Can this bacterial phenomenon be explained?

[sixty_reefer]

As part of a experiment on the effects of decomposition of organics I’ve added a defrost shrimp (5g) to a vessel with 100ml of tank water containing a very small amount of carbohydrates.

The goal of the experiment was to analyse the ammonia rise from the shrimp and the slow decomposition process due to low o2 in a small volume of water although it seems that the carbohydrates affected the end result in a way that I can’t understand, the ammonia rise never happened and instead ammonia was instantly converted to nitrite (1ppm plus) and nitrite converted to nitrates (220 plus)

question:

how did this happened, organic Carbon can only be useful during the denitrification process for denitrifying bacteria to my knowledge, nitrifying bacteria can’t use organic carbon in order to accelerate the nitrifying cycle and heterotrophic nitrifying bacteria is poorly studied for me to make any conclusion.

I have repeated the test with just tap water and the shrimp and the result at 8 hours was just the expected small amount of ammonia, it seems that the carbohydrates were the culprit.

Some more info on the experiment

experiment 1
Decomposition of a shrimp in a small vessel

• Duration 18 hours
• 5 grams defrosted cooked shrimp
• 100 ml tank water collected just after dosing reef actif
• ammonia 0 pppm
• nitrite 0
• nitrates 15ppm
• ph 8.1

Results:

• Shrimp weigh 5 grams
• ammonia 0 ppm
• nitrite above 1 ppm
• nitrates 220 ppm plus (system runs at 15ppm
• ph 6

comments:

There was no weight loss, visually the shrimp still looked fresh, there was a cloudiness of the water in the vessel although ammonia was 0 ppm.
I was hoping to have an ammonia reading to illustrate this part of the test that would show the results of slow decomposition is ammonia. It seems that somehow the carbon source transformed all the ammonia to nitrite and nitrite to nitrates

will tag @MnFish1 and @Lasse to hear they’re views although if anyone knows this phenomenon or what I may have missed out please chime in.

pictures from experiment 1

0 hours beginning of experiment

18 hours end of experiment

18 hours end of experiment

18 hours ammonia 0 ppm

18 hours nitrite 1ppm plus

nitrates at 18 hours 220 plus (system nitrate 15ppm)

 

[TangerineSpeedo]

I would repeat the test to see if you can repeat the results. I would also duplicate the experiment with a raw shrimp.
I believe that Reef Active in combination with existing bacteria in the water column is enough to handle the slow breakdown of the cooked shrimp. Also temperature could be a factor in the reduction of the breakdown of the shrimp.

 

[sixty_reefer]

I would repeat the test to see if you can repeat the results. I would also duplicate the experiment with a raw shrimp.
I believe that Reef Active in combination with existing bacteria in the water column is enough to handle the slow breakdown of the cooked shrimp. Also temperature could be a factor in the reduction of the breakdown of the shrimp.

I think it’s worth to try and repeat experiment 1 as you say it left me with so many questions that were unexpected
I’ve redone it with just normal water and a shrimp and the result was just ammonia as expected.

I've done another experiment in my tank to illustrate that O2 makes a difference in the decomposition process and that one was straight forward
Only thing I can’t understand is what happened in the small vessel

Experiment 2
Decomposition of a shrimp in a reef tank

• Duration 18 hours
• 6 grams defrosted cooked shrimp
• 25 gallons reef tank (currently at full dose of actif)

results:

• shrimp weight 3 grams (lost 3 grams)

comments:

The shrimp was left on a corner of the system for 18 hours, today at the end of the experiment the shrimp had obvious signs of advanced decomposition (see pics and video) and it lost 3 grams in weight

pictures and videos experiment 2

0 hours before it was added to the sys

18 hours after being added to the system

shrimp in the system 18 hours (before being removed for weight check

 

[taricha]

There was no weight loss, visually the shrimp still looked fresh, there was a cloudiness of the water in the vessel although ammonia was 0 ppm.
I was hoping to have an ammonia reading to illustrate this part of the test that would show the results of slow decomposition is ammonia.

The ammonia test reaction failed because there's too much stuff in the water.

18 hours ammonia 0 ppm

This, in my opinion is not a zero ammonia result - it's a blew-up-the-ammonia-test result.

For the salycilate ammonia test method to work, you have chlorine combine with the ammonia to form monochloramine. From Hach documentation...

