Nitrogen assimilation (anabolism) by bacteria

[Dan_P]

The aquarium is stable at 5mg/l,

How is the nitrate being measured?

 

[sixty_reefer]

How is the nitrate being measured?

With a nitrate test kit. As a note I’m not adding nitrate to the tank, I’m adding early forms of nitrogen

This is how the protein skimmer looks like, taking in account that there is no fish it seems to be working well.

 

[Dan_P]

It can, if it’s collecting bacteria that uses nitrogen to create body mass.

How old was the skimmate in which you measured 100 ppm nitrate?

Have you confirmed that organisms in the aquarium water can convert ammonia to nitrate by adding ammonia to a sample of aquarium water and measuring nitrite concentration over time? The length of time should be equal to the age of the skimmate you tested for nitrate.

 

[Dan_P]

With a nitrate test kit.

What test kit are you using?

 

[sixty_reefer]

How old was the skimmate in which you measured 100 ppm nitrate?

Since the beginning of the experiment although on Sunday I’ve started taking a few ml and spot feed a feather star. In hope that this method is producing enough microbes to feed one.

Have you confirmed that organisms in the aquarium water can convert ammonia to nitrate by adding ammonia to a sample of aquarium water and measuring nitrite concentration over time? The length of time should be equal to the age of the skimmate you tested for nitrate.

Yes, that was the first thing I did, as I set up the aquarium with cycled media and water, I’ve used Dr. Tim’s ammonium chloride to ensure normal nitrogen cycle was working.

 

[sixty_reefer]

What test kit are you using?

https://www.jbl.de/?mod=products&func=detail&id=8706&country=gb&lang=en#2408217
I only use JBL, they the most precise in the hobby after Hanna imo. Ive compared them agains ICP and agains Salifer and API.

 

[Dan_P]

Since the beginning of the experiment although on Sunday I’ve started taking a few ml and spot feed a feather star. In hope that this method is producing enough microbes to feed one.

That is pretty old skimmate, and likely if it has not been stored in the freezer, no longer represents what is in the aquarium water.

Yes, that was the first thing I did, as I set up the aquarium with cycled media and water, I’ve used Dr. Tim’s ammonium chloride to ensure normal nitrogen cycle was working.

I was more interested in the aquarium water itself because it is unlikely to process ammonia.

Ok, we will have to assume the skimmate is carbon deficient which rotted, producing ammonia. We will also assume that there has been enough time to grow a sufficiently large population of nitrifying bacteria to generate 100 ppm of nitrate. This means that no conclusion can be drawn from the fact that 100 ppm nitrate was observed in the skimmer. Right?

 

[sixty_reefer]

That is pretty old skimmate, and likely if it has not been stored in the freezer, no longer represents what is in the aquarium water.

It’s around 10 to 12 days old, the production of foam only started after the organic carbon was introduced.

I was more interested in the aquarium water itself because it is unlikely to process ammonia.

Ok, we will have to assume the skimmate is carbon deficient which rotted, producing ammonia. We will also assume that there has been enough time to grow a sufficiently large population of nitrifying bacteria to generate 100 ppm of nitrate. This means that no conclusion can be drawn from the fact that 100 ppm nitrate was observed in the skimmer. Right?

I’m not sure if I understand the above.

 

[Dan_P]

https://www.jbl.de/?mod=products&func=detail&id=8706&country=gb&lang=en#2408217
I only use JBL, they the most precise in the hobby after Hanna imo. Ive compared them agains ICP and agains Salifer and API.

OK thanks. I needed a rough idea about the precision of the numbers you are reporting. I will be attaching a +/- 50% when I think about the test results.

 

[Dan_P]

I’m not sure if I understand the above.

No prob. Let me expand on this.

Since a skimmer cannot concentrate nitrate, the large concentration you measured has to come from somewhere else. I assume the likely source is from the oxidation of ammonia by bacteria. The ammonia comes from bacterial action on organic nitrogen in the skimmate. When the C:N ratio of this organic matter is too low for complete consumption of the nitrogen by the bacteria, the bacteria excretes the unneeded ammonia. Ammonia is typically not rapidly convert to nitrate in aquarium water, but enough time, the few nitrifying bacteria in aquarium water collected by the skimmate will grow to a sufficiently large number to convert the ammonia to the observed 100 ppm nitrate.

 

[sixty_reefer]

No prob. Let me expand on this.

Since a skimmer cannot concentrate nitrate, the large concentration you measured has to come from somewhere else. I assume the likely source is from the oxidation of ammonia by bacteria. The ammonia comes from bacterial action on organic nitrogen in the skimmate. When the C:N ratio of this organic matter is too low for complete consumption of the nitrogen by the bacteria, the bacteria excretes the unneeded ammonia. Ammonia is typically not rapidly convert to nitrate in aquarium water, but enough time, the few nitrifying bacteria in aquarium water collected by the skimmate will grow to a sufficiently large number to convert the ammonia to the observed 100 ppm nitrate.

It’s plausible to happen, I wouldn’t be able to confirm it as it would be out of my scope of abilities.

 

[sixty_reefer]

No prob. Let me expand on this.

Since a skimmer cannot concentrate nitrate, the large concentration you measured has to come from somewhere else. I assume the likely source is from the oxidation of ammonia by bacteria. The ammonia comes from bacterial action on organic nitrogen in the skimmate. When the C:N ratio of this organic matter is too low for complete consumption of the nitrogen by the bacteria, the bacteria excretes the unneeded ammonia. Ammonia is typically not rapidly convert to nitrate in aquarium water, but enough time, the few nitrifying bacteria in aquarium water collected by the skimmate will grow to a sufficiently large number to convert the ammonia to the observed 100 ppm nitrate.

There is also a second hypothesis, a few years back I’ve noticed that my test kits do react to organics. In that experiment I was removing sand from my tank, collecting the excess organic trapped in the sand and diluting them into 0ppm water, I noticed in those tests that my test kits react to organic in the water and it would show nitrates value in line with the amount of organic added to the test solution.

This means as I diluted 1ml of my protein skimmer contents with 9 ml of 0 ppm new water that the result could just be a indicator of a large concentration of organic material in the collection cup and not actually nitrate.


I believe that for clarity this needs to be mentioned.

 

[Dan_P]

There is also a second hypothesis, a few years back I’ve noticed that my test kits do react to organics. In that experiment I was removing sand from my tank, collecting the excess organic trapped in the sand and diluting them into 0ppm water, I noticed in those tests that my test kits react to organic in the water and it would show nitrates value in line with the amount of organic added to the test solution.

This means as I diluted 1ml of my protein skimmer contents with 9 ml of 0 ppm new water that the result could just be a indicator of a large concentration of organic material in the collection cup and not actually nitrate.


I believe that for clarity this needs to be mentioned.

i doubt the chemistry in a nitrate test kit would react with organic matter in any meaningful way. What you observed was probably a case of the sample being contaminated with nitrite or nitrate.

 

[sixty_reefer]

i doubt the chemistry in a nitrate test kit would react with organic matter in any meaningful way. What you observed was probably a case of the sample being contaminated with nitrite or nitrate.

It was a weird reaction, not to sure what happened, as with this one, we can only have indications on what could possibly be happening, its one of them that we will probably never really know what’s doing what. In the meantime I’ll carry on with it, see where it takes me.

Nitrates here’s my conclusion:
Both vessels are in RO, vessel on the right is control to illustrate that there is no nitrates presence, vessel on the left is the same with a large piece of macro algae. 5ppm of nitrates extracted from a piece of macro algae under 10 minutes. I don’t mind doing a full 10 minute video on the phosphates or nitrates test.

 

[taricha]

So the overall scope of the experiment is to look at how bacteria, when fed a carbon source will either assimilate aquarium nitrogen into biomass, or do something else with the nitrogen like respire it, denitrify etc.

The idea is to try to catch the bacteria incorporating water NO3 into biomass.

The data to support that is that 1) NO3 will disappear from the water, and 2) that testable nitrogen will hopefully accumulate in the skimmer, from bacteria collected there.

So here's how I'd expect it to go:
as you gradually feed in Carbon, some portion of the fed carbon will be used for biomass (growth) by the bacteria - the cells will also contain nitrogen and phosphorus.
If the skimmer is actually good at removing bacteria from the water, then you will be collecting a sample of organics in the skimmer including bacterial mass (but also some proteins etc). Then you have to wait for some amount of remineralization/decomposition to take place so that some bacterial biomass nitrogen turns into testable nitrogen - ammonia and maybe nitrite, nitrate if theres enough O2 in the skimmer cup.
The remineralization will not be close to 100%, so actual nitrogen balance accounting where you numerically compare what's removed from the water vs what shows up in biomass will not be possible. But it's not necessary to do a 1:1 accounting, you can simply demonstrate that feeding carbon removes NO3 from water and some N shows up in the skimmer.

(before I ramble more about experimental details and tweaks, @sixty_reefer do I have the picture mostly right?)

 

[taricha]

Here's a thing I did where I wanted to actually do quantitative biomass C,N,P accounting.
I had to do it in freshwater, because the heated digestions I had that can totally remineralize C, N, and P from bacterial biomass don't work in saltwater. I also had to run much higher nutrients, and get much bigger cloudy bloom than anything applicable to aquaria, because of the poor sensitivity of the tests. I also used sources of C and N that aren't really aquarium-relevant in the large concentrations I used (glutamine and glucose)

But what I was interested in was if I was recovering all the C,N,P from the bacterial bloom by the digestions.

For the blue data, I tested the cloudy water (including bacterial nutrients). For the red data, I filtered the water through 0.2um syringe filter to remove particulates and get only the remaining dissolved nutrients in the water.

you can see that the amount of C,N,and P in the filtered out particulates was increasing as the bloom got bigger, and the total N & P stayed the same. But the total Carbon didn't because some carbon got oxidized for energy instead of used for biomass.

here's my numerical accounting.

the bottom two lines are a mole accounting of the bacterial biomass (calculated from what was removed by filtration).

I wouldn't expect any of this to work the same under reef aquarium conditions, but the overall picture might be approximately useful.

 

[sixty_reefer]

So the overall scope of the experiment is to look at how bacteria, when fed a carbon source will either assimilate aquarium nitrogen into biomass, or do something else with the nitrogen like respire it, denitrify etc.

The idea is to try to catch the bacteria incorporating water NO3 into biomass.

The data to support that is that 1) NO3 will disappear from the water, and 2) that testable nitrogen will hopefully accumulate in the skimmer, from bacteria collected there.

So here's how I'd expect it to go:
as you gradually feed in Carbon, some portion of the fed carbon will be used for biomass (growth) by the bacteria - the cells will also contain nitrogen and phosphorus.
If the skimmer is actually good at removing bacteria from the water, then you will be collecting a sample of organics in the skimmer including bacterial mass (but also some proteins etc). Then you have to wait for some amount of remineralization/decomposition to take place so that some bacterial biomass nitrogen turns into testable nitrogen - ammonia and maybe nitrite, nitrate if theres enough O2 in the skimmer cup.
The remineralization will not be close to 100%, so actual nitrogen balance accounting where you numerically compare what's removed from the water vs what shows up in biomass will not be possible. But it's not necessary to do a 1:1 accounting, you can simply demonstrate that feeding carbon removes NO3 from water and some N shows up in the skimmer.

(before I ramble more about experimental details and tweaks, @sixty_reefer do I have the picture mostly right?)

As I’ve started the experiment I was thinking similar to your thoughts, although I’m experimenting with a different type of organic carbon, its a particular SLDOC that remains im the water column for longer than the usual LDOC ( vodka and vinegar) my observation so far it was that it don’t seem to reduce nitrate (No3) as long as early forms of nitrogen are available, such as ammonium and amino acids, only as ammonium and amino acids are exhausted it will start reducing No3.
It’s being difficult to understand why that is happening as this is not how we usually see organic carbon act in a reef environment, we add organic carbon to target No3 not to target earlier forms of nitrogen. Hence the suspicion on the bacteria stimulated by this type of organic carbon having the ability to assimilate early forms of nitrogen. I can’t come to any certain conclusions as of yet.

 

[sixty_reefer]

Here's a thing I did where I wanted to actually do quantitative biomass C,N,P accounting.
I had to do it in freshwater, because the heated digestions I had that can totally remineralize C, N, and P from bacterial biomass don't work in saltwater. I also had to run much higher nutrients, and get much bigger cloudy bloom than anything applicable to aquaria, because of the poor sensitivity of the tests. I also used sources of C and N that aren't really aquarium-relevant in the large concentrations I used (glutamine and glucose)

But what I was interested in was if I was recovering all the C,N,P from the bacterial bloom by the digestions.

For the blue data, I tested the cloudy water (including bacterial nutrients). For the red data, I filtered the water through 0.2um syringe filter to remove particulates and get only the remaining dissolved nutrients in the water.

you can see that the amount of C,N,and P in the filtered out particulates was increasing as the bloom got bigger, and the total N & P stayed the same. But the total Carbon didn't because some carbon got oxidized for energy instead of used for biomass.

here's my numerical accounting.

the bottom two lines are a mole accounting of the bacterial biomass (calculated from what was removed by filtration).

I wouldn't expect any of this to work the same under reef aquarium conditions, but the overall picture might be approximately useful.

That’s a interesting data, in this experiment I’m keeping a Nitrate and phosphate in a constant reef environment, Nitrate at 5mg/l and phosphate at 0.4 mg/l I was hoping to see phosphate to reduce in a certain ratio to Nitrogen use although it’s not happening so far I’m on day 15 of the experiment.
And the only nutrients that I believe that are being used it’s early forms of nitrogen and organic carbon, phosphate hasn’t moved since the beginning of the test same for Nitrate with the exception of two situations. Maybe they will only be affected if Nitrate was to be affected.
In a way I suspect that I’m using organic carbon to maintain a fixed residual nitrate by exporting the earlier forms of nitrogen before they have a chance to become nitrate.
Potentially a similar behaviour to how macro or micro algae and coral would help control nitrate but using bacteria in a controlled way instead.

 

[taricha]

my observation so far it was that it don’t seem to reduce nitrate (No3) as long as early forms of nitrogen are available, such as ammonium and amino acids, only as ammonium and amino acids are exhausted it will start reducing No3.

So just to be clear, you won't be able to say much of anything about what's happening if your media or food contains forms of N you aren't testing for.
If you are interested in different forms of N being taken up (ammonia vs NO3) then you'll need to measure them both (and nitrite too). And expend some effort on using water/foods that has only forms of N that you are actually checking for.
Simpler to just do nitrate. But if the NO3 vs ammonia interests you then sure. Just needs to have more stuff measured to get a clear picture.

 

[sixty_reefer]

So just to be clear, you won't be able to say much of anything about what's happening if your media or food contains forms of N you aren't testing for.
If you are interested in different forms of N being taken up (ammonia vs NO3) then you'll need to measure them both (and nitrite too). And expend some effort on using water/foods that has only forms of N that you are actually checking for.
Simpler to just do nitrate. But if the NO3 vs ammonia interests you then sure. Just needs to have more stuff measured to get a clear picture.

I’ve been using raw products instead of food.

I’m using a bottle of mixed forms of nitrogen that 10 ml is equivalent to 2 mg/l of Nitrate in 24 hours. The daily dose it’s below next to the date, the daily dose of organic carbon is also next to the date in grams.

I wanted to be able to know how much is being added of each product to be able to have a vague idea of what’s being used.

I’ve observed a balance of 4 ml of early forms of nitrogen to 0.02 grams of organic carbon so far, I’ve them increased organic carbon to 0.04 grams and nitrate started to be reduced, a day after I’ve increased nitrogen to 8 ml and nitrate regained balance at 5mg/l residual.

Phosphate hasn’t decreased since the 10 of October. My observation so far is that apartment phosphate will only decrease if Nitrate itself decrease that’s how carbon dosing works but it seems that if only nitrogen is used phosphate will not be affected.


10- October (2ml day - 0.02 grams day)
No3 5 mg/l
Po4 0.4 mg/l
Kh 8
Ph 8.2
O2 10 mg/l

12- October (4ml day - 0.02 grams day)
No3 1 mg/l
Po4 0.4 mg/l
Ph 8.2

16- October (4ml day - 0.02 grams day)
No3 5 mg/l
Po4 0.4 mg/l
Ph 8.2
Kh 9.5
O2 10 mg/l

18- October (4ml day - 0.04 grams day)
No3 5 mg/l
Po4 0.4 mg/l
Ph 8.2
Kh 9.5

20- October (8ml day - 0.04 grams day)
No3 1 mg/l
Po4 0.4 mg/l
Ph 8.2
Kh 9.5

22- October (8ml day - 0.04 grams day
No3 5 mg/l
Po4 0.4 mg/l
Ph 8.2
Kh 10.5
O2 10 mg/l

24- October (8ml day - 0.04 grams day
No3 5mg/l
Po4 0.4 mg/l
Ph 8.2
Kh 10.5
O2 10 mg/l