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

[taricha]

(post 3 updated with procedure)

Procedure Part 1 - Ammonia only:
This - Part 1 - is meant to be pretty restrictive to clearly distinguish those sources with and without classic chemoautotrophic nitrifiers. Bacteria products that don't process ammonia under these conditions will have their particular needs met in subsequent parts. No food/organic carbon - including liquid media in bottle, no light nor substrate surfaces aside from the glass and water. All they get is moving saltwater, ammonia, O2, moderate temps and time.
Water is new Instant Ocean at 1.026 S.G. Stock solution is prepared from Ammonium Chloride. On day 0, 0.5ppm total ammonia is added - over the next 2-3 days, more stock is added to ramp up to 2.0ppm total ammonia added.
60 mL of the water is added to 100mL glass Kimax bottles (bottles will then have about 50mL of airspace - abundant O2 for these conditions). After inoculation, bottles are placed on their side on an orbital shaker at ~60rpm 24/7 in the dark.

Ambient temps are 72-78F. Bottles will be opened every 1-3 days and samples for chemical tests are taken with sterilized disposable pipette tips to avoid cross contamination. After sampling, bottles are shaken vigorously for ~3 seconds to aerate O2 to back near maximum or break up any surface films that might try to form.
For this part, since both random contamination and saltwater tank contamination do nothing to ammonia for ~a month - samples are not sterilized. All materials are simply washed in soap and water, and rinsed in tap + distilled like a hobbyist might do.

Inoculation:
The bottled bacteria products are vigorously shaken for 30 seconds before opening. They are then poured into 15mL centrifuge tubes and centrifuged at 4000 RPM for ~60 sec.
The media from the bottle is then poured off leaving the visible bacteria pellet behind. The pelleted bacteria are resuspended in the new mixed saltwater, and the bottles are then inoculated with the resuspended bacteria. The amount of bacteria added for all products will be 3x the minimum amount recommended (since some do not give a maximum amount recommended "pour the whole bottle".)
Sand products have their media removed similarly by being scooped into a beaker and the new saltwater is poured over and gently swirled around so the water moved but not the sand. The new saltwater is poured off and sand - enough to cover the bottom - is added to inoculate the bottles.

Measurements:
Total ammonia - modified API
Nitrite - API
pH - API
Will all be measured colorimetrically and values calculated from calibrations done on those test kits.
After nitrite is zero - either by being fully processed or none ever produced - Nitrate - hanna high range will be measured to compare to the expected NO3 from the Ammonia processed.



(Procedure Part 2 - fish food: ....later)

 

[MnFish1]

(post 3 updated with procedure)

Procedure Part 1 - Ammonia only:
This - Part 1 - is meant to be pretty restrictive to clearly distinguish those sources with and without classic chemoautotrophic nitrifiers. Bacteria products that don't process ammonia under these conditions will have their particular needs met in subsequent parts. No food/organic carbon - including liquid media in bottle, no light nor substrate surfaces aside from the glass and water. All they get is moving saltwater, ammonia, O2, moderate temps and time.
Water is new Instant Ocean at 1.026 S.G. Stock solution is prepared from Ammonium Chloride. On day 0, 0.5ppm total ammonia is added - over the next 2-3 days, more stock is added to ramp up to 2.0ppm total ammonia added.
60 mL of the water is added to 100mL glass Kimax bottles (bottles will then have about 50mL of airspace - abundant O2 for these conditions). After inoculation, bottles are placed on their side on an orbital shaker at ~60rpm 24/7 in the dark.

Ambient temps are 72-78F. Bottles will be opened every 1-3 days and samples for chemical tests are taken with sterilized disposable pipette tips to avoid cross contamination. After sampling, bottles are shaken vigorously for ~3 seconds to aerate O2 to back near maximum or break up any surface films that might try to form.
For this part, since both random contamination and saltwater tank contamination do nothing to ammonia for ~a month - samples are not sterilized. All materials are simply washed in soap and water, and rinsed in tap + distilled like a hobbyist might do.

Inoculation:
The bottled bacteria products are vigorously shaken for 30 seconds before opening. They are then poured into 15mL centrifuge tubes and centrifuged at 4000 RPM for ~60 sec.
The media from the bottle is then poured off leaving the visible bacteria pellet behind. The pelleted bacteria are resuspended in the new mixed saltwater, and the bottles are then inoculated with the resuspended bacteria. The amount of bacteria added for all products will be 3x the minimum amount recommended (since some do not give a maximum amount recommended "pour the whole bottle".)
Sand products have their media removed similarly by being scooped into a beaker and the new saltwater is poured over and gently swirled around so the water moved but not the sand. The new saltwater is poured off and sand - enough to cover the bottom - is added to inoculate the bottles.

