Problems Due to Bacterial Imbalances? AquaBiomics Test Results.

[hart24601]

Are total bacterial counts part of the test? I remember a few advanced aquarist articles that showed overall counts varying pretty dramatically and unsurprisingly with husbandry techniques such as skimmer/no skimmer, carbon dosing, perhaps Refugium (can’t remember that one). Having total population counts seem quite important to this conversation, does tank that have 10x more bacteria have greater diversity or are a few types becoming more dominant- and is that diversity still there just harder to see via PCR because the much more prevalent species amplifying so much more readily because that tank has 10 or 20 times more bacteria. And which is better? Or does it matter at all. You can nano drop to see the amount of DNA extracted but ideally using something like a flow cytometer.

Ah here is the link and it was looking at GAC and uv as well.

Feature Article: Bacterial Counts in Reef Aquarium Water: Baseline Values and Modulation by Carbon Dosing, Protein Skimming, and Granular Activated Carbon Filtration

What are the bacteria populations in the water column of reef tanks, and how does that value compare with bacterial counts in authentic reef water? Does carbon dosing indeed increase water column bacteria populations (i.e., is growth carbon limited)? Does mechanical filtration (protein skimming...

www.advancedaquarist.com

 

[AquaBiomics]

Are total bacterial counts part of the test? I remember a few advanced aquarist articles that showed overall counts varying pretty dramatically and unsurprisingly with husbandry techniques such as skimmer/no skimmer, carbon dosing, perhaps Refugium (can’t remember that one). Having total population counts seem quite important to this conversation, does tank that have 10x more bacteria have greater diversity or are a few types becoming more dominant- and is that diversity still there just harder to see via PCR because the much more prevalent species amplifying so much more readily because that tank has 10 or 20 times more bacteria. And which is better? Or does it matter at all. You can nano drop to see the amount of DNA extracted but ideally using something like a flow cytometer.

Ah here is the link and it was looking at GAC and uv as well.

Feature Article: Bacterial Counts in Reef Aquarium Water: Baseline Values and Modulation by Carbon Dosing, Protein Skimming, and Granular Activated Carbon Filtration

What are the bacteria populations in the water column of reef tanks, and how does that value compare with bacterial counts in authentic reef water? Does carbon dosing indeed increase water column bacteria populations (i.e., is growth carbon limited)? Does mechanical filtration (protein skimming...

www.advancedaquarist.com

This is an important question.

The answer is no, this method (the same one used by the Earth Microbiome Project and the Coral Microbiome Project) doesnt provide direct cell counts. The methods for doing so accurately require fairly expensive equipment that I havent purchased yet. I do of course have access to DNA quantification (I use fluorescence, even more sensitive the absorbance based instruments like the NanoDrop) but the low concentrations of DNA produced here don't quantify well. qPCR would be the ideal in terms of cost vs throughput. I hope to add that to the lab at some point.

For now, we will have to settle for relative abundance, like most studies using this technology. I'd like to add the capability to do absolute quantification when I can.

You also raise an important point about some sequences potentially amplifying better than others. While I can't make any absolute, 100% guarantees, I will say that I am standing on the shoulders of giants here -- a large community of researchers has spent considerable time developing primers (DNA molecules used during preparation of samples) that are in principle more or less universal, and amplify all microbes more or less equally well. I haven't seen any new revisions to these primers since about 2012, so the community seems to have settled on the use of these.

 

[hart24601]

This is an important question.

The answer is no, this method (the same one used by the Earth Microbiome Project and the Coral Microbiome Project) doesnt provide direct cell counts. The methods for doing so accurately require fairly expensive equipment that I havent purchased yet. I do of course have access to DNA quantification (I use fluorescence, even more sensitive the absorbance based instruments like the NanoDrop) but the low concentrations of DNA produced here don't quantify well. qPCR would be the ideal in terms of cost vs throughput. I hope to add that to the lab at some point.

