Bacteria ratios, putting a end to it!

[Reefahholic]

NO3 can have a lag effect too.

This is what would happen if you dosed glucose in a tank without anoxic areas for denitrification to happen, I would imagine

this is one of the reasons the tank is bare, there’s no way anyone can say that denitrifying bacteria is removing the nutrients or interfering with the results

 

[sixty_reefer]

A drop of 0.2 ppm in 10 hrs would cause most Acropora to RTN or STN. That’s a huge drop.

It only dropped that fast because a bloom was induced, this is just to show that the bacteria that uses carbon to build biomass is also utilising phosphates. If something like this was made in a tank it could potentially kill the live stock as well as this blooms deplete oxygen, although if the dose is increased properly as its a standard procedure with carbon dosing this kind of numbers would be seen in a working reef aquarium also.

 

[sixty_reefer]

NO3 can have a lag effect too.

Only on nitrates, phosphates won’t be affected by nitrites.
Phosphates reduction is the centre of the experiment, nitrates is just to show that they are being reduced.

 

[MnFish1]

As some of you may know already, I have been discussing extensively on bacterial ratios and the effects on po4.
today I decided that the subject it’s getting a bit tiring to me, so am going to do what is logical

I’ve pulled my testing tank from the garage and will put a end to the discussion trough testing.

Main goal here is:

Does carbon dosing reduces phosphates and if it does what’s the ratio?

for testing:

10 litres tank water (2.6 gallons)
Phosphates will aim at 0.5 ppm
Nitrates will aim at 20ppm
Carbon source will be vodka 1ml

The reason I am going for 1ml vodka in 10 litres of water is to try and create a bloom, this should create some reliable test results in less than 24hours.


anyone got any opinions or objections on the way the test is going to be carried out before I start I don’t use Hanna for po4 testing although I am happy for others that have a Hanna to replicate the test?

edit:
The display tank is 100 litres and it has a 2ml vodka dosing daily

I have another 'question' about the experiment - and I don't know if you have any access to pure cultures of various bacteria - BUT - another issue is lets say you add the vodka and get 'a bloom' - a bloom of 'what'? This is important because different heterotrophic bacteria and fungi have completely different pathways of ammonia AND nitrate reduction - and also varying rates of PO4 removal.

PO4 by definition will drop because as new bacteria are created they require phosphorous to form, live and reproduce. However, depending on the bacteria thats most responsible for the 'bloom' - the ratio of nitrate/Po4 may be totally different.

I hope you understand the point - which is - without a bacterial inoculation of some type it's going to be difficult to tell whats going on. For example - you could inoculate with a presumed heterotrophic bacterial mixture (See Dr. Reef's experiments - to see the 'presumed heterotrophic mixtures' - if you cannot get a pure isolate of say Pseudomonas, etc.

You can ignore these comments if you've already addressed this potential issue - I just haven't seen it

 

[Reefahholic]

The bloom would be mostly Heterotrophic correct?

 

[MnFish1]

It only dropped that fast because a bloom was induced, this is just to show that the bacteria that uses carbon to build biomass is also utilising phosphates. If something like this was made in a tank it could potentially kill the live stock as well as this blooms deplete oxygen, although if the dose is increased properly as its a standard procedure with carbon dosing this kind of numbers would be seen in a working reef aquarium also.

Another question - sorry but it's a separate topic that again I haven't seen asked/answered. What is your definition of 'a bloom'. Is it cloudy water? Is it merely that PO4 is dropping meant that bacteria must be growing - and thats 'a bloom'. Someone alluded to this earlier - that eventually, when PO4, etc become depleted the dying bacteria themselves will provide nutrients for more bacteria (though obvious over time the numbers will decrease) - I believe you answered this with the fact that its going to be a short duration experiment.

I might suggest that somehow - you decide at what point to measure your ratio's - because IMHO - if you measure during the logarithmic growth phase as compared to the so-called 'stationary phase' or 'death phase' you also may get totally different ratios.

 

[MnFish1]

The bloom would be mostly Heterotrophic correct?

Yes - but thats thousands of different potential species of bacteria. Even if there were nitrifiers/denitrifiers present the doubling time of heterotrophs in the short term would out compete in all likelihood.

 

[MnFish1]

PS for those interested in this topic here is an article - @sixty_reefer this might help explain some of what I was getting at in a more concise manner. https://www.sciencedirect.com/science/article/pii/S0038071722000682

 

[sixty_reefer]

I have another 'question' about the experiment - and I don't know if you have any access to pure cultures of various bacteria - BUT - another issue is lets say you add the vodka and get 'a bloom' - a bloom of 'what'? This is important because different heterotrophic bacteria and fungi have completely different pathways of ammonia AND nitrate reduction - and also varying rates of PO4 removal.

PO4 by definition will drop because as new bacteria are created they require phosphorous to form, live and reproduce. However, depending on the bacteria thats most responsible for the 'bloom' - the ratio of nitrate/Po4 may be totally different.

I hope you understand the point - which is - without a bacterial inoculation of some type it's going to be difficult to tell whats going on. For example - you could inoculate with a presumed heterotrophic bacterial mixture (See Dr. Reef's experiments - to see the 'presumed heterotrophic mixtures' - if you cannot get a pure isolate of say Pseudomonas, etc.

You can ignore these comments if you've already addressed this potential issue - I just haven't seen it

You are right although we still on the first part of the experiment, the issue we been having so far is that many say that carbon dosing won’t affect phosphates, the theory I’ve wrote has been extremely criticised on those grounds that are near to be proven wrong. hopefully this will end up today, as for which bacteria is doing the work I think I would have to try and find a way, unfortunately I don’t have a facility over here like aquabiomics, wend I started last night it was a theory today it looks like it’s more evidence based and more work will need to be done

 

[Reefahholic]

Yes - but thats thousands of different potential species of bacteria. Even if there were nitrifiers/denitrifiers present the doubling time of heterotrophs in the short term would out compete in all likelihood.

How many of those thousands do we typically see in marine aquariums?

Correct, Heterotrophic bacteria can divide in 20 minutes and would outcompete the other bacteria.

 

[sixty_reefer]

The bloom would be mostly Heterotrophic correct?

I set the scenario to be only heterotrophic, I don’t see a way that autotrophic bacteria could influence results

 

[sixty_reefer]

How many of those thousands do we typically see in marine aquariums?

Correct, Heterotrophic bacteria can divide in 20 minutes and would outcompete the other bacteria.

Agree nitrifying is 16 hours to multiply

 

[sