Looking for the Magic in Bottled Bacteria (and in our tanks)

[taricha]

But I think a major component that is missing and isn't quite the same is the fact that the bacteria need something to host in ...

I.e. you can't have a Saltwater aquarium without some type of rock, bio-filter media.
...

Like technically you're not really activating any of the bacteria, ...

I just would like to see test done with some bio media or something that could be sterile when added.

Thanks for the skepticism and questions. It helps think through what I probably know and what I don't.

It's a good question: how applicable is a test in tubes and bottles - to what these products are supposed to do in an aquarium?
A few quick points.
First and foremost: these bottle bacteria ARE activating - Fig 3,4,5 & 6 all show bottle bacteria activating, consuming O2, digesting proteins etc. So they are getting what they need in order to grow.
Secondly, when the products activate under rich conditions with plenty of O2 available, they cloud the water. So they seem to do fine without needing to be surface bound.
Also, remember these are not nitrifiers - which are predominantly surface associated.
These product descriptions indicate these bacteria will get at the food wherever it is : "Digestion of uneaten/undigested food, excreta, detritus, and other latent organic material, resulting in cleaner and healthier aquarium substrate"

So if I have a dusting of food on the bottom of the bottle, it ought to be able to find it.

(later issues of gas exchange and O2 depletion will be addressed)

 

[Dan_P]

Part 2: The Sleeper has Awakened: What does it do when it wakes?

In Part 1 we saw that just because an aquarium food source contains nutrients and native aquarium bacteria consume it, doesn't mean it'll trigger growth of bottled products (post 1), but the bottled bacteria are viable and can be awakened by some sources, even aquarium sources - very rich ones at least (post 13).
The data thus far pushes 3 big questions to the front for me.

1. Is there a "richness gap" between what nutrients seem needed to activate the bacteria and what exists in our tanks?
2. Does native Aquarium bacteria more thoroughly decompose food matter than bottle bacteria?
3. Is there a multi-day lag time of inactivity with bottle bacteria compared to native bacteria?

number 2 is the hardest, so we'll look at the others for now.

Let's check the timing response of bottle bacteria and native aquarium bacteria.
This is with LB media - the bacterial growth medium used Figure 3 but diluted to 0.5% this time instead of 1%.
Details: 20mL tubes with Aquarium water spiked to 0.5% LB Media. Heat-killed by covered pot boiling 30 minutes. Then inoculated with bacteria, sealed and opened at test time. Each data point is a replicate tube - not one sample opened on multiple days.

Figure 5: LB Media diluted to 0.5% Inoculated with Waste Away, MicroBacter7, and Aquarium Water

Oxygen consumption (from oxidized Carbon), Ammonia production (from breaking down proteins), and pH drop (CO2 and/or other acid production) all tell the same story. Every sample inoculated with aquarium water showed activity between day 0.7 and 1.7, while the Waste Away samples showed activity by day 3.7. MicroBacter7 really wasn't interested in the stuff and showed no activity even after nearly 6 days.
(PO4 is complicated because the medium likely contains a lot of P in forms other than PO4 that don't show up on a test, so decrease of PO4 could happen by bacterial growth, and increase of PO4 could happen by breakdown of the organic P forms to inorganic PO4.)

So it looks like right at a 3 day lag for the bottled bacteria compared to aquarium bacteria, which themselves had maybe a half-day lag - though that is understandable as I don't normally feed my tank laboratory bacterial growth media

Here's another test run done with a high level of fish flake (the same flake that got no response in the first post)
Details: 150mL flasks with Aquarium water and 100mg/L fish flake (53mg/L protein). Heat-killed by pressure canner. Then inoculated with bacteria, and sealed until samples withdrawn and tested at days 2, 4 & 7. This method lost sterilization after 8 days and was improved on later. I'd take it with a grain of salt, but it is consistent with other results.

Figure 6: Fish Flake 100mg/L (53mg/L protein) Inoculated with Waste Away, Pristine, Live Rock Enhance, Heat-Killed Pristine, and Aquarium Water

Oxygen consumption shows activity from Pristine and Live Rock Enhance between day 2 and 4, and from Waste Away after day 4. Ammonia production shows the exact same story - with the additional information that the aquarium bacteria releases far more ammonia from protein breakdown than the bottle products.
All this data is also consistent with a near-immediate response from aquarium bacteria but a 2-4 day lag time from the bottle bacteria products. (A separate test with same food & concentration also had MicroBacter7 showing activity at day 4.)

This gives a different interpretation to the lack of activity in the earlier fish flake test in post 1, Fig 1
Maybe the issue is a long delay and not just a lack of richness. Even if the fish flake (16mg/L protein) in that first test were rich enough, the hold time for that first test was only 2.5 days.

So where do we stand?
Thus far, in every sample that wakes the bottled bacteria (Fig 3, 5 & 6), the activity is only detectable with a 2-4 day lag behind the native aquarium bacteria. And that's with bacterial growth media or 3 weeks' worth of fish food (50mg/L protein).
On the "richness" question, a mere 5 days' worth of fish food (16mg/L protein) showed no activity in bottle bacteria in 2.5 days, but it's unknown if it wasn't enough food, or if hold time was too short and activity would've appeared after 3-4 days.

Nice data in part 2. Like my situation with The Lord if the Rings movie, I can’t decide if I like the first or second part better.

A thought. Lag time and bacteria number. Could we account for the slow starts of the bottled bacteria as long lag times and/or a smaller number of bacteria? The aquarium water consortium is an up and running culture with possibly very numbers. Could this fact make aquarium water look like it is a formidable opponent?

Are you finding any pH effects when you run cultures in BOD experiments instead of with a headspace?

 

[taricha]

I just would like to see test done with some bio media or something that could be sterile when added.

oops. forgot to add, this is a good idea (I'm skeptical I'd see a difference) but it's easily done, and I'll add something like this in future. I've been surprised before!

 

[terraincognita]

Maybe it won’t?

maybe some grow/duplicate faster than others.

who knows.

I think adding bio media could show a bigger difference.

maybe in some 5gs with a simple HOB.

 

[Alenya]

Studies have shown that in beginning a sourdough bread starter from commercial yeast, that after a week or so the commercial yeast species is almost entirely replaced with the yeast species present in the local environment.

Do you think there may be an element of this happening in starting with a commercial bacteria and it being replaced over time by local bacteria?

 

[taricha]

Studies have shown that in beginning a sourdough bread starter from commercial yeast, that after a week or so the commercial yeast species is almost entirely replaced with the yeast species present in the local environment.

Do you think there may be an element of this happening in starting with a commercial bacteria and it being replaced over time by local bacteria?

good question.
In these sealed and heat-killed tests, I think I'm getting whatever was in the bottles, and only that. I base that on using the bottle product raw or heat-killed as a control. See figure 4 for example. The heat-killed bottle products do nothing at all for the full 5 days while the raw products all give a response.
In tests where I add any amount of aquarium water (I've gone as low as ~1/90th of a drop) - it always acts like all my other aquarium water additions. If I do bottle product and aquarium water? ...we'll see.

 

[ScottR]

@flampton is correct that there are no nitrifiers / tank cyclers doing anything important here. (Aside: in a flask of fish flake, aquarium water took 4.5-5 weeks to cycle)

But the nitrifiers aren't the ones we're talking about when we think about what happens to uneaten food, dead organisms, nameless grunge in our tank.
for @Variant 's point "Beneficial" is I suppose subject to interpretation.

Let me try to set goals for my bacteria
I'll tell you what I want to happen to food and waste that go in my tank - I want it to disappear totally! However, since that is impossible, I want what remains to be empty of nutrients that will drive nuisance growth. I'd like organic carbon to have already been consumed, and the same for nitrogen stores - so that nuisance algae are not fed by the particles that remain in the tank.

So if I have bacteria that consume nutrients C, N, P from fish food and digest it - either by converting it to their biomass or kicking it into the water, then I'm happy.

In that sense, my tiny doses of aquarium water are "beneficial" to my goals.

I’ve partially read through this thread and picked this post out. In your opinion, would nitrifying bacteria additions be wise or not? I know that nitrifying bacteria don’t just disappear and a cycle won’t give up on an established tank. But would adding them occasionally help to break down nutrients faster and more efficiently? Or is it all done in vain?

 

[ScubaFish802]

Have you thought about adding Vibrant to this list for testing? It is advertised as the catch all solution to the problems you initially stated. Bulk Reef Supply did a 6(?) tank test and it cleared just about everything that was thrown at it Here.

I also am curious as others have asked how your products arrived (insulated with hot/cold packs similar to Turbostart? Or completely unregulated for example

 

[SMSREEF]

It seems the lag time you are seeing in the results for bottled bacteria is the amount of time the spores are taking to activate. I do not claim to fully understand the info in the article below, but it may help explain what you are seeing in the results. It also may suggest a possible amino acid addition to speed up the activation.

article link here: https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/jam.12343#jam12343-fig-0004

A major feature of Stage I is the enormous heterogeneity in the process, with some spores taking ≤10 min to complete Stage I, while others take many hour or even days (Setlow et al. 2012). The reason for this heterogeneity appears to be huge variation between individual spores in the time, termed ‘Tlag’, between germinant addition and initiation of rapid CaDPA release.