For now, we will have to settle for relative abundance, like most studies using this technology. I'd like to add the capability to do absolute quantification when I can.

You also raise an important point about some sequences potentially amplifying better than others. While I can't make any absolute, 100% guarantees, I will say that I am standing on the shoulders of giants here -- a large community of researchers has spent considerable time developing primers (DNA molecules used during preparation of samples) that are in principle more or less universal, and amplify all microbes more or less equally well. I haven't seen any new revisions to these primers since about 2012, so the community seems to have settled on the use of these.

Appreciate the response and look forward to seeing the ongoing results!

 

[Dan_P]

I'm assuming the time of day reference alludes to fishes feeding during the day? I think I can get around the question of low DOC v. low bacteria populations. I'll spike a sample with sugar and compare results to an unspiked sample.

Quick follow up on BOD discussion. First three observations to confirm that freshly prepared Instant Ocean might be diluent for high levels of DOC, e.g., skimmate. It is not bad but will try again with sterilized Instant Ocean. 4 th observation is start of work to home in on medium nutrition content to serve as a standard for assessing the activity of the WasteAway seed that I am using. Last two runs demonstrate why I add a WasteAway seed to my BOD samples: little BOD if I don’t. This in itself is worthy of a study. My Triton NDOC test shows low TOC and nitrogen. An AquaBiomics test might be in order.

 

[AquaBiomics]

Quick follow up on BOD discussion. First three observations to confirm that freshly prepared Instant Ocean might be diluent for high levels of DOC, e.g., skimmate. It is not bad but will try again with sterilized Instant Ocean. 4 th observation is start of work to home in on medium nutrition content to serve as a standard for assessing the activity of the WasteAway seed that I am using. Last two runs demonstrate why I add a WasteAway seed to my BOD samples: little BOD if I don’t. This in itself is worthy of a study. My Triton NDOC test shows low TOC and nitrogen. An AquaBiomics test might be in order.

Really interesting. To make sure I'm understanding the treatments, what was the source of nutrients added in the +nutrient treatments?

If I'm thinking about this correctly does this show that your tank contains 'refractory' DOC that is not readily consumed by existing microbes in the tank water, but is consumed by the added microbes in wasteaway?

These physiological measurements are really important, I look forward to reading more about them.

 

[Dana Riddle]

Quick follow up on BOD discussion. First three observations to confirm that freshly prepared Instant Ocean might be diluent for high levels of DOC, e.g., skimmate. It is not bad but will try again with sterilized Instant Ocean. 4 th observation is start of work to home in on medium nutrition content to serve as a standard for assessing the activity of the WasteAway seed that I am using. Last two runs demonstrate why I add a WasteAway seed to my BOD samples: little BOD if I don’t. This in itself is worthy of a study. My Triton NDOC test shows low TOC and nitrogen. An AquaBiomics test might be in order.

Very interesting! I received the nitrite reagents Saturday and now can begin work on getting my observations written. Your 'tank' BOD is pretty much in line with my tank. As for the bacterial products, I found the 'cycle booster' products contained a lot of ammonia. I'll have to give this some thoughts - appreciate your dedication!

 

[Dan_P]

Really interesting. To make sure I'm understanding the treatments, what was the source of nutrients added in the +nutrient treatments?

If I'm thinking about this correctly does this show that your tank contains 'refractory' DOC that is not readily consumed by existing microbes in the tank water, but is consumed by the added microbes in wasteaway?

These physiological measurements are really important, I look forward to reading more about them.

PO4 and glutamine were the nutrients which I just happen to have solutions of. The standard nutrients for testing seed bacteria are glucose and glutamic acid.

With regards to the presence of “refractory DOC”, great question! I don’t know. I have been debating how to interpret whether the data indicates something about the nature of the DOC or the make up of the bacterial population In the water or is something missing in my water to help the resident bacteria to consume it. This last notion is mixed up with that of the bacteria type in the water column, i.e., they are fussy eaters or need supplements to assimilate the carbon. At the moment, I may punt and just monitor the BOD with and without seed bacteria. If it is like everything else I trend, there is not going to be constant difference nor correlation between the two BOD’s.

