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Old 01/01/2018, 06:51 AM   #1
Belgian Anthias
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Reflects before you begin dosing carbohydrates

Nitrate assimilation

Nitrate assimilation is subjected to regulation that may differ depending on the organism. In general, nitrate assimilation is controlled at the transcriptional level by nitrate and nitrite induction and by ammonium repression.
In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs.
In cyanobacteria, the transcription factor represses nitrate assimilation genes when ammonium is present whereas it activates transcription of these genes at a high
carbon/nitrogen ratio.
The nitrate-assimilation process begins with the transport of nitrate into the cell. Nitrate is further reduced to nitrite in a two-electron reaction by a cytoplasmic molybdenum containing nitrate reductase followed by a six-electron nitrite reduction to produce ammonia.
The assimilatory reduction of nitrate to ammonium is an energetically expensive process since it requires eight electrons and complex prosthetic groups for the nitrate and nitrite reductase enzymes, in addition to the active nitrate transport. In order to avoid this energetic cost under unnecessary environmental conditions, bacteria have evolved a strict control of the expression of the Nas system. Thus expression of the Nas genes is subjected to dual control based on specific nitrate or nitrite induction, and ammonium repression, by a general nitrogen-regulatory system. http://www.baharini.eu/baharini/doku...traat_reductie

When adding carbohydrates for the first time not a lot will change to the nitrate level. Ammonia, normally nitrified by autotrophs, is used first . Only when the supply of ammonia is insufficient nitrate will be used. When dosing is continued nitrification will be suppressed due to ammonia availability as the heterotrophs grow a lot faster. Heterotroph ammonium reduction ( assimilation) produces 40 x more bio mass compared to autotroph ammonia reduction. ( http://www.baharini.eu/baharini/doku...onium_reductie ) As a result less nitrate will be produced. The moment the C:N ratio increases due to dosing and increased ammonium take up ( and less or no nitrate production) nitrate may be used for celsynthesis. Nitrate is also taken up for respiration. Nitrate take up for respiration is not suppressed by ammonia. The nitrate level may be depleted fast at high C:N ratio. All this implements a very low nitrification and denitrification capacity.
When biopellets are used in a reactor there is always a high C:N ratio available.

An aquarium system that is kept in balance by carbohydrate dosing has a limited carrying capacity. Such systems are vulnerable for a system crash due to the well known new tank syndrome.
Once started dosing it may be difficult to stop. Dosing should be build off during a period of at least 15 days.

Please comment.


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Old 01/01/2018, 08:23 AM   #2
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Well that made my head feel funny lol. Question though, you keep saying Carbohydrates, do you mean Carbon? Never heard of Carbohydrate dosing.


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Old 01/01/2018, 09:07 AM   #3
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Thanks for sharing your knowledge. Some of the technical jargon in your post is beyond my level.

Assuming that you meant organic carbon by "carbohydrates", what you stated makes sense and is in fact well established in the literature. It is well known that reef organisms have preference for ammonia over nitrates for their nitrogen source. Daniel Knop and Michael Fatherree, for example, mention this phenomenon in their respective books on giant clams.

I also agree with your statement that "Once started dosing it may be difficult to stop". I think if one decides to stop dosing organic carbon, he/she should do so in small steps instead of in a big bang approach. Last summer, I experimented with a commercial product that contains marine-based biopolymers in it. It is marketed as Reef Actif by Tropic Marin. Even though I knew that I should decommission the use of this product slowly when my experiment finally finished at the end of last October, I went against my own knowledge and stopped using it at once. My reef system did not crash, but it was seriously destabilised in terms of nutrients and their ratio. It took nearly two months to regain its balance and it is still not quite there yet.

Moreover, in case you do not know, there is another commercial product marketed by Tropic Marin called NP-Bacto-Balance. It contains among other ingredients (e.g. organic carbon) organic and inorganic forms of phosphates and inorganic nitrates. The main idea is to prevent complete nitrogen and phosphorus depletion providing that the product is used according to its dosing instructions. I guess that there are ways of countering some of the disadvantages of organic carbon dosing, which you correctly pointed out in your post. It is possible to have a healthy reef that relies on organic carbon dosing. My system has been relying on it for the past three years. However, I will update this thread if it ever crashes one day.

Happy new year.

PS. The links you provided in your post are not accessible.


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Old 01/01/2018, 01:19 PM   #4
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Well that made my head feel funny lol. Question though, you keep saying Carbohydrates, do you mean Carbon? Never heard of Carbohydrate dosing.
Organic carbon. What is added to the system are carbohydrates as ethanol (wodka), sucrose (sugar) , acetic acid ( vinegar).


