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

[taricha]

The purpose of this thread is to try to measure and understand how bottled bacteria might work in our tanks. Not the cyclers / nitrifiers - that's well covered here (bacteria in a bottle, Myth or Fact). I'm talking about the bacteria that are meant to help with digestion and breakdown of food, waste, films, organics, and nameless grunge in established aquaria. In addition to wanting this material gone for reasons of appearance, by removing this material hobbyists hope that nuisance growth such as cyano, dinos, and algae can be reduced.
It's often assumed that bacterial activity isn't easily testable, so we throw up our hands and say these questions are beyond reach of the hobbyist - but maybe pour in another bottle just in case .
In reality, there are many things that bacteria do that are very measurable, as we'll see. Are some of those measurable things the stuff the bottles say, and the things we are looking for? Cleaner, less grungy and nuisance-algae free tanks?

I still don't know some of the answers here - and I'm sure some things will be wrong. But how bacteria (bottles and native aquarium) behave in our tanks is important and fascinating, and we can know much more than many assume. Look forward to questions and comments.
So let's take a look!

Some of the bacterial products used here include:

• Waste Away
• MicroBacter 7
• MicroBacter Clean
• Pristine (Seachem)
• Live Rock Enhance (dry powder)

Part 1: Waking up Sleeping Beauty

If the bottled bacteria are going to be helpful, then they'll need to become active from whatever sedentary state they are in - rather than a state that keeps them viable for months in a bottle. Can we wake them up, and can we tell if they've been woken up?
(In an earlier thread Waste Away: Is it really bacterial? Or chemical? What does it do? - I tried a LOT and failed - to find Waste Away active in aquarium-like conditions. But let's try harder, and more carefully.)

Let's feed some ground up fish flake into bottles of aquarium water and check to see if we can wake up bottled bacteria. So we can see the bottled bacteria better, let's sterilize the whole mess first.

Details: 150mL flasks with aquarium water + 30mg/L of ground fish flake (roughly 5-10x a daily feed of my system.) Everything was autoclaved at 121C for 30 minutes. All bottles, stoppers, pipettes etc, were autoclaved, then autoclave door was partially opened and allowed to sit overnight - to cool and re-absorb oxygen into the water to near but not 100% O2 saturation (~7mg/L O2).
One treatment got a recommended dose of sterilized (autoclave) Waste Away. "WA (ster)"
Second treatment got the same dose of raw WA out of the bottle. "WA (raw)"
Third treatment got aquarium water in same volume as recommended WA dose + recommended dose of autoclaved WA. "Aq+WA(ster)"
Duplicates of each.

everything was stoppered and held for 2.5 days.

Figure 1: Fish Flake 30mg/L (16mg/L protein) Inoculated with Waste Away, Sterilized Waste Away, and Aquarium Water + Sterilized Waste Away

We have three different markers for bacterial activity.

1. The amount of dissolved oxygen in the water decreases as bacteria consume organics and oxidize Carbon to CO2.
2. Ammonia is released as proteins etc are processed - either by breaking them down for their carbon and releasing N, or by taking them in to build cells and releasing whatever excess N isn't used for biomass.
3. Phosphate is consumed as cells multiply and build biomass. (It's also released over time by fish food, but here more is consumed than released.)

All 3 markers tell us the same story. The autoclaved bottled bacteria do nothing as expected, but the raw bacteria straight out of the bottle look exactly like the autoclaved ones! Meanwhile native aquarium bacteria are behaving as expected, multiplying, breaking down fish food and consuming oxygen.

No Dice. Let's try a different food, and add another bacterial product to see if we have better luck.

This is 5% skimmate from my system, sterilized and added nothing "Cntl", Waste Away "WA", MicroBacter 7 "MB7", and Aquarium water "Aq".
It was filtered (0.45 micron) and diluted to 5% with aquarium water. Done in 20mL test tubes. I found that boiling ~30min in a covered pot works as well as autoclaving for these sterilization purposes. To ensure enough viable inoculum in the small volume test tube, I centrifuged the bacteria spores out of Waste Away and MicroBacter 7, and poured off the media - then resuspended in distilled water - generating a cloudy liquid of suspended spores - and dosed. This way I could add 10x recommended dose of spores without the bottled media interfering.

In addition to the Oxygen consumption and Ammonia production, bacterial digestion also lowers pH through creation of CO2 and sometimes the production of other acids.

Figure 2: Skimmate diluted to 5% Inoculated with Waste Away, MicroBacter7, and Aquarium Water

All 3 markers, Oxygen, Ammonia, and pH tell us the same story again, now with two different products. The bottled bacteria aren't activated - indistinguishable from sterile control, and the same dose of aquarium water as the bottle product introduces bacteria that do all the things we'd expect.
Bonus, if you look at the picture in the bottom right - during one of the chemical tests, a shake step created a nice foam in the inactive samples - as might be expected with skimmate, the proteins foam quite well. But the samples that got Aquarium bacteria did not foam - pointing toward the proteins having been broken down / consumed.

What will it take to wake these guys up? Are there maybe no viable bacteria at all? These sorts of tests and those in the earlier mentioned thread focused on WA had me thinking "maybe not?"
However, I now know there are absolutely viable bacteria in these products, so what will it take to activate bottled bacteria? Next...

Conclusion of Part 1: Waking up Sleeping Beauty (For real this time) See Post 13

Part 2: The Sleeper has Awakened: What does it do when it wakes? See Post 38

 

[brandon429]

following
curious if claimed overall results in aquaria are false attributes and to what mechanism the real credit goes if any

 

[flampton]

Yeah following as well. I'm going to be doing something similar as I'm developing my own bacterial product (s). I'm going to try and push this field forward including a guaranteed cfu count per bottle before expiration and shpiing fresh cultures.

 

[Rick Mathew]

The purpose of this thread is to try to measure and understand how bottled bacteria might work in our tanks. Not the cyclers / nitrifiers - that's well covered here (bacteria in a bottle, Myth or Fact). I'm talking about the bacteria that are meant to help with digestion and breakdown of food, waste, films, organics, and nameless grunge in established aquaria. In addition to wanting this material gone for reasons of appearance, by removing this material hobbyists hope that nuisance growth such as cyano, dinos, and algae can be reduced.
It's often assumed that bacterial activity isn't easily testable, so we throw up our hands and say these questions are beyond reach of the hobbyist - but maybe pour in another bottle just in case .
In reality, there are many things that bacteria do that are very measurable, as we'll see. Are some of those measurable things the stuff the bottles say, and the things we are looking for? Cleaner, less grungy and nuisance-algae free tanks?

I still don't know some of the answers here - and I'm sure some things will be wrong. But how bacteria (bottles and native aquarium) behave in our tanks is important and fascinating, and we can know much more than many assume. Look forward to questions and comments.
So let's take a look!

Some of the bacterial products used here include:

• Waste Away
• MicroBacter 7
• MicroBacter Clean
• Pristine (Seachem)
• Live Rock Enhance (dry powder)

Part 1: Waking up Sleeping Beauty

If the bottled bacteria are going to be helpful, then they'll need to become active from whatever sedentary state they are in - rather than a state that keeps them viable for months in a bottle. Can we wake them up, and can we tell if they've been woken up?
(In an earlier thread Waste Away: Is it really bacterial? Or chemical? What does it do? - I tried a LOT and failed - to find Waste Away active in aquarium-like conditions. But let's try harder, and more carefully.)

Let's feed some ground up fish flake into bottles of aquarium water and check to see if we can wake up bottled bacteria. So we can see the bottled bacteria better, let's sterilize the whole mess first.

Details: 150mL flasks with aquarium water + 30mg/L of ground fish flake (roughly 5-10x a daily feed of my system.) Everything was autoclaved at 121C for 30 minutes. All bottles, stoppers, pipettes etc, were autoclaved, then autoclave door was partially opened and allowed to sit overnight - to cool and re-absorb oxygen into the water to near but not 100% O2 saturation (~7mg/L O2).
One treatment got a recommended dose of sterilized (autoclave) Waste Away. "WA (ster)"
Second treatment got the same dose of raw WA out of the bottle. "WA (raw)"
Third treatment got aquarium water in same volume as recommended WA dose + recommended dose of autoclaved WA. "Aq+WA(ster)"
Duplicates of each.

everything was stoppered and held for 2.5 days.

We have three different markers for bacterial activity.

1. The amount of dissolved oxygen in the water decreases as bacteria consume organics and oxidize Carbon to CO2.
2. Ammonia is released as proteins etc are processed - either by breaking them down for their carbon and releasing N, or by taking them in to build cells and releasing whatever excess N isn't used for biomass.
3. Phosphate is consumed as cells multiply and build biomass. (It's also released over time by fish food, but here more is consumed than released.)

All 3 markers tell us the same story. The autoclaved bottled bacteria do nothing as expected, but the raw bacteria straight out of the bottle look exactly like the autoclaved ones! Meanwhile native aquarium bacteria are behaving as expected, multiplying, breaking down fish food and consuming oxygen.

No Dice. Let's try a different food, and add another bacterial product to see if we have better luck.

This is 5% skimmate from my system, sterilized and added nothing "Cntl", Waste Away "WA", MicroBacter 7 "MB7", and Aquarium water "Aq".
It was filtered (0.45 micron) and diluted to 5% with aquarium water. Done in 20mL test tubes. I found that boiling ~30min in a covered pot works as well as autoclaving for these sterilization purposes. To ensure enough viable inoculum in the small volume test tube, I centrifuged the bacteria spores out of Waste Away and MicroBacter 7, and poured off the media - then resuspended in distilled water - generating a cloudy liquid of suspended spores - and dosed. This way I could add 10x recommended dose of spores without the bottled media interfering.

In addition to the Oxygen consumption and Ammonia production, bacterial digestion also lowers pH through creation of CO2 and sometimes the production of other acids.

All 3 markers, Oxygen, Ammonia, and pH tell us the same story again, now with two different products. The bottled bacteria aren't activated - indistinguishable from sterile control, and the same dose of aquarium water as the bottle product introduces bacteria that do all the things we'd expect.
Bonus, if you look at the picture in the bottom right - during one of the chemical tests, a shake step created a nice foam in the inactive samples - as might be expected with skimmate, the proteins foam quite well. But the samples that got Aquarium bacteria did not foam - pointing toward the proteins having been broken down / consumed.

What will it take to wake these guys up? Are there maybe no viable bacteria at all? These sorts of tests and those in the earlier mentioned thread focused on WA had me thinking "maybe not?"
However, I now know there are absolutely viable bacteria in these products, so what will it take to activate bottled bacteria? Next...

Conclusion of Part 1: Waking up Sleeping Beauty (For real this time)

Well done!!

 

[taricha]

I'm developing my own bacterial product (s). I'm going to try and push this field forward including a guaranteed cfu count per bottle before expiration and shipping fresh cultures.

ooh. Glad this thread is attracting some quality eyeballs and comments.
"fresh cultures" in the sense of in-exponential growth phase (or very near to it) seems a really relevant idea here. One that I'll come back to.

 

[Dan_P]

Oh you cruel author!!! Here I am reading this very interesting narrative, being dragged along by the plot, and bang! “To be continued”! You and the Game of Thrones producers should arrested for cruelty!!!

...OK, I am better now. Great “Nova” series in the making. I find your persistence investigating bottled bacteria refreshing.

Hurry up with the next installment! I need to know whether Prince Charming Is going to do his thing???

 

[SPS2020]

Were the bacteria shipped in a temperature controlled container? I.E., insulated with cool pack. I ask because I've recently ordered MicroBacter XLM and it shipped during 90+ degree days with no cool pack and I doubt live bacteria could survive (en masse) a ride in the back of a delivery vehicle for x number of days at such temperatures. At least from my experience with this and two more bottles of XLM...very slow start of cycle 21 days in.

 

[ichthyogeek]

Oh now this is interesting. My question, is how did you manage to centrifuge/autoclave? Those are expensive, unless you're using the salad spinner/whirligig and Instant Pot.

 

[flampton]

Well you don't need the bacteria in exponential phase unless they have a strong programmed cell death in stationary. Basically it will be strain dependent, especially if you consider spore formers which are the most ideal for long term packaging.

Oh and if anyone else wants to experiment and you don't have a autoclave you can still experiment by using Tyndallization.

 

[Dan_P]

Well you don't need the bacteria in exponential phase unless they have a strong programmed cell death in stationary. Basically it will be strain dependent, especially if you consider spore formers which are the most ideal for long term packaging.

Oh and if anyone else wants to experiment and you don't have a autoclave you can still experiment by using Tyndallization.

OK, I looked it up. Did not know this process had a name.

 

[taricha]

Were the bacteria shipped in a temperature controlled container?

I thought I had a bad bottle to blame for some of my results, and got duplicate. Later the bottles showed activity so no concern there.

Basically it will be strain dependent, especially if you consider spore formers which are the most ideal for long term packaging.

