Prodibio BioDigest Likely Has No Effect On Ammonia Oxidation and Does Not Help with Waste Reduction

[mcgullen]

For years, many aquarists and I religiously dose our tanks with Prodibio BioDigest, in reliance of the claims the product, through a "community" of "bacteria in optimal proportion" would help with "a quick set up of the biological filtration" and with the nitrifying and denitrifying process in general.

My recent research on the product makes me doubt all these claims. Through this thread, I invite R2R members and Prodibio to examine the results and join the discussion. I truly hope we can demonstrate some level of usefulness on the product, instead of continuing to dose it without knowing what's really going on.

1. 16S rRND Sequencing on Prodibio BioDigest

I have 16S rRNA sequencing results of BioDigest on hand. I am not entirely sure whether I could get into trouble for publishing them, given that Prodibio website explicitly says that their bacterial composition is "confidential." I am also withholding the results for now out of courtesy to Prodibio.

But what I can say is that BioDigest does not appear to contain any nitrifying bacteria in the strict sense. In other words, it does not contain any autotrophic bacteria at all, not to mention strain from the Nitrobacteraceae family. This is consistent with the findings of @Dr. Reef study on Bottled Bacteria, where Prodibio could not reduce ammonia level without organic carbon help.

So BioDigest wasn't really "helping with a quick set up of biological filtration" in ways that reasonable consumers who read this claim would have hoped. It appears that Prodibio was aiming to be technically correct but misleading, hoping that no one would bother to dig deeper.

2. Prodibio BioDigest Likely Doesn't Help with Ammonia Oxidation

One would have hoped that, even if BioDigest does not contain true nitrifying bacteria, at least its heterotrophic strains could still consume ammonia when rapidly multiplies.

Well, it didn't.

I tested 3 times with the following setup:

1 gallon of fresh seawater in a nano tank with no filter media.
Heavy aeration
1 ml of TM All-for-Reed was added for trace minerals.
Phosphate was adjusted to 0.1 ppm with KH2PO4.
Ammonia was adjusted to 1.0 ppm (NH3-N) with NH3CL.
0.1g, 0.5g, and 1.0g of brown sugar was added in the first, second, and third run, respectively

Prodibio BioDigest did not bring down ammonia in any of the three runs within 48 hours. Nor did BioDigest beat the control tank in any of the run. In the second run after 72 hours, both the control tank and the BioDigest tank became cloudy, with ammonia starting to drop. This suggests that external heterotrophic bacteria started to multiply and brought ammonia down. I suspect this is what happened in @Dr. Reef study. When he added fish food and witnessed ammonia starting to drop in the BioDigest tank, it was probably some naturally occurring Bacillus strains that were already in the tank water doing the job.

3. Prodibio BioDigest Likely Doesn't Help with Nitrate Reduction

Determined to find at least one concrete case where BioDigest could help, I turned to the claim that it reduces "nitrates and phosphates." The 16s rRNA results do support this claim as there is a Pseudomonas strain known to be an aerobic denitrifican present in the vial in high concentration.

Except that it didn't work.

I again tested 3 times with the following setup:

1 gallon of changed out seawater from refugium.
Heavy aeration
1 ml of TM All-for-Reed was added for trace minerals.
Phosphate was adjusted to 0.1 ppm with KH2PO4.
Nitrates tested to be about 10 ppm (NO3-N).
0.1g, 0.5g, and 1.0g of brown sugar was added in the first, second, and third run, respectively.

Again, with 48 hours on each run, nitrates never went down. Nor did phosphate.

4. What's Next

Could someone, including Prodibio, provide one scenario (that'd actually happen in home aquaria) where BioDigest can demonstrably help with, well, anything? Please also do not require the use of Biotim, as it introduces yet another black box shrouded in mystery.

I would very much like to measure an improvement in any major water parameter that can be convincingly attributed to BioDigest. That would be a much better ending to this story than accepting that I and many others have been paying for an expensive placebo all these years.

Thanks.

McGullen

 

[Kenneth Wingerter]

Interesting. Following.

 

[Dr. Reef]

You are very much on point. I never noticed any change in ammonia dosing Prodibio. I also came to same conclusion that it might be heterotrophic bacteria (almost in most brands I tested) rather than true nitrifying bacteria.
I also forwarded my findings to Prodibio and excat setup of my experiment.
I was emailed back with a response that Prodibio will replicate my experiment and get back to me with results.
I never heard back from them.

