Not all Cycling Bacteria are created equal. Who's who, and what do they need?

[taricha]

Fritz 900 says it is basically Fritz 9, but 15x more concentrated, but the preference for it to be stored refrigerated throws me off. I have a bit of a theory as to the refrigeration thing - possibly something to do with just slowing down metabolism versus actual dormancy, but am not quite sure.

What's your idea about them being different - in some way other than just more cells/mL?
Are you thinking that the shelf-stable vs refrigeration is pointing to some notable difference other than just concentration?
This is one reason I centrifuge them and pour off the media - I don't trust a dark colored smelly media as being chemically inert. There could be a lot of carbon source in there, which could make nitrification harder to recognize. My guess is that many cells/mL without preservatives - the refrigeration just slows decomposition and protects the bacteria from decomposition and byproducts.

 

[Dan_P]

From Fritz; (Dr Reef thread);

Well, this bit o’ information saves a whole bunch of time

Thanks

 

[taricha]

Recent study on bottled bacteria:

Efficacy of quick-start nitrifying products in controlled fresh-water aquaria

Quick-start nitrifying products (QSNP) are sold to aquarium hobbyists with the claim that the nitrogen cycle is rapidly initiated allowing for instant…

www.sciencedirect.com

Interesting. A bit lower success rate than in our saltwater products so far....
"Four out of 5 QSNP failed to reduce TAN concentrations from baseline levels over the 14-day test period. One of the QSNP appears to significantly be able to reduce TAN concentrations in controlled freshwater aquaria."

The Tetra product that lowered ammonia also produced nitrate, so sounds like classic nitrification.
Another product "Cycle" (maybe Fluval Cycle) had higher NO3, but no ammonia decrease which sounds like some NO3 in the bottle - you can find this in our nitrifier bottles too. Biospira and one and only doses straight from the bottle add some NO3 before they do anything to ammonia.

This paper was entirely a test of nitrification from just ammonia.

...On the other hand, here's where API (Quick Start) shared their own study for how their product works with fish food as input. I'll probably use it to help thinking about that phase of this exercise.
https://apifishcare.com/pdfs/products-us/quick-start/quick-start-study.pdf

 

[Paul B]

I take some mud from here and throw it in. Reef is 52 years old, no problems yet but I hear at 53 years, problems could pop up.

This is manufactured by "Mother Nature" and is free, but I can put it in a bottle with a nice picture of a French Angelfish on it and charge $29.95

 

[Azedenkae]

I'm a scientist as well. IMHO - just multiplying the Fritz 9 by 15 is not an accurate way to do it for at least a couple reasons. And they are different products - used for different things right? My GUESS would be (though I think when @Dr. Reef was doing his experiments, Fritz explained the difference - which I don't remember) - that either they are different bacteria - or the lower concentration allows them to be stored differently.

EDIT - addition: When a person takes a product and dilutes it or concentrates it to 'prove something' - there are many possible points of error. In other words - there is no way to prove that Fritz 9 vs 900 works better or worse using this concentrating experiment IMHO.

From the way you are phrasing things, I can't help but think you are reading far more into what I aim to get from the experiment I suggested, than what I want to know - and all I want to know is what happens if @taricha multiplies Fritz 9 by 15 times in their experiment. I am not looking to see if one product works better or worse in practice, or if all factors are equal - though I am certainly looking to see how they compare specifically in this case with how @taricha performs their experiment. And yes, there are certainly a lot of factors which can impact why one would work better or worse here, but I have assumed none of it. All I want to see at this point is how they compare specifically in this experiment. I am well aware of not reading more into it than there is once the results come in.

I agree it would be interesting (and very simple) to compare ammonia consumption (and nitrite consumption) of the two products at recommended concentrations, and as you suggested, at adjusted concentrations.

Aye, just interested in seeing what it looks like.

From Fritz; (Dr Reef thread);

Last I sent them an email, they did say it was the same species, so good to see they are sticking to their guns.

Interesting that the implication seems to be that refrigeration is the 'default' to keep the product as viable as quickly as possible, and the longer storage for Fritz 9 is pretty much because they decided Fritz 9 is the far slower product anyways and kinda like 'yeah whatever'.

What's your idea about them being different - in some way other than just more cells/mL?
Are you thinking that the shelf-stable vs refrigeration is pointing to some notable difference other than just concentration?
This is one reason I centrifuge them and pour off the media - I don't trust a dark colored smelly media as being chemically inert. There could be a lot of carbon source in there, which could make nitrification harder to recognize. My guess is that many cells/mL without preservatives - the refrigeration just slows decomposition and protects the bacteria from decomposition and byproducts.