I think a lot of this deals with spore formers. Some of these products explicitly say they contain spores - some specifically bacillus. Live Rock Enhance says bacillus subtilis and bacillus licheniformis. Others have descriptions of activity that sound a lot like the described behavior of those bacillus species.

also relevant to your Question, @SPS2020 bacillus spores can tolerate heat up to boiling and so are at no risk of losing viability to warm summer shipping.
The nitrifiers in the product you mentioned are a different kettle of fish.

 

[taricha]

Oh now this is interesting. My question, is how did you manage to centrifuge/autoclave? Those are expensive, unless you're using the salad spinner/whirligig and Instant Pot.

you can get a centrifuge for cheap if you aren't bothered by knobs that fall off, the wife's pressure canner can also run 120C at 15 PSI, or bug some friends in science lab.

 

[taricha]

Conclusion of Part 1: Waking up Sleeping Beauty (for real this time)

When we left off in post 1 we had tried fish flake and skimmate, and our bottled bacteria products did precisely nothing. Not consuming PO4 to build biomass, not consuming Oxygen (oxidizing Carbon for energy), and not breaking down proteins etc to release N, seemingly not waking up.
Makes us worried if there are viable bacteria in the bottles. Let's go way out of the box and try something that isn't an aquarium thing at all, but a common bacterial growth medium - LB media.

Tank water was spiked to 1% LB media, heat-killed and added treatments of killed Waste Away "WA(st)", raw Waste Away "WA", or Aquarium water "Aq"
150mL flasks (day 3 measurements in extra 20mL test tubes). "heat-killed" media and Waste Away were done by covered 30 min boiling in tightly covered pot. In order to allow more growth with less restriction from O2 in this rich media, 10% air space was left in each sample, so O2 could diffuse in slowly.

Figure 3: LB Media diluted to 1% Inoculated with Waste Away, Heat-Killed WA, and Aquarium Water

And Voila! we have vigorous activity. By day 5 we have complete O2 consumption by the bottled (and aquarium) bacteria and plenty of Ammonia production. Bonus: bottom left you can see turbidity (cloudiness) illustrated with a black background, a home depot puck light, and a white plastic cap diffuser. The scattering from the bacteria in the water is very evident in the samples that grew vs the killed control. Bottom right is the scattering quantified with a digital photo app.
One thing we might notice here is that the amount of O2 consumption and ammonia production seem faster in the native aquarium bacteria than in the bottle bacteria product. Additionally, the ammonia production (a marker for breakdown of compounds with excess N) reaches far higher with native aquarium bacteria. Worth thinking about, but let's leave these here for now and see if they show up again later.

Ok, a 1% dilution of a bacterial growth media can activate bottle bacteria, but can we achieve that with something more plausible for an aquarium? We failed with fish food, but maybe it just wasn't rich enough? And let's look at a bunch of bottle bacteria products.

So here's Frozen Cube (Emerald Entree) 50mg/L of protein.
Treatments are:
Heat-killed: Pristine, Waste Away, MicroBacter7 listed as "Pri (Ster)" etc
Raw recommended dose: WA, MB7, Aq water, my old (maybe dead) bottle of WA, Pri
Two additional controls got nothing.
I ground up (mortar and pestle) a measured amount (50mg/L protein) to a paste and added to Instant Ocean. Agitated the bottle to make it uniform as I split into all the ~20mL tubes. Heat-killed with 30 min covered boil, and added bacteria treatments.

Figure 4: Frozen Food Cube 50 mg/L protein Inoculated with several raw or killed bacteria products, and Aquarium Water

Ooh boy! we asked for activity, and here we've got it by the buckets. Every replicate of every product came alive while the killed versions of the same products act just like blank controls. Even my old bottle of WA, that I was worried about, still perked up. All consumed O2, and all did....stuff to the ammonia reading. Digestion of Nitrogen-rich compounds could release excess N, and building biomass could consume N, so both are possible.
Total ammonia test chemistry reacts with ammonia and proteins and aminos, so the initial levels / controls are not really much ammonia - but interfering N in proteins etc. The increase of ammonia after digestion is real and almost all actually ammonia.

Notice that again the native aquarium bacteria were the most aggressive oxidizers of carbon - O2 in sealed tubes was fully depleted - and the largest producers of ammonia. (The 2nd WA sample looks nothing like any other bacterial product sample and looks a lot like Aq bacteria so lets think of it as likely contamination - oops.) Other than that, it's quite interesting how different the bottle product results look from the native Aq bacteria when presented with the same nutrients.

