Looking for thoughts on organic carbon dosing and nitrate

[GARRIGA]

I’m not a scientist therefore not disciplined in proper testing procedures but if purpose to establish how this occurs in an aquarium environment then doesn’t maturity of that environment matter. Are the tests going to extend years into the future as mature media might behave differently than that which is new and will different media be used. My understanding being that contact time affects amount of DO left post nitrification therefore that which has prolonged contact time might behave differently as to how biofilms a few centimeters in respond vs several father down the path that molecule of water must travel. Might turn out that deeper down that corridor that molecule of water travels the ratio of N produced might be considerably lower vs that removed than the 7:1 per the study quoted.

At least that’s how I understand it. Something needs to reduce/remove DO plus a carbon source to facilitate denitrification. Unless all biofilms behave the same regardless how much actual DO exists outside them.

 

[Dan_P]

I’m not a scientist therefore not disciplined in proper testing procedures but if purpose to establish how this occurs in an aquarium environment then doesn’t maturity of that environment matter. Are the tests going to extend years into the future as mature media might behave differently than that which is new and will different media be used. My understanding being that contact time affects amount of DO left post nitrification therefore that which has prolonged contact time might behave differently as to how biofilms a few centimeters in respond vs several father down the path that molecule of water must travel. Might turn out that deeper down that corridor that molecule of water travels the ratio of B produced might be considerably lower vs that removed than the 7:1 per the study quoted.

At least that’s how I understand it. Something needs to reduce/remove DO plus a carbon source to facilitate denitrification. Unless all biofilms behave the same regardless how much actual DO exists outside them.

For me, your observations capture the complexity of the topic.

 

[Lasse]

I’m not a scientist therefore not disciplined in proper testing procedures but if purpose to establish how this occurs in an aquarium environment then doesn’t maturity of that environment matter. Are the tests going to extend years into the future as mature media might behave differently than that which is new and will different media be used. My understanding being that contact time affects amount of DO left post nitrification therefore that which has prolonged contact time might behave differently as to how biofilms a few centimeters in respond vs several father down the path that molecule of water must travel. Might turn out that deeper down that corridor that molecule of water travels the ratio of N produced might be considerably lower vs that removed than the 7:1 per the study quoted.

At least that’s how I understand it. Something needs to reduce/remove DO plus a carbon source to facilitate denitrification. Unless all biofilms behave the same regardless how much actual DO exists outside them.

IMO - its important to differ between biofilm and substrate. When you talk about a biofilm - you normally mean a thin layer in the interface between two media. Its clear that deep in the substrate things can happen and also can internal DOC be produced that favour denitrification. In my case - I´m sure that I have rather high nitrification in the bottom of my reversed flow deep sand bed because I pump a lot of oxygen rich water into the plenum and denitrifikation in the middle of the same sand bed there the oxygen is depleted - but the biofilm responsible for the nitrification is IMO not so active in the denitrification.

Sincerely Lasse

 

[GARRIGA]

IMO - its important to differ between biofilm and substrate. When you talk about a biofilm - you normally mean a thin layer in the interface between two media. Its clear that deep in the substrate things can happen and also can internal DOC be produced that favour denitrification. In my case - I´m sure that I have rather high nitrification in the bottom of my reversed flow deep sand bed because I pump a lot of oxygen rich water into the plenum and denitrifikation in the middle of the same sand bed there the oxygen is depleted - but the biofilm responsible for the nitrification is IMO not so active in the denitrification.

Sincerely Lasse

Was referring to a biofilm as you described yet assume it might behave differently depending on available DO. The latter affected by what happened previously. For example, excluding additions of DO, deeper the water has traveled through that substrate then I'm expecting a reduction of DO due to prior nitrification.

 

[Lasse]

For example, excluding additions of DO, deeper the water has traveled through that substrate then I'm expecting a reduction of DO due to prior nitrification.

That´s true DO is depleted rather fast in an nitrifying biofilm - below 100 µm there was not enough of oxygen left for nitrification.

