Bacterial analysis of reef tanks

[MrAnderson]

I'm considering offering a service for the reefing community but I'm not sure anyone would want it. I literally thought of this just yesterday, so bear with me as I explain.

 

People go crazy trying to ID the odd snail, crab, polyp etc. Sometimes a definitive ID can't be made. However, the urge to know EXACTLY what is in your tank seems to be of great curiosity to many. Now that we know that Nitrobacter and Nitrosomonas aren't the major nitrogen cycle bacteria in reef environments, would people be curious about the identifications of the bacteria making up the community in their tanks? In nature, every bacterial community is unique, with highly varied populations and species between them. (http://www.pnas.org/content/108/34/14288.short) This tells us that each tank's bacterial community, with it's own unique seeding material and selective conditions, would be unique.

 

The deliverable would be a survey of bacterial populations in a tank, along with a diversity measurement. Natural live rock reef biofilm bacterial communities usually consist of somewhere between 200-500 different species. It's impossible to identify them all, but just as a survey of 1000 households is statistically sufficient to determine values for national political polls, a survey of about 50 samples is sufficient statistically to sketch out a bacterial community. The bacterial IDs would be made using 16S analysis with DNA sequencing. Each sequence would be analyzed for a known "match" via GenBank. For example, here's a close match for one of the samples from my live rock samples, a purple, sulfur-reducing bacteria from the Order Chromatiales:

 

http://www.ncbi.nlm.nih.gov/nucleotide/169788200?report=genbank&log$=nuclalign&blast_rank=90&RID=E6BSZM5T01R

 

If this were someone's sample, this would be one of 50 IDs, and this particular sample had a diversity value of approximately 425 different species (this was from the interior, anoxic portion of live rock from Manado, Indonesia.)

 

One could gauge the microbiological health of the tank by the diversity value, and the 50 species/genus-level identifications give a qualitative idea of the community composition.

 

I find this type of thing extremely interesting, but I don't know that others share this level of curiosity.

 

Because this is a molecular-level identifi cation and lab materials and reagents (and DNA sequencing) are expensive, the price would be in the hundreds of dollars. I don't expect to be overwhelmed by demand lol, but I am curious if anyone out there would be interested. Obviously this would be for the serious hobbyist and not for everyone.

 

Share thoughts, ask questions!

[RayWhisperer]

I'd be interested in this, if done on a large enough scale as to limit cost prohibitiveness. However, I'm wondering exactly how much rock would be required. While you posted a portion taken from the interior, anoxic part. Roughtly how large a portion and how deep?

 

As well as proper handling of the rock after collection. As I believe (read this as do not know) there could be a lot of contamination from the interior parts being exposed to other factors like an oxygen rich environment, or even air. Unless, of course, you are suggesting whole rocks be sent. In which case I'd have to decline, as I only have 3 large pieces in my system.

[Reeftech]

This would be cool to have one sample from each state.Then we could see if there are any factors of temperature and elevations that play a role in the survival of some of these bacterias.

 

Also since the east coast and west coast get specimens that are local it will also show different strains.

 

Do you think a used filter floss would be just as sufficient as a piece of rock?Im sure if floss was left in a tank for a week or so it will have collected the same bacteria.

 

This is just my theory on the subject.

[MrAnderson]

I'd be interested in this, if done on a large enough scale as to limit cost prohibitiveness. However, I'm wondering exactly how much rock would be required. While you posted a portion taken from the interior, anoxic part. Roughtly how large a portion and how deep?

 

As well as proper handling of the rock after collection. As I believe (read this as do not know) there could be a lot of contamination from the interior parts being exposed to other factors like an oxygen rich environment, or even air. Unless, of course, you are suggesting whole rocks be sent. In which case I'd have to decline, as I only have 3 large pieces in my system.

i just use a scraping of material about the size of a pea. there are various ways to insure proper handling - some clinical diagnostic sample collection is done by patients at home using sterile collection materials and solid instructions.

 

for the interior portion my grad student cracked open the rock in a hood and scraped using flame-sterilized scrapers

This would be cool to have one sample from each state.Then we could see if there are any factors of temperature and elevations that play a role in the survival of some of these bacterias.

 

Also since the east coast and west coast get specimens that are local it will also show different strains.

 

Do you think a used filter floss would be just as sufficient as a piece of rock?Im sure if floss was left in a tank for a week or so it will have collected the same bacteria.

 

This is just my theory on the subject.

filter floss would be fine, but a rock scraping would be ideal. bacteria found in the water column is a subset of those found on live rock.

 

 

You might be better off with a "Gillette" type business model. Offer the testing services at a loss, but offer cultures of bacteria that might be missing from the Client's system for sale at an insane profit to make up for it. It doesn't seem like it does anyone any good to know that their system has bacteria "a", "b" and "c", but is missing bacteria "x", "y" and "z", if you can't do anything about it.

this isn't a typical business startup. i already have an active academic lab and resources to keep it running. this was intended to be a side project in addition to offering commercial genomic services, generally to other researchers. for those that are familiar with academic science, i have a research lab and basically have permission to start up a core facility.

 

hey, if there's insufficient interest, that's that. no skin off my nose, either way i go back to lab and keep doing my job.

[RayWhisperer]

So, A. Would you be able to use a small rock? I'm just not sure at what depth within the rock the anoxic zone is. I understand it can vary greatly depending on things like porosity. I'm just wondering if I could yank out a 1"x2" piece of rubble from one of the back chambers and have an adequate anoxic area within such a small rock.

[MrAnderson]

it doesn't have to be from the inside of a rock, i just threw that in there because it was a convenient example of a sample from a study we did comparing bacteria on the surface of the rock with those inside.

 

however, as reeftech pointed out, the range of sample types to choose can get crazy if you get curious about numerous variables. i think the least invasive sample would be a simple surface scraping and it can come from anywhere in the tank.

 

 

also, blueprintguy, that IS a great suggestion, but not really viable in this case. almost all of the bacterial species present on live rock is VBNC - viable, but nonculturable. i couldn't make commercial cultures representing natural populations if i wanted to.

[lakshwadeep]

If I had the money, I would like this a lot. Bacterial 16S analysis would hopefully also give relative abundances. That's what some people in a lab I'm rotating with do for the metagenome of tunicates and sponges (I'm not sure exactly what was the method). You might look at how the Sargasso Sea metagenomic sampling was done, which have added a large amount of information to databases. Also, archaea are a possibility, and one of the first members (and culturable, no less) of a proposed new clade of ammonia-oxidizing archaea (again upsetting the standard model of the nitrogen cycle) was actually identified in a Seattle aquarium.

http://www.nature.com/nature/journal/v437/n7058/full/nature03911.html

 

I think using sand may be an easier and possibly more homogenous option for sampling. Then again, rocks may have again some unique environments. Overall, I think first finding the diversity would be important, including detailed documentation of the stocking/age/origin. If the dataset is large enough, perhaps some correlations can be made in terms of tank health and genetic diversity.

[metrokat]

I'm just going to bump this thread because Mr. Anderson rocks.

[TheUnfocusedOne]

Aww you deleted my post. Why you gotta hate on me A?

 

 

Srsly though, I like this idea. I was thinking about doing something like this a while back but I don't have access to any of the equipment. Do you guys have a sequencer?

[MrAnderson]

If I had the money, I would like this a lot. Bacterial 16S analysis would hopefully also give relative abundances. That's what some people in a lab I'm rotating with do for the metagenome of tunicates and sponges (I'm not sure exactly what was the method). You might look at how the Sargasso Sea metagenomic sampling was done, which have added a large amount of information to databases. Also, archaea are a possibility, and one of the first members (and culturable, no less) of a proposed new clade of ammonia-oxidizing archaea (again upsetting the standard model of the nitrogen cycle) was actually identified in a Seattle aquarium.

http://www.nature.com/nature/journal/v437/n7058/full/nature03911.html

 

I think using sand may be an easier and possibly more homogenous option for sampling. Then again, rocks may have again some unique environments. Overall, I think first finding the diversity would be important, including detailed documentation of the stocking/age/origin. If the dataset is large enough, perhaps some correlations can be made in terms of tank health and genetic diversity.

 

i have a student working up a methodology for Archaea, I think we're pretty much there, he just needs to do the cloning. But yes, the nitrogen cycle in marine environments is completely misunderstood. Thanks for reinforcing that point, because I do think this type of analysis can be interesting and useful.

 

sand might be a good idea but i haven't tried it. so far i've been working on biofilms but it might be worth a try.

 

Aww you deleted my post. Why you gotta hate on me A?

 

 

Srsly though, I like this idea. I was thinking about doing something like this a while back but I don't have access to any of the equipment. Do you guys have a sequencer?

 

because you wrote "this is a dumb idea", dumdum. this isn't the lounge and you're lucky i don't timeout you.

[TheUnfocusedOne]

im not a dumdum...

[Hans Dorn]

 

also, blueprintguy, that IS a great suggestion, but not really viable in this case. almost all of the bacterial species present on live rock is VBNC - viable, but nonculturable. i couldn't make commercial cultures representing natural populations if i wanted to.

What does this say w.r.t all the bacteria supplements you can buy for your tank?

[MrAnderson]

i left that unsaid lol... but yeah, they're not representative of any natural bacterial communities or populations.

[tibbsy07]

This is a great idea! I've got a little thing going right now trying to make glycerol stocks of the scrapings from my rock so I can seed future tanks. I'm not worried about not being able to culture it in the lab, as I'm only taking the samples and freezing them. And if I'm only using it to start a cycle, I'm not too worried about the glycerol. The glycerol in my yeast stocks hasn't hurt my homebrew

 

How cost effective would this really be, though? Sequencing here is still a thousand dollars at least, ignoring any reagents needed for the isolation process. What kind of sequencing are you doing? I am most definitely interested.

 

i left that unsaid lol... but yeah, they're not representative of any natural bacterial communities or populations.

THANK YOU! Finally. I hate these products.

[TheUnfocusedOne]

You could also see how populations of bacteria shift overtime within a single tank. That might be easier to start off with, and would let you do some method refinement before jumping in. I would guess there would be a winnowing process that would reduce the diversity of the bacteria overtime.

 

Who knows, maybe this part of the reason why some people have issues sustaining older tanks? A lack of genetic diversity would make them less able to react to significant and abrupt changes.

[MrAnderson]

This is a great idea! I've got a little thing going right now trying to make glycerol stocks of the scrapings from my rock so I can seed future tanks. I'm not worried about not being able to culture it in the lab, as I'm only taking the samples and freezing them. And if I'm only using it to start a cycle, I'm not too worried about the glycerol. The glycerol in my yeast stocks hasn't hurt my homebrew

 

How cost effective would this really be, though? Sequencing here is still a thousand dollars at least, ignoring any reagents needed for the isolation process. What kind of sequencing are you doing? I am most definitely interested.

 

THANK YOU! Finally. I hate these products.

 

 

sequencing is cheap, like a few bucks per sample. that's a great idea making the glycerol stocks - where do you store them?

 

You could also see how populations of bacteria shift overtime within a single tank. That might be easier to start off with, and would let you do some method refinement before jumping in. I would guess there would be a winnowing process that would reduce the diversity of the bacteria overtime.

 

Who knows, maybe this part of the reason why some people have issues sustaining older tanks? A lack of genetic diversity would make them less able to react to significant and abrupt changes.

 

what in the world makes you think i don't already have fully worked out protocols?? do you really think i'm that stupid to offer commercial services for something i'm not expert in? i've been doing this for years, have hundreds of published sequences. jesus... i'm taking this to pms.

 

i don't want this to sound dicky, but i'm offering services in this thread, not seeking help. i've already done nutrient-loading as well as time-course experiments and have seen how populations shift over time, under various conditions. that's why i'm offering this service int he first place! quiet in the back of the class please. please keep the thread on topic.

[tibbsy07]

I just store them in our lab's -80 C. I have a little freezer box that doesn't take up much space (like 36 cryo tubes), so the boss doesn't care. Once I graduate I'll have to figure out what to do with it.

 

Only a few bucks per sequence? That's great! To be honest, we don't much much sequencing other than for plasmids and stuff that we're checking for a gene or something. I was thinking full genome sequencing, which can be pretty expensive (relatively), and that might just be our facility.

[MrAnderson]

yeah this is just 16S gene sequencing, wgs isn't necessary at all.

[Veng]

For me knowing what was in my tank would interest me, but only for about 5-10$ Maybe 20$ Beyond that, I’d be more interested in a frag.

 

On the other hand, if you could sell a scientifically proven cycle in a bottle you could make some money.

[markalot]

Doesn't Dr Tim do this?

 

I agree with the bottle ... I could not comprehend the importance of bacteria in my tank and certainly wouldn't pay money for an analysis I could not understand. Give me some magic juice in a bottle and tell me all my problems will be solved.

 

It's interesting that tanks ever have any diversity, if they do. Most live rock is half dead anyway and most of the nitrifying bacteria comes out of the air. How about people that start up with dead rock and a dead shrimp or other artificial ammonia source? I suppose the theory is that each tanks achieves a biologically diverse peak at some point in it's life and then that diversity starts to go downhill as the tank ages. Proof of this would be ideal.

[Reeftech]

Hey maybe some one has some tyree le bacteria i would trade for my ora captive bred bacteria. .HA just kidding.

 

Seriously this guy is offering to anyone that wants to know this stuff an analysis that will allow people to know what is actually in a bacteria strain.This may help to better explain the cycle of a tank or it may also confuse new hobbyists at the same time.

[lakshwadeep]

Doesn't Dr Tim do this?

 

I agree with the bottle ... I could not comprehend the importance of bacteria in my tank and certainly wouldn't pay money for an analysis I could not understand. Give me some magic juice in a bottle and tell me all my problems will be solved.