Measurements:
Total ammonia - modified API
Nitrite - API
pH - API
Will all be measured colorimetrically and values calculated from calibrations done on those test kits.
After nitrite is zero - either by being fully processed or none ever produced - Nitrate - hanna high range will be measured to compare to the expected NO3 from the Ammonia processed.



(Procedure Part 2 - fish food: ....later)

One comment - would consider adding a small amount of PO4 - since even obligate nitrifiers will require Some PO4. If using freshly made seawater there may not be enough.

 

[taricha]

phosphate is also a worthwhile consideration if bacteria are unresponsive to ammonia under the test conditions.

 

[taricha]

Here's Group 1 - the first 10 test samples.
1 - "Sterile": heat killed by boiling -not quite sterile, but close enough for our purposes.
2 - nothing : No additions of any bacterial sources, but random contamination that would occur when mixing new saltwater etc will be present
3 & 4 - tankwater: replicates each inoculated with a drop of water from my tank.
(because 1,2,3,& 4 will all do nothing to ammonia in any short time frame, these are all essentially negative controls just with different details. Previous fiddling around suggests that around 3-4 weeks, my tank water should start to show cycling activity on the ammonia. I'll treat this as a sort of minimum detectable baseline for nitrification.)

5 & 6 - Biospira (old but not expired bottle): contains "Nitrosospira, Nitrosomonas, Nitrospira and Nitrococcus" (this is a positive control - if it doesn't work, then I botched something badly.)

7 & 8 - Seachem Stability: "Stability® will rapidly and safely establish the aquarium biofilter in freshwater and marine systems[...] Stability® is formulated specifically for the aquarium and contains a synergistic blend of aerobic, anaerobic, and facultative bacteria which facilitate the breakdown of waste organics, ammonia, nitrite, and nitrate....
The necessary conditions for the growth of Stability's™ bacteria strains encompass a very broad range. When other bacteria begin to die off (usually from high organic loads caused by the undetected death of an organism), Stability™ simply becomes more effective."

9 & 10 - Nature's Ocean Bio-Activ Live Aragonite sand
"Collected in its natural environment and packaged in its natural state
Just place in tank, add saltwater [...] then add fish, no waiting required.
Instant ammonia cycling
Contains natural live marine bacterial biofilm."

 

[taricha]

And here's the data for Group 1:
Boiled "sterilized", Nothing, Tank water, Biospira, Seachem Stability, and Nature's Ocean Bio-Active Live sand.



Total Ammonia and Nitrite

Both replicates for Biospira (yellow) and the Nature's Ocean bagged sand (orange) processed away the ammonia in the left chart, and no other treatments had any effect on the total ammonia measurements. Likewise, the Biospira and the Nature's Ocean samples also produced significant (but very different) amounts of Nitrite in sync with the ammonia removal. (I was on vacation from day 4-10).

Final Nitrate value and trend of pH

The left chart shows the amount of NO3 measured at day 20. All samples except those that processed away ammonia showed zero Nitrate. The amount of final nitrate for the biospira and nature's ocean samples was within Hanna stated uncertainty of the maximum NO3 possible from oxidizing ~2.5 ppm total ammonia.
The pH on the right chart has an uncertainty of at least +-0.05, so with that in mind - there's no clear activity from any of the samples other than the Biospira and Nature's Ocean. The process of oxidizing ammonia to NO2 and NO2 to NO3 are both pH-lowering processes, so this data is in agreement with the other 3 measures. (The boiled "sterile" sample has a different pH than the other samples that did nothing due to changes in the seawater caused by the boiling process.)

Commentary
Biospira is entirely as expected - even a bottle near the expiration date shows some ammonia oxidized to nitrite within 24 hr, and the rate is fast enough to handle accumulating ammonia early in a new lightly fed / stocked system.
Whether the Nature's Ocean Live Sand result is surprising or not depends on your prior expectations of those bags of wet sand sitting on the shelf in your LFS. I personally did not expect it to have classic nitrifiers at all, but the data is clear, it does. The rate is far slower than biospira - but the mechanism of ammonia removal is the same. Since the ammonia processing didn't begin until ~day 9, the "Instant Cycling" isn't really supported by this part of the test. However this product advice does make sense with the data provided "During the first month introduce no more than 1" of fish per 5 gallon saltwater."