If you have a ton of rotting shrimp in the water there are a bunch of compounds other than ammonia that will react with the chlorine in the test kit: proteins, amino acids, etc - and there may not be enough chlorine left to generate the proper reactions. Hence the solution is very pale and not really yellow.

In fact you can blow an ammonia test with just way too much ammonia. Also the pH may be below range for the test to function, since you rotted the shrimp in tap water, the pH could be quite acidic.
see below from another hach ammonia test documentation

I bet if you do a serial dilution on the sample with zero ammonia tank water:
1/3, 1/10, 1/30, 1/100 .... then some of those diluted samples will test positive for ammonia.

 

[sixty_reefer]

The ammonia test reaction failed because there's too much stuff in the water.




This, in my opinion is not a zero ammonia result - it's a blew-up-the-ammonia-test result.

For the salycilate ammonia test method to work, you have chlorine combine with the ammonia to form monochloramine. From Hach documentation...

If you have a ton of rotting shrimp in the water there are a bunch of compounds other than ammonia that will react with the chlorine in the test kit: proteins, amino acids, etc - and there may not be enough chlorine left to generate the proper reactions. Hence the solution is very pale and not really yellow.

In fact you can blow an ammonia test with just way too much ammonia. Also the pH may be below range for the test to function, since you rotted the shrimp in tap water, the pH could be quite acidic.
see below from another hach ammonia test documentation

I bet if you do a serial dilution on the sample with zero ammonia tank water:
1/3, 1/10, 1/30, 1/100 .... then some of those diluted samples will test positive for ammonia.

Thank you, am sure that a simple explanation like you mentioned could be plausible and I think that I will do that once i repeat the experiment.
Any thoughts on why nitrite was 1ppm plus and nitrate around 220 plus? for a minute I thought that the ammonia had been converted to nitrite and nitrates and the reason I couldn’t detect it, some error from my side that I also cant explain.

 

[taricha]

Any thoughts on why nitrite was 1ppm plus and nitrate around 220 plus?

NO2 interference on a NO3 kit can be as low as like 5:1 for stuff like hanna, or it can be like 100:1 for something like API.
I don't know the interference amounts on your NO3 kit. But if NO2 is high then it's best to throw up your hands and just say "lots of NO2, so can't measure NO3"

In general, if you run chemical tests on highly concentrated samples like your "shrimp soup" then unless it is well-behaved with dilutions - then I wouldn't trust it.

So if you can do something like sample, 1/3 sample, and 1/10 sample give a number, 1/3 of the number, and 1/10 of the number then I would feel okay about the test kit result.

(highly concentrated samples complicate testing)

 

[sixty_reefer]

NO2 interference on a NO3 kit can be as low as like 5:1 for stuff like hanna, or it can be like 100:1 for something like API.
I don't know the interference amounts on your NO3 kit. But if NO2 is high then it's best to throw up your hands and just say "lots of NO2, so can't measure NO3"

I agree nitrite will affect overall nitrates although what I find confuseing is that ammoniaconverted to nitrite

In general, if you run chemical tests on highly concentrated samples like your "shrimp soup" then unless it is well-behaved with dilutions - then I wouldn't trust it.

This is not the first time I’m observing this phenomenon wend using this carbohydrate, in another thread with a larger volume of water (5 gallons) I’ve observed a full working nitrogen cycle in just 10 hours, this is why I am here to see if folks know why a carbohydrate (organic carbon) would accelerate the process.

This is a link to the 10 hours results from the other experiment that are similar although ammonium chloride was used instead of a shrimp

Post in thread '72h cycle and avoiding the ugly stage'
https://www.reef2reef.com/threads/72h-cycle-and-avoiding-the-ugly-stage.952413/post-10873088

So if you can do something like sample, 1/3 sample, and 1/10 sample give a number, 1/3 of the number, and 1/10 of the number then I would feel okay about the test kit result.


(highly concentrated samples complicate testing)

 

[taricha]

There is such a thing as heterotrophic nitrification, where heterotroph bacteria use a carbon source and convert ammonia to NO2 and NO3, but it's not a one-to-one conversion. Usually a large amount of ammonia will be taken in and a much smaller amount of NO2 and NO3 (counting N) will appear.
If the carbon source runs out, those bacteria will just sit there and stare at the ammonia.

Many cycling bacteria sold (brightwell, etc) are actually this heterotroph type of nitrifier. Biospira, one and only, and fritz seem to be classical nitrifiers.

 

[sixty_reefer]

There is such a thing as heterotrophic nitrification, where heterotroph bacteria use a carbon source and convert ammonia to NO2 and NO3, but it's not a one-to-one conversion. Usually a large amount of ammonia will be taken in and a much smaller amount of NO2 and NO3 (counting N) will appear.
If the carbon source runs out, those bacteria will just sit there and stare at the ammonia.

Many cycling bacteria sold (brightwell, etc) are actually this heterotroph type of nitrifier. Biospira, one and only, and fritz seem to be classical nitrifiers.

Heterotrophic nitrification would make sense to me, I just didn’t consider the hypothesis as I’ve been told several times that is rare in home aquaria.

my experiments come from looking into implementing biofloc technology into reef systems that claims that heterotrophic bacteria stimulated by carbohydrates are the sole responsible for decomposition of organic matter and ammonia reduction.

on the larger system it stalled at 14-18 hours prova due to the carbon source running out could justify it as you mentioned, I’ve been told that it could be the heterotrophic and nitrifying bacteria competition for space as a reason for the stall.
In the small vessel experiment the water was milky white as you would expect from a heterotrophic bacteria bloom.

if all the above checks this could mean that actif is not just a carbon source for decomposing bacteria but also a carbon source for heterotrophic nitrifying bacteria? As biofloc claims

 

[brandon429]

I appreciate Taricha's detailed response on why/how nondigital test kits can misread and be impacted by common cycling approaches, I'm currently linking this thread/his explanation to several ongoing cycle study threads.

we have been collecting misreads in red sea and api kits for a long, long time and it's nice to see some detailing on how that happens sometimes.

 

[brandon429]

I find it patently amazing we were instructed to cycle with ammonia levels nearly certain to overpower the test kits cyclers use. it was specifically the sellers of bottle bac who told us to dose 2ppm/high levels of ammonia, then we can track out about ten thousand instances of people buying replacement bottle bac for apparently dead bacteria, as indicated by the overdriven/adulterated test kits. what a functional $ feedback loop was created.

 

[taricha]

Heterotrophic nitrification would make sense to me, I just didn’t consider the hypothesis as I’ve been told several times that is rare in home aquaria.

Since like half the tank starter products are heterotrophs, I don't think you could call the process rare.
BUT... in an established tank without intentional carbon dosing, heterotrophs might take a back seat to autotrophs and photosynthetic uptake. Maybe. Depends on the Carbon inputs to the system.

on the larger system it stalled at 14-18 hours prova due to the carbon source running out could justify it as you mentioned

If heterotrophs run out of carbon, ammonia won't be processed. But also if O2 is eaten up then no oxidation of ammonia to NO2/NO3 can happen by anybody.

I’ve been told that it could be the heterotrophic and nitrifying bacteria competition for space as a reason for the stall.

Space? not a relevant limit for competition. Even a flat film on bare glass can house far far more bacteria than needed to process all the ammonia in reef tanks.
O2 competition, you bet.
This fantastic paper is all about what happens when you take a nitrifying biofilter and feed organic carbon to it.
Nitrifying biofilms deprived of organic carbon show higher functional resilience to increases in carbon supply
The bacterial populations shift, but not really because of space - mostly because of O2 competition. (or I guess you could say competition for the space where O2 is). The classic nitrifiers have an absolute need for O2. The heterotrophs have other tricks they can do - respire NO3 for example - so low O2 causes population shifts toward heterotrophs and away from classic nitrifiers.

In the small vessel experiment the water was milky white as you would expect from a heterotrophic bacteria bloom.

and cloudy water indicates O2 will be a limiting resource unless measures are specifically taken to aggressively aerate.

if all the above checks this could mean that actif is not just a carbon source for decomposing bacteria but also a carbon source for heterotrophic nitrifying bacteria?

Those two groups "decomposers" and "heterotrophic nitrifiers" can be one and the same.
Here's a couple of papers on bacillus strains doing heterotrophic nitrification when fed carbon.
Nitrogen removal capability through simultaneous heterotrophic nitrification and aerobic denitrification by Bacillus sp. LY

and
Aerobic nitrification–denitrification by heterotrophic Bacillus strains

This second paper shows Bacillus subtilis and Bacillus licheniformis using Carbon sources to make NO3, while simultaneously also gassing off some nitrogen as N2. Those species of bacteria are some that are in bacterial "grunge eater"/ "decomposer" products in the hobby.

Too bad the conditions in the experiment look nothing like anything our tanks might see.
Otherwise it might be really easy to show that bottled bacterial products are effective and helpful!

 

[sixty_reefer]

Since like half the tank starter products are heterotrophs, I don't think you could call the process rare.
BUT... in an established tank without intentional carbon dosing, heterotrophs might take a back seat to autotrophs and photosynthetic uptake. Maybe. Depends on the Carbon inputs to the system.


If heterotrophs run out of carbon, ammonia won't be processed. But also if O2 is eaten up then no oxidation of ammonia to NO2/NO3 can happen by anybody.


Space? not a relevant limit for competition. Even a flat film on bare glass can house far far more bacteria than needed to process all the ammonia in reef tanks.
O2 competition, you bet.
This fantastic paper is all about what happens when you take a nitrifying biofilter and feed organic carbon to it.
Nitrifying biofilms deprived of organic carbon show higher functional resilience to increases in carbon supply
The bacterial populations shift, but not really because of space - mostly because of O2 competition. (or I guess you could say competition for the space where O2 is). The classic nitrifiers have an absolute need for O2. The heterotrophs have other tricks they can do - respire NO3 for example - so low O2 causes population shifts toward heterotrophs and away from classic nitrifiers.



and cloudy water indicates O2 will be a limiting resource unless measures are specifically taken to aggressively aerate.


Those two groups "decomposers" and "heterotrophic nitrifiers" can be one and the same.
Here's a couple of papers on bacillus strains doing heterotrophic nitrification when fed carbon.
Nitrogen removal capability through simultaneous heterotrophic nitrification and aerobic denitrification by Bacillus sp. LY

and
Aerobic nitrification–denitrification by heterotrophic Bacillus strains

This second paper shows Bacillus subtilis and Bacillus licheniformis using Carbon sources to make NO3, while simultaneously also gassing off some nitrogen as N2. Those species of bacteria are some that are in bacterial "grunge eater"/ "decomposer" products in the hobby.

Too bad the conditions in the experiment look nothing like anything our tanks might see.
Otherwise it might be really easy to show that bottled bacterial products are effective and helpful!

Thank you for this, I will read the shared papers as they seem to be the information missing.

experice 2 on post #3 was made in my system at full dose of TM actif it showed a 3 gram lost on a 6 gram shrimp in 18 hours.

looking at the positive side of the results (although they have some flows) it seems that actif is working in two ways.
1. Reducing organic matter and redirecting those nutrients to bacteria mass (experiment 2) that can be removed from a system by organisms that prey on bacteria, protein skimmer or water changes.
2. Reducing ammonia by transforming it into nitrite and nitrate. (Experiment 1) that are forms that nuisance will require to use more energy to revert them to useful ammonia

Just like some bottled bacteria with the aim to clean a system, I believe they work due to the organic carbon content in addition to possibly having bacteria.

on another thread we are implementing actif as a way to reduce or stall the growth of nuisance photosynthetic organisms (something I’ve observed in my reef with the continuous dosing of actif), if the thought that ammonia and organic nutrients are large contributing aspect of nuisance blooms then bottled bacteria and actif would be a good nutrient to boost competition.

 

[gbroadbridge]

Since like half the tank starter products are heterotrophs, I don't think you could call the process rare.
BUT... in an established tank without intentional carbon dosing, heterotrophs might take a back seat to autotrophs and photosynthetic uptake. Maybe. Depends on the Carbon inputs to the system.


If heterotrophs run out of carbon, ammonia won't be processed. But also if O2 is eaten up then no oxidation of ammonia to NO2/NO3 can happen by anybody.


Space? not a relevant limit for competition. Even a flat film on bare glass can house far far more bacteria than needed to process all the ammonia in reef tanks.
O2 competition, you bet.
This fantastic paper is all about what happens when you take a nitrifying biofilter and feed organic carbon to it.
Nitrifying biofilms deprived of organic carbon show higher functional resilience to increases in carbon supply
The bacterial populations shift, but not really because of space - mostly because of O2 competition. (or I guess you could say competition for the space where O2 is). The classic nitrifiers have an absolute need for O2. The heterotrophs have other tricks they can do - respire NO3 for example - so low O2 causes population shifts toward heterotrophs and away from classic nitrifiers.



and cloudy water indicates O2 will be a limiting resource unless measures are specifically taken to aggressively aerate.