 

[Dan_P]

Very interesting! I received the nitrite reagents Saturday and now can begin work on getting my observations written. Your 'tank' BOD is pretty much in line with my tank. As for the bacterial products, I found the 'cycle booster' products contained a lot of ammonia. I'll have to give this some thoughts - appreciate your dedication!

Heads up!

I am having second thoughts about recommending the use of WasteAway as a seed to BOD measurements. I looked at the effect of adding WasteAway to pasteurized Instant Ocean. I ran duplicate BOD flasks and measured oxygen in triplicate. I am a bit disappointed with my “sterile” technique, but that aside, there is a surprisingly large BOD with WasteAway addition. To make matters more confusing, the BOD was smaller for the same size addition of WasteAway to tank water. I am moving on from WasteAway for now. I hope @taricha figures this out

BOD5 Pasteurized Instant Ocean 0.83 +/- 0.33 ppm
BOD5 Pasteurized Instant Ocean+WasteAway 3.27 +/- 0.46 ppm

BOD5Tank Water 0.47 +/- 0.33 ppm
BOD5 Tank Water+WasteAway 1.77 +/- 0.33 ppm

 

[MnFish1]

id love to see someone with a year old+ tank with only dry rock. Im approaching the 10 month mark on a dry rock only tank.

I have results from such a tank. I dont know if @AquaBiomics wants to share the results of our experiment - I sent in 8 samples of various areas in the tank - In one - I simply took a sample of the tank water. In another I took a sample after having used my hand to stir up the water some - not to the point of cloudiness - but just over the rocks - here are the results (all using dry rock tank is about 4 years old) -

Here is the report after stirring up the water:


1000124

• This sample has established a new record as the most diverse sample I've ever tested, with over 800 different types of microbes detected.
• The balance score is quite low, indicating a different community composition than the typical water sample. If you examine the family abundance barplot you see that this sample has higher than usual levels of Flammeovirgaceae. This group is associated with marine sediments.
• While the levels of ammonia-oxidizing microbes are on the lower end of the typical range, the diversity is unusually high, with three different groups represented. Nitrite oxidizing bacteria are present at high levels.
• Cyanobacteria are present at high levels and very high diversity relative to their typical abundance in water + biofilm samples.
• No evidence of bacterial pathogens of fish or corals.
• I would guess this sample was taken from the sediments, rocks, or a similar part of the aquarium.

Here is the report taken just from the top of the tank:

1000127

• These samples had high and very similar diversity scores (410-469), showing a more diverse community than about 3/4 of the tanks I've sampled.
• These samples showed very low and similar balance scores (0.03 - 0.08), indicating a community that is very different than the typical reef tank. I think it's important to recognize that different doesn't necessarily mean unhealthy; this score indicates "atypical" rather than "unhealthy".
• The composition of the communities in all three samples is nearly identical (part 2).
• All show elevated levels of Flammeovirgaceae, a group associated with marine sediments. And they show lower than usual levels of Pelagibacteraceae, a free-living group that is abundant in the open ocean. They also show lower than usual levels of Alteromonadaceae, Flavobacteriaceae, and Vibrionaceae. Together, these differences suggest a very different nutrient environment in your tank than many other reef tanks. You can read more about these groups here.
• All three samples contain detectable ammonia-oxidizing microbes, although the types of AOB detected differ among samples. 2 out of 3 samples contain detected nitrite oxidizing bacteria, while they were not detected in the third. This suggests these groups were present at very low levels, highlighting the challenges of detecting rare bacterial types. This is why I always caution people that a lack of detection is not the same thing as absence.
• Two out of three samples showed no cyanobacteria, while the their showed moderate levels of a common type. I would not interpret any of these results as a cause for concern.
• All three samples were free of known bacterial pathogens of fish or corals. This is far from a universal result; perhaps 1 in 10 tanks has a detectable pathogen. So to have 3 clear tests seems to be convincing evidence these are not present in your system.