It is interesting that the final nitrate for both Biospira and the Nature's Ocean is exactly (within error) what you would expect from the classical ammonia->NO2->NO3 pathway - indicating that no other processes like assimilation were significant in doing anything to ammonia. It's also interesting that the intermediate nitrite accumulated in the Biospira is so much lower than in the Nature's Ocean although the amount of final NO3 is the same. This seems to indicate that in the Nature's Ocean, the ammonia oxidizers are active first, and the Nitrite oxidizers lag behind - while in Biospira some portion of the NO2 must be oxidized to NO3 at nearly the same time as the ammonia is being oxidized to NO2. Either the separate populations of ammonia oxidizers and nitrite oxidizers are working at the same time or biospira contains some nitrifying organisms capable of doing both ammonia and nitrite oxidation. This is unusual, but is called complete ammonia oxidation - comammox - and has been found in some nitrospira in recirculating aquaculture system bioflters (Complete nitrification: insights into the ecophysiology of comammox Nitrospira) so it can't be ruled out in Biospira which is known to contain nitrospira.

As for the non-responders: the negative controls of "sterile", "nothing", and tank water made no measurable changes to ammonia over the 20 day time frame. And Seachem Stability was indistinguishable from those. This is consistent with the presumption that this product contained only heterotroph nitrifiers, and would do nothing when presented with ammonia only. We'll see if it behaves differently later, when fed fish food.

 

[Azedenkae]

And here's the data for Group 1:
Boiled "sterilized", Nothing, Tank water, Biospira, Seachem Stability, and Nature's Ocean Bio-Active Live sand.



Total Ammonia and Nitrite

Both replicates for Biospira (yellow) and the Nature's Ocean bagged sand (orange) processed away the ammonia in the left chart, and no other treatments had any effect on the total ammonia measurements. Likewise, the Biospira and the Nature's Ocean samples also produced significant (but very different) amounts of Nitrite in sync with the ammonia removal. (I was on vacation from day 4-10).

Final Nitrate value and trend of pH

The left chart shows the amount of NO3 measured at day 20. All samples except those that processed away ammonia showed zero Nitrate. The amount of final nitrate for the biospira and nature's ocean samples was within Hanna stated uncertainty of the maximum NO3 possible from oxidizing ~2.5 ppm total ammonia.
The pH on the right chart has an uncertainty of at least +-0.05, so with that in mind - there's no clear activity from any of the samples other than the Biospira and Nature's Ocean. The process of oxidizing ammonia to NO2 and NO2 to NO3 are both pH-lowering processes, so this data is in agreement with the other 3 measures. (The boiled "sterile" sample has a different pH than the other samples that did nothing due to changes in the seawater caused by the boiling process.)

Commentary
Biospira is entirely as expected - even a bottle near the expiration date shows some ammonia oxidized to nitrite within 24 hr, and the rate is fast enough to handle accumulating ammonia early in a new lightly fed / stocked system.
Whether the Nature's Ocean Live Sand result is surprising or not depends on your prior expectations of those bags of wet sand sitting on the shelf in your LFS. I personally did not expect it to have classic nitrifiers at all, but the data is clear, it does. The rate is far slower than biospira - but the mechanism of ammonia removal is the same. Since the ammonia processing didn't begin until ~day 9, the "Instant Cycling" isn't really supported by this part of the test. However this product advice does make sense with the data provided "During the first month introduce no more than 1" of fish per 5 gallon saltwater."

It is interesting that the final nitrate for both Biospira and the Nature's Ocean is exactly (within error) what you would expect from the classical ammonia->NO2->NO3 pathway - indicating that no other processes like assimilation were significant in doing anything to ammonia. It's also interesting that the intermediate nitrite accumulated in the Biospira is so much lower than in the Nature's Ocean although the amount of final NO3 is the same. This seems to indicate that in the Nature's Ocean, the ammonia oxidizers are active first, and the Nitrite oxidizers lag behind - while in Biospira some portion of the NO2 must be oxidized to NO3 at nearly the same time as the ammonia is being oxidized to NO2. Either the separate populations of ammonia oxidizers and nitrite oxidizers are working at the same time or biospira contains some nitrifying organisms capable of doing both ammonia and nitrite oxidation. This is unusual, but is called complete ammonia oxidation - comammox - and has been found in some nitrospira in recirculating aquaculture system bioflters (Complete nitrification: insights into the ecophysiology of comammox Nitrospira) so it can't be ruled out in Biospira which is known to contain nitrospira.