Those two groups "decomposers" and "heterotrophic nitrifiers" can be one and the same.
Here's a couple of papers on bacillus strains doing heterotrophic nitrification when fed carbon.
Nitrogen removal capability through simultaneous heterotrophic nitrification and aerobic denitrification by Bacillus sp. LY

and
Aerobic nitrification–denitrification by heterotrophic Bacillus strains

This second paper shows Bacillus subtilis and Bacillus licheniformis using Carbon sources to make NO3, while simultaneously also gassing off some nitrogen as N2. Those species of bacteria are some that are in bacterial "grunge eater"/ "decomposer" products in the hobby.

Too bad the conditions in the experiment look nothing like anything our tanks might see.
Otherwise it might be really easy to show that bottled bacterial products are effective and helpful!

Interesting research - thanks for posting.

 

[Dan_P]

As part of a experiment on the effects of decomposition of organics I’ve added a defrost shrimp (5g) to a vessel with 100ml of tank water containing a very small amount of carbohydrates.

The goal of the experiment was to analyse the ammonia rise from the shrimp and the slow decomposition process due to low o2 in a small volume of water although it seems that the carbohydrates affected the end result in a way that I can’t understand, the ammonia rise never happened and instead ammonia was instantly converted to nitrite (1ppm plus) and nitrite converted to nitrates (220 plus)

question:

how did this happened, organic Carbon can only be useful during the denitrification process for denitrifying bacteria to my knowledge, nitrifying bacteria can’t use organic carbon in order to accelerate the nitrifying cycle and heterotrophic nitrifying bacteria is poorly studied for me to make any conclusion.

I have repeated the test with just tap water and the shrimp and the result at 8 hours was just the expected small amount of ammonia, it seems that the carbohydrates were the culprit.

Here are a few thoughts.

@taricha is correct. The nitrate test results if a sample containing nitrite are unusable.

The nitrite could have arisen from ammonia on the shrimp. This is possible because of the huge ratio of shrimp to water. The observed 1 ppm nitrite is equivalent to about 0.33 ppm ammonia or 33 micrograms. Not much spread over the entire shrimp

If the estimate for the ammonia content of the shrimp is correct, it would correspond to the observed small amount of ammonia in the freshwater version of the experiment.

The only mystery for me in this experiment is why you have ammonia oxidizing bacteria in the aquarium water. Is this a new aquarium? Are you dosing bacteria to this system?

 

[Randy Holmes-Farley]

I have repeated the test with just tap water and the shrimp and the result at 8 hours was just the expected small amount of ammonia, it seems that the carbohydrates were the culprit.

Unless you see a different result between tank water dosed with and without Reef Actif, I would not assume it is playing a substantial role in what you are seeing. There are lots and lots of differences between tap water and tank water.

 

[sixty_reefer]

The only mystery for me in this experiment is why you have ammonia oxidizing bacteria in the aquarium water. Is this a new aquarium? Are you dosing bacteria to this system?

The system the water come from is nearly a year old and haven’t seen any type of bottled bacteria in all its life.
The system has a daily maintenance dose of actif that will be feeding all this bacteria (feeding the bacteria instead of dosing the bacteria) this sample was taken around 10 minutes after dosing the system.

 

[brandon429]

We demonstrated ability to cycle a 200 gallon 100% dry setup in 20 days using solely aquarium water, I expect aob to be found in aquarium water because it's all high shear water full of rafts sloughed off live rock, containing aob from its surfaces. Even if aquabiomics disagrees, for charge, I expect every drop of tank water from a reef to have transmissible filter bacteria in every case.

 

[sixty_reefer]

Unless you see a different result between tank water dosed with and without Reef Actif, I would not assume it is playing a substantial role in what you are seeing. There are lots and lots of differences between tap water and tank water.

I agree, I may try with some water from the LFS to see if any difference, maybe try and replicate both simultaneously and add more volume to the water.

 

[sixty_reefer]

Redoing the hole thing side by side with LFS water just to check for testing error.
Doing it in 500ml container this time.