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

[taricha]

Here's Group 3 data.

I'm really enjoying how many different cycling curve shapes I'm getting from the different nitrifying products.

Group 3 Total Ammonia and Nitrite

Fritz Turbo start, and Caribsea Ocean Direct responded to ammonia-only quickly and cleared it in about the same time, but the sand started slower and sped up while fritz slowed way down at the very end after the fastest initial activity - note how the curve really flattens compared to the live sand.
On NO2, the sand cleared it shortly after clearing ammonia, while Fritz was much slower on NO2 oxidation and there was still trace amount at 34 days, with a slowing consumption of nitrite as well.

The negative controls of "sterilized" and Tank water did precisely nothing to ammonia over the 33 days, and MB7 and PNS substrate sauce looked like the do-nothings.

Group 3 Final Nitrate and pH

Both Fritz and Caribsea OceanDirect had measured final NO3 that were in agreement with classic nitrification - the amount of NO3 produced is within error of what would be expected from total conversion of all ammonia to NO3.

The pH shows the same 2 products as being active as the ammonia & NO2 data: Caribsea OceanDirect sand and Fritz all had clear downward pH trends during the nitrification.

Commentary
A couple more non-responders in this group - MB7 and PNS substrate sauce join the non-responder products from earlier rounds: API Quick Start and Seachem Stability as products that do not seem to activate under the restrictive conditions of just ammonia and moving water in the dark. The Live Sands continue to impress - I'm going to drop the air quote "Live sand" from now on, all 3 live sand products available locally processed ammonia and nitrite in textbook fashion. Ocean Direct was a bit faster than Caribsea's other sand product arag-alive. In fact, only Biospira cleared ammonia+nitrite faster than OceanDirect. Fritz turbostart did what it is known for - extremely rapid immediate ammonia oxidiation. It seems to lose interest little bit as the ammonia levels get really low. I expected this from heterotrophs, but apparently Fritz is a type of nitrifier that really excels at high ammonia levels, but isn't so active against low levels. Fun thought: if you take Randy's suggestion to heart - that nitrifiers represent a competitor to coral nutrition and should be viewed as "bad guys" in an established reef tank, then this behavior from Fritz may actually be just what you want: aggressive against high levels, but doesn't care about low levels of ammonia, leaving it for corals. (I don't agree with the "bad guy" premise, but that's a diff thread). This slow-down of Fritz turbostart for low-level ammonia and nitrite was also observed by @Dan_P a couple of years ago. We might also speculate that if it's very slow in uptake against the low levels in a reef tank, it's highly likely that it eventually fades to nothing and other strains replace it over time. Other true nitrifier products measured so far did not indicate any notable slow-down at low concentrations.

I'm a bit surprised at the number of people in threads who use MB7 as cycling bacteria (BRS apparently promoted it for that purpose in videos?), but I'm confident it's one of the heterotroph types, and so addition of food later should be much more illuminating.

 

[Dan_P]

Here's Group 3 data.

I'm really enjoying how many different cycling curve shapes I'm getting from the different nitrifying products.

Group 3 Total Ammonia and Nitrite

Fritz Turbo start, and Caribsea Ocean Direct responded to ammonia-only quickly and cleared it in about the same time, but the sand started slower and sped up while fritz slowed way down at the very end after the fastest initial activity - note how the curve really flattens compared to the live sand.
On NO2, the sand cleared it shortly after clearing ammonia, while Fritz was much slower on NO2 oxidation and there was still trace amount at 34 days, with a slowing consumption of nitrite as well.

The negative controls of "sterilized" and Tank water did precisely nothing to ammonia over the 33 days, and MB7 and PNS substrate sauce looked like the do-nothings.

Group 3 Final Nitrate and pH

Both Fritz and Caribsea OceanDirect had measured final NO3 that were in agreement with classic nitrification - the amount of NO3 produced is within error of what would be expected from total conversion of all ammonia to NO3.

The pH shows the same 2 products as being active as the ammonia & NO2 data: Caribsea OceanDirect sand and Fritz all had clear downward pH trends during the nitrification.