I wonder if there's a difference between keeping the bacteria cold - i.e. just slowing down their metabolism versus actually letting the nitrifiers go dormant.

 

[MnFish1]

Last I sent them an email, they did say it was the same species, so good to see they are sticking to their guns.

Interesting that the implication seems to be that refrigeration is the 'default' to keep the product as viable as quickly as possible, and the longer storage for Fritz 9 is pretty much because they decided Fritz 9 is the far slower product anyways and kinda like 'yeah whatever'.

I have no problem with looking at the products side by side. IMHO - both of the bottles contain dormant bacteria. It would seem to me you would want to do a couple more trials 1)Fritz 9, 2) Fritz 900, 3) fritz 9 (concentrated) and 4) Fritz 900 (Diluted 1:15) in addition to the other controls.

IMHO there should also be some other controls, i.e. I do not think its correct to spin the bacteria down for these products, unless you don't do a similar experiment 'un-spun' - which would answer the question 'is there a carbon source or something else' in the bottle. If you merely spin the bacteria, sure you can know that you've taken all of the liquid out, but you do not know whether 1) the bacteria/spores are damaged 2) If the bottle with the media (unseen) works better - that would suggest that there is something in the solution promoting growth.

 

[taricha]

This is manufactured by "Mother Nature" and is free, but I can put it in a bottle with a nice picture of a French Angelfish on it and charge $29.95

And now I'll think about that mud next time I look at a bottle of $40 One and Only on the LFS shelf.

I wonder if there's a difference between keeping the bacteria cold - i.e. just slowing down their metabolism versus actually letting the nitrifiers go dormant.

Oh, I see. Now I get the shape of your question. Other nitrifying products rely on nutrient deprivation to stabilize the bacteria in the bottle, but Fritz for their concentrated product uses refrigeration.
So the bacteria are coming back from two different stasis mechanisms, and 9 vs 900 gives you likely the same bacteria under those two different storage conditions.

 

[taricha]

Here's an update on a loose end - how long it takes for a drop of my tank water to process the ammonia in these containers. I had previously said that I expected it to be ~30 days based on my recollection of earlier messing around I've done.

Previously my tank water has shown nitrification around day 30 or so. So the Seachem stability would need to be several days faster than that, for me to think that it has some nitrifiers beyond just random aquarium water contamination

Now, the tank water samples in Group 1 finally have done enough to make their timing clear.

One of the two Tank Water replicates began processing ammonia around day 54, and the other replicate around day 62. Neither show any signs of NO2 oxidation.
The "sterilized", the "Nothing", and the Seachem Stability samples are clearly even less responsive than a drop of my tank water under these conditions. None of them are showing anything even going out this far in time.
I was unsure how long these samples would be able to go before simply random contamination would cycle all the bottles, but apparently the answer is... a very long time. Which increases my confidence that even slow responders that take 20 or 30 days are not the result of random contamination, but are actually in the material added.

 

[MnFish1]

Here's an update on a loose end - how long it takes for a drop of my tank water to process the ammonia in these containers. I had previously said that I expected it to be ~30 days based on my recollection of earlier messing around I've done.


Now, the tank water samples in Group 1 finally have done enough to make their timing clear.

One of the two Tank Water replicates began processing ammonia around day 54, and the other replicate around day 62. Neither show any signs of NO2 oxidation.
The "sterilized", the "Nothing", and the Seachem Stability samples are clearly even less responsive than a drop of my tank water under these conditions. None of them are showing anything even going out this far in time.
I was unsure how long these samples would be able to go before simply random contamination would cycle all the bottles, but apparently the answer is... a very long time. Which increases my confidence that even slow responders that take 20 or 30 days are not the result of random contamination, but are actually in the material added.

Nice job. Curious - the Stability 'bottle'did you also spin down that one? Did you spin it fast and long enough to get spores?

 

[Dan_P]

Here's an update on a loose end - how long it takes for a drop of my tank water to process the ammonia in these containers. I had previously said that I expected it to be ~30 days based on my recollection of earlier messing around I've done.


Now, the tank water samples in Group 1 finally have done enough to make their timing clear.

