Nitrogen assimilation (anabolism) by bacteria

[sixty_reefer]

@taricha, @Dan_P

How could I identify the above?
I’ve tried a few things in the past and they really haven’t worked out well although this time around is starting to look that it may be a strong evidence of the existence of anabolism in a aquarium filtration if presented with different types of organic carbon.

In the last experiment I did, in the first week I’ve added 0.5 ml of vinegar’s just to confirm what happened last time we’re taricha pointed out correctly that the hole system did become anoxic allowing the hole tank becoming a ground for bacteria to respire No3. I’ve made this in hope to verify that there was no anoxic areas in the system were they could use organic carbon.

Part two I’ve allowed the bloom to stop, 2 days of a small haze and I’ve introduced a particulate form of organic carbon, this carbon under the microscope seems to be floating around the tank for a few days before it’s consumed.

My observation so far have been that the tank has a demand for nitrogen that goes in line with the dose of particulate organic carbon. I’ve recently doubled the dose of carbon and nitrogen demand increase by two fold to be able to keep nitrate detectable at 5ppm, there hasn’t been a demand for phosphate since the test started or if it has it wasn’t significant to see it in the test kits (very accurate in past tests).
The water has been cristal clear with a fairly high ph (8.2) and oxygen levels have been at 10 mg/l plus.


Just wondering what additional test I should be doing in order to determine if I’m in the right way?

 

[Randy Holmes-Farley]

Let's back up please, and explain what you are trying to do.

First and most important, is this a reef tank, and if so, how do you intend to demonstrate it is bacteria using both the carbon and the nitrogen, assuming you had a way to measure them?

 

[sixty_reefer]

Let's back up please, and explain what you are trying to do.

First and most important, is this a reef tank, and if so, how do you intend to demonstrate it is bacteria using both the carbon and the nitrogen, assuming you had a way to measure them?

hi, I’m trying to determine on what method bacteria is using nitrogen in my current experiment.

I’m using organic carbon to increase Numbers in the tank but their consumption of nutrients is not matching past experiments, more in particular phosphate is not being used in significant amounts.

The experiment is being done in a 15 gallon AIO with just media and water from a cycled mature reef, the hole experiment is being done in a dark environment also.

The results has been a large amount of pelagic bacteria and a single cell organism that I’ve not been able to identify so far.

This was the result of the mix, a vast amount of floating organics that under the microscope look like this at 4 magnifications. The single cell organism seem to prey on the bacteria. The carbon I’ve used it’s also in there as the still organic matter.


Just trying to understand the results of the experiment. As in the past every time I experimented with organic carbon phosphate had a small impact.

 

[Randy Holmes-Farley]

You don't give any phosphate data so I cannot comment on whether it is reasonable or test error or a real effect such as denitrification taking place.

 

[sixty_reefer]

You don't give any phosphate data so I cannot comment on whether it is reasonable or test error or a real effect such as denitrification taking place.

Thank you for helping me look it the results.

From the 10- October were trough my notes the system found a balance. Before it was a little touch and go to balance the nutrients, experiment started on the 2th October.

The nitrogen used is 10ml is equivalent to 2ppm of nitrate in 50 litres of saltwater in 24 hours.

Carbon was added by weight with a estimated 0.02 grams added per day.

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

There was no phosphate added to the test since 8th of October and the test are accurate, I’ve been using it for years agains ICP and during the first week they were all over until it decided not to move.
The doses were altered after testing.

Also I’m not understanding why the Kh keeps rising a few days after the organic carbon is increased.

 

[Randy Holmes-Farley]

What nitrogen source? Consumption of dosed nitrate boosts alkalinity.

 

[sixty_reefer]

What nitrogen source? Consumption of dosed nitrate boosts alkalinity.

NitroPlus | Zlements

www.zlements.com

On this experiment I’ve used the above, as it says to wait 24 hours to test, I’ve assumed that it was a mix of nitrogen. They don’t specify the content. It was locally available so thought in trying it.

My knowledge is limited in this area, wend you say “consumed” do you mean that nitrifying bacteria is respiring No3?
How can I go around this and stop the alkalinity from rising?

