Participating in the free microbiome analysis (PNWMAS April meeting)

[SuncrestReef]

1 minute ago, Lexinverts said:

Interesting. What do you keep nitrate and phosphate at in that tank?

NO3:  4 - 8 ppm (measured by Red Sea Pro which doesn't give any granularity between those two values)
PO4:  0.02 - 0.03 ppm (measured by Hanna ULR Phosphorus HI 736)

I'm trying to reduce nitrates to combat a bubble algae outbreak.

[Lexinverts]

6 minutes ago, SuncrestReef said:

NO3:  4 - 8 ppm (measured by Red Sea Pro which doesn't give any granularity between those two values)
PO4:  0.02 - 0.03 ppm (measured by Hanna ULR Phosphorus HI 736)

I'm trying to reduce nitrates to combat a bubble algae outbreak.

John, you're clearly an outlier. 😜

[SuncrestReef]

4 minutes ago, EMeyer said:

This forum needs a "mind blown" reaction. Cause, /mind blown

My dry rock + bacteria in a bottle experimental tanks have very low scores, despite strong nitrification

My gut says there is more to your success than dry rock + Dr Tims, but can't presume to know what!

In my case I'm guessing it's random luck.  I have added inverts from 3 stores, corals from 5 stores and 11 individuals, and fish from 3 stores.  The only other possible source of life was from a bag of "live sand" I added while my tank was still cycling because I decided I wanted a deeper sand bed but I didn't realize the extra bag of sand I ordered wasn't dry sand.

[SuncrestReef]

6 minutes ago, Lexinverts said:

John, you're clearly an outlier. 😜

Where do you keep your NO3 and PO4?

[SuncrestReef]

Here's the breakdown of microbial families in my tank.  I'm all about the Pelagibacteraceae. 🤓

[EMeyer]

22 minutes ago, Lexinverts said:

Robert....I'm guessing

Robert's tanks are interesting in their own way. His are some of the tanks I'd like to discuss in a future post. Considering how happy his corals always look, the differences between his tanks and others are really interesting. 

[EMeyer]

10 minutes ago, Lexinverts said:

John, you're clearly an outlier. 😜

I'll be a pain in the [language filter] semantics guy and say that while he's an outlier for diversity (highest score in the group) he is the opposite of an outlier for balance (the relative abundance of the bacterial families in his sample is very similar to the average; R2 = 0.7)

other tanks show high diversity but are very imbalanced. Or vice versa. 

I find these two metrics interesting because they are not correlated, a tank can be diverse, balanced, both, or neither...

 

[Lexinverts]

4 minutes ago, SuncrestReef said:

Where do you keep your NO3 and PO4?

Most of my tanks are similar to yours nutrient-wise. In my high nutrient tank that had the high diversity score, my nitrate runs about 20 ppm. I haven't tested phosphate in that tank in a while, so I will have to check it out.

[SuncrestReef]

2 minutes ago, EMeyer said:

I'll be a pain in the [language filter] semantics guy and say that while he's an outlier for diversity (highest score in the group) he is the opposite of an outlier for balance (the relative abundance of the bacterial families in his sample is very similar to the average; R2 = 0.7)

other tanks show high diversity but are very imbalanced. Or vice versa. 

I find these two metrics interesting because they are not correlated, a tank can be diverse, balanced, both, or neither...

 

Here's that summary:

[Lexinverts]

2 minutes ago, EMeyer said:

I'll be a pain in the [language filter] semantics guy and say that while he's an outlier for diversity (highest score in the group) he is the opposite of an outlier for balance (the relative abundance of the bacterial families in his sample is very similar to the average; R2 = 0.7)

other tanks show high diversity but are very imbalanced. Or vice versa. 

I find these two metrics interesting because they are not correlated, a tank can be diverse, balanced, both, or neither...

 

Yes, my balance score was low for my high diversity tank. Interesting!

[Lexinverts]

15 minutes ago, SuncrestReef said:

In my case I'm guessing it's random luck.  I have added inverts from 3 stores, corals from 5 stores and 11 individuals, and fish from 3 stores.  The only other possible source of life was from a bag of "live sand" I added while my tank was still cycling because I decided I wanted a deeper sand bed but I didn't realize the extra bag of sand I ordered wasn't dry sand.

Did you dip those corals that were acquired from 5 stores and 11 individuals? I've always wondered what coral dips did to bacteria...

[SuncrestReef]

Eli, I know you probably need some sleep after all your work on this, but overall did our samples provide data you were roughly expecting, or were you completely surprised by the results?

[SuncrestReef]

Just now, Lexinverts said:

Did you dip those corals that were acquired from 5 stores and 11 individuals? I've always wondered what coral dips did to bacteria...