As for the non-responders: the negative controls of "sterile", "nothing", and tank water made no measurable changes to ammonia over the 20 day time frame. And Seachem Stability was indistinguishable from those. This is consistent with the presumption that this product contained only heterotroph nitrifiers, and would do nothing when presented with ammonia only. We'll see if it behaves differently later, when fed fish food.

Awesome! Pretty much as expected, yeah. If you can, can you keep the sterilized, saltwater, and Seachem Stability experiments going on for a while? Seachem Stability allegedly do contain some nitrifiers, just that they also contain non-nitrifying bacteria. I'd be keen to see if eventually the alleged nitrifiers would start to do their thing, and how long it takes.

 

[brandon429]

I am astounded, floored and amazed at whoever wrote a common cycling chart decades ago. they preempted all these proofing studies with such an amazing observation.

what you provided above is amazing verification of the natural timing of things, truly amazing work.

It wouldn't be too hard to find the post made by aquabiomics, under his original name Eli, that stated here on the site he'd tested nature's ocean sand and found zero nitrifying bacteria. from that I interpret he's over-ascribing the powers of his test (that statement was made before his company came about for profit, but he was using the same mechanism for testing) or he happened to get a bad batch. anyone taking his test at face value as absolute proof of anything is perhaps being misled. I'm saying that because your results are so shocking different.

 

[brandon429]

what you found with stability is so helpful to our cycle threads as well/avoid it

 

[C4ctus99]

what you found with stability is so helpful to our cycle threads as well/avoid it

For fishless skip cycle, yes. For fish in skip cycle I’ve used it numerous times without issue

 

[taricha]

Awesome! Pretty much as expected, yeah. If you can, can you keep the sterilized, saltwater, and Seachem Stability experiments going on for a while? Seachem Stability allegedly do contain some nitrifiers, just that they also contain non-nitrifying bacteria. I'd be keen to see if eventually the alleged nitrifiers would start to do their thing, and how long it takes.

Yes. I intend to check the tank water and other non-responding samples until they begin to cycle. 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 ( which is hard to prevent in my work space. )

 

[brandon429]

Cactus what you've seen is possibly the pH protection aspect of free ammonia Dan mentions so often
(initial setups tend to be lower in pH than tuned, adjusted aged setup with higher pH where ammonia present would burn/harm badly, low pH suppresses the consequence of free ammonia)

am I misreading that graph above: stability is useless/ am bad at graphs I sure might be

 

[taricha]

what you found with stability is so helpful to our cycle threads as well/avoid it

I agree with the below. No justification to use something like this on fishless cycle when others are so solid.

For fishless skip cycle, yes. For fish in skip cycle I’ve used it numerous times without issue

I look forward to seeing what it does presented with fish-in-like conditions.

It wouldn't be too hard to find the post made by aquabiomics, under his original name Eli, that stated here on the site he'd tested nature's ocean sand and found zero nitrifying bacteria. from that I interpret he's over-ascribing the powers of his test (that statement was made before his company came about for profit, but he was using the same mechanism for testing) or he happened to get a bad batch. anyone taking his test at face value as absolute proof of anything is perhaps being misled. I'm saying that because your results are so shocking different.

I don't recall him being definitive, but I do remember him saying he wasn't impressed with the bagged live sand products.
But this makes more sense with added context.
First, aquabiomics later decided that they were missing the nitrifying community in too many people's systems, so added a direct swab as a component of the test.
Secondly, think about what these results say. It took 9 days for a visible response to ammonia. Normal established systems would be far faster than that. And the method usually detects low nitrifying community counts in established systems. So how small must the initial community in the bag of sand have been for it to take 9 days of exponential growth before it could detectably move ammonia? I have no doubt that the initial population would pose a challenge to an aquabiomics style test to measure, but after 9 days of growth in response to ammonia, it would've easily measured that.

 

[C4ctus99]

Cactus what you've seen is possibly the pH protection aspect of free ammonia Dan mentions so often
(initial setups tend to be lower in pH than tuned, adjusted aged setup with higher pH where ammonia present would burn/harm badly, low pH suppresses the consequence of free ammonia)

am I misreading that graph above: stability is useless/ am bad at graphs I sure might be

@taricha did not add a phosphate source, which my understanding is needed to activate the nitrifiers in stability (taricha?)


I had a coral beauty, two clowns, and two barnacle blennies in a 5 gallon bucket for 36 hour then 72 hour time periods using stability without any ammonia issues. Not recommended at all, but was what I had at the time.