Sincerely Lasse

 

[GARRIGA]

That´s true DO is depleted rather fast in an nitrifying biofilm - below 100 µm there was not enough of oxygen left for nitrification.

Sincerely Lasse

I’m speaking more of the available DO to that biofilm early in the nitrification process vs late. Deep sand bed biofilm will be exposed to less DO at the bottom than near the surface and that is where I think the dynamics within that biofilm change.

Surface DSB biofilm exposed to greater amounts of DO will out produce nitrates removed yet when surrounded with less oxygen nitrates will be removed at a quicker pace than produced. Seems only logical that production of nitrates will taper off as DO is less available to convert ammonia and nitrites. Granted denitrification converts nitrates to nitrite and as I understand it can produce ammonia but there should be no further reduction from ammonia to nitrite than to nitrates. My thought process.

 

[biom]

Possible, and happening in a reef tank are not the same, however. Artificially changing the situation by 100 fold (bulk ammonia concentration, and hence the input rate of nitrate to the deeper layers, and reduction of O2 within the biofilm) to do a test may lead to inaccurate extrapolation to real reef tank situations.

Who is saying it is happening in a reef tank?
If something is clear from the discussion so far, is that nobody knows what is happening. So we are picking information from the Net (in a very unprofessional and non-systematical way I would say because this is a hobby to all of us), about how it happened in other systems (eel farms, wastewater treatment, etc.) and laboratory experiments to discuss if it is possible to apply to a reef aquarium.

 

[Lasse]

I’m speaking more of the available DO to that biofilm early in the nitrification process vs late. Deep sand bed biofilm will be exposed to less DO at the bottom than near the surface and that is where I think the dynamics within that biofilm change.

Surface DSB biofilm exposed to greater amounts of DO will out produce nitrates removed yet when surrounded with less oxygen nitrates will be removed at a quicker pace than produced. Seems only logical that production of nitrates will taper off as DO is less available to convert ammonia and nitrites. Granted denitrification converts nitrates to nitrite and as I understand it can produce ammonia but there should be no further reduction from ammonia to nitrite than to nitrates. My thought process.

Still - I´m talking about the process in a single biofilm (as the article do) Biofilm for me is a film that cover the area of a medium and its local. Every grain in a sand bed has its own biofilm - IMO - there its organisms differ according to the environmental conditions. I agree with you that denitrification occurs down in a sand bed but what I (and the article) is discussing is the process in an individual bio film. In the article - the biofilm was 200 µm thick

Sincerely Lasse

 

[Randy Holmes-Farley]

Who is saying it is happening in a reef tank?
If something is clear from the discussion so far, is that nobody knows what is happening. So we are picking information from the Net (in a very unprofessional and non-systematical way I would say because this is a hobby to all of us), about how it happened in other systems (eel farms, wastewater treatment, etc.) and laboratory experiments to discuss if it is possible to apply to a reef aquarium.

I'm certainly good with that conclusion, but Lasse seems to be saying that denitrification was happening in reef tanks, and he was giving evidence that it could using these papers. Maybe I was misunderstanding his position. If he is claiming it "may or may not be happening", I'm fine with that.

 

[Lasse]

and he was giving evidence that it could using these papers. Maybe I was misunderstanding

You partly missunderstand my position. I still convinced that denitrification take place in reef tanks like mine with help of dosing DOC but my standpoint according the paper linked by @biom is - IMO - that this paper prove that the theory that many persons in the reefing community had put forward (nitrification/denitrification in the same biofilm has an importance for the nitrate concentrations) is false. The paper shows that nitrification rate in this experiment is 7 time higher than the denitrification rate which means that the NO3 in the water column will rise - not decrease.