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Old 01/01/2018, 02:07 PM   #5
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Thanks for sharing your knowledge.

PS. The links you provided in your post are not accessible.
Happy new year!

In that case you must log in or register. Makazi Baharini is a closed dokuwiki.

NP-BACTO-BALANCE: Can not find out how much usable organic carbon it contains. It claims creating an optimal nitrate-phosphate balance. It would be interesting to know how it is able to do so and what this optimal balance should be. For biosynthesis ( assimilation) this would be in weight +- 10 to 1.


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Old 01/01/2018, 02:12 PM   #6
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Happy new year!

In that case you must log in or register. Makazi Baharini is a closed dokuwiki.

NP-BACTO-BALANCE: Can not find out how much usable organic carbon it contains. It claims creating an optimal nitrate-phosphate balance. It would be interesting to know how it is able to do so and what this optimal balance should be. For biosynthesis ( assimilation) this would be in weight +- 10 to 1.
That's correct. In long term use (just under three years), I have achieved 10:1 ratio.

I was informed by Hans-Werner Balling that Tropic Marin "have chosen a harmless simple organic N compound that releases ammonia after hydrolysis to avoid the potentially dangerous ammonia itself. The organic N compound is quite stable but can be hydrolyzed by enzymes of corals and bacteria quickly."


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Old 01/01/2018, 03:03 PM   #7
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By providing additional carbohydrates a cycle is created from NH4 to NH4. Only +- 30% of the cultivated biomass will be removed by a skimmer. http://www.baharini.eu/baharini/doku...iwitafschuimer. The rest must be consumed. it becomes part of the food cycle.
To reduce 1 g NH4-N, +- 8 g protein is produced, the equivalent of 20 grams food containing 30% protein. When the produced protein is consumed some is used to increase the total bioload but most of it will be released as ammonia and a bit ureum. To reduce 1gram nitrate by assimilation also 0,1gram phosphate is needed. http://www.baharini.eu/baharini/doku...onium_reductie This may become an issue in biopellet reactors.
Once regular dosing carbohydrates, most of the dose is used to remove ammonia which is the result of previous doses.

It must be clear that when additional carbohydrates are provided this must be taken in account for the total food supply. A refugium can be added to help consume the produced bacterial biomass by organisms which may be used as a food souce.
It must also be clear that when the organic carbon supply is interrupted biomass will decay. Organisms which depend on it will die. On that moment nitrification capacity will be insufficient .

Heterotrop ammonia reduction produces a lot of biomass, 40x more as nitrification. This is done every cycle from NH4 to NH4. It is a fact that +- 2% of the bacterial biomass will be lost as it is not recyclable and will be left as detritus.
The growt may also become an issue when biofilters or DSB are used. They may clog. But at a high maintained C:N ratio these filters are not needed.


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Old 01/01/2018, 03:40 PM   #8
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It must be clear that when additional carbohydrates are provided this must be taken in account for the total food supply. A refugium can be added to help consume the produced bacterial biomass by organisms which may be used as a food souce.

It must also be clear that when the organic carbon supply is interrupted biomass will decay. Organisms which depend on it will die. On that moment nitrification capacity will be insufficient .
What about increasing the amount of activated carbon already used or even perhaps start to fluidise it to increase its efficiency of adsorption rate of bacterial mass? Activated carbon was shown to be more efficient in removing total dissolved organics than a protein skimmer.


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Old 01/01/2018, 03:59 PM   #9
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Nitrate assimilation
Nitrate assimilation is subjected to regulation that may differ depending on the organism. In general, nitrate assimilation is controlled at the transcriptional level by nitrate and nitrite induction and by ammonium repression.
Many systems are controlled at this level. No arguement here

Quote:
In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs.
In cyanobacteria, the transcription factor represses nitrate assimilation genes when ammonium is present whereas it activates transcription of these genes at a high carbon/nitrogen ratio.
Without a reference to the study, I will take your word for it, though this seems reasonable. Complementary chromatic adaptation has been described for cyanobacteria and there seems to be a link between color of the organism and nitrogen source. No surprise then about the level of control but the notion that a high C to N ratio is a trigger is interesting. Again, no reference, so, I will take your word for now.