Commentary
A couple more non-responders in this group - MB7 and PNS substrate sauce join the non-responder products from earlier rounds: API Quick Start and Seachem Stability as products that do not seem to activate under the restrictive conditions of just ammonia and moving water in the dark. The Live Sands continue to impress - I'm going to drop the air quote "Live sand" from now on, all 3 live sand products available locally processed ammonia and nitrite in textbook fashion. Ocean Direct was a bit faster than Caribsea's other sand product arag-alive. In fact, only Biospira cleared ammonia+nitrite faster than OceanDirect. Fritz turbostart did what it is known for - extremely rapid immediate ammonia oxidiation. It seems to lose interest little bit as the ammonia levels get really low. I expected this from heterotrophs, but apparently Fritz is a type of nitrifier that really excels at high ammonia levels, but isn't so active against low levels. Fun thought: if you take Randy's suggestion to heart - that nitrifiers represent a competitor to coral nutrition and should be viewed as "bad guys" in an established reef tank, then this behavior from Fritz may actually be just what you want: aggressive against high levels, but doesn't care about low levels of ammonia, leaving it for corals. (I don't agree with the "bad guy" premise, but that's a diff thread). This slow-down of Fritz turbostart for low-level ammonia and nitrite was also observed by @Dan_P a couple of years ago. We might also speculate that if it's very slow in uptake against the low levels in a reef tank, it's highly likely that it eventually fades to nothing and other strains replace it over time. Other true nitrifier products measured so far did not indicate any notable slow-down at low concentrations.

I'm a bit surprised at the number of people in threads who use MB7 as cycling bacteria (BRS apparently promoted it for that purpose in videos?), but I'm confident it's one of the heterotroph types, and so addition of food later should be much more illuminating.

OK MB7 goes on my list of questionable/potential snake oil bottled bacteria products. Oh yeah, fantastic work as usual.

As for “nitrifying bacteria are coral enemies”, I am now thinking about a subset of new aquaria that could skip the cycling process altogether ( this subset could be the systems that validate @brandon429 approach to reefing) Definitely in this subset are the coral only systems with a sprinkling of clean up crew. Stretching the boundary line a bit, a low number of little fish added after the coral are growing could be OK. The hidden variable here is micro algae and coral as ammonia removers. Eventually nitrifying bacteria will move in (Maybe. I need to test this).

 

[brandon429]

I fully believe that will work too, you've described not much challenge loading at all in that particular setup

 

[taricha]

OK MB7 goes on my list of questionable/potential snake oil bottled bacteria products.

and how are enzymes not digested immediately by bacteria in the aquarium?

one day I'll get around to measuring the amount of digestible organic carbon in a dose of MB7. I imagine it is significant and relevant to people's experience with the product, much like Waste Away.

Stretching the boundary line a bit, a low number of little fish added after the coral are growing could be OK. The hidden variable here is micro algae and coral as ammonia removers.

If I run a liter of my tank water through a glass fiber filter, I can put the filter under the microscope and find countless microalgae cells captured on the filter. If I place the filter in a solvent I can extract plenty of chlorophyll to measure by absorbance and fluorescence.
I have no reason to think 1L of LFS water would be all that different, So when a product says "add fish on day 1" in all likelihood - they are also saying "add single cell photosynthetic algae on day 1"

 

[taricha]

Here's the data on this idea about taking a good bottle and subjecting to temperature extremes to see if it slows a little bit, or catastrophic loss (99%+) or not at all. I think this one came out pretty close to conventional wisdom.

Potential Side-trip: "dud"-ify some good bottles.

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)

Here's Ammonia and Nitrite.... ( I changed the numbers - 1 is nothing, 2 is room temp biospira, 3 is Ice bath etc.) The high temp exposed ones are marked with stars.

