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

[taricha]

My hypothesis is that there are very different types of Bacteria that are being used to "cycle" tanks to prevent ammonia accumulation, and it's not entirely clear which products contain which group, and it's also poorly understood what the requirements are for one of these groups to do their job.
The intention is not for this to be a thread about this product "works" and that one "doesn't work". I believe that all of the products likely contain bacteria that are known to process away ammonia under certain conditions, but I think the conditions needed for different products to process ammonia is essentially unknown to us and those conditions may be very different from one product to another.

One group of these starters are the classic nitrifiers or chemoautotrophs - fancy word for saying they get their energy from the oxidation of ammonia->NO2->NO3. Others can oxidize ammonia and produce some NO2 and NO3 if they have an organic carbon source for energy. This energy via organic carbon may be supplied by fish food/waste and they may be able to keep ammonia processed away with only normal feeding. These are the heterotroph nitrifiers. There's also bacteria that are simply heterotrophs that do no nitrification. They consume organic carbon as a food and take in ammonia that they need for the nitrogen in their biomass, but do not process any ammonia to released nitrogen as NO2/NO3/N2 etc.

The chemoautotrophs, the traditional cycling bacteria are very well documented: Dr. Reef's "Myth or Fact" thread demonstrated that Biospira, Dr. Tim's One and Only, and Fritz Turbostart are clearly in this group, and the hobby conventional wisdom is that they may be the only hobby starter products that fall into this category. Interestingly, there are some other products that have descriptions and instructions that make them sound like this type (MicroBacter Start XLM - says you can fishless cycle with ammonia only) but we do not know if they are or not.
That means that the rest of the cycling products that aren't this type are doing something else, need some other food source, and we don't actually know what conditions they need to do the job we expect them to do. For example, I can buy fish food that is ~30% protein or ~55% protein - if the organic carbon in fish food is being used as energy to process the ammonia from protein breakdown - then presumably some bacteria may be able to do the job adequately at a low protein feed % but not at a high protein feed %.

The general intention of the experiment is to determine the conditions needed by these bacteria to process ammonia.
The first part will be: by running them against additions of only ammonia (from NH4Cl), those who can process ammonia with no light or organic carbon and produce NO3 at a near nitrogen-balance to the ammonia consumed are the classic nitrifiers in the category with Biospira, One and Only, and Fritz Turbostart.
The second part will be: determining which other products can substantially or completely keep ammonia from common fish food processed away.
The last part will be: for those products who do not keep ammonia from fish food substantially controlled in part 2, determining what additional help they need to make that happen - different food, additional carbon source, light, helper bacteria from some other source like the fish? Are some wastewater bacteria that need water nutrients more like wastewater treatment to do anything substantial?

Edit:
Results for the ammonia-only tests of 1st 3 groups.
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

 

[taricha]

I'm open to suggestions for starter bacterial products to test that may be interesting or widely used in the hobby. Here's the ones I intend to do at the moment.

BioSpira, Dr Tim's One and Only, Fritz turbostart will be used as positive controls. (If they don't work, then something in the experiment has gone badly sideways.)
API Quick Start
MicroBacter Start XLM
MicroBacter 7
Aquavitro Seed
Seachem Stability
TLC StartSmart Complete (very gimmicky one that my LFS stocks a ton of)
Nature's Ocean (live bagged sand)
Caribsea Arag-Alive (live bagged sand)
PNS Substrate Sauce

 

[taricha]

(placeholder post about procedure specifics)
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)

 

[brandon429]

that is a great representative spread of what the hobby is using, perfect.

 

[MnFish1]

My hypothesis is that there are very different types of Bacteria that are being used to "cycle" tanks to prevent ammonia accumulation, and it's not entirely clear which products contain which group, and it's also poorly understood what the requirements are for one of these groups to do their job.
The intention is not for this to be a thread about this product "works" and that one "doesn't work". I believe that all of the products likely contain bacteria that are known to process away ammonia under certain conditions, but I think the conditions needed for different products to process ammonia is essentially unknown to us and those conditions may be very different from one product to another.