Yes, I dip every coral before it goes in the tank.  I still ended up with aiptasia, red bugs, bristle worms, asterina stars, stomatella snails, bryopsis, ulva lactuca, and bubble algae.  None of these were introduced on purpose.  If all of these critters survived the dip, I'm sure bacteria weren't deterred.

[Lexinverts]

1 minute ago, SuncrestReef said:

Yes, I dip every coral before it goes in the tank.  I still ended up with aiptasia, red bugs, bristle worms, asterina stars, stomatella snails, bryopsis, ulva lactuca, and bubble algae.  None of these were introduced on purpose.  If all of these critters survived the dip, I'm sure bacteria weren't deterred.

We'll need to interrogate you at some point regarding your bacterial husbandry. You must be doing something right! 😎

[EMeyer]

10 minutes ago, SuncrestReef said:

Eli, I know you probably need some sleep after all your work on this, but overall did our samples provide data you were roughly expecting, or were you completely surprised by the results?

it was a pleasant surprise that there is a core microbiome shared among different tanks. I can talk more about this in a future thread; it requires data. But its not just "every tank is a unique snowflake". There is a strong pattern. Your tank is a good example. 

And some tanks that participants logged as having problems were also major outliers with low scores, which is cool... I will be following up with them. 

Also, these score show interesting patterns in experimental tanks... very low scores in brand new tanks, and big differences between dry rock, live rock, and bacteria in a bottle....

I would file all of that under "promising and hoped for"

the most head scratching part is the small number of tanks with very different microbiomes but no reports of problems. 

Its a complex enough question I cant trust my gut to figure it out, I will have to do some stats. and its likely more samples are needed. This is my initial impression.

 

 

Edited September 5, 2019 by EMeyer

[SuncrestReef]

1 minute ago, EMeyer said:

it was a pleasant surprise that there is a core microbiome shared among different tanks. I can talk more about this in a future thread; it requires data. But its not just "every tank is a unique snowflake". There is a strong pattern. Your tank is a good example. 

And some tanks that participants logged as having problems were also major outliers with low scores, which is cool... I will be following up with them. 

Also, these score show interesting patterns in experimental tanks... very low scores in brand new tanks, and big differences between dry rock, live rock, and bacteria in a bottle....

I would file all of that under "promising and hoped for"

the most head scratching part is the small number of tanks with very different microbiomes but no reports of problems. 

Its a complex enough question I cant trust my gut to figure it out, I will have to do some stats. and its likely more samples are needed. This is my initial impression.

 

 

Very cool.  I'm definitely interested in seeing your future write ups and further data analysis.

Thank you so much for including us in your project!  Please let us know if you ever want additional samples to see how things change over time.

[youcallmenny]

This sample was taken before I fixed my lights @EMeyer if that tells you anything.  This balance was the result of an improper lighting photo period and spectrum.  My Triton-ICP test taken almost the same day came back kind of weird too.  Now that things are rocking and rolling I'd love, and be willing to pay for, another test.  Please keep me in mind for the next round, this is hugely fascinating to me.  Thank you for the inclusion.   Here are all of the out of tolerance readings from the ICP test.  Hopefully this is helpful to you. 

 

[albertareef]

8 hours ago, youcallmenny said:

This sample was taken before I fixed my lights @EMeyer if that tells you anything.  This balance was the result of an improper lighting photo period and spectrum.  My Triton-ICP test taken almost the same day came back kind of weird too.  Now that things are rocking and rolling I'd love, and be willing to pay for, another test.  Please keep me in mind for the next round, this is hugely fascinating to me.  Thank you for the inclusion.   Here are all of the out of tolerance readings from the ICP test.  Hopefully this is helpful to you. 

 

This correlative data (ICP with bacterial profile) could prove really interesting in the long run.  BTW - you and John are all about the Pelagi!

[EMeyer]

Hi everyone,

I've been working for a couple days on an update and am excited to share the results. I hope this new analysis shows the potential for this kind of data to shed light on specific groups of bacteria that aquarists are interested in. These are new dimensions to think about.... previously we've focused on overall patterns; now we'll look at specific groups of microbes aquarists care about (whether they realize it or not)

I'm uploading these now.... within a few minutes, you can check out your updated reports to see new info about specific groups of interest in your samples:

• Nitrifying bacteria
• Cyanobacteria
• Pathogens

https://aquabiomics.com/view-reports/

This is just the beginning of exploring the functional meaning of these microbiomes. A few highlights:

 

Your tanks' microbiomes are almost completely free of known pathogens

Most samples lacked any evidence of known coral or fish pathogens. A few samples showed a low level of fish pathogens. 

 

But some of them do have relatively high levels of cyanobacteria

A few samples showed unusually high cyanobacterial levels compared to the rest. I see that some of these were noted as having cyanobacteria when you registered your samples. I'll be curious to hear if the cyano scores you received match with your impressions of your tank at the time or after sampling. 