The tube that spent hours in an ice bath was basically indistinguishable from the room temp control by the end, which makes sense because refrigeration is often encouraged.
The "slight freeze" tube that was placed in temps just below freezing (fresh water froze, but the saltwater biospira media did not) was notably slowed down but still active at maybe 50%.
The tube that spent 2 milder days in my trunk (temps in trunk over 100F) seemed almost the same as the tube that spent 5 days in the trunk with 3 of those holding ~120F in the trunk. Both the tubes that spent time in the heat looked similar to the "slight freeze" - around half of ammonia oxidation rate was lost.
The sample that was hard frozen 1x at 0F overnight, and the one that was thawed and refrozen (3x total freezes) in the freezer seem to have lost all or nearly all function. No clear detectable activity from them at 6 days. If they aren't completely dead, they are really really close. They no longer act like bottles of biospira.
(I'll keep watching the one that got a hard freeze 1x as it looks like it may be starting to barely produce nitrite, but in practice - even if it eventually came alive, you'd still want to get a different bottle if yours froze solid.)

 

[Dan_P]

Here's the data on this idea about taking a good bottle and subjecting to temperature extremes to see if it slows a little bit, or catastrophic loss (99%+) or not at all. I think this one came out pretty close to conventional wisdom.



Here's Ammonia and Nitrite.... ( I changed the numbers - 1 is nothing, 2 is room temp biospira, 3 is Ice bath etc.) The high temp exposed ones are marked with stars.

The tube that spent hours in an ice bath was basically indistinguishable from the room temp control by the end, which makes sense because refrigeration is often encouraged.
The "slight freeze" tube that was placed in temps just below freezing (fresh water froze, but the saltwater biospira media did not) was notably slowed down but still active at maybe 50%.
The tube that spent 2 milder days in my trunk (temps in trunk over 100F) seemed almost the same as the tube that spent 5 days in the trunk with 3 of those holding ~120F in the trunk. Both the tubes that spent time in the heat looked similar to the "slight freeze" - around half of ammonia oxidation rate was lost.
The sample that was hard frozen 1x at 0F overnight, and the one that was thawed and refrozen (3x total freezes) in the freezer seem to have lost all or nearly all function. No clear detectable activity from them at 6 days. If they aren't completely dead, they are really really close. They no longer act like bottles of biospira.
(I'll keep watching the one that got a hard freeze 1x as it looks like it may be starting to barely produce nitrite, but in practice - even if it eventually came alive, you'd still want to get a different bottle if yours froze solid.)

Takes a beating and still seems to out perform Dr Tim’s

 

[CBonito]

Awesome thread Taricha.

 

[Idech]

This is incredible work, thank you !

It would be interesting when you’re done to get a summary listing all products in order of days to reduce ammonia to 0 (sorry if I missed it).

Also, is it too late to add a product ? Aquaforest BioS is readily available around here and it would be nice to know.

 

[taricha]

It would be interesting when you’re done to get a summary listing all products in order of days to reduce ammonia to 0 (sorry if I missed it).

Sure. I meant to post a table summary, but I was waiting until after group 3.

Also, is it too late to add a product ? Aquaforest BioS is readily available around here and it would be nice to know.

maybe. I'll look into it.

 

[taricha]

Here's a summary of results so far... (Will update 1st post.)
The color groupings are arbitrary, and the days shouldn't be taken as definitive. Changes in flow, temp, surfaces etc could make these faster. These are responses under the most restrictive conditions - simply ammonia, gently moving water, and time.

Links to data posts...
Group 1: post 45
Group 2: post 64 and post 96
Group 3: post 141

Main experimental stuff remaining:
Group 4 is currently running (including a different bottle of One and Only).
Then the non-responders in the Ammonia-Only round (those that do not process ammonia alone) will be fed fish food to determine if they can control ammonia when there's a carbon source present.
Small side-tests to illustrate applicability to hobby systems:
Compare centrifuged to raw out of the bottle for some of the slow responders to check that the settle-able solids are indeed where the active nitrifiers are.
Compare performance for a few of the responders when added to a small tank (10L) to see if it's similar enough to their performance in my 60mL sample jars.

 

[Azedenkae]

On the topic of feeding heterotrophs, I wonder if supplying glucose might be preferable?

Not all heterotrophs can consume glucose, but well, chances are they probably do lol.