One of the two Tank Water replicates began processing ammonia around day 54, and the other replicate around day 62. Neither show any signs of NO2 oxidation.
The "sterilized", the "Nothing", and the Seachem Stability samples are clearly even less responsive than a drop of my tank water under these conditions. None of them are showing anything even going out this far in time.
I was unsure how long these samples would be able to go before simply random contamination would cycle all the bottles, but apparently the answer is... a very long time. Which increases my confidence that even slow responders that take 20 or 30 days are not the result of random contamination, but are actually in the material added.

Does a sample of your sand, skimmed off the surface, do well removing ammonia from solution?

 

[taricha]

Does a sample of your sand, skimmed off the surface, do well removing ammonia from solution?

On these scales, yes. A scoop of the top surface of my sand when tested in the past would consume 0.1-0.2ppm ammonia per day initially, so a slight drop and NO2 production could be detected within the first 24 hours. So the weeks and weeks that it takes to see the same for an addition from the water says something about where more of my nitrifying activity is.

 

[MnFish1]

On these scales, yes. A scoop of the top surface of my sand when tested in the past would consume 0.1-0.2ppm ammonia per day initially, so a slight drop and NO2 production could be detected within the first 24 hours. So the weeks and weeks that it takes to see the same for an addition from the water says something about where more of my nitrifying activity is.

This entire experiment does not make sense - in at least one sense. You cannot use little vials- shaken to make any flames second - spinning the solution out of the solutions - makes no sense. The goal of this forum was to post an experimental method - then - get critique and agreement after discussion - In this case there are a number of vials tumbling - that may (or may not) have anything to do with reef aquaria - yet - without vetting - here it is. Presuming to be the end result?

 

[taricha]

Potential Side-trip: "dud"-ify some good bottles.
Take a biospira confirmed to work and leave it in the trunk of my car for a couple of days that will reach ~100F.
Take another and freeze it for a day or two.

Do either of those cause catastrophic loss of function (99+% activity drop) - or is this shipping excuse for dud bottles just an unjustified free pass?

My presumption is that freeze is a hard kill, but heat is just a modest loss of activity. (there's probably some papers that would save me the trouble of this side trip)

It turns out the papers by themselves don't actually spare us the side-trip. I was hoping definitive demonstrations that nitrifiers are killed by freezing, but instead...
Cold Adapted Nitrosospira sp.: A Potential Crucial Contributor of Ammonia Oxidation in Cryosols of Permafrost-Affected Landscapes in Northeast Siberia

"The 16S rRNA based microbial community analysis of the soil samples revealed a high diversity at the phylum level with Proteobacteria, Bacteriodetes, Actinobacteria, and Chloroflexi as dominant groups (60% and 90% relative abundance, Figure 3). OTUs classified as nitrifying taxa revealed a relative abundance of 6.2% of the total reads in the sample from the polygon rim (PR5–15) and 0.6% in the sample of the floodplain (FP0–5) (Table 2).
Nitrosospira as AOB could be identified as the main nitrifier with a relative abundance of 50% to 80% of the reads classified as nitrifiers (Figure 4). Nitrospira were the predominant NOB with up to 34% as nitrifier reads, e.g., in the cliff (C150). Only a few AOA could be detected with a relative abundance between 0.2% and 3.4% of all nitrifier reads (Figure 4)."

So the genus nitrospira and nitrosospira that appear on the label in a bottle of Biospira (and probably in other products) can also be found in siberian permafrost near the surface. (Also some archaea too.)
Obviously, our strains of these nitrifiers are expected to be from tropical lineages that should be very different from the permafrost types. But, it's certainly not clear from this that we should expect a freeze to be an automatic kill. We might actually need to check that bit of conventional wisdom.

BTW, a search on google scholar for "permafrost nitrifiers" is interesting. It's well researched, because wastewater treatment in northern europe needs strains of nitrifiers that can operate in a biofilter all the way down to the edge of freezing, so a bunch of strains have been studied for how well they eat ammonia and nitrite at super cold 1C and 4C etc. Crazy.


Other side-excursions, I've got a couple of aquabiomics tests laying around, and I'm thinking about whether it's worth sending in a test from the Tank Water samples, where a single drop of tank water eventually multiplied and processed the ammonia around day 60 (post 128). It's probably just a few cells of the usual stuff that slowly expanded in populations, but it would be really cool (but unlikely) if this cultured up a "large" population of Archaea somehow.


Group 3 is rolling along: Fritz 900, Microbacter7, PNS substrate sauce, and Caribsea OceanDirect live sand. Like the other groups, some are done already, and some have done nothing.