 

[Randy Holmes-Farley]

Any disappearance of nitrate that you dose will raise alkalinity.

 

[Randy Holmes-Farley]

If N is added as amino acids or ammonium bicarbonate, there will not be an alk effect unless nitrate is rising.

 

[sixty_reefer]

If N is added as amino acids or ammonium bicarbonate, there will not be an alk effect unless nitrate is rising.

I believe they are a mix, I’ve ensured that there was enough media to transform nitrogen into nitrate. But I can’t be certain if all is getting to nitrate or being used before that happens.

From your explanation and looking at my notes above, I think I can see the alkalinity increase after my nitrates drop, as I’ve increased organic carbon and a day after increased the nitrogen to match, I’m going to try and increase nitrogen first next time around and a day after increase organic carbon to see if that stops the alkalinity increase.
Or should I do it at the same time? Double the carbon and nitrogen in the same day?
So far I think the balance it’s around 4ml per 0.02 grams, I’ve doubled the dose to 8ml to 0.04 grams and it seems to stay stable again at 5 mg/l Nitrate, just no phosphate drop so far.

 

[Dan_P]

What nitrogen source? Consumption of dosed nitrate boosts alkalinity.

Thinking about this experiment with the data that has been shared…

If dosed nitrate is not being consumed, can we say that alkalinity will not increase, all else being held constant? But,…

Can nitrate disappear from solution without an increase in alkalinity? Although,…

Can the standard hobby kit detect an alkalinity change when 4 ppm nitrate is consumed? And,…

Can the standard hobby kit detect a decrease in PO4 when the nitrogen from 4 ppm NO3 is converted to biomass?

To clarify, this post only seems like I have turned the table on you in the chemistry question of the day.

 

[Dan_P]

@taricha, @Dan_P

How could I identify the above?

Following along.

I just asked Randy for some information for us to consider concerning your observations.

 

[sixty_reefer]

Thinking about this experiment with the data that has been shared…

If dosed nitrate is not being consumed, can we say that alkalinity will not increase, all else being held constant? But,…

Can nitrate disappear from solution without an increase in alkalinity? Although,…

I wasn’t aware of this until yesterday, I’m going to read on the subject and try and become a little more aware of how this works.

Can the standard hobby kit detect an alkalinity change when 4 ppm nitrate is consumed? And,…

Can the standard hobby kit detect a decrease in PO4 when the nitrogen from 4 ppm NO3 is converted to biomass?

My test kits only detect a 0.1 mg/l decrease or increase if the phosphate consumption it’s only a 0.01 mg/l it would take me 10 days to be able to see it in my kit. That’s not helping the experiment although I have a feeling that will be much less than that as phosphate has been at 0.4 mg/l for around 12 days.

To clarify, this post only seems like I have turned the table on you in the chemistry question of the day.

The experiment started as trying to see what happens to a biological filter as phytoplankton degrades in the water column, in theory phytoplankton is one of the reasons why some waters become more nutrient rich than others. The products used are also of the shelf at most LFS worldwide and could be replicated by anyone that wishes to verify the current observations.

 

[sixty_reefer]

Following along.

I just asked Randy for some information for us to consider concerning your observations.

I’ve been reading this morning on the different relations and I believe I’ve found a test that could indicate or rule out anabolism.

As one bacteria is removing N2 from the tank and the other is assimilating it into biomass, I thought if I was to collect some water from the tank (100ml) and add (10ml) from the bacteria that I’m collecting in the protein skimmer to it. Allow one week or two to pass (allowing the bacteria to die) and test for nitrate, if the nitrate in the water didn’t change it would be normal denitrification and if the nitrates were to rise it could potentially indicate anabolism.

Does it sound plausible?

 

[sixty_reefer]

Ive performed another test this evening

My reagents works with 10ml of tank water to perform a test.

I’ve added 9 ml new water with 1ml of water from the protein skimmer.

The result was 10 mg/l, times 10 for dilution, should be around 100 mg/l in the protein skimmer.
Dividing 100mg/l by 50l (tank volume) it comes at 2 mg/l of nitrate exported from the tank per each 10ml of protein skimmer water.