 

It's interesting that tanks ever have any diversity, if they do. Most live rock is half dead anyway and most of the nitrifying bacteria comes out of the air. How about people that start up with dead rock and a dead shrimp or other artificial ammonia source? I suppose the theory is that each tanks achieves a biologically diverse peak at some point in it's life and then that diversity starts to go downhill as the tank ages. Proof of this would be ideal.

 

Where are you getting all this information? From the label on a bottle? Dr. Tim does not say what is specifically in his bottles, and you blindly just buy them assuming both his theory is right and that he has applied his theory to have significant numbers of bacteria in the bottle, which might be sitting on shelves for months.

 

What does half-dead rock mean? It doesn't take many individual bacteria to reestablish populations. How do you know nitrifying bacteria come mostly from the air? Why did it take using live rock for people to successfully keep corals and other previously impossible livestock? Have you ever seen a tank with only dry rock and dead shrimp? It is far from a healthy tank until there are some live rock introduced through frags or other sources.

[MrAnderson]

like I said, this isn't for everyone and I didn't think there would be much demand. those that are interested I imagine would be advanced reefers with high budgets. the cost would have been comparable to a decent-sized colony of coral, or a moderately-rare fish, mid-hundreds.

 

i don't see any real demand, so i think those points are moot anyhow.... and like i said, i do this everyday for my research anyhow so my own interests are satisfied. since this would have been brokered by the university, any profit would have gone to the university so it's not like i would make any money from it. i would have gained esteem at my institution and with administrators, but that's really all

 

i just thought some would appreciate being able to get data typically inaccessible to reefers. that's really why i offered my expertise and lab. it's not like you can find out this information any other way, or anywhere else.

[markalot]

Where are you getting all this information? From the label on a bottle? Dr. Tim does not say what is specifically in his bottles, and you blindly just buy them assuming both his theory is right and that he has applied his theory to have significant numbers of bacteria in the bottle, which might be sitting on shelves for months.

 

What does half-dead rock mean? It doesn't take many individual bacteria to reestablish populations. How do you know nitrifying bacteria come mostly from the air? Why did it take using live rock for people to successfully keep corals and other previously impossible livestock? Have you ever seen a tank with only dry rock and dead shrimp? It is far from a healthy tank until there are some live rock introduced through frags or other sources.

 

There is no information in my post. I am simply saying that Dr Tim claims to have analyzed the good stuff and bottled it. Mr A is willing to actually run tests and show results, but in the end most people won't be able to read them (that's my opinion).

 

I'm not getting your second paragraph at all. Half dead rock means you purchase live rock when an aquarium starts and get the ammonia spike due to all the dieoff, since most rock sold as live rock is just barely alive. Indeed what does that mean? I have no idea, do you? What bacteria lives, what dies? What's left? How does this population change over time?

 

Finally you claim people can't run a a healthy tank using dead rock and cycling it with a dead shrimp or other source of ammonia. That's an interesting claim.

[metrokat]

So the scope of Mr Andersons OP is beyond my understanding or need in this hobby, but I'm posting anyway. I hope Mr. Anderson won't put me ovah his knee. Hmm, maybe that is the purpose. Anyway.

 

Since Dr Tims came into the picture on this thread: My bottle of Dr. Tims Waste-Away miracle cure all is on its way to me. I ordered it because 2 days ago I had a nasty cruddy slimy snotty build up in my sump. The skimmer went bananas, the surface was filthy, there were thick copious looking bubbles on the surface, I expected the worse. At that point my best guess was that it was reef slime, which is caused by an over abundance or explosion of one kind of bacteria. Usually caused by deodorizers, cooking, alcohol etc and my cleaning lady had been rather over zealous that very day.

 

Research pointed to Dr Tims along with water changes, peroxide, opening windows and similar things. I did them all. The problem has subsided but it got me thinking about Mr. Anderson. (not that it takes much to do that anyway, just saying).

 

Simply knowing what bacteria is in my system is like getting a blood test and finding out my cholesterol level without knowing if it is good or bad. Maybe I find out my cholesterol is bad, but then what am I going to do about that without the advice of a doctor, in this case someone who can tell me okay you have bacterial strains A & B but you really need to add C & D.

 

So that is the magic of Dr Tims. He never says whats in the bottle only that it will fix problem A or B or this or that. Is it possible that it has what is in Microbacter7 or ZeoBak or BioSpira? Maybe it has sugar and other liquid carbon fuel. Who knows. So I'll just keep eating my probiotic yogurt and hope that it really has what the packaging says.

 

The point is that if I could take the service Mr Anderson is offering and be able to apply it to increase the success of my tank I would be interested.

 

What is needed is part 2 of the equation: After the analysis, what is the recommendation?

I believe providing a custom bottle of bacterial soup would be a killer service.