 

[taricha]

A thought. Lag time and bacteria number. Could we account for the slow starts of the bottled bacteria as long lag times and/or a smaller number of bacteria? The aquarium water consortium is an up and running culture with possibly very [high] numbers. Could this fact make aquarium water look like it is a formidable opponent?

Just for kicks I poked around bionumbers to get a numerical feel for what exponential growth from almost no cells to cloudy water bloom that consumes all oxygen would look like.
Bacillus subtilis mass 150 femtograms, at ~50% Carbon that's 6.3x10^-15 moles C. Water going from fully oxygenated to zero would be about 0.21 millimoles of O2, and if we ballpark guess that half the consumed carbon is oxidized to CO2 and the other half builds biomass then we've built biomass of 0.21 millimoles Carbon. That predicts 3.3 *10^10 cells in a liter.

Exponential growth of 3.3 * 10^10 cells requires 35 doublings. The peak dividing time for b. subtilis is estimated anywhere from 30 min in LB media to 120 min in other media. At 30 minutes, that would only take 17.5 hours, at 120 minutes, that would take 3 days.

So yes, if there's a minimal number of initial viable cells then maybe the entire lag time can be attributed to the time for exponential growth.

Are you finding any pH effects when you run cultures in BOD experiments instead of with a headspace?

Yes, I found surprisingly large drops in pH - under 7 sometimes - (and ORP drop too) in sealed BOD tests with no airspace, but including airspace and mixing caused this to mostly or entirely disappear. I'll dig up some data later.

 

[taricha]

Have you thought about adding Vibrant to this list for testing? It is advertised as the catch all solution to the problems you initially stated. Bulk Reef Supply did a 6(?) tank test and it cleared just about everything that was thrown at it Here.

I also am curious as others have asked how your products arrived (insulated with hot/cold packs similar to Turbostart? Or completely unregulated for example

Vibrant is a weird animal. centrifuging accumulates no solid pellet. So I can't confidently separate bacteria in it from the bottle media like all the others. Also at fairly modest levels it interferes with some chemical tests like PO4.
Could it be tested? yes, but it'd be a slightly different approach from the others.

I got these bottles from LFS, amazon, and BRS - in the same way many reefers would. They do activate so they aren't "dead." Spore formers are not like nitrifiers and can handle temps up to boiling.

 

[flampton]

Vibrant is a weird animal. centrifuging accumulates no solid pellet. So I can't confidently separate bacteria in it from the bottle media like all the others. Also at fairly modest levels it interferes with some chemical tests like PO4.
Could it be tested? yes, but it'd be a slightly different approach from the others.

I got these bottles from LFS, amazon, and BRS - in the same way many reefers would. They do activate so they aren't "dead." Spore formers are not like nitrifiers and can handle temps up to boiling.

How many G are you pulling? If you're not pulling anything down at 4K+ then there is either no bacteria or too few for a visual pellet. Looking at the ingredients I see that it is basically carbon(acetate/aspartate) and nitrogen dosing(aspartate). And what's interesting is if you're not getting a pellet the cfu count is low and thus to be effective the bacteria will have to expand.

Which means they are in your tank. So after using vibrant once you could potentially just make a aspartate,acetate soln for pennies and dose that.

 

[taricha]

I’ve partially read through this thread and picked this post out. In your opinion, would nitrifying bacteria additions be wise or not? I know that nitrifying bacteria don’t just disappear and a cycle won’t give up on an established tank. But would adding them occasionally help to break down nutrients faster and more efficiently?

I haven't experimented on nitrifiers. That is a well worn pathway by others on this board, and so I'll defer to them.
So we're talking about what happens when food falls to the sand and gets missed, or fish poo or dead organisms - animal and algal. Organics. The rate and degree which those are going to get broken down: consumed for biomass, or remineralized to simple inorganic forms of N, P, and C (ammonia, PO4, CO2 etc) might depend on a system's heterotrophic bacteria - which is what these bottles are about.
It certainly won't depend on how big the nitrifier crew is.

There's a separate answer to your question that's very interesting but way outside the scope of this thread. Reef tanks handle ammonia in two ways - the textbook nitrogen cycle with nitrifiers, or with carbon dose-fueled heterotrophs (and algae, but let's keep it simple). Aquabiomics has found (article) that tanks can be VERY different - some have lots of nitrifiers, some where they're nearly undetectable.
Which path is "better"? Dunno, but if it were believed that nitrification is a better path for handling ammonia and avoiding nuisance growth, then a good bottle of nitrifiers to jump-start the population might be warranted. Maybe after a tank went off of carbon dosing, took down an algae scrubber, or after doing something stupid and damaging the biofilter, or when massively increasing the livestock load. Nitrifier bottles are so good, and nitrifiers grow so slowly, that a bottle really can change the behavior of the system in that respect.