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The nitrate-assimilation process begins with the transport of nitrate into the cell. Nitrate is further reduced to nitrite in a two-electron reaction by a cytoplasmic molybdenum containing nitrate reductase followed by a six-electron nitrite reduction to produce ammonia.
The assimilatory reduction of nitrate to ammonium is an energetically expensive process since it requires eight electrons and complex prosthetic groups for the nitrate and nitrite reductase enzymes, in addition to the active nitrate transport. In order to avoid this energetic cost under unnecessary environmental conditions, bacteria have evolved a strict control of the expression of the Nas system. Thus expression of the Nas genes is subjected to dual control based on specific nitrate or nitrite induction, and ammonium repression, by a general nitrogen-regulatory system. http://www.baharini.eu/baharini/doku...traat_reductie
Link does not work but this is well known.

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When adding carbohydrates for the first time not a lot will change to the nitrate level. Ammonia, normally nitrified by autotrophs, is used first . Only when the supply of ammonia is insufficient nitrate will be used. When dosing is continued nitrification will be suppressed due to ammonia availability as the heterotrophs grow a lot faster. Heterotroph ammonium reduction ( assimilation) produces 40 x more bio mass compared to autotroph ammonia reduction. ( http://www.baharini.eu/baharini/doku...onium_reductie )
This is published in the aquaculture literature

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As a result less nitrate will be produced. The moment the C:N ratio increases due to dosing and increased ammonium take up ( and less or no nitrate production) nitrate may be used for celsynthesis. Nitrate is also taken up for respiration. Nitrate take up for respiration is not suppressed by ammonia. The nitrate level may be depleted fast at high C:N ratio. All this implements a very low nitrification and denitrification capacity.
The potential risks of robbing Peter to pay Paul does not seem to be addressed in the aquarium carbon dosing discussions. Maybe most aquarists get away with a reduced denitrication capacity. In low nutrient systems like reef aquaria, maybe the risk is not so high.

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When biopellets are used in a reactor there is always a high C:N ratio available.

An aquarium system that is kept in balance by carbohydrate dosing has a limited carrying capacity. Such systems are vulnerable for a system crash due to the well known new tank syndrome.
Once started dosing it may be difficult to stop. Dosing should be build off during a period of at least 15 days.
The carbon dosing system vulnerability conjecture needs support. It is reasonable and something that I have wondered about but could be absolutely wrong. Yes, switching back and forth, between non-dosing to dosing or dosing to non-dosing, with minimal accumulation of ammonia or nitrate would take time for the population of organisms to readjust. But to say that one regimen has a higher “carrying capacity” needs support. It might be amenable to an stoichiometric calculation.


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Old 01/01/2018, 07:23 PM   #10
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An aquarium system that is kept in balance by carbohydrate dosing has a limited carrying capacity. Such systems are vulnerable for a system crash due to the well known new tank syndrome.
Once started dosing it may be difficult to stop. Dosing should be build off during a period of at least 15 days.
I don't know of any evidence supporting this statement. A number of people have posted otherwise as far as stopping the dosing, due to the capacity to consume ammonia. Both the carbon-consuming organisms and the standard microbes can grow, given enough ammonia, and they might not overlap physically.


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Old 01/02/2018, 01:59 AM   #11
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Many systems are controlled at this level. No arguement here



Without a reference to the study, I will take your word for it, though this seems reasonable. Complementary chromatic adaptation has been described for cyanobacteria and there seems to be a link between color of the organism and nitrogen source. No surprise then about the level of control but the notion that a high C to N ratio is a trigger is interesting. Again, no reference, so, I will take your word for now.



Link does not work but this is well known.



This is published in the aquaculture literature



The potential risks of robbing Peter to pay Paul does not seem to be addressed in the aquarium carbon dosing discussions. Maybe most aquarists get away with a reduced denitrication capacity. In low nutrient systems like reef aquaria, maybe the risk is not so high.



The carbon dosing system vulnerability conjecture needs support. It is reasonable and something that I have wondered about but could be absolutely wrong. Yes, switching back and forth, between non-dosing to dosing or dosing to non-dosing, with minimal accumulation of ammonia or nitrate would take time for the population of organisms to readjust. But to say that one regimen has a higher “carrying capacity” needs support. It might be amenable to an stoichiometric calculation.
The links should work. If an access denied page is shown one must register and log in to get access to the wiki. All references are included in the wiki articles and most references can be consulted. The wiki is written in Dutch, some pages are translated in English. Most references used are in English.

In LNS a caveat may be the competition for available phosphate and other building materials certainly when the doses do not correspond the needs.
In LNS the doses may be responsible for most of the biomass production, more as normal feedings. As the small amount ammonia is immediately taken up when it becomes available most LNS do have very little or no nitrification capacity. Not a problem as long as the ammonia take up rate will be maintained.