 

[MnFish1]

They’re absolutely on benthic surfaces, the test doesn’t rule that out. I’m estimating .01% of the tank surface area is swabbed for assessment and if the nitrifers are deeper into a biofilm than cotton can penetrate the test doesn’t rule them out. The test is complex such that I need to go reread to see if they have been measured in tanks but I missed it

Chunk out a small section of rock. Pestle it up into mud water using steel and a clean work area, I’m curious to see if the test can’t find nitrifiers and if not-where they exist to handle our fish bioload

-any genus that accomplishes nitrification would be confirming to find and amazing if they’re not being found in benthic microfilm layers. The sheer number of nanos that perform 100% water changes and kill no fish attest they’re on surfaces in some arrangement tbd

It would be neat to measure where the genomics system can find nitrifying bacteria...aim the sampling as required and try and post them as measures found. Over time as reports pattern we can gain info. Once we clearly find and zone where the nitrifiers are then we can know which bacteria were associated with them and see how big of a scope the test measures

Check out my results - 'from the water column'

 

[brandon429]

ok re reading right now thanks for heads up

 

[MnFish1]

By the way - in the experiment that @AquaBiomics and I did - I sent 8 unknown samples - one swab and 1 liquid sample from 'unknown' sources. I will say - though I'm waiting for the results for the 'water part' vs the 'swab part' separated out - which I think will be very helpful given the way I did the sampling - One of the samples was merely the 'fixative' - i.e. a negative control - and as expected - nothing was found bacteria wise. Another sample was from my RO water and a swab of my RO tank - Another was an 'unknown' that I'm not going to tell him yet - but - both the RO - and the other 'unknown' - were accurately identified as 'not being from a reef tank... So congrats to @AquaBiomics

Without telling him 'which samples are 'what'. I took 1 sample from output of my skimmer - and one from the skim mate, One from the bottom of the tank and a swab of rock - as well as the other samples I already discussed

 

[AquaBiomics]

I have results from such a tank. I dont know if @AquaBiomics wants to share the results of our experiment - I sent in 8 samples of various areas in the tank - In one - I simply took a sample of the tank water. In another I took a sample after having used my hand to stir up the water some - not to the point of cloudiness - but just over the rocks - here are the results (all using dry rock tank is about 4 years old) -

Here is the report after stirring up the water:


1000124

• This sample has established a new record as the most diverse sample I've ever tested, with over 800 different types of microbes detected.
• The balance score is quite low, indicating a different community composition than the typical water sample. If you examine the family abundance barplot you see that this sample has higher than usual levels of Flammeovirgaceae. This group is associated with marine sediments.
• While the levels of ammonia-oxidizing microbes are on the lower end of the typical range, the diversity is unusually high, with three different groups represented. Nitrite oxidizing bacteria are present at high levels.
• Cyanobacteria are present at high levels and very high diversity relative to their typical abundance in water + biofilm samples.
• No evidence of bacterial pathogens of fish or corals.
• I would guess this sample was taken from the sediments, rocks, or a similar part of the aquarium.

Here is the report taken just from the top of the tank:

1000127

• These samples had high and very similar diversity scores (410-469), showing a more diverse community than about 3/4 of the tanks I've sampled.
• These samples showed very low and similar balance scores (0.03 - 0.08), indicating a community that is very different than the typical reef tank. I think it's important to recognize that different doesn't necessarily mean unhealthy; this score indicates "atypical" rather than "unhealthy".
• The composition of the communities in all three samples is nearly identical (part 2).
• All show elevated levels of Flammeovirgaceae, a group associated with marine sediments. And they show lower than usual levels of Pelagibacteraceae, a free-living group that is abundant in the open ocean. They also show lower than usual levels of Alteromonadaceae, Flavobacteriaceae, and Vibrionaceae. Together, these differences suggest a very different nutrient environment in your tank than many other reef tanks. You can read more about these groups here.
• All three samples contain detectable ammonia-oxidizing microbes, although the types of AOB detected differ among samples. 2 out of 3 samples contain detected nitrite oxidizing bacteria, while they were not detected in the third. This suggests these groups were present at very low levels, highlighting the challenges of detecting rare bacterial types. This is why I always caution people that a lack of detection is not the same thing as absence.
• Two out of three samples showed no cyanobacteria, while the their showed moderate levels of a common type. I would not interpret any of these results as a cause for concern.
• All three samples were free of known bacterial pathogens of fish or corals. This is far from a universal result; perhaps 1 in 10 tanks has a detectable pathogen. So to have 3 clear tests seems to be convincing evidence these are not present in your system.

Thanks for bringing this back up.

Sorry for the delay - my only hesitation in writing up our experiment was that I havent had a chance to run the separate analysis of swabs vs filters that we discussed.

Very interesting to hear your description of how these samples were generated. That is quite an effect from stirring up your tank! I always advise people to sample an undisturbed aquarium based on previous studies that showed water changes changed the water community. But honestly this is an even bigger effect than I'd have expected.

The second sample you list in this post, I discussed along with the other two that showed nearly identical profiles. Since we're discussing this now, can you clarify whether those three were replicates?

I will get back to the comparison of swab vs filters, and the identification of your other mystery samples, as soon as I can.

 

[AquaBiomics]

By the way - in the experiment that @AquaBiomics and I did - I sent 8 unknown samples - one swab and 1 liquid sample from 'unknown' sources. I will say - though I'm waiting for the results for the 'water part' vs the 'swab part' separated out - which I think will be very helpful given the way I did the sampling - One of the samples was merely the 'fixative' - i.e. a negative control - and as expected - nothing was found bacteria wise. Another sample was from my RO water and a swab of my RO tank - Another was an 'unknown' that I'm not going to tell him yet - but - both the RO - and the other 'unknown' - were accurately identified as 'not being from a reef tank... So congrats to @AquaBiomics

Without telling him 'which samples are 'what'. I took 1 sample from output of my skimmer - and one from the skim mate, One from the bottom of the tank and a swab of rock - as well as the other samples I already discussed

Great! Thanks, this has been really interesting exploring unknown samples. Now you've got me all curious again about these, I will have to move this analysis back to the front burner.

 

[MnFish1]

Thanks for bringing this back up.

Sorry for the delay - my only hesitation in writing up our experiment was that I havent had a chance to run the separate analysis of swabs vs filters that we discussed.

Very interesting to hear your description of how these samples were generated. That is quite an effect from stirring up your tank! I always advise people to sample an undisturbed aquarium based on previous studies that showed water changes changed the water community. But honestly this is an even bigger effect than I'd have expected.

The second sample you list in this post, I discussed along with the other two that showed nearly identical profiles. Since we're discussing this now, can you clarify whether those three were replicates?

I will get back to the comparison of swab vs filters, and the identification of your other mystery samples, as soon as I can.

I think its best to do once the swab/liquid comes back

 

[MnFish1]

Thanks for bringing this back up.

Sorry for the delay - my only hesitation in writing up our experiment was that I havent had a chance to run the separate analysis of swabs vs filters that we discussed.

Very interesting to hear your description of how these samples were generated. That is quite an effect from stirring up your tank! I always advise people to sample an undisturbed aquarium based on previous studies that showed water changes changed the water community. But honestly this is an even bigger effect than I'd have expected.

The second sample you list in this post, I discussed along with the other two that showed nearly identical profiles. Since we're discussing this now, can you clarify whether those three were replicates?

I will get back to the comparison of swab vs filters, and the identification of your other mystery samples, as soon as I can.

I think its best to do it after the swab/filter comes back - but no they were not replicates

 

[taricha]

Great discussion here! missed it earlier.