Why I believe that it is denitrification that is responsible for my results in my tank and not assimilatory nitrate reduction, Dissimilatory nitrate reduction to ammonium we can discuss in another thread I will stress once again that I have not say that denitrification take place in every aquarium - what I said before (and still believe) is that the main question in the first post can be answered like this - dose DOC and get lag phase before NO3 drop significantly - its likely denitrification that have kicked in. Below is my post # 226 there I think I clarify my standpoint

As I try to explain in my long post - I do not see how a mass calculation can prove a bacterial assimilatory nitrate reduction of large amount of NO3 in organisms with an atomic N/P ration lower than 17:1 (between the indexfinger and the thumb) This based on the food input. Till this comes also the N input that we can´t control - N fixation. Short - there will be other nutrient limitation that stops the growth of the bacterial biomass before. Obvious - P will be consumed in the form of PO4 and even if you add P (as PO4) to the system - you by yourself rise the question about trace elements.

Note - I have not say that denitrification is always the major factor for nitrat reduction in our aquarium - the only thing I have pointed out is that if you in an aquarium with high NO3 concentrations (let us say above 5 mg/L) and normal PO4 concentrations (around 0.1 mg/L) dose DOC and get lag phase before NO3 drop significantly - its likely denitrification that have kicked in. This is because the denitrification process has low sensitivity for limitation of other compound than DOC and NO3.

DOC is essential in most system in order to get denitrification started in two major pathways. To create a large enough fast growing aerobic heterotrophic bacteria population that create oxygen depletion in certain spots and force the facultative aerobics to use NO3 as electron acceptor in the cellular respiration. Than this happens - the DOC´s importance swing to serve as electron donator in the anaerobic cellular respiration that use NO3 instead of O2.

Other type of assimilatory nitrate reduction (microalgae, corals, fungi and macroalgae) can play a major roll and be the most important nitrate reduction processes in many aquariums. If the population the organisms are large enough and that no other nutrient or trace element limitations occur. In a mass balance point of view - macroalgae is maybe the most interesting organism here - its internal atomic N/P ration is around 30 (correspond to a NO3/PO4 ratio (by weight) around 20 if NO3 is the only N source). The uptake ratio of N/P is higher than my calculated input ratio for both dry and natural food.

Also processes that reduce the NH4/NH3 concentration like heavy aeration at high pH and the anammox can reduce the net production of NO3 in the nitrification process, Direct uptake of NH4/NH3 by algae, corals and other photosynthetic organism reduce the NO3 production as well.


This was the question (my bold). My answer is that if there is high NO3 concentrations compared with PO4 concentrations in the start of DOC dosing and there is a lag period - is likely the denitrification that´s responsible for the reduction of NO3. This because the ratio of inputted atomic N and P together with internal N fixation exceeds the bacteria internal atomic ratio by far. If there is a too high atomic ratio between NO3-N and PO4-P - in the start - a growth of bacteria biomass can´t solve the problem by itself - its need other NO3-N export or/and an other P import pathway (dosing P)

Sincerely Lasse

I will also stress that I do not think the bioblocks or other porous material can cause any denitrification rate that matter in a reef tank - at least not if there is no DOC dosing.

Sincerely Lasse

 

[Randy Holmes-Farley]

- IMO - that this paper prove that the theory that many persons in the reefing community had put forward (nitrification/denitrification in the same biofilm has an importance for the nitrate concentrations) is false. The paper shows that nitrification rate in this experiment is 7 time higher than the denitrification rate which means that the NO3 in the water column will rise - not decrease.

OK, thanks.

However, what if organics are higher (due to organic carbon dosing), nitrate in the bulk water is higher (say, 50 ppm), and ammonia in the water is much lower (say, 0.01 ppm), wouldn't it be plausible that the balance might shift to more denitrification than nitrification?

That said, I'm not presently convinced the paper actually showed any denitrification as opposed to ordinary nitrate consumption to form biomolecules.

 

[HomebroodExotics]

OK, thanks.

However, what if organics are higher (due to organic carbon dosing), nitrate in the bulk water is higher (say, 50 ppm), and ammonia in the water is much lower (say, 0.01 ppm), wouldn't it be plausible that the balance might shift to more denitrification than nitrification?

