?

 

[Aqua Man]

They did do water changes. Although I think they only did actual cleaning a couple times. Some of the ugly grew back quickly in some tanks.

 

[taricha]

Maybe bacteria does not play as big a role as thought?

The answer depends on your expectations.
Some say bacteria has no importance, others say it has to be part of the tank maturation consideration.
Worth thinking about. Not sure where on the scale I would put it right now.

 

[Thales]

As i recall, they didn't do anything to address the die off from the live rock they used which would obviously feed algae like crazy...this struck me as somewhat odd since most people would change the water if it were their tank which would greatly reduce algal growth... doesn't it seem like the results might not accurately represent a typical scenario for this tank?

I think people are too focused on algal problems, especially with new systems - kind of having all the drywall dilivered to a build site before the foundation is even poured (This analogy needs work).
I also think there is no typical, and that folks extend and confuse the establishment of a new system by taking bits and pieces from different philosophies and trying to use them together without enough understanding of what is supposed to happen and how different ideas may stifle each other.
IMO, starting up a tank is different from starting the display phase of the tank, and that if people more understood that, they would have an easier and more fruitful time establishing a reef system..

 

[-XENOMORPH-]

Knowing that I know nothing......
I started 2 yrs ago with dry rock and live sand. Bottle bacteria, pods, phyto etc....i am trying... but the struggle is real.. dam DINOS!!!! .. never again! I'm now saving up a few bucks to buy live rock with all hitchhikers, good and bad, from tamPAbay saltwater. THE MORE bacteria and hitchhikers, the better. A la natural!!!!