I should probably put together a table to at a glance see who's done what so far in the bottles with the "fishless cycle" part of the test.

 

[Dan_P]

It turns out the papers by themselves don't actually spare us the side-trip. I was hoping definitive demonstrations that nitrifiers are killed by freezing, but instead...
Cold Adapted Nitrosospira sp.: A Potential Crucial Contributor of Ammonia Oxidation in Cryosols of Permafrost-Affected Landscapes in Northeast Siberia

"The 16S rRNA based microbial community analysis of the soil samples revealed a high diversity at the phylum level with Proteobacteria, Bacteriodetes, Actinobacteria, and Chloroflexi as dominant groups (60% and 90% relative abundance, Figure 3). OTUs classified as nitrifying taxa revealed a relative abundance of 6.2% of the total reads in the sample from the polygon rim (PR5–15) and 0.6% in the sample of the floodplain (FP0–5) (Table 2).
Nitrosospira as AOB could be identified as the main nitrifier with a relative abundance of 50% to 80% of the reads classified as nitrifiers (Figure 4). Nitrospira were the predominant NOB with up to 34% as nitrifier reads, e.g., in the cliff (C150). Only a few AOA could be detected with a relative abundance between 0.2% and 3.4% of all nitrifier reads (Figure 4)."

So the genus nitrospira and nitrosospira that appear on the label in a bottle of Biospira (and probably in other products) can also be found in siberian permafrost near the surface. (Also some archaea too.)
Obviously, our strains of these nitrifiers are expected to be from tropical lineages that should be very different from the permafrost types. But, it's certainly not clear from this that we should expect a freeze to be an automatic kill. We might actually need to check that bit of conventional wisdom.

BTW, a search on google scholar for "permafrost nitrifiers" is interesting. It's well researched, because wastewater treatment in northern europe needs strains of nitrifiers that can operate in a biofilter all the way down to the edge of freezing, so a bunch of strains have been studied for how well they eat ammonia and nitrite at super cold 1C and 4C etc. Crazy.


Other side-excursions, I've got a couple of aquabiomics tests laying around, and I'm thinking about whether it's worth sending in a test from the Tank Water samples, where a single drop of tank water eventually multiplied and processed the ammonia around day 60 (post 128). It's probably just a few cells of the usual stuff that slowly expanded in populations, but it would be really cool (but unlikely) if this cultured up a "large" population of Archaea somehow.


Group 3 is rolling along: Fritz 900, Microbacter7, PNS substrate sauce, and Caribsea OceanDirect live sand. Like the other groups, some are done already, and some have done nothing.

I should probably put together a table to at a glance see who's done what so far in the bottles with the "fishless cycle" part of the test.

Maybe pilot the temperature effect by splitting a bottle amongst a bunch of capped test tubes, expose the tubes to different temperatures and time at temperature and then dilute contents with IO containing ammonia.

 

[taricha]

While I wait on Fritz 900 in group 3 to finish clearing nitrite...
Here's the temp extreme test on biospira.

Maybe pilot the temperature effect by splitting a bottle amongst a bunch of capped test tubes, expose the tubes to different temperatures and time at temperature and then dilute contents with IO containing ammonia.

Exactly so. Biospira split into test tubes:
1 - Room temp
2 - Ice-water bath for several hours
3 - barely freezing overnight (freshwater freeze but not the saltwater biospira medium)
4 - overnight hard freeze (0F) in the freezer
5 - 3x cycle of hours of hard freeze (0F) and thaw.
6 - 2 milder days (mid 80's F outside) in trunk of my car
7 - 5 days : 2 milder days + 3 hotter ones (upper 90's F outside, ~120F measured in trunk)

A first early peak at 24 hours shows some activity from all the treatments except bottles 4 & 5 - hard freeze (0F). Also it looks like all the temp treatments slowed activity somewhat relative to the tube kept room temp.

Hard data in a couple of days....

 

[Dan_P]

While I wait on Fritz 900 in group 3 to finish clearing nitrite...
Here's the temp extreme test on biospira.


Exactly so. Biospira split into test tubes:
1 - Room temp
2 - Ice-water bath for several hours
3 - barely freezing overnight (freshwater freeze but not the saltwater biospira medium)
4 - overnight hard freeze (0F) in the freezer
5 - 3x cycle of hours of hard freeze (0F) and thaw.
6 - 2 milder days (mid 80's F outside) in trunk of my car
7 - 5 days : 2 milder days + 3 hotter ones (upper 90's F outside, ~120F measured in trunk)

A first early peak at 24 hours shows some activity from all the treatments except bottles 4 & 5 - hard freeze (0F). Also it looks like all the temp treatments slowed activity somewhat relative to the tube kept room temp.