I do not claim to fully understand the info in the article below, but it may help explain what you are seeing in the results. It also may suggest a possible amino acid addition to speed up the activation.

article link here: https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/jam.12343#jam12343-fig-0004

A major feature of Stage I is the enormous heterogeneity in the process, with some spores taking ≤10 min to complete Stage I, while others take many hour or even days (Setlow et al. 2012). The reason for this heterogeneity appears to be huge variation between individual spores in the time, termed ‘Tlag’, between germinant addition and initiation of rapid CaDPA release.

Thank you for bringing this up. I read either that article or some of the material it pulled from.

Would you believe that straight amino acids (glutamine, glutamic acid) did not reliably germinate these spores?

On the lag being super-variable, it keeps nagging at me. If the observable multi-day lag were because some spores can germinate in minutes and some in days, then why are all the bottle bac lag times in the 2-4 day range? Maybe the lag explains some of the variation within that range and the exponential growth time from a very very small number of cells explains the overall existence of the observed multi-day "lag" in the first place.

 

[flampton]

Just for kicks I poked around bionumbers to get a numerical feel for what exponential growth from almost no cells to cloudy water bloom that consumes all oxygen would look like.
Bacillus subtilis mass 150 femtograms, at ~50% Carbon that's 6.3x10^-15 moles C. Water going from fully oxygenated to zero would be about 0.21 millimoles of O2, and if we ballpark guess that half the consumed carbon is oxidized to CO2 and the other half builds biomass then we've built biomass of 0.21 millimoles Carbon. That predicts 3.3 *10^10 cells in a liter.

Exponential growth of 3.3 * 10^10 cells requires 35 doublings. The peak dividing time for b. subtilis is estimated anywhere from 30 min in LB media to 120 min in other media. At 30 minutes, that would only take 17.5 hours, at 120 minutes, that would take 3 days.

So yes, if there's a minimal number of initial viable cells then maybe the entire lag time can be attributed to the time for exponential growth.


Yes, I found surprisingly large drops in pH - under 7 sometimes - (and ORP drop too) in sealed BOD tests with no airspace, but including airspace and mixing caused this to mostly or entirely disappear. I'll dig up some data later.

You're not getting a 10E10 solution utilizing your method. You're probably hitting at best low 10E8. You're having the culture grow under restricted oxygen and stationary. How deep are you running the cultures? There will be a oxygen concentration gradient to consider as well and microenvironments were the bacteria are running different programs so to speak

If you want to mimic your tank in these experiments you'll need to either shake your cultures or run them very thin. Don't worry about measuring O2 consumption. Just look at culture turbidity and compare visually.

Your LB + saltwater has too high of an osmotic pressure. You need to modify by excluding the salt. So tryptone and ye with the saltwater. I haven't looked at the parameters but you probably should use 90% saltwater to adjust for the molarity of the tryptone and ye. Which btw will by definition not be LB anymore When you change a recipe you can name it something else.

I think you have shown what you wanted to that bacterial products have some amount of bacteria.(except possibly Vibrant)

Unfortunately just seeing growth does not help us at all. Bacteria use nutrients and divide. But are they useful to the aquarium? That question has never been scientifically or even casually answered by the probiotic bacteria companies. They just say believe it and well people believe it because bacteria are cool, interesting, and can be dangerous. (Remember we are not talking about the nitrogen cycle bacteria, that science has been established for years and is easily testable)

 

[taricha]

How many G are you pulling? If you're not pulling anything down at 4K+ then there is either no bacteria or too few for a visual pellet. Looking at the ingredients I see that it is basically carbon(acetate/aspartate) and nitrogen dosing(aspartate). And what's interesting is if you're not getting a pellet the cfu count is low and thus to be effective the bacteria will have to expand.

Which means they are in your tank. So after using vibrant once you could potentially just make a aspartate,acetate soln for pennies and dose that.

4k rpm 10min, in 15ml syringe tubes. Vibrant is super optically clear compared to all the products I've messed with here. Everything else forms a very obvious pellet.
Vibrant wouldn't be the only product that has important active ingredients in the media if that were the case. fully characterizing what's in the liquid media is probably well outside my ability, hence my reluctance to try to test it too much.