The "carrying capacity" is the capacity of the system to reduce ammonia. Normally 2/3 is reduced by nitrification.
The "carrying capacity" is decisive for the maximum bioload.
When organic carbon dosing is used to reduce ammonia the carrying capacity is easily adjusted but the system may become completely dependable of these doses and the corresponding bacterial growth which growth is dependable of a lot of other factors and parameters.


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Old 01/02/2018, 03:37 AM   #12
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I don't know of any evidence supporting this statement. A number of people have posted otherwise as far as stopping the d, due to the capacity to consume ammonia. Both the carbon-consuming organisms and the standard microbes can grow, given enough ammonia, and they might not overlap physically.
Of coarse all bacteria may grow when given enough ammonia and other building materials but when the usable organic carbon is matched the heterotrops will use up most of the ammonia leaving very little or nothing for the nitrifiers and other organisms because of the very high grow rate. This is supported by all approved publications I have read about ammonia reduction in aquaculture systems . http://www.baharini.eu/baharini/doku...onium_reductie More evidence I do not need.
In zero emission marine aquaculture systems ( ZMAS) using carbohydrates dosing to maintain a high C:N ratio no or little nitrate is produced.

Which are the standard microbes? Nitrifiers and denitrifiers?
How one can avoid the bacteria to overlap "physically"?

For me It is logic that when ammonia is removed by assimilation this ammonia is not available for nitrification and denitrification. It is a fact that autotrophs are suppressed by heterotroph growth.


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Old 01/02/2018, 04:56 AM   #13
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more to think about.

It takes about 1 day for heterotrops to double there biomass. it takes about 15 days for autotrophs ( nitrifiers) to double there biomass. This means that at least 15 days are needed to double the nitrifying capacity when for some reason the heterotroph growth can not be maintained and longer to become normal.

All recipes for carbohydrate dosing are based on the nitrate level. But it is not nitrate that triggers the bacterial growth, it is ammonia by priority..

In nature and in aquaria usable organic carbon is limited available. This way making it possible most organic material can be recycled and the nitrogen cycle can be completed. Nitrogen is send back were it came from and is not stored in biomass.
What happens when organic carbon is unlimited available?


In LNS or VLNS dosing must be correct, corresponding to the needs. Otherwise not all of it may be used for growth due to not enough building material. The moment building material as phosphate is delivered the competition for the building material may end up in a real war. Guess who will win!?
In general in LNS where the balance is maintained by carbon dosing a high C:N ratio is available.
Are we going to assume that doses are ok or is there a way to be sure the dosing is correct.?
What do we know about the hierarchy between organisms in using building materials?

Commercial products which contain polymers and other building materials are available but how to dose these products if one thus not know how many organic carbon is actually released or used? Following the guidelines assuming it will be ok?

Biopellets used in a reactor: the carbon source is unlimited available in the reactor but the other building materials are not.

The question asked when biofilters are used for ammonia reduction is:"What to do with the nitrate produced" As nitrogen can easily be removed from the system to where it came from this problem can be solved.

The question asked when carbohydrates are dosed for ammonia reduction may be:"What to do with the biomass produced?"
In aquaculture systems the biomass is harvested and a new batch is started.
When algae scrubbers are used for assimilative ammonia reduction the produced biomass is harvested.


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Old 01/02/2018, 05:21 AM   #14
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You have not answered my previous question about the use of activated carbon to harvest the produced biomass.


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Old 01/02/2018, 05:59 AM   #15
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The links should work. If an access denied page is shown one must register and log in to get access to the wiki. All references are included in the wiki articles and most references can be consulted. The wiki is written in Dutch, some pages are translated in English. Most references used are in English.

How do I register? What’s the Dutch word I am looking to click to register?
Maybe you could cut and paste the references into this post so we can at least read the science behind conjectures.


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Old 01/02/2018, 06:14 AM   #16
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It takes about 1 day for heterotrops to double there biomass. it takes about 15 days for autotrophs ( nitrifiers) to double there biomass. This means that at least 15 days are needed to double the nitrifying capacity when for some reason the heterotroph growth can not be maintained and longer to become normal.
Yes, the heterotrophs can grow quickly, even to the point that they serve as food in shrimp aquaculture.

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All recipes for carbohydrate dosing are based on the nitrate level. But it is not nitrate that triggers the bacterial growth, it is ammonia by priority..
I wondered about this. This is probably the explanation for the delay in nitrate reduction when dosing is started.