In my opinion and experience, the 3 brands that tested good for me all the time and that I can assume to be true nitrifying bacteria (according to my experiments) came to be
1. Fritz Turbostart 900 (the best)
2nd & 3rd place shared by Dr Tim's One and only and Bio Spira( if you can find a fresh bottle)

 

[brandon429]

I had been discussing some skepticism here recently on the matters you're mentioning

Thoughts on adding to the cycling tank

With my tank cycle started I am thinking of adding some PNS substrate starter bacteria or Aquaforest Life Bio Fil to the system for more bacteria diversity. Anyone have any thoughts or experience with this?

www.reef2reef.com

 

[Muffin87]

It is possible that when they say it contains live bacteria, what they mean actually is that it contains "live" spores of bacteria?

Not saying it does, just trying to imagine a scenario where they weren't outright lying.

 

[mcgullen]

It is possible that when they say it contains live bacteria, what they mean actually is that it contains "live" spores of bacteria?

Not saying it does, just trying to imagine a scenario where they weren't outright lying.

Yes, it most likely contains live spores. But regardless, the product must have some core function. What's the core function of BioDigest other than moving money from me to Prodibio? I have tried everything, hoping that it would "work" in any sense of that word. It just didn't work at all.

 

[Muffin87]

Yes, it most likely contains live spores.

Well if it contains live spores it would take a lot longer than 48 hours.
In fact, when cycling a tank Biodigest requires "StopAmmo", which sequesters most of the ammonia, but not all of it.

I had an interest in StopAmmo because it can be used with copper. I'm not an expert on the Prodibio line!

 

[taricha]

Lets look at this one, as I think it has the best chance to show something....

3. Prodibio BioDigest Likely Doesn't Help with Nitrate Reduction

Determined to find at least one concrete case where BioDigest could help, I turned to the claim that it reduces "nitrates and phosphates." The 16s rRNA results do support this claim as there is a Pseudomonas strain known to be an aerobic denitrifican present in the vial in high concentration.

Except that it didn't work.

I again tested 3 times with the following setup:

1 gallon of changed out seawater from refugium.
Heavy aeration
1 ml of TM All-for-Reed was added for trace minerals.
Phosphate was adjusted to 0.1 ppm with KH2PO4.
Nitrates tested to be about 10 ppm (NO3-N).
0.1g, 0.5g, and 1.0g of brown sugar was added in the first, second, and third run, respectively.

Again, with 48 hours on each run, nitrates never went down. Nor did phosphate.

I think given that you have some pseudomonas, you are looking for nitrate reduction - here reduction meaning the chemical process that's opposite of oxidized. So NO3 -> NO2 So you should be able to do essentially what you've done but don't do an NO3 test - do a Nitrite, NO2 test instead.
The issue is that the bacteria may have been reducing some NO3 to NO2, but with an NO3 test, the interference with NO2 present really confuses things - it might even make NO3 look like it went up.
The NO2 test, on the other hand would be definitive. If you have a saltwater sample with NO3 and no NO2, then NO2 gets produced, you can be sure there is nitrate reduction occuring.
https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01:_Labs/1.26:_Nitrate_Reduction

 

[taricha]

1. 16S rRND Sequencing on Prodibio BioDigest

I have 16S rRNA sequencing results of BioDigest on hand. I am not entirely sure whether I could get into trouble for publishing them, given that Prodibio website explicitly says that their bacterial composition is "confidential." I am also withholding the results for now out of courtesy to Prodibio.

But what I can say is that BioDigest does not appear to contain any nitrifying bacteria in the strict sense. In other words, it does not contain any autotrophic bacteria at all, not to mention strain from the Nitrobacteraceae family. This is consistent with the findings of @Dr. Reef study on Bottled Bacteria, where Prodibio could not reduce ammonia level without organic carbon help.

I agree, and this is what I would have expected from the Bottled Bacteria, myth or fact thread. True chemoautotroph nitrifier products are few (namely the 3 listed earlier). The rest will require a carbon source.

2. Prodibio BioDigest Likely Doesn't Help with Ammonia Oxidation

One would have hoped that, even if BioDigest does not contain true nitrifying bacteria, at least its heterotrophic strains could still consume ammonia when rapidly multiplies.

Well, it didn't.

I tested 3 times with the following setup:

1 gallon of fresh seawater in a nano tank with no filter media.
Heavy aeration
1 ml of TM All-for-Reed was added for trace minerals.
Phosphate was adjusted to 0.1 ppm with KH2PO4.
Ammonia was adjusted to 1.0 ppm (NH3-N) with NH3CL.
0.1g, 0.5g, and 1.0g of brown sugar was added in the first, second, and third run, respectively

If I were going to try to get these guys to oxidize ammonia, look at something like this.

Ammonium transformed into nitrous oxide via nitric oxide by Pseudomonas putida Y-9 under aerobic conditions without hydroxylamine as intermediate

They fed the bacteria acetic acid / acetate as a carbon source. See if vinegar neutralized with kalk gives some result. Start at 1mL vinegar / gallon and try 3x and 10x that amount.