 

[taricha]

You're not getting a 10E10 solution utilizing your method. You're probably hitting at best low 10E8. You're having the culture grow under restricted oxygen and stationary. How deep are you running the cultures? There will be a oxygen concentration gradient to consider as well and microenvironments were the bacteria are running different programs so to speak

If you want to mimic your tank in these experiments you'll need to either shake your cultures or run them very thin. Don't worry about measuring O2 consumption. Just look at culture turbidity and compare visually.

Your LB + saltwater has too high of an osmotic pressure. You need to modify by excluding the salt. So tryptone and ye with the saltwater. I haven't looked at the parameters but you probably should use 90% saltwater to adjust for the molarity of the tryptone and ye. Which btw will by definition not be LB anymore When you change a recipe you can name it something else.

I think you have shown what you wanted to that bacterial products have some amount of bacteria.(except possibly Vibrant)

Unfortunately just seeing growth does not help us at all. Bacteria use nutrients and divide. But are they useful to the aquarium? That question has never been scientifically or even casually answered by the probiotic bacteria companies. They just say believe it and well people believe it because bacteria are cool, interesting, and can be dangerous. (Remember we are not talking about the nitrogen cycle bacteria, that science has been established for years and is easily testable)

Whoa. Thanks a ton. Lots to think through here.
Quick Question. I'm not really trying to mimic all processes of my tank. I'm just focused on the re-mineralization from organics to their breakdown products.
How important is shaking, gas exchange etc. to that?

 

[flampton]

Whoa. Thanks a ton. Lots to think through here.
Quick Question. I'm not really trying to mimic all processes of my tank. I'm just focused on the re-mineralization from organics to their breakdown products.
How important is shaking, gas exchange etc. to that?

Extremely! The destruction of organics is always performed faster aerobically as O2 is by far the best electron acceptor, and aerobic heterotrophs are going to be the majority of what is growing in a working aquarium.

O2 is so important to most everything we need in an aquarium and that is why I'm actually concerned about the O2 in my tank because I'm at 7000 ft above sea level so have only 85% of the O2 at Sea level

There is always the small anaerobic niche that contains bacteria that converts nitrate to nitrogen gas but that is not really the detritus cleaning we are talking about and they're not the bacteria in the bottled products.

 

[flampton]

Now the objective with heterotrophs is not cycling or remineralization of nutrients at all. You're not looking for N2 production, CO2 production, and btw phosphate has no cycle. Phosphate is phosphate is phosphate.

So utilizing these products you want a good export process in place as well decent tissue import processes. So we're talking skimmer and food chain e.g. SPS capture the bacteria and move the nutrients into their tissue... Basically anything that consumes bacteria and moves the nutrients to their tissues

Edit: so yes you're looking for some CO2 production from bottled bac as they need to be breathing!!... That was silly thing for me to say

 

[SMSREEF]

Would you believe that straight amino acids (glutamine, glutamic acid) did not reliably germinate these spores?

On the lag being super-variable, it keeps nagging at me. If the observable multi-day lag were because some spores can germinate in minutes and some in days, then why are all the bottle bac lag times in the 2-4 day range? Maybe the lag explains some of the variation within that range and the exponential growth time from a very very small number of cells explains the overall existence of the observed multi-day "lag" in the first place.

I would believe that glutamate didn’t based on the article. It also did say germinants are not exclusively amino acids. However if I was gonna try one to speed up activation, maybe L-alanine.

there is a patented application for pond bacteria spore activation, uses “A nutrient-germinant composition according to another preferred embodiment of the invention comprises one or a combination of two or more L-amino acids, optionally D-glucose (which increases the binding affinity of L-amino acids for their cognate receptors in the spore coat), HEPES sodium salt (a biological buffer to provide the proper pH for spore germination)”

Method for Improving Quality of Aquaculture Pond Water Using a Nutrient Germinant Composition and Spore Incubation Method - NCH Corporation

<div p-id="p-0001">A method for improving the quality of pond water used in aquaculture applications by adding to the pond water active bacteria that are preferably germinated from spores on site usin

www.freepatentsonline.com

But, you did support these bottles have bacteria spores in them. That’s pretty huge! And it’s something I was skeptical about.
I think the time necessary to activate (although bothersome) is not a big deal in the long run.
I think that bottled bacteria (Not the live stored in the fridge) will all have a lag compared to the already active from our tanks. I think in order to send them across the country and have spores survive, they probably need to be superdormant spores. Maybe it’s a 1 day or 2 day activation and then they need to logarithmically replicate like you had written in a previous post.

I really enjoy reading these threads because I learn so much. Thanks for doing this Taricha, I can’t wait to see what you do next.

 

[Paston1]

If I was to send Prodibio biogest would you be up for running another test?