So bottled bacteria products absolutely contain viable bacteria that can grow. But so far we've seen growth on rich laboratory bacterial media and a fish food level of ~50mg/L protein - when I only feed an average of ~3mg/L protein to my tank on a daily basis. (And earlier 30mg/L of flake - 16mg/L protein showed no activity.)

To my mind these are the big Qs after what we've seen so far.

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

Next: We'll see if some of those questions are within our reach and take a look at specific bottle bacteria claims to try to match those to experimental situations.

 

[Variant]

@taricha Can you tell me if I'm interpreting your experiment incorrect?

So if you used aquarium water to see how it compares to bottled bacteria, would the results you're seeing prove that beneficial bacteria does indeed exist in the water column? Often times, I hear/read how beneficial bacteria is primarily on surfaces versus water borne. I recently watched the 2019 MACNA video of Dr.Tim's take on growing bacteria and he had stated how aquarium water doesn't have beneficial bacteria and that if you took water from a mature aquarium and dumped it in a new tank, you only just added dirty water.

Your results seem to show that the water itself has benefit. Am I wrong?

 

[blasterman]

To my mind these are the big Qs after what we've seen so far.

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

In my opinion a yes on all of those. These type of bacteria are controlled by the ecology of a tank and hit an equilibrium based on that ecology. That's why tanks often hit static nitrate levels and regardless of how many water changes you do that equilibrium level will return. Also why these products are pointless. A bottle of sleeping bacteria isn't going to alter the balance in your tank. The added bacteria typically just die.

 

[brandon429]

Connecting Established Reef To New Tank

EDIT: In conclusion, if using the method I did - connecting an established reef to a new system with dry rock (clean), I recommend the following: 1. Dose your new tank prior to connecting to your old tank with NO3 and PO4 enough to raise to your established tank levels. 2. Take your skimmer...

www.reef2reef.com

sets proof that not only does water column water have plenty of nitrifiers on rafts, transmittable, they are in numbers sufficient enough to cycle within cycling chart timeframes.
*as I re read T is focusing on substrate-reducing bacteria not nitrifying strains for this experiment

that above is only for cyclers, but its in response to Dr Tims findings on cycling bacteria.

aquabiomics has also measured nitrifying/cycling strains of bacteria in water samples from Jon Malkerson if I recall the thread correctly. multi-point confirms are getting stronger

 

[flampton]

@taricha Can you tell me if I'm interpreting your experiment incorrect?

So if you used aquarium water to see how it compares to bottled bacteria, would the results you're seeing prove that beneficial bacteria does indeed exist in the water column? Often times, I hear/read how beneficial bacteria is primarily on surfaces versus water borne. I recently watched the 2019 MACNA video of Dr.Tim's take on growing bacteria and he had stated how aquarium water doesn't have beneficial bacteria and that if you took water from a mature aquarium and dumped it in a new tank, you only just added dirty water.

Your results seem to show that the water itself has benefit. Am I wrong?

So this is the thing, what defines beneficial bacteria? Are we talking about the cyclers, the guys that convert ammonia to nitrite and those that convert nitrite to nitrate, or are we talking about the bacteria that are important for carbon dosing?

The water will have many of the latter but few of the former. That's why the input reef just mentioned took twenty days which is not all that impressive. If you got a truly high concentration of fresh cultures of the cyclers you can cycle a tank immediately. Basically you would apply the culture directly to the rocks and place in the aquarium. I might do this if I have time at work just for fun.

 

[Variant]

So this is the thing, what defines beneficial bacteria? Are we talking about the cyclers, the guys that convert ammonia to nitrite and those that convert nitrite to nitrate, or are we talking about the bacteria that are important for carbon dosing?

The water will have many of the latter but few of the former. That's why the input reef just mentioned took twenty days which is not all that impressive. If you got a truly high concentration of fresh cultures of the cyclers you can cycle a tank immediately. Basically you would apply the culture directly to the rocks and place in the aquarium. I might do this if I have time at work just for fun.

doesn't the OP's experiment show that the bacteria present in the established aquarium water is more effective at becoming 'activated' and begin the denitrification process via the ammonia charts he's showing?

 

[flampton]

doesn't the OP's experiment show that the bacteria present in the established aquarium water is more effective at becoming 'activated' and begin the denitrification process via the ammonia charts he's showing?

No all it shows is you get ammonia production which is not the important step in the cycle. Most bacteria excrete ammonia after consuming amino acids for energy

 

[flampton]

And as for the OPs flake food might have a preservative that would inhibit the bacterial growth. What was the ingredient list? Anything jump out at you taricha?