As someone who has battled cyano in one of my tanks for almost 2 years, I am also intrigued by the thought of comparing notes on the type of cyanobacteria we're dealing with (I've broken it down by family here). Perhaps this will lead to clarity on how to win the battle (in my case, nitrate dosing made a huge difference, but that was trial and error)

 

Your tanks vary widely in levels of nitrifying bacteria

Finally, I was surprised to see this much variation in nitrifying bacteria. Since I bet some of you will be surprised too, I thought this was worth discussing. 

Nitrifying groups include ammonia oxidizing bacteria and archaea (AOB & AOA), and nitrite oxidizing bacteria (NOB). Almost all tanks showed comparably high levels of AOB & AOA. But there was substantial variation in NOB. Many samples showed clear evidence for one or more types of NOB, but almost as many showed no evidence of NOB at all. 

To be clear, I searched extensively for nitrite oxidizing bacteria. I even looked for the fifth clade that has only recently been discovered (Nitrotoga). I tried! And will keep exploring to see if there is something I have missed. 

But I suspect the key to understanding this variation may lie in considering that DNA sequencing measures the composition of the community, and there is a finite limit to detection of rare types. If one type "blooms" to a high abundance, the probably that rare types will not be detected increases (assuming the number of DNA sequences per sample remains constant). Like any measurement, there is a limit of detection for this analysis. In this case, its approximately 1 DNA sequence per 10,000 (i.e. 0.1 ppt or 100 ppm). 

Bottom line - if your sample shows up as having no NOB, I do not interpret that as absence of NOB. I interpret it as meaning that NOB are present at very low levels in your tank relative to other kinds of microbes in your tank. 

This variation is surprising to me. When we encounter surprising patterns there can be a tendency to assume they result from errors. And I am still digging to see if there is some way my code is missing something. But I will raise two points that I believe support this observation:

• Most of the known NOB were found in some tanks. So their absences from other tanks is not a systematic error in the method (e.g. gaps in the database or uneven DNA extraction efficiencies). This is a genuine difference among tanks; I just don't believe its presence vs absence. I think its quantitative. 
• Occasionally I have put a bunch of fish in a tank that previously had a low bioload. I've seen very minor ammonia spikes from this, but much larger nitrite spikes. This would be consistent with a lower population of NOB than AOB. 

 

Anyway, as always, I will be curious to hear what you think!

 

Edited September 9, 2019 by EMeyer

[SuncrestReef]

Thanks for the update, and new detailed reports!  I'm relieved to see I have no pathogens, and my cyano levels are low.  I will sleep better tonight!

[albertareef]

Thanks Eli!  Do you think the relatively low level of NOB in some cases could be related to the sampling paradigm - i.e. since it was from the water column vs. substrate.  Any evidence that these forms of bacteria are less likely to "shed" into the column?  Just curious...

[EMeyer]

9 hours ago, albertareef said:

Thanks Eli!  Do you think the relatively low level of NOB in some cases could be related to the sampling paradigm - i.e. since it was from the water column vs. substrate.  Any evidence that these forms of bacteria are less likely to "shed" into the column?  Just curious...

This is a question I wondered about too. I only have a single comparison, because I screwed up one of my sand samples. But based on this single comparison, it appears you are correct and sampling affects the NOB metric.

I see ~50-fold lower AOB+AOA in the water column than in the sand. The sand had detectable NOB while the water did not. Even in the sand, NOB is ~60 times less abundant than AOB+AOA. So it makes sense that if all the sand microbes are reduced by ~50 fold in the water column then the less abundant NOB would become undetectable. 

Things to think about. Its easy to change the sampling protocol. Its just important that we have something everyone can do consistently. 

• Sampling sand isnt possible for people with bare bottoms. 
• I fail to imagine any way of sampling live rock associated microbes consistently. 

Water that flows constantly through a biofilm-covered aquarium seems to be the one thing all reefers have available to sample in an easy manner... and for about half of the tanks sampled it showed both AOB and NOB... I do think the variation we see here is real, but its variation in amounts rather than presence/absence. Still pondering this one. 

Any feedback welcome!

[obrien.david.j]

Eli, One feedback.  Your reports include Sample Date, but they don't include Report Generation Date.   As you're updating the coding and report generations, I'd love to see a Report Date (or Report Coding Rev) added.  So I can ask you questions about a specific .pdf revision.   

Overall, my report is a Low Balance Score  (am I the lowest?)  With a huge "Bacteriovoracaceae", and almost non-existent "Flavobacteriaceae" & "Pelagibacteraceae".   Any idea what that means?  (BTW, I accept an answer of I-Don't-Know.    That's I'm here to support learning.) 