 

[taricha]

On the topic of feeding heterotrophs, I wonder if supplying glucose might be preferable?

Not all heterotrophs can consume glucose, but well, chances are they probably do lol.

I'm thinking I'm going to use this as the inspiration for the heterotroph phase of the experiment. Basically model fish-in cycling.
https://apifishcare.com/pdfs/products-us/quick-start/quick-start-study.pdf

I won't actually put fish in the tiny sample jars, obviously. But I will use fish water from a bag of fish from my LFS.
The part that I wonder about is that the fish water will be full of heterotrophs already, and so I'm not convinced that the addition of the bottle products will be a strong enough effect to see some difference vs the activity of the heterotrophs. But I guess that's what the experiment is for.

 

[taricha]

Update to this data on the temp extremes, and it's a fun one (to me, anyway)....

Here's the data on this idea about taking a good bottle and subjecting to temperature extremes to see if it slows a little bit, or catastrophic loss (99%+) or not at all. I think this one came out pretty close to conventional wisdom.



Here's Ammonia and Nitrite.... ( I changed the numbers - 1 is nothing, 2 is room temp biospira, 3 is Ice bath etc.) The high temp exposed ones are marked with stars.

The tube that spent hours in an ice bath was basically indistinguishable from the room temp control by the end, which makes sense because refrigeration is often encouraged.
The "slight freeze" tube that was placed in temps just below freezing (fresh water froze, but the saltwater biospira media did not) was notably slowed down but still active at maybe 50%.
The tube that spent 2 milder days in my trunk (temps in trunk over 100F) seemed almost the same as the tube that spent 5 days in the trunk with 3 of those holding ~120F in the trunk. Both the tubes that spent time in the heat looked similar to the "slight freeze" - around half of ammonia oxidation rate was lost.
The sample that was hard frozen 1x at 0F overnight, and the one that was thawed and refrozen (3x total freezes) in the freezer seem to have lost all or nearly all function. No clear detectable activity from them at 6 days. If they aren't completely dead, they are really really close. They no longer act like bottles of biospira.
(I'll keep watching the one that got a hard freeze 1x as it looks like it may be starting to barely produce nitrite, but in practice - even if it eventually came alive, you'd still want to get a different bottle if yours froze solid.)

The Biospira sample that got a single "hard freeze" in 15mL centrifuge tube overnight in the freezer at 0F - actually ended up processing ammonia AND nitrite just fine, it was merely significantly delayed. See Yellow data below.

The amount of NO2-N accumulation is clearly lower than the ammonia-N consumed (like the other biospira samples) - indicating plenty of active nitrite oxidizers in this sample too. In fact, although the onset of ammonia processing was delayed by ~7 days, after that - the rate of ammonia consumed and nitrite produced looks almost identical to rate of the other active samples.
The sample that got 3x cycles of hours to overnight hard (0F) freezes and thaws still looks dead by comparison.

Heh, maybe if everyone used biospira, more people might have the @brandon429 perspective - that nitrification is unavoidable, fast, and so reliable it's not even worth measuring.

 

[brandon429]

I use the no measuring approach on any of the bacteria after ten days wait and after some feed was provided and we never dose 2 ppm ammonia, I advise cycles opposite of yours and in this group there aren’t any fails on seneye. I’m constantly trying to avoid mb7 in cycles, when they’re using it I try and add five more days to the wait period

I prefer the fish food as the feed and not any ammonia added in my testless threads or they can add a few drops and that’s the totality of ammonia used

even partially compromised bacteria work just fine on two clownfish inside a tank’s dilution after ten days wait and not much loading they have to process, from any brand. I don’t buy the timeframes stated when it’s not seneye you know I’m an eternal skeptic for the lag times of non digital test kits even though I think you’re a pro with api. If it’s not a seneye graph I think it’s estimated just a little and I don’t mean seneye discs laid out and guessed at for the color…am meaning a digital seneye graph printed out from the machine and those data plots used to make the graph. For sure this thread has provided very interesting data for sure this is no slight to the work on file it’s more detailed than any I’ve seen on the matter.