That said, I'm not presently convinced the paper actually showed any denitrification as opposed to ordinary nitrate consumption to form biomolecules.

Why would denitrification NOT occur in a reef tank just as it would in any other waste management application?

 

[Lasse]

However, what if organics are higher (due to organic carbon dosing), nitrate in the bulk water is higher (say, 50 ppm), and ammonia in the water is much lower (say, 0.01 ppm), wouldn't it be plausible that the balance might shift to more denitrification than nitrification?

If we for the moment ignore the possibility of aerobic denitrification - it works as below IMO

If you dose DOC - you alter the whole system. You will favour heterotrophic aerob bacteria and they will concur out/disable the more slow growing nitrification bacteria even from the surface of the biofilm. Depending on time and dose - the biofilm will be total free from at least active nitrobacter/nitrospira populations (they become dormant). No complete nitrification. This oxygen loving heterotrophs will deplete the film of oxygen and at the depth in the biofilm where O2 becomes the limited factor for heterotrophic growth -> facultative heterotroph aerobs start to use NO3 and DOC in the respiration instead of the absent oxygen.

You get no or very low nitrification in the biofilm but denitrification.

This is what happens in a SBR reactor. First you run the batch reactor with high oxygen and inorganic carbon (HCO3/CO3) - next step is to lower the oxygen and rise the DOC concentration. The same biofilm will in the first stage remove BOD (organic carbon) and when BOD is low - perform nitrification. The second stage will give denitrification. Can this be done in a reef aquarium - 1 day DOC dosing/ 1 day no DOC dosing - maybe but you need to rinse your substrate from lose organics (read heterotopic biofilm) before the nitrification step

That said, I'm not presently convinced the paper actually showed any denitrification as opposed to ordinary nitrate consumption to form biomolecules.

Maybe so - but for me - it is a prove that a nitrification biofilm produce more NO3 than it consume. The opposite (or equal production/consumption) has been a myth for many years in the reefing community.

Sincerely Lasse

 

[Randy Holmes-Farley]

Why would denitrification NOT occur in a reef tank just as it would in any other waste management application?

It might, but there are critical differences.

Wastewater often has high ammonia, high nitrate, and high organics.

Those are not necessarily true in reef tanks.

Second, waste water treatment often uses processes not present in reef aquaria:

"There are two main process configurations for denitrification filters commercially available, downflow and upflow continuous backwash filters"

https://www.epa.gov/sites/default/files/2019-08/documents/denitrifying_filters_fact_sheet_p100il79.pdf

 

[Randy Holmes-Farley]

Maybe so - but for me - it is a prove that a nitrification biofilm produce more NO3 than it consume. The opposite (or equal production/consumption) has been a myth for many years in the reefing community.

I don't think the detailed level of suggesting a single biofilm either does or does not allow denitrification, is a "myth" that many reefers are even aware of. It's a rather esoteric concept compared to what reefers generally discuss which is happening down in sand beds or rock pores, which does not necessarily relate to what is happening a biofilm open to the bulk aerated water.

 

[Lasse]

Wastewater often has high ammonia, high nitrate, and high organics.

Incoming Municipal sewers are normally very low to zero in nitrate. High ammonia, high PO4 and high BOD5 or 7 is a rule. Depending on construction and regulation - outgoing water - beside low PO4 and BOD5 or 7 - it can be high in NH4/NH3, high in NO3 or low in both. In Sweden today is nitrogen removal mandatory in many waste water treatment plants - but that´s normally not that way in many countries

I don't think the detailed level of suggesting a single biofilm either does or does not allow denitrification, is a "myth" that many reefers are even aware of. It's a rather esoteric concept compared to what reefers generally discuss which is happening down in sand beds or rock pores, which does not necessarily relate to what is happening a biofilm open to the bulk aerated water.