Otherwise I was in the "green range" of other items.  Except fish pathogens, with a high "Photobacterium damselae".  But, my fish are all doing fine.

 

One question I don't remember you asking us when submitting the sample is, "how long has your tank been effectively up and running, and have you dosed any medications in the last 12 months."   I've been up for > 10 years, but Yes I've dosed Interceptor Plus (for Red Bugs) and Bendazole Fenbendazole Powder (for Blue Clove Polyps) within 12 months of giving you the sample.

Thank you for continuing to look across these samples.   Reef Keeping is about Learning!

-David J. O'Brien

 

AquaBiomics report 2019-09-09 Update - 1000037.pdf

 

[EMeyer]

On 9/9/2019 at 10:26 PM, obrien.david.j said:

Eli, One feedback.  Your reports include Sample Date, but they don't include Report Generation Date.   As you're updating the coding and report generations, I'd love to see a Report Date (or Report Coding Rev) added.  So I can ask you questions about a specific .pdf revision.   

Thanks David, this makes sense. I've added that to my code, future updates will include a report date and previous versions will get pushed into an "archive" folder on the reports page. 

On 9/9/2019 at 10:26 PM, obrien.david.j said:

Overall, my report is a Low Balance Score  (am I the lowest?)  With a huge "Bacteriovoracaceae", and almost non-existent "Flavobacteriaceae" & "Pelagibacteraceae".   Any idea what that means?  (BTW, I accept an answer of I-Don't-Know.    That's I'm here to support learning.) 

Not the lowest! 

I'm finishing up a writeup on the major families found in these data. This is almost ready. 

The short answer is - bacterial communities in the ocean are mostly determined by dissolved nutrients. Not just NO3 etc, but complex and largely unmeasured things like the specific types of DOC... I (and I believe We) dont know the specific answer with regards to aquaria. This is the subject for my next experiment beginning soon - how does the type and amount of DOC (as used in carbon dosing) affect the microbiome?

On 9/9/2019 at 10:26 PM, obrien.david.j said:

Otherwise I was in the "green range" of other items.  Except fish pathogens, with a high "Photobacterium damselae".  But, my fish are all doing fine.

Yeah, this was interesting. Part of my goal here is to establish a baseline. I expected there to be some low level of background pathogens in our tanks. Overall we only found a couple of pathogens, at very very low levels, in a few tanks. 

The first thing I want to emphasize is that a few subspecies in this group are pathogens, but not all. Still, this bug is clearly a member of the species, which includes some of the most important in marine aquaculture. But it could be a less pathogenic variety. I can dig deeper on this and get back to you. It may be that 16S sequence data (what we used) cannot distinguish these subspecies; that happens sometimes. 

I have not previously investigated this specific pathogen yet but I will be, since it is one of the few that showed up at all. My initial survey shows this pathogen causes Pasteurellosis, or Photobacteriosis. Symptoms include loss of appetite, reduced activity, lesions and hemorrhages around eyes or mouth, lesions and tubercles on internal organs, eventually leading to death. Sounds fun. The diseases caused by bacteria in this species are considered some of the most dangerous among established and emerging diseases in fish aquaculture. The literature says they are ubiquitous throughout the aquaculture industry. 

It can infect a broad range  of fish species, although older fish become tolerant due to maturation of their immune systems. Interestingly there is good evidence that infection requires iron, and this can be deleted in marine aquariums. Antibiotics and other chemotherapies have been used in fish aquaculture to control this disease, although antibiotic resistance has developed in some strains. 

I base this on a brief reading of the following two free references on this subject that are available for anyone who is curious to learn more

• http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.731.3326&rep=rep1&type=pdf
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058529/

Whatever the case, its clear that your tank was an outlier in having such a high frequency of this bug (which ranged from absent to 10x lower than yours in most samples). The observation of such high levels without visible symptoms is an important observation to remember if we see P. damselae in other tanks. I will also be curious to compare your specific sequence data with other members of the group that show up in tanks WITH disease symptoms. 

I will be curious to hear if it shows up again in retesting (see upcoming post, I'll be inviting re-sampling of the same tanks with my updated sampling kit)

On 9/9/2019 at 10:26 PM, obrien.david.j said:

One question I don't remember you asking us when submitting the sample is, "how long has your tank been effectively up and running, and have you dosed any medications in the last 12 months."   I've been up for > 10 years, but Yes I've dosed Interceptor Plus (for Red Bugs) and Bendazole Fenbendazole Powder (for Blue Clove Polyps) within 12 months of giving you the sample.

Another great point. I've added "How long has your tank been up and running?" to the sample registration form.

 

Thanks for the feedback, all...

[obrien.david.j]

Eli - Thanks for the detailed responses.  I look forward to the next evolution of your analysis.