 

[taricha]

Had a few bottles free and space on the shaker, so...
Sidequest #2 in progress.

When you do test FritzZyme TurboStart 900, I think it'd also be good to test FritzZyme TurboStart 700, and then both with fresh rather than saltwater. One study had found that nitrifiers specifically adapted to saltwater can still function pretty decently in fully fresh water, but not vice versa.

I took saltwater and lake water and dosed each to ~2ppm total ammonia and split each into 3 containers: Nothing, Fritz 700, & Fritz 900.

We'll check the expectation that the saltwater strains can muddle through in fresh water, while the freshwater strain is totally shut down by saltwater. (Of no relevance to the hobby, just curiosity, and it should be easy and quick. )

 

[brandon429]

which of the bottle brands tested would not be able to carry a basic reef bioload (two clowns, some snails, a few frags + their commanded feed) after dosing any of the brands with a pinch of fish food and waiting 15 days to begin

as a sum-summary of all the data which brands still fail on day 15/unable to process any ammonia

surface area + 15 days wait on fish food alone is going to carry a decent cycle ability, for ammonia control, hence the 10 day ammonia line on all cycling charts/it would have been neat to have just tank water + some feed + 15 days as a baseline here but I'm aware the study was for slow v fast bottle bac brands.

 

[taricha]

which of the bottle brands tested would not be able to carry a basic reef bioload (two clowns, some snails, a few frags + their commanded feed) after dosing any of the brands with a pinch of fish food and waiting 15 days to begin

Ask me again after I have run some bacteria against fish flake samples.
But right now I can tell you some of the classic nitrifier products are fast enough that it doesn't matter what the source of ammonia is, whether fish flake or ammonia drops, they can keep up with the low load of a tank start.

 

[taricha]

Side quest #2

When you do test FritzZyme TurboStart 900, I think it'd also be good to test FritzZyme TurboStart 700, and then both with fresh rather than saltwater. One study had found that nitrifiers specifically adapted to saltwater can still function pretty decently in fully fresh water, but not vice versa. I presume it does not apply to every case, but would still be interesting to see for FritzZyme.

I took saltwater and lake water and dosed each to ~2ppm total ammonia and split each into 3 containers: Nothing, Fritz 700, & Fritz 900.

We'll check the expectation that the saltwater strains can muddle through in fresh water, while the freshwater strain is totally shut down by saltwater. (Of no relevance to the hobby, just curiosity, and it should be easy and quick. )

Here's the result. First, in saltwater....

There may be some activity by the freshwater Fritz 700 in salt (slowly accumulating tiny amount of nitrite), but it's very small, and doesn't seem useful in this context.

And in freshwater....

The reverse - the Fritz saltwater 900 shows a tiny flush of activity judged by NO2, but it's barely above noise and doesn't seem useful.
It seems neither the fresh nor salt versions are acceptable substitutes for the other in a pinch.

 

[Azedenkae]

Side quest #2




Here's the result. First, in saltwater....

There may be some activity by the freshwater Fritz 700 in salt (slowly accumulating tiny amount of nitrite), but it's very small, and doesn't seem useful in this context.

And in freshwater....

The reverse - the Fritz saltwater 900 shows a tiny flush of activity judged by NO2, but it's barely above noise and doesn't seem useful.
It seems neither the fresh nor salt versions are acceptable substitutes for the other in a pinch.

Very interesting! Thanks a bunch for testing this out! Yeah I agree with your assessment.

Now I am curious how nitrification continued to occur in one of my tanks when I converted it from salt to fresh... I wonder if there's a difference between how nitrifiers may respond if already established in a tank versus dumped straight into the tank... But anyways, best not go even further down the rabbit hole lol.

 

[taricha]

Now I am curious how nitrification continued to occur in one of my tanks when I converted it from salt to fresh... I wonder if there's a difference between how nitrifiers may respond if already established in a tank versus dumped straight into the tank

I can imagine that if it was embedded in biofilms there might be enough surrounding aqueous film to blunt the osmotic shock.
Whereas I just dumped them into freshwater.

But anyways, best not go even further down the rabbit hole lol.

I'll try