Hard data in a couple of days....

You are such a tease. Just for that I am not going to suggest that we also look at the effect of the various algicide products (Algaefix, ChemiClean, etc.) on AOB and NOB. I won’t even mention that the Pharma cocktail I am testing with algae seems to have no effect on AOB. So there

 

[taricha]

Just for that I am not going to suggest that we also look at the effect of the various algicide products (Algaefix, ChemiClean, etc.) on AOB and NOB. I won’t even mention that the Pharma cocktail I am testing with algae seems to have no effect on AOB.

I remember that while I had decent amounts of Algaefix in my tank, aquabiomics found normal to above average levels of AOB and NOB.
I kind of chuckle at the various medications that say "will not harm biofilter," like that's some kind of indicator of it being gentle and harmless. I mean, true, sure - but biofilters can take so much abuse that's really not saying much. Heck, Eli found that the brown jelly cipro treatments didn't hurt the tank biofilters.

 

[Dan_P]

I remember that while I had decent amounts of Algaefix in my tank, aquabiomics found normal to above average levels of AOB and NOB.
I kind of chuckle at the various medications that say "will not harm biofilter," like that's some kind of indicator of it being gentle and harmless. I mean, true, sure - but biofilters can take so much abuse that's really not saying much. Heck, Eli found that the brown jelly cipro treatments didn't hurt the tank biofilters.

Could be that the good guys are gram negative and many of the bad guys are gram positive.

 

[taricha]

Group 3:
(Group 1 samples are here in post 44, Group 2 samples are in post 63)

Here's Group 3 - the next 10 samples:

1 - "Sterile": heat killed by boiling - not quite sterile, but close enough for our purposes.
2 - tank water: inoculated with a drop of water from my tank.
(these are negative controls like in the first two rounds. They should do nothing over any reasonable time frame of interest.)

3 & 4 Frtitz Turbo Start 900: This is the positive control for group 3. Generally considered the fastest reducer of high ammonia. "FritzZyme® TurboStart® 900 contains the same proven saltwater strains of effective, live nitrifying bacteria as FritzZyme® 9 but at 15 times the concentration....TurboStart® completely cycles aquariums in five days or less, allowing for safe, immediate addition of livestock....TurboStart® 900 has an expiration date and a 4-month refrigerated shelf life to ensure viable, effective product.

5 & 6 - MicroBacter 7 : "MicrōBacter7 is a selective complex of extremely effective microbes and enzymes that rapidly reduces the concentrations of organic nitrogen, ammonia, nitrite, nitrate, phosphate, and organic carbon in all marine and freshwater ecosystems." This seems to be a widely used cycling bacteria, although there is little reason for it to be so widely used. It's not even Brightwell's designed best cycling product (MB Start XLM).

7 & 8 - PNS Substrate Sauce: "Specially formulated for cycling and conditioning new saltwater aquaria.Removes ammonia, nitrite and phosphate (assimilation). Removes nitrate (denitrification).Compliments "traditional" cycling (i.e., nitrifying) bacteria such as Nitrobacter and Nitrosomonas. Occupies very different niche than nitrifying bacteria (e.g., anaerobic, heterotrophic and light-loving)." This is not expected to do anything to ammonia under these conditions, this is just a baseline for comparison to later heterotrophic activity.


9 & 10 - Caribsea Ocean Direct live sand:
"preserves real live sand with its own original bacteria. Every breathable bag of Ocean Direct™ is alive with up to 1000 times more beneficial bacteria than other preservation methods. Every grain of Ocean Direct™ is coated with bacteria and encapsulated by capillary action in a thin film of real ocean water."

 

[Dan_P]

5 & 6 - MicroBacter 7 : "MicrōBacter7 is a selective complex of extremely effective microbes and enzymes that rapidly reduces the concentrations of organic nitrogen, ammonia, nitrite, nitrate, phosphate, and organic carbon in all marine and freshwater ecosystems." This seems to be a widely used cycling bacteria, although there is little reason for it to be so widely used. It's not even Brightwell's designed best cycling product (MB Start XLM).

I wonder if so e of the enzymes they are refering to are related to the ones added to laundry detergent and how are enzymes not digested immediately by bacteria in the aquarium?