One group of these starters are the classic nitrifiers or chemoautotrophs - fancy word for saying they get their energy from the oxidation of ammonia->NO2->NO3. Others can oxidize ammonia and produce some NO2 and NO3 if they have an organic carbon source for energy. This energy via organic carbon may be supplied by fish food/waste and they may be able to keep ammonia processed away with only normal feeding. These are the heterotroph nitrifiers. There's also bacteria that are simply heterotrophs that do no nitrification. They consume organic carbon as a food and take in ammonia that they need for the nitrogen in their biomass, but do not process any ammonia to released nitrogen as NO2/NO3/N2 etc.

The chemoautotrophs, the traditional cycling bacteria are very well documented: Biospira, Dr. Tim's One and Only, and Fritz Turbostart are believed to be the only hobby starter products that we are confident fall into this category. Interestingly, there are some other products that have descriptions and instructions that make them sound like this type (MicroBacter Start XLM) but we do not know if they are or not.
That means that the rest of the cycling products that aren't this type are doing something else, need some other food source, and we don't actually know what conditions they need to do the job we expect them to do. For example, I can buy fish food that is ~30% protein or ~55% protein - if the organic carbon in fish food is being used as energy to process the ammonia from protein breakdown - then presumably some bacteria may be able to do the job adequately at a low protein feed % but not at a high protein feed %.

The general intention of the experiment is to determine the conditions needed by these bacteria to process ammonia.
The first part will be: by running them against additions of only ammonia (from NH4Cl), those who can process ammonia with no light or organic carbon and produce NO3 at a near nitrogen-balance to the ammonia consumed are the classic nitrifiers in the category with Biospira, One and Only, and Fritz Turbostart.
The second part will be: determining which other products can substantially or completely keep ammonia from common fish food processed away.
The last part will be: for those products who do not keep ammonia from fish food substantially controlled in part 2, determining what additional help they need to make that happen - different food, additional carbon source, light, helper bacteria from some other source like the fish? Are they wastewater bacteria that need water nutrients more like wastewater treatment to do anything substantial?

You seem to be ignoring a previous experiment done by @Dr. Reef that answered this question. Of course - I don't want to jump onto your thread - claiming that someone is trying to dupe the reefing community - and then making up an experiment that deems to show it - But, IMHO - its been done..

 

[MnFish1]

I'm open to suggestions for starter bacterial products to test that may be interesting or widely used in the hobby. Here's the ones I intend to do at the moment.

BioSpira, Dr Tim's One and Only, Fritz turbostart will be used as positive controls. (If they don't work, then something in the experiment has gone badly sideways.)
API Quick Start
MicroBacter Start XLM
MicroBacter 7
Aquavitro Seed
Seachem Stability
TLC StartSmart Complete (very gimmicky one that my LFS stocks a ton of)
Nature's Ocean (live bagged sand)
Caribsea Arag-Alive (live bagged sand)
PNS Substrate Sauce

This seems odd - since I was involved in the previous experiment - many of the products you're recommending are not useful for cycling.

 

[brandon429]

I'm happy to see any replication of the experiment from new sources. no matter who does these, Ill be comparing their day ten ammonia logs to the ammonia line from a cycling chart. I'll also be watching to see if they use non digital or digital test kits, lag time/speed of report never agree between the two so I'm always left to guess what digital ones would read...but I'm hungry for any snippet of information. we can find a few hundred digital nh3 cycling threads to peruse that weren't part of any controlled study, they were people with a seneye and a display and some bottle bac and some ammonia... and those snippets count just the same. the amalgam mix of all the outcomes is the big pie I like to think about when posting in cycling threads

I want as many people doing this experiment as we can possibly get. if there are changes to rules to be made it'll come from pattern works like these. Taricha Dan and Randy are among the few where non-digital ammonia readings taken seem very reliable: controls confounds very well compared to res publica

 

[MnFish1]

My hypothesis is that there are very different types of Bacteria that are being used to "cycle" tanks to prevent ammonia accumulation, and it's not entirely clear which products contain which group, and it's also poorly understood what the requirements are for one of these groups to do their job.
The intention is not for this to be a thread about this product "works" and that one "doesn't work". I believe that all of the products likely contain bacteria that are known to process away ammonia under certain conditions, but I think the conditions needed for different products to process ammonia is essentially unknown to us and those conditions may be very different from one product to another.

One group of these starters are the classic nitrifiers or chemoautotrophs - fancy word for saying they get their energy from the oxidation of ammonia->NO2->NO3. Others can oxidize ammonia and produce some NO2 and NO3 if they have an organic carbon source for energy. This energy via organic carbon may be supplied by fish food/waste and they may be able to keep ammonia processed away with only normal feeding. These are the heterotroph nitrifiers. There's also bacteria that are simply heterotrophs that do no nitrification. They consume organic carbon as a food and take in ammonia that they need for the nitrogen in their biomass, but do not process any ammonia to released nitrogen as NO2/NO3/N2 etc.

The chemoautotrophs, the traditional cycling bacteria are very well documented: Biospira, Dr. Tim's One and Only, and Fritz Turbostart are believed to be the only hobby starter products that we are confident fall into this category. Interestingly, there are some other products that have descriptions and instructions that make them sound like this type (MicroBacter Start XLM) but we do not know if they are or not.
That means that the rest of the cycling products that aren't this type are doing something else, need some other food source, and we don't actually know what conditions they need to do the job we expect them to do. For example, I can buy fish food that is ~30% protein or ~55% protein - if the organic carbon in fish food is being used as energy to process the ammonia from protein breakdown - then presumably some bacteria may be able to do the job adequately at a low protein feed % but not at a high protein feed %.

The general intention of the experiment is to determine the conditions needed by these bacteria to process ammonia.
The first part will be: by running them against additions of only ammonia (from NH4Cl), those who can process ammonia with no light or organic carbon and produce NO3 at a near nitrogen-balance to the ammonia consumed are the classic nitrifiers in the category with Biospira, One and Only, and Fritz Turbostart.
The second part will be: determining which other products can substantially or completely keep ammonia from common fish food processed away.
The last part will be: for those products who do not keep ammonia from fish food substantially controlled in part 2, determining what additional help they need to make that happen - different food, additional carbon source, light, helper bacteria from some other source like the fish? Are they wastewater bacteria that need water nutrients more like wastewater treatment to do anything substantial?

BTW - Yes - it's been shown already - different bacteria (heterotrophs - vs autotrophs) that are being used to cycle a tank. However - the exact parameters are not established - But - as already described by @Dr. Reef there may be a phosphorous requirement - in addition to ammonia, etc. All that said - I think this has already been confirmed.

 

[MnFish1]

I'm happy to see any replication of the experiment from new sources. no matter who does these, Ill be comparing their day ten ammonia logs to the ammonia line from a cycling chart. I'll also be watching to see if they use non digital or digital test kits, lag time/speed of report never agree between the two so I'm always left to guess what digital ones would read...but I'm hungry for any snippet of information. we can find a few hundred digital nh3 cycling threads to peruse that weren't part of any controlled study, they were people with a seneye and a display, and those snippets count just the same. I want as many people doing this experiment as we can possibly get.

LOL. of course

 

[MnFish1]

I'm happy to see any replication of the experiment from new sources. no matter who does these, Ill be comparing their day ten ammonia logs to the ammonia line from a cycling chart. I'll also be watching to see if they use non digital or digital test kits, lag time/speed of report never agree between the two so I'm always left to guess what digital ones would read...but I'm hungry for any snippet of information. we can find a few hundred digital nh3 cycling threads to peruse that weren't part of any controlled study, they were people with a seneye and a display, and those snippets count just the same. I want as many people doing this experiment as we can possibly get.

I hope that the people replicating the experiment - are actually doing so. In other words - for example - a physics teacher vs a microbiologist may make a difference

 

[C4ctus99]

This seems odd - since I was involved in the previous experiment - many of the products you're recommending are not useful for cycling.

What do you mean by useful for cycling? With fish, without fish, one week without them with? Or just general cycling?

I can only speak from my experience, but I’ve used seachem many many times, including a five gallon bucket with 4 gallons of water and 5 fish, without issue

 

[Azedenkae]

Here's the deal in my view. It depends on how you define cycling - if it is to establish (robust) nitrification, then only some products are suitable FritzZyme, Bio-Spira, etc. These contain only nitrifiers.

Other products like Stability, etc., contains non-nitrifying heterotrophs (either to the exclusion of nitrifiers or also including them). Regardless, the presence of heterotrophs make them unsuitable for cycling, in my view. Non-nitrifying heterotrophs often outcompete nitrifiers and that kinda defeats the purpose.

However, if one defines cycling, as establishing some sort of method of biological ammonia removal, then that's another story. Because while nitrification is the process whereby ammonia is consumed as an energy source, ammonia can also be consumed as a nitrogen source. However, the latter is for growth, i.e. requiring the consumption of other substrates constantly for ammonia consumption to be kept up.

It's like incinerating garbage versus having a garbage dump. Nitrification is analogous to the former - you can constantly accept more garbage (to a degree, but let's ignore that) and it all just gets incinerated. Consumption of ammonia as a nitrogen source is the latter - the dump just gets larger and larger, and either cause problems, or a huge chunk has to be removed.

 

[Dan_P]

You seem to be ignoring a previous experiment done by @Dr. Reef that answered this question. Of course - I don't want to jump onto your thread - claiming that someone is trying to dupe the reefing community - and then making up an experiment that deems to show it - But, IMHO - its been done..

Maybe @Dr. Reef will drop in and add some useful comments.

 

[brandon429]

I can't wait to see brightwell stuff benched against Dr Tims, biospira and fritz. Dr. Reef may have already done those but I don't recall the p sauce etc, this study seems to incorporate the newer strains people are fervently being sold to use during cycling. I find this study a helpful update to Dr. Reef's thread, not an equal study.

*to avoid trolling this thread we should wait for results from T now. any experiment Taricha or Dan wants to run: we should take notes. On all reef forums there aren't two other posters with better input in my opinion for the matter. even if the matter was a direct copy of prior studies from others, you want these guys running them with the controls they use, it's phenomenal. new things can easily be found even in direct repeats

I can't wait to see how Ken's substrate doser tests out.

 

[MnFish1]

Maybe @Dr. Reef will drop in and add some useful comments.

Yes - agreed - the point of 'repilicating' an experiment is following the protocol exactly as the first person did it. That was my point.

 

[MnFish1]

I'm happy to see any replication of the experiment from new sources. no matter who does these, Ill be comparing their day ten ammonia logs to the ammonia line from a cycling chart. I'll also be watching to see if they use non digital or digital test kits, lag time/speed of report never agree between the two so I'm always left to guess what digital ones would read...but I'm hungry for any snippet of information. we can find a few hundred digital nh3 cycling threads to peruse that weren't part of any controlled study, they were people with a seneye and a display and some bottle bac and some ammonia... and those snippets count just the same. the amalgam mix of all the outcomes is the big pie I like to think about when posting in cycling threads

I want as many people doing this experiment as we can possibly get. if there are changes to rules to be made it'll come from pattern works like these. Taricha Dan and Randy are among the few where non-digital ammonia readings taken seem very reliable: controls confounds very well compared to res publica

Brandon - it was good old me - that suggested this forum. If you took the time to read the comments at the top of the thread - and the recommendations for posting, you would see that the initial purpose was to get other peoples opinions on experimental design. I actually wrote the text that @Daniel@R2R wrote. So - if you have a problem - again - talk to the administrators of the site. Nothing I said was critical - I only pointed out that a similar experiment that was vetted by multiple people has already been done - including microbiologists.

 

[MnFish1]

What do you mean by useful for cycling? With fish, without fish, one week without them with? Or just general cycling?

I can only speak from my experience, but I’ve used seachem many many times, including a five gallon bucket with 4 gallons of water and 5 fish, without issue

I meant - exactly what was shown in the prior experiment. 1. Most people equate cycling with nitrifying bacteria (oligotrophy). Nitrosomonas, etc etc etc. 2. the previous experiment showed that many 'bacteria products' for cycling PROBABLY contained heterotrophs - since they only result in ammonia reduction in the presence of other substances (containing PO4 and C. BTW - You've used Seachem "what" - I have used Stability multiple times as well,. I was merely trying to point out that there is a difference between autotrophs and heterotrophs - and when one does the experiment, one needs to be attentive to those issues

 

[Azedenkae]

I meant - exactly what was shown in the prior experiment. 1. Most people equate cycling with nitrifying bacteria (oligotrophy). Nitrosomonas, etc etc etc. 2. the previous experiment showed that many 'bacteria products' for cycling PROBABLY contained heterotrophs - since they only result in ammonia reduction in the presence of other substances (containing PO4 and C. BTW - You've used Seachem "what" - I have used Stability multiple times as well,. I was merely trying to point out that there is a difference between autotrophs and heterotrophs - and when one does the experiment, one needs to be attentive to those issues

I have personally noticed with products like Seachem Stability and Microbacter7, that the conversion is not characteristic of nitrification. I.e. that in the dark (i.e. no growth of phototrophs), the decrease in ammonia did not corresponding with an expected increase in nitrite/nitrate.

I also noticed that if I dose just ammonia, sometimes I may see a decline - sharply in fact, but then complete and sudden absence of any further decrease. Again, in line with the idea that there are heterotrophs and they could rapidly consume ammonia in conjunction with organic substrates, but then the moment organic substrates are depleted, ammonia is no longer consumed.

So yeah, totally agreed that one has to be very considerate about how one does the experiments. Autotrophs versus heterotrophs mainly (and also lithotrophs versus organotrophs I suppose), but also amongst autotrophs, chemoautotrophs versus photoautotrophs.

 

[Dr. Reef]

I am feeling hot in here. lol
@MnFish1 @brandon429 I love you both, I personally know you both and I have worked on such expoeriments with you both. And I know on what side fo the coin you both tend to lean. Both are good people so lets share in the good info.

to OP, So i did just that few years back and @brandon429 Brightwell MB something i dont remember the name was part of the study.
There were 12 products if i recal. 8 from USA and 3 from Europe and 1 from Hong Kong.
Fritz Turbostart 900 ranked highest in dropping ammoni to 0 in any situation i throw at it. from off the chart to 0 in few days while others strugged or failed or stalled. Then more controlled 4-6ppm to 0 in 3 days in a 5 gal tank.
2nd position was tied between Bio Spira and Dr Tims one and only. Only problem with Bio Spira is to get a fresh bottle as I was lucky enough for IO to send me fresh bottle for free to test, hobbyists may not be that lucky.
Dr Tims worked well as well with no freshness issue like bio spira.

These above 3 I can confidently say are true nitrifying bacteria in my opinion. Rest all in my opnion and per my study seems like they are hetrtrophic or in simple terms sludge removers.

These other products stalled in sterile tank with just ammonia in them. Thats how I conducted the experiments. Hetrotrophis bacteria or sludge removers need carbon and phosphurus source to survive and in my studt 5 gal tanks there wanot much for them to live off. Thus stalling every time till i introduced some fish food later on like 3-5 days of stalled cycle and they kick back in and brought ammonia back down to 0.

I think it all depends on how and how you define a cycle. If you are in a hurry to setup a tank and wat to put fish in the first day after setting up the tank then first 3 mentioned will do the job just fine. Setup a tank dose Fritz or Bio spira or Dr Tim and add fish.
Others will also do the same but intially added bacteria will fade out over time making room for true nitrifying bacteria to colonize and take over. Basicaly they keep ammonia at bay.

All products will process ammonia faster if:
1. Salinity is low more like 1.015-1.017
2. pH and Alk needs to be high
3. Carbon source needs to present like in form of food or live fish
4. Phosphurus needs to be present.

This type of cycle is INSTANT CYCLE or EMERGENCY Hospital tank type, for perfect result good old method of introducing deli shrimp or some ammonia to tank and let it stew for a month is the best robust method.

 

[revhtree]

The gatekeeping has to stop. We're just going to start thread banning without warning. This goes for people who consistently argue and won't let others have an opinion without making it personal. I was going to say it nicely and add the word, please, but that hasn't worked before. Last and final warning and I have instructed our mod team to just thread ban instantly and to report for further action depending on the offense.

Sheesh I hate to even have to type something like that but it really is the last straw. For those of you who are abiding by the TOS, THANK YOU and please don't take offense to this message.