From what I’ve gathered reading through taricha, dan, and mnfish, there are two types of nitrifiers and one needs phosphates from fish food/poop to be able to break down ammonia

 

[taricha]

am I misreading that graph above: stability is useless/ am bad at graphs I sure might be

To reiterate, this round is meant to prevent activity by heterotrophs, so only the classic chemoautotroph nitrifiers are detected.
I've done nothing yet that says anything about how these might respond to fish food etc. So, to be determined.

 

[taricha]

@taricha did not add a phosphate source, which my understanding is needed to activate the nitrifiers in stability (taricha?)

The organic carbon source for energy/ growth is the more crucial missing peice in ammonia-only cycle for heterotrophs. (Fish food will provide both carbon/ energy and phosphorous.)

 

[Azedenkae]

Yes. I intend to check the tank water and other non-responding samples until they begin to cycle. 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 ( which is hard to prevent in my work space. )

Sounds like a plan! I fully agree. Keep us updated!

 

[taricha]

I intend to check the tank water and other non-responding samples until they begin to cycle. 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 ( which is hard to prevent in my work space. )

Here's the update on the non-responders in Group 1 at day ~33 - still no activity from my tank water, or the other negative controls, and no activity from Seachem Stability either.

This is just more clarity that the biospira and Nature's Ocean responses were far far different from the non-responders.
I keep saying that in the past my tank water showed some nitrification around day ~30, but nothing apparent in this case, so we'll see.


Meanwhile, Group 2 (One and Only, MB Start XLM, API QuickStart, Caribsea Arag-alive) has been up and going with a couple of products clearly responding so far and others not yet.

 

[Azedenkae]

Here's the update on the non-responders in Group 1 at day ~33 - still no activity from my tank water, or the other negative controls, and no activity from Seachem Stability either.

This is just more clarity that the biospira and Nature's Ocean responses were far far different from the non-responders.
I keep saying that in the past my tank water showed some nitrification around day ~30, but nothing apparent in this case, so we'll see.


Meanwhile, Group 2 (One and Only, MB Start XLM, API QuickStart, Caribsea Arag-alive) has been up and going with a couple of products clearly responding so far and others not yet.

Quick Q - do you still have any of the responders from group 1? Nitrifiers are known to be super resilient, I'd be keen to see if you can just re-dose ammonia and see immediate activity from them again.

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.

 

[taricha]

Quick Q - do you still have any of the responders from group 1? Nitrifiers are known to be super resilient, I'd be keen to see if you can just re-dose ammonia and see immediate activity from them again.

nope! I bleach-washed those already in preparation for group 3. :-(
But I'll have responders for group 2 that I can do this with as a side study. You thinking leave them churning without ammonia / nitrite for a month, then see what they can do when re-presented with the same ammonia challenge?

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.

Interesting off-label use idea. Worth looking at. Agree that what I've read aligns with your prediction, the saltwater nitrifier ought to muddle through and handle the freshwater, but the freshwater one ought to struggle or fail entirely at full salinity.

I feel like the cycling products that say "fresh or saltwater" and "add fish immediately" are really interesting superheroes and I definitely want to see what they are doing up close.

 

[Azedenkae]

But I'll have responders for group 2 that I can do this with as a side study. You thinking leave them churning without ammonia / nitrite for a month, then see what they can do when re-presented with the same ammonia challenge?

While we are at it, I'd try to split them across three designs:
1. Churning
2. No churning
3. Churning, no oxygen
4. No churning, no oxygen (?)

(4) would be preferable, but I suppose not absolutely necessary to do. Not sure how you'd do anoxic conditions though. XD I am a dry lab not a wet lab microbiologist lol.

I feel like the cycling products that say "fresh or saltwater" and "add fish immediately" are really interesting superheroes and I definitely want to see what they are doing up close.

Yep! I mean I've been lazy, but I could also check out some nitrifier genomes in IMG/M or similar to see if there's any reason. My hunch is that like many other prokaryotes adapted to saltwater conditions, saltwater-adapted nitrifiers produce (organic) osmoregulatory molecules, rather than having their molecular machinery modified to adapt to intracellular saline conditions. In which case shifting to a less saline environment just means transcriptional downregulation of genes producing said osmoregulators, as opposed to have their molecular machinery just cease to function. And freshwater-adapted nitrifiers simply do not produce such molecules, either having never been to in the first place or losing the capacity to do so.