I have seen this statement - nitrification in the upper layer of a biofilm and simultaneous denitrification in the deeper layer in the same biofilm in many threads on R2R - among them this thread. The short distance between production and "consumption" use to be a common phrase. Many "magical" bio cubes says work this way.

I do not think there is a long distance between our opinions. I´m rather sure that low NO3 concentration in many mature reef tanks with low fish load but high biomass of corals is due to assimilatory nitrate reduction by corals, algae and other photosynthetic organisms (ans some others too) - not denitrification. However in my tank there I never remove any organics or do WC - I need to adress a high efficiency denitrification system. One of the reasons why I think that denitrification is one of the major pathways for N removal in my system is that I have construct my reverse flow deep sand bed after the book for effective denitrification systems - old school and here is my measurement for the last months

Last 3 weeks

Note I use Hanna High NO3 checker. Its plus minus 2 mg/L on every measure point. I´ll test with their low method tomorrow.



Sincerely Lasse

 

[Lasse]

I did measurements both with Hanna High NO3 checker and their Low NO3 checker. Result 0.7 and 1.66. I would say that their high NO3 checker is good enough for normal measurements if its accuracy of ± 2 mg/L is included in the calculations. I will compare these meters with an Oceamo analysis later this month. My NO2 (Hanna checker ULR Nitrite) is around 0,02 mg/L NO2 (converted from 6 ppb NO2-N)

Sincerely Lasse

 

[biom]

Is it discussion now - if there is denitrification happening or not in a "normal" reef tank?
And Randy says it is not happening and Lasse is saying it is happening at least in the deep sand bed or in filtration as the one his tank?
But this could be tested if in a tank with zero ammonia and 50 ppm nitrate is added DOC and drop of nitrate is observed, then :
a/ if there is denitrification happening then significant concentration of nitrite should emerged at some point in the tank water and to follow the same curve as nitrate but shifted in time?
b/ if there is no significant nitrite detected - then we have other process - assimilation?

In Lasse's case he had drop of nitrate from 25 ppm to 1 ppm in 10 days and he had only 0.02 ppm NO2 measured. Lasse did you have measured nitrite as well during drop of nitrate observed?

 

[Lasse]

Denitrification will not aiways create NO2 in the water. Only if it is incomplete. A well working system will transfer NO3 seemless into N2.

Sincerely Lasse

 

[taricha]

@biom I posted this earlier in the thread but this is pretty influential on my thinking of what's going on here, of why most people see little NO3 drop, but how much potential NO3 drop could occur in spaces where organic carbon and NO3 get into low O2 spaces.

When Dan dosed acetate he saw 40 moles C to 1 mole NO3 decrease.
And Lasse saw something like 2 moles C to 1 mole NO3.

So I did some data mining from the last 3 pages of @Lasse build thread. And I already considered Lasse's setup to be a best possible case - but crunching the numbers looks impossibly good.
Like 2 moles of Carbon removes 1 mole of Nitrogen, good.

Left chart is where I took different stretches of Lasse's data where he did various set volumes of 8% ethanol added fro a few days at a time.
When he doesn't dose any, NO3 climbs at between 2.5 to 4 ppm NO3 per day.
When he doses 18mL/day through his sand, NO3 drops around 2.5-3ppm per day.
The amount dosed is in mL of 8% ethanol (I interpret this as like 1/5 vodka) into 310L system.
The right chart is me trying to convert all those to moles/L of Carbon and Nitrogen.
(I'm not even sure that data is theoretically possible. Maybe Lasse can spot my error)


@Dan_P data with dumping vinegar into the water was more in the real world, like 40 moles C per 1 mole NO3 removed.

see follow-up post 165 also for putting this result in context to denitrification equation etc.

if there is denitrification happening then significant concentration of nitrite should emerged at some point in the tank water and to follow the same curve as nitrate but shifted in time?

In this paper Lasse linked, it shows how NO2 produced can be very small even for large NO3 decrease (fig 2).

Here is a comparison between different DOC as electron donors in denitrification(PDF)

Sincerely Lasse