Also heterotroph nitrifiers are almost always tested at higher ammonia than the couple of ppm that hobbists use.
from the linked paper
"NH4+ dramatically decreased from 177.33 mg/L to 1.05 mg/L, and the decline rate reached 99.41% after 24 h of incubation."
so maybe I'd try more like 5 to 10ppm ammonia instead of 1ppm.

 

[mcgullen]

I agree, and this is what I would have expected from the Bottled Bacteria, myth or fact thread. True chemoautotroph nitrifier products are few (namely the 3 listed earlier). The rest will require a carbon source.


If I were going to try to get these guys to oxidize ammonia, look at something like this.

Ammonium transformed into nitrous oxide via nitric oxide by Pseudomonas putida Y-9 under aerobic conditions without hydroxylamine as intermediate

They fed the bacteria acetic acid / acetate as a carbon source. See if vinegar neutralized with kalk gives some result. Start at 1mL vinegar / gallon and try 3x and 10x that amount.

Also heterotroph nitrifiers are almost always tested at higher ammonia than the couple of ppm that hobbists use.
from the linked paper
"NH4+ dramatically decreased from 177.33 mg/L to 1.05 mg/L, and the decline rate reached 99.41% after 24 h of incubation."
so maybe I'd try more like 5 to 10ppm ammonia instead of 1ppm.

Got it. Will try 5ppm and vinegar as carbon source.

Also back to report that Prodibio BioDigest with a carbon ratio of C:N did nothing to changed out seawater nitrate level (measured at 16) after 24 hours. No trace of nitrite detected either.

So for all intents and purposes, and despite all theories on how it could have worked, Prodibio remains useless for now.

For users of this product, ask yourselves one question: have you actually measured any tangible improvement? If so, please let me know and I will try my best to replicate it.

 

[taricha]

Also back to report that Prodibio BioDigest with a carbon ratio of C:N did nothing to changed out seawater nitrate level (measured at 16) after 24 hours. No trace of nitrite detected either.

Usually the ratios are like 5 or 10 to 1 Carbon to Nitrogen in the tests I've seen showing heterotrophs can denitrify. Interesting that you didn't see it do this - if you repeat this with just aquarium water, you'll find that it can absolutely denitrify some NO3 to detectable NO2 over 48 hours if given a carbon source and allowed to deplete O2. I've done it with my tank water, and others have too.


Just for sake of discussion, and because I find these things interesting, let me say that when I looked at bottled heterotroph bacteria in order to do anything - took several days (like 3+) before they showed detectable activity. below is a quoted section from this post (click for more details)

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.

Which sets up a really interesting puzzle. If heterotrophs in aquarium water are fast and active, and those coming out of bottles have lag (for several possible reasons: spores, preservatives, low nutrient resting states) and if the things that heterotrphs in bottles are supposed to do, aquarium native bacteria can already also do - then how is a hobbyist ever supposed to detect that the bottled heterotroph bacteria do something meaningful?

 

[mcgullen]

Has there ever been official information on lag time of BioDigest? With the product being marketed as live bacteria, I would expect it to work within 12 hours. So far, I have zero luck getting it to work on ammonia, nitrate or phosphate in that time span. Strange given that I do see live bacteria with 16S sequencing. Also many strains are not spore forming. So it's not like that it needs to sporulate.

 

[EeyoreIsMySpiritAnimal]

...denitrify some NO3 to detectable NO2

But the definition of denitrification is: "the microbial process of reducing nitrate and nitrite to gaseous forms of nitrogen, principally nitrous oxide (N2O) and nitrogen (N2)."

As I have always understood it, it is not converting nitrate to nitrite...

 

[mcgullen]

To clarify, Prodibio BioDigest does have a blend of live bacteria. I can see the blend from 16s sequencing. They are not false marketing that.

I am just struggling to prove on how these bacteria actually help with tangible improvements (lower ammonia, nitrate or phosphate) in real aquarium settings.

So far, tests that simulate real world scenarios all failed to elicit beneficial responses clearly attributable to BioDigest.

That’s clearly impossible as the live bacteria in BioDigest must do something. If anyone can come up with a set of parameters and goals for me to try, that would be greatly appreciated.

A summary of two main tests that have failed so far. Model tanks are 1 gallon tanks cleaned with ClO2 tablets and then thoroughly rinsed and neutralized with sodium thiosulfate. Parameters are from colorimeters.