I’ve performed the same test with just new water and that come to 0 mg/l

Could this test support the idea? I may have messed up somewhere in my maths. But if the nitrogen in this experiment tank was being used by normal denitrification (N2 gas), I should in theory not have such a high concentration of nitrate in the protein skimmer. Unless I’m not doing something right in this test.

 

[Randy Holmes-Farley]

Thinking about this experiment with the data that has been shared…

If dosed nitrate is not being consumed, can we say that alkalinity will not increase, all else being held constant? But,…

Can nitrate disappear from solution without an increase in alkalinity? Although,…

Can the standard hobby kit detect an alkalinity change when 4 ppm nitrate is consumed? And,…

Can the standard hobby kit detect a decrease in PO4 when the nitrogen from 4 ppm NO3 is converted to biomass?

To clarify, this post only seems like I have turned the table on you in the chemistry question of the day.

I don’t know what is in the product you are using, but the most likely N additives won’t boost alk.

 

[sixty_reefer]

I don’t know what is in the product you are using, but the most likely N additives won’t boost alk.

I think you answered dans thoughts on the overall experiment. I’ve understood that part and reading on it today it’s the same for both types, only the usage of nitrates cause the alkalinity rise. from what I read the only difference between them is that one creates biomass and the other removes it from the system via gas. There was also something about anabolism increasing o2 that I will try and read more on it.

 

[Dan_P]

I’ve been reading this morning on the different relations and I believe I’ve found a test that could indicate or rule out anabolism.

As one bacteria is removing N2 from the tank and the other is assimilating it into biomass, I thought if I was to collect some water from the tank (100ml) and add (10ml) from the bacteria that I’m collecting in the protein skimmer to it. Allow one week or two to pass (allowing the bacteria to die) and test for nitrate, if the nitrate in the water didn’t change it would be normal denitrification and if the nitrates were to rise it could potentially indicate anabolism.

Does it sound plausible?

Some technical questions before plausibility consideration:

What nutrients are in the aquarium water?
Are nutrients added during the experiment?
How is the aquarium water sterilized?
Why is 10 mL rather than one drop of skimmer collected bacteria added?
Why is nitrate measured only once after one or two weeks?
If nitrate does not change, how do we know that the bacteria just did grow?
If nitrate does not change, how do we know that the water contains bacteria capable of reducing nitrate, or generating nitrate?

 

[Dan_P]

Ive performed another test this evening

My reagents works with 10ml of tank water to perform a test.

I’ve added 9 ml new water with 1ml of water from the protein skimmer.

The result was 10 mg/l, times 10 for dilution, should be around 100 mg/l in the protein skimmer.
Dividing 100mg/l by 50l (tank volume) it comes at 2 mg/l of nitrate exported from the tank per each 10ml of protein skimmer water.

I’ve performed the same test with just new water and that come to 0 mg/l

Could this test support the idea? I may have messed up somewhere in my maths. But if the nitrogen in this experiment tank was being used by normal denitrification (N2 gas), I should in theory not have such a high concentration of nitrate in the protein skimmer. Unless I’m not doing something right in this test.

What is the measured nitrate concentration in the aquarium?
Are you measuring the nitrate in the water generated by the collapsed skimmer foam?
The skimmer cannot concentrate nitrate.

 

[sixty_reefer]

What is the measured nitrate concentration in the aquarium?

The aquarium is stable at 5mg/l, it has been since the 10th of October a clear indication that the daily addition of nitrogen is being used by a organism in the tank before it can become Nitrate.

More evidence of that was the two observed situations of alkalinity rise, were I’ve increased organic carbon first and a day later increase nitrogen, allowing the bacteria to reduce nitrate as energy. Giving a indication that the strain present in my tank will prefer nitrogen but it will use nitrate if early forms of nitrogen not enough in the tank.
A day after this happened my nitrate was 1mg/l, that only happened twice in the experiment so far.

Are you measuring the nitrate in the water generated by the collapsed skimmer foam?
The skimmer cannot concentrate nitrate.

It can, if it’s collecting bacteria that uses nitrogen to create body mass.
Once this bacteria dies the nitrogen becomes available again in the water column through nutrient recycling.