Perhaps in some tanks, the eukaryotes (from algae to animals) take up so much of the N that little is left for microbial populations, which correspondingly decline in size.

An analogous situation is with a freshwater planted tank. When your plant density gets really high the plants take over a substantial portion of the biological filtration for the tank. I know this, because back in my planted tank days, I could sometimes see an ammonia spike in my planted tank if I trimmed too many plants, too fast. That was a surprise!

This might be a distinction without much difference, but I wonder if in a well-established system the bacteria comprising the biofilm of the livestock itself (think coral mucus) becomes the central hub of ammonia oxidation / nitrification?
"During daytime, the mucus conditions are oxic, and nitrification takes place via Archaea similar to N. maritimus. During night-time, the conditions in the mucus layer are anoxic and denitrification takes place via denitrifying Archaea. Nitrogen products (nitrite and nitrate) can thus be recycled and assimilated into the coral holobiont." - Global distribution and diversity of coral-associated Archaea and their possible role in the coral holobiont nitrogen cycle

Can we do something similar for nitrite using BOD5? The reason I ask is that NOB are undetectable in a greater number of tanks than AOB/AOA. And as people have been discussing, other organisms (like algae and some animals) actively compete for ammonia. It would be interesting to test for nitrite oxidizing activity in your water, if its possible to do something analogous to the ammonia test.

I've done a couple of quick looks at processing of elevated nutrients in a bottle of tank water kept in the dark. The full cycle appears in just a few days: ammonia production, ammonia to nitrite, nitrite to nitrate. All happen in measurable amounts in a few days to a week.
I think @Dan_P has seen similar fairly quick "cycling" in just tank water.

In another I took a sample after having used my hand to stir up the water some - not to the point of cloudiness - but just over the rocks - here are the results (all using dry rock tank is about 4 years old) -

Here is the report after stirring up the water:

That was a fun test and a really cool result.

 

[MnFish1]

Thanks for bringing this back up.

Sorry for the delay - my only hesitation in writing up our experiment was that I havent had a chance to run the separate analysis of swabs vs filters that we discussed.

Very interesting to hear your description of how these samples were generated. That is quite an effect from stirring up your tank! I always advise people to sample an undisturbed aquarium based on previous studies that showed water changes changed the water community. But honestly this is an even bigger effect than I'd have expected.

The second sample you list in this post, I discussed along with the other two that showed nearly identical profiles. Since we're discussing this now, can you clarify whether those three were replicates?

I will get back to the comparison of swab vs filters, and the identification of your other mystery samples, as soon as I can.

That will be important

 

[MnFish1]

Great discussion here! missed it earlier.



This might be a distinction without much difference, but I wonder if in a well-established system the bacteria comprising the biofilm of the livestock itself (think coral mucus) becomes the central hub of ammonia oxidation / nitrification?
"During daytime, the mucus conditions are oxic, and nitrification takes place via Archaea similar to N. maritimus. During night-time, the conditions in the mucus layer are anoxic and denitrification takes place via denitrifying Archaea. Nitrogen products (nitrite and nitrate) can thus be recycled and assimilated into the coral holobiont." - Global distribution and diversity of coral-associated Archaea and their possible role in the coral holobiont nitrogen cycle


I've done a couple of quick looks at processing of elevated nutrients in a bottle of tank water kept in the dark. The full cycle appears in just a few days: ammonia production, ammonia to nitrite, nitrite to nitrate. All happen in measurable amounts in a few days to a week.
I think @Dan_P has seen similar fairly quick "cycling" in just tank water.


That was a fun test and a really cool result.

But if one is wanting to decide the bacteria types in his/her tanks - it seems like that would be important....

 

[Cv111]

I find this information extremely interesting, and wonder if there are practical implications to be gleaned from it...

I'm late to the party, but nitrifying bacteria are not in the water. They form biofilms on the rockwork. You'd need to resuspend those before taking your sample to see high abundance. Most likely you will only get a little bit in the water itself.