1. Freshwater, ammonia at 1.0 ppm and 0.5ppm, respectively, sucrose and ethanol as carbon with C:N at 10 to 1, phosphate at 0.1ppm, and TM Reef for All as trace mineral supplement. Goal is to see lower ammonia within 48 hours with 1 standard vial of BioDigest. Ammonia did not change at all.

2. Saltwater from water change, nitrate at 16 ppm, nitrite at 0.007 ppm, phosphate at 0.2ppm, ammonia at below 0.01 ppm, sucrose and ethanol as carbon with C:N at 10:1. Goal is to see lower nitrates and phosphate within 48 hours of adding 1 standard vial of BioDigest. There was no significant change.

 

[taricha]

But the definition of denitrification is: "the microbial process of reducing nitrate and nitrite to gaseous forms of nitrogen, principally nitrous oxide (N2O) and nitrogen (N2)."

As I have always understood it, it is not converting nitrate to nitrite...

Not primarily, no. You're correct that usually the focus is the eventual off-gassing of Nitrogen as N2O or N2. But some NO2 usually gets made along the way...
"As shown in Fig. 1, aerobic denitrifiers can conduct an aerobic respiratory process in which nitrate is converted gradually to N2 (NO3−→ NO2−→ NO →N2O → N2), using nitrate reductase (Nar or Nao), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos)."
Aerobic denitrification: a review of important advances of the last 30 years

 

[Randy Holmes-Farley]

FWIW, there would not need to be any nitrifying bacteria for a bacterial product to reduce ammonia and nitrate in a reef tank. Simple growth and metabolism will take them up.

As I noted in a different thread yesterday...

I would in some ways equate constantly adding bacteria such as Biodigest with organic carbon dosing.

In both methods, bacteria are increasing in the water and potentially taking up nutrients, trace elements, and maybe reducing levels of other organic matter.

 

[mcgullen]

To clarify, Prodibio BioDigest does have a blend of live bacteria. I can see the blend from 16s sequencing. They are not false marketing that.

I am just struggling to prove on how these bacteria actually help with tangible improvements (lower ammonia, nitrate or phosphate) in real aquarium settings.

So far, tests that simulate real world scenarios all failed to elicit beneficial responses clearly attributable to BioDigest.

That’s clearly impossible as the live bacteria in BioDigest must do something. If anyone can come up with a set of parameters and goals for me to try, that would be greatly appreciated.

A summary of two main tests that have failed so far. Model tanks are 1 gallon tanks cleaned with ClO2 tablets and then thoroughly rinsed and neutralized with sodium thiosulfate. Parameters are from colorimeters.

1. Freshwater, ammonia at 1.0 ppm and 0.5ppm, respectively, sucrose and ethanol as carbon with C:N at 10 to 1, phosphate at 0.1ppm, and TM Reef for All as trace mineral supplement. Goal is to see lower ammonia within 48 hours with 1 standard vial of BioDigest. Ammonia did not change at all.

2. Saltwater from water change, nitrate at 16 ppm, nitrite at 0.007 ppm, phosphate at 0.2ppm, ammonia at below 0.01 ppm, sucrose and ethanol as carbon with C:N at 10:1. Goal is to see lower nitrates and phosphate within 48 hours of adding 1 standard vial of BioDigest. There was no significant change.

I used the freshwater setup again and added 0.1g of sodium acetate per gallon. Both the control and the exoeriment tanks showed bacterial bloom and lowered ammonia. So I can't attribute this result to BioDigest. There is still no definitive proof on how BioDigest actually helps.

 

[taricha]

I used the freshwater setup again and added 0.1g of sodium acetate per gallon. Both the control and the exoeriment tanks showed bacterial bloom and lowered ammonia.

This is sort of more what I would expect. The claims on bottled bacteria are not crazy, because there are likely many bacteria in the bottles and in our systems that can do those things if you give them the right food. The complication is that it means it's hard to know that the bacteria specifically from the bottle are the ones that did it.

 

[mcgullen]

This is sort of more what I would expect. The claims on bottled bacteria are not crazy, because there are likely many bacteria in the bottles and in our systems that can do those things if you give them the right food. The complication is that it means it's hard to know that the bacteria specifically from the bottle are the ones that did it.

I tried the saltwater setup with 0.1g of sodium acetate per gallon. Both tank showed lowered nitrate. So it is impossible to tell if it's BioDigest doing the work.

 

[Randy Holmes-Farley]

As taricha stated, that's basically my expectation as well and is the basis for organic carbon dosing. I dosed vinegar, which supplies the same chemical (acetate) to the bacteria and to a wide array of organisms in a reef tank, including corals, sponges, etc.

As an aside, it's very challenging in a reef tank to distinguish direct acetate uptake effects on higher organisms to consumption of bacteria or other microorganisms by those same higher organisms.