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In nature and in aquaria usable organic carbon is limited available. This way making it possible most organic material can be recycled and the nitrogen cycle can be completed. Nitrogen is send back were it came from and is not stored in biomass.
What happens when organic carbon is unlimited available?
A lot of carbon goes into energy requirements, production of CO2. I think something like more than 50% of the carbon consumed goes to CO2. This is one factor preventing aquaria from turning into sewage plants.


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Old 01/02/2018, 01:12 PM   #17
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For me It is logic that when ammonia is removed by assimilation this ammonia is not available for nitrification and denitrification. It is a fact that autotrophs are suppressed by heterotroph growth.
This has nothing to do with carrying capacity, at least as far as I use the term. Maybe you mean something else, but I take it to mean the upper limit of the ammonia that the tank can process per unit time.


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Old 01/02/2018, 01:33 PM   #18
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IAll recipes for carbohydrate dosing are based on the nitrate level. But it is not nitrate that triggers the bacterial growth, it is ammonia by priority..
I'm not sure why this matters. The end result is the consumption of nitrate, and I think we've all seen that dosing requires per-tank tuning.


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Old 01/02/2018, 01:34 PM   #19
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By providing additional carbohydrates a cycle is created from NH4 to NH4. Only +- 30% of the cultivated biomass will be removed by a skimmer.
I don't see any reason to believe that we know how much of the cultivated biomass will be exported. Clearly, in practice, tanks generally can export as much as is needed. Some tanks certainly have issues with carbon dosing, for reasons that aren't clear.


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Old 01/02/2018, 05:22 PM   #20
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I'm not sure why this matters. The end result is the consumption of nitrate, and I think we've all seen that dosing requires per-tank tuning.
I am not familiar with how one determines how much carbon to add to an aquarium but for aquaculture, the amount of carbohydrate added is based on the stoichiometric balance of C:N that is required by heterotrophs to assimilate all the nitrogen in the feed. I always assumed that dosing aquaria is more an empirical exercise, starting low and increasing. As you say, per-tank tuning. I will bet no one doses carbon to balance the C:N ratio in what they add to the tank. Would be an interesting experiment though.

As for heterotrophs consuming nitrate because we carbon dose, has this been proven in an aquarium this is what actually happens? Might another explanation be that nitrate production is minimized because of increased ammonia assimilation by hetertrophic bacteria and the nitrate already produced is simply converted to nitrogen by autotrophs?


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Old 01/02/2018, 05:37 PM   #21
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I don't see any reason to believe that we know how much of the cultivated biomass will be exported. Clearly, in practice, tanks generally can export as much as is needed. Some tanks certainly have issues with carbon dosing, for reasons that aren't clear.
Why don’t we know how much biomass is exported? Even public and commercial aquariums don’t know?


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Old 01/02/2018, 05:41 PM   #22
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As for heterotrophs consuming nitrate because we carbon dose, has this been proven in an aquarium this is what actually happens? Might another explanation be that nitrate production is minimized because of increased ammonia assimilation by hetertrophic bacteria and the nitrate already produced is simply converted to nitrogen by autotrophs?
Interesting point! It's possible that the reduction in the nitrate level is indeed due to decreased nitrate production, resulting in a declining level due to standard denitrification. It'd be hard to be sure what's happening, but I'll be more careful in my wording in the future.


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Old 01/02/2018, 05:48 PM   #23
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Why don’t we know how much biomass is exported? Even public and commercial aquariums don’t know?
I don't know of any way to measure it, in practice. It can leave the tank (or nutrient flow, at least) in a number of ways. The various components of the biomass can be skimmed or be exported via water changes, denitrification, outgassed as carbon dioxide, or deposited as phosphate, etc, in coral skeletons or coralline. The last case isn't technically export from the system, but it does remove the nutrients from the nutrient flow in the tank, at least for some period of time.

A lot of things can be skimmed, and characterizing what comes out of the skimmer back into what was in the tank would be non-trivial, in my opinion. One guess is that carbon dosing removes nutrients by feeding bacteria that release skimmable organics. It's also likely that at least some of the carbon is consumed by bacteria that are themselves skimmed, but I don't know how much that would be.


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Old 01/02/2018, 06:15 PM   #24
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, but I'll be more careful in my wording in the future.
Me too.


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Old 01/02/2018, 06:26 PM   #25
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denitrification, outgassed as carbon dioxide, or deposited as phosphate, etc,
Maybe the determination is along the lines of measuring the metabolic rate of animals but do it for the entire aquarium. This feels like a serious Google research project.


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