Problems Due to Bacterial Imbalances? AquaBiomics Test Results.

[sarcophytonIndy]

I think the “Typical sample” comes from an average from tanks tested. There’s no saying if a certain test is better than other at the moment is just out of curiosity but some patterns start to emerge from tanks tested.

Right, but my point is that the "average tank tested" may be very different than the average tank in existence. I'm guessing a lot of newbie tanks won't be included.

 

[brandon429]

The sampling method matters tremendously, when sending in samples are people including benthic/substrate swabs or scrapes, or are all water samples just topwater sent in

Getting the bioslicks that house nitrifiers wouldn't be challenging if scraped portions of slicks get analyzed.

We have work threads where we do very harsh things to reef tanks like tap rinse an entire sandbed, or drain a tank to the sand and leave rocks exposed thirty mins

The nitrifiers are so locked in, we know they won't die/we never lose tanks to recycles so to me that says basic substrate swabbing won't dislodge them, some form of scrape will be required to assess biofilm insulation

 

[lexinverts]

The sampling method matters tremendously, when sending in samples are people including benthic/substrate swabs or scrapes, or are all water samples just topwater sent in

Getting the bioslicks that house nitrifiers wouldn't be challenging if scraped portions of slicks get analyzed.

We have work threads where we do very harsh things to reef tanks like tap rinse an entire sandbed, or drain a tank to the sand and leave rocks exposed thirty mins

The nitrifiers are so locked in, we know they won't die/we never lose tanks to recycles so to me that says basic substrate swabbing won't dislodge them, some form of scrape will be required to assess biofilm insulation

Good point about sampling method being important. The sampling protocol was made simple so that everyone could easily sample their tanks in exactly the same way.

The sampling method includes both a water sample and a swab of the biofilm on the return pipe.
Results show that both water and biofilm samples include "benthic" bacteria. Benthic/surface living bacteria are shed into the water column in recirculating aquariums, so, with both the water sample and the swab, they are sampled in two different ways.

 

[AquaBiomics]

I received results from AquaBiomics showing bacteria populations in my reef tank. As it turned out, this test shows my tank has bacteria indicative of a mature reef - a total of 442 bacteria types were found with a diversity score of .92 (on a scale of 0 to 1.0.) Interestingly, nitrifying bacteria are practically non-existent. Coral and fish pathogens were not found as well.
I tend to think using Florida live rock is at least partially responsible for the diversity score. Has anyone had this test done where only dry rock was used?

Hi Dana,

Thanks for sharing your results. I was especially curious to read your report because of your use of live phytoplankton. Here are some thoughts on yours --

Your tank has an interesting community -- like the average tank, and like many marine environments (especially open ocean and other low nutrient habitats), yours has a high level of Pelagibacteraceae. We've been discussing this group in posts about others' results... this group is interesting to me because quantitatively its one of the most abundant groups in the ocean, so the very low levels of this group in some tanks represents a big difference from the communities corals encounter in nature.

Unlike the average, your tank has much higher levels of Alteromonadaceae. But I should point out that this is one of the most variable families, ranging from from 0.02% - 73%. This group is known to use a broad range of dissolved nutrients, and blooms in high glucose conditions. So I suspect variation in this group may reflect nutrient conditions in our tanks, especially DOC which few of us measure. (Although it strikes me that maybe you do! Have you measured DOC in your tanks?)

In the open ocean, microbial communities appear to be driven largely by the kind of algal communities that bloom and then lyse, releasing species-specific mixes of nutrients for the microbes. (https://www.nature.com/articles/ismej2015105) So I suspect that your dosing of live phytoplankton is contributing to the dominance of these two groups.

It would be really interesting to know how different kinds of microalgae would affect the microbiome, that might be a great natural way of adjusting our tanks' microbiomes.

--

The low levels of ammonia-oxidizing microbes (AOA & AOB) and nitrite oxidizing bacteria (NOB) in your sample may be unexpected but they reflect a trend I've seen in many tanks. Including my own home tanks (apparently I've done a better job of cultivating the right microbes in my experiments than in my hobby).

I want to add one detail about this finding for context -- both of your samples (water and biofilm) contained plenty of DNA and amplified very well. In fact, we got double the expected coverage for your biofilm sample, so the low levels don't result from undersampling the community.

I'm starting to think that while nitrifying microbes play important roles in establishing a tank, the amount of ammonia and nitrite processed through these populations, and therefore their population sizes, varies widely across tanks. Whether the competition is from large coral colonies, macroalgae in refugiums, or as in your case from live phytoplankton, prokaryotes in a mature tank have to compete with a bunch of other hungry mouths.

 

[AquaBiomics]

The sampling method matters tremendously, when sending in samples are people including benthic/substrate swabs or scrapes, or are all water samples just topwater sent in

Getting the bioslicks that house nitrifiers wouldn't be challenging if scraped portions of slicks get analyzed.

We have work threads where we do very harsh things to reef tanks like tap rinse an entire sandbed, or drain a tank to the sand and leave rocks exposed thirty mins

The nitrifiers are so locked in, we know they won't die/we never lose tanks to recycles so to me that says basic substrate swabbing won't dislodge them, some form of scrape will be required to assess biofilm insulation

I do see your point, but think you may be underestimating how much material a swab removes when its scraped across a pipe biofilm. Depending on the pipe, we can get some pretty thick, visible chunks. I agree that the biofilm is too tough to be entirely removed by washing or swabbing, but the goal here is just to get enough of the biofilm to analyze. Some of the swabs I get back are pretty gross. Makes me glad I'm in the aquarium microbiome business instead of the human stool microbiome business.

 

[Dana Riddle]

Hi Dana,

Thanks for sharing your results. I was especially curious to read your report because of your use of live phytoplankton. Here are some thoughts on yours --

Your tank has an interesting community -- like the average tank, and like many marine environments (especially open ocean and other low nutrient habitats), yours has a high level of Pelagibacteraceae. We've been discussing this group in posts about others' results... this group is interesting to me because quantitatively its one of the most abundant groups in the ocean, so the very low levels of this group in some tanks represents a big difference from the communities corals encounter in nature.

Unlike the average, your tank has much higher levels of Alteromonadaceae. But I should point out that this is one of the most variable families, ranging from from 0.02% - 73%. This group is known to use a broad range of dissolved nutrients, and blooms in high glucose conditions. So I suspect variation in this group may reflect nutrient conditions in our tanks, especially DOC which few of us measure. (Although it strikes me that maybe you do! Have you measured DOC in your tanks?)

In the open ocean, microbial communities appear to be driven largely by the kind of algal communities that bloom and then lyse, releasing species-specific mixes of nutrients for the microbes. (https://www.nature.com/articles/ismej2015105) So I suspect that your dosing of live phytoplankton is contributing to the dominance of these two groups.

It would be really interesting to know how different kinds of microalgae would affect the microbiome, that might be a great natural way of adjusting our tanks' microbiomes.

--

The low levels of ammonia-oxidizing microbes (AOA & AOB) and nitrite oxidizing bacteria (NOB) in your sample may be unexpected but they reflect a trend I've seen in many tanks. Including my own home tanks (apparently I've done a better job of cultivating the right microbes in my experiments than in my hobby).

I want to add one detail about this finding for context -- both of your samples (water and biofilm) contained plenty of DNA and amplified very well. In fact, we got double the expected coverage for your biofilm sample, so the low levels don't result from undersampling the community.

I'm starting to think that while nitrifying microbes play important roles in establishing a tank, the amount of ammonia and nitrite processed through these populations, and therefore their population sizes, varies widely across tanks. Whether the competition is from large coral colonies, macroalgae in refugiums, or as in your case from live phytoplankton, prokaryotes in a mature tank have to compete with a bunch of other hungry mouths.

Thank you for your response - I need to read through all your posts, take notes, and educate myself. As for your question about DOC, there is an easy way (albeit crudely) to estimate it. It's the 5-day Biochemical Oxygen Demand (BOD5) where oxygen consumption within a 300 ml sample held at 20C in darkness is determined. The EPA allows reporting of Total Organic Carbon (TOC) when a correlation with BOD5 has been established. I haven't tested my current aquarium since multiple previous testing never had a DO drop >2 mg/L - they were always 0.2 mg/L or so. I'll take a look and report back when the test is completed. I need use that expensive DO meter that is just using up bench space.

 

[Scott Campbell]

Thank you for your response - I need to read through all your posts, take notes, and educate myself. As for your question about DOC, there is an easy way (albeit crudely) to estimate it. It's the 5-day Biochemical Oxygen Demand (BOD5) where oxygen consumption within a 300 ml sample held at 20C in darkness is determined. The EPA allows reporting of Total Organic Carbon (TOC) when a correlation with BOD5 has been established. I haven't tested my current aquarium since multiple previous testing never had a DO drop >2 mg/L - they were always 0.2 mg/L or so. I'll take a look and report back when the test is completed. I need use that expensive DO meter that is just using up bench space.

One of the interesting observations looking at posted results is that so many tanks end up with one to two dominant strains of bacteria. And your results appear to be the most extreme of any posted. Just eyeballing it - it appears two strains of bacteria represent 90+% of your total bacteria. Have no idea what that means - but seems rather amazing given the diversity of strains also represented in your tank.

 

[Dan_P]

Thank you for your response - I need to read through all your posts, take notes, and educate myself. As for your question about DOC, there is an easy way (albeit crudely) to estimate it. It's the 5-day Biochemical Oxygen Demand (BOD5) where oxygen consumption within a 300 ml sample held at 20C in darkness is determined. The EPA allows reporting of Total Organic Carbon (TOC) when a correlation with BOD5 has been established. I haven't tested my current aquarium since multiple previous testing never had a DO drop >2 mg/L - they were always 0.2 mg/L or so. I'll take a look and report back when the test is completed. I need use that expensive DO meter that is just using up bench space.

Time of day that you take a BOD sample might matter. Also, if you are not seeding your BOD test, you do not know whether the low BOD is because of low DOC, of low pelagic bacteria numbers or both.

@taricha has recently been using BOD in his substrate studies and is generating interesting results measuring oxygen levels with an O2 meter. He might have something to this discussion.

The conundrum we face with regard to DOC measurements of any kind is how they correlate to anything that is going on in the aquarium. DOC, like NO3 and PO4, might only be an imperfect surrogate marker for activity that is mostly happening on the surfaces of the aquarium. We probably need to start developing methods to measure what is happening on rock surfaces and within the substrate to really understand the “health” of the aquarium, where probably by no coincidence, most nuisance organisms seem to gather.

 

[Dana Riddle]

Time of day that you take a BOD sample might matter. Also, if you are not seeding your BOD test, you do not know whether the low BOD is because of low DOC, of low pelagic bacteria numbers or both.

@taricha has recently been using BOD in his substrate studies and is generating interesting results measuring oxygen levels with an O2 meter. He might have something to this discussion.

The conundrum we face with regard to DOC measurements of any kind is how they correlate to anything that is going on in the aquarium. DOC, like NO3 and PO4, might only be an imperfect surrogate marker for activity that is mostly happening on the surfaces of the aquarium. We probably need to start developing methods to measure what is happening on rock surfaces and within the substrate to really understand the “health” of the aquarium, where probably by no coincidence, most nuisance organisms seem to gather.

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.

 

[AquaBiomics]

Right, but my point is that the "average tank tested" may be very different than the average tank in existence. I'm guessing a lot of newbie tanks won't be included.

I think you're absolutely right, the hobbyists and professional coral growers who have contributed to this database are far from amateurs, they're people deep enough down the reefing rabbit hole to want to take part in something like this! So although there are some young tanks in the database I doubt there are any newbie reefers in the database.

It makes me think I should add a regularly updated table: descriptive statistics of tanks in the database. It seems that things like range of tank ages, etc. can be described without violating anyone's privacy.
Hopefully a table can describe this better than a sentence; I'll link it back here when its finished.

 

[AquaBiomics]

Thank you for your response - I need to read through all your posts, take notes, and educate myself. As for your question about DOC, there is an easy way (albeit crudely) to estimate it. It's the 5-day Biochemical Oxygen Demand (BOD5) where oxygen consumption within a 300 ml sample held at 20C in darkness is determined. The EPA allows reporting of Total Organic Carbon (TOC) when a correlation with BOD5 has been established. I haven't tested my current aquarium since multiple previous testing never had a DO drop >2 mg/L - they were always 0.2 mg/L or so. I'll take a look and report back when the test is completed. I need use that expensive DO meter that is just using up bench space.

That's awesome, I ask about DOC levels and you just happen to have a DO meter on hand and the experience to use it for estimating DOC. I'll be curious to hear what you find.

Thinking more about this, it seems timing of the phytoplankton dosing is important for both the microbial and DOC measurements. I dont remember, do you add this continuously or in regular e.g. daily additions?

 

[Dana Riddle]

That's awesome, I ask about DOC levels and you just happen to have a DO meter on hand and the experience to use it for estimating DOC. I'll be curious to hear what you find.

Thinking more about this, it seems timing of the phytoplankton dosing is important for both the microbial and DOC measurements. I dont remember, do you add this continuously or in regular e.g. daily additions?

The phyto additions are made at least twice daily (I thaw two Mysis cubes in the green water and feed in the am and pm, plus a random addition sometime during the day. It will be easy enough to take samples throughout the day. I'll get to work on sterilizing the glassware.
Have you (or have you considered) making a presentation at MACNA or any other major conference?

 

[Crotalus]

I've always heard that nitrifying bacteria resides on surfaces and not in the water column. I guess this test bears that out.

 

[brandon429]

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

 

[AquaBiomics]

The phyto additions are made at least twice daily (I thaw two Mysis cubes in the green water and feed in the am and pm, plus a random addition sometime during the day. It will be easy enough to take samples throughout the day. I'll get to work on sterilizing the glassware.
Have you (or have you considered) making a presentation at MACNA or any other major conference?

Haven't done one yet but I did a fair amount of public speaking during my academic time, it'd be fun to present this stuff at MACNA or similar.

Really looking forward to hearing how the DOC measurements go!

 

[AquaBiomics]

I've always heard that nitrifying bacteria resides on surfaces and not in the water column. I guess this test bears that out.

I find them in both. I've tested water and biofilm in a bunch of tanks now, and I find both ammonia-oxidizing and nitrite-oxidizing bugs in both the water and the biofilm. Their relative abundance in these two communities differs, and sometimes theyre detected in one but not the other.

While I do detect them in both fractions, I don't see this as challenging the standard understanding that most nitrifying microbes are surface-associated. The long studied differences in structure and motility of different microbial families supports that view. I think some of these cells are just periodically dislodged from surfaces in by water flow, enough to be picked up in the sample, while others are truly free living.

 

[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.

I observed a spike in BOD before feeding as well as after. Maybe the fish were excited when I showed up to sample the water just before feeding time. I am repeating the time of day study several more times.

Would spiking sugar occur every time you do a BOD observation to correct the BOD result for varying levels of bacteria?

 

[Dana Riddle]

I observed a spike in BOD before feeding as well as after. Maybe the fish were excited when I showed up to sample the water just before feeding time. I am repeating the time of day study several more times.

Would spiking sugar occur every time you do a BOD observation to correct the BOD result for varying levels of bacteria?

Adding sugar (or other carbon source) would verify the presence of carbonaceous bacteria, as long as there is a substantial drop in dissolved oxygen over the course of 5 days. I would also add a chemical that inhibits nitrification to another sample. So, a control sample to determine oxygen consumption in a 'straight' 300 ml sample. Another 300 ml sample with sugar added to see if DO drop is limited by bacteria or biodegradable substances. A third sample spiked with ammonia to determine the presence of nitrifying bacteria.

 

[Dan_P]

Adding sugar (or other carbon source) would verify the presence of carbonaceous bacteria, as long as there is a substantial drop in dissolved oxygen over the course of 5 days. I would also add a chemical that inhibits nitrification to another sample. So, a control sample to determine oxygen consumption in a 'straight' 300 ml sample. Another 300 ml sample with sugar added to see if DO drop is limited by bacteria or biodegradable substances. A third sample spiked with ammonia to determine the presence of nitrifying bacteria.

I follow you. I hadn’t thought about ammonia spiking.

 

[PSXerholic]

Thanks! I'll take a look!

Do so, I joined and will perform tests prior and after a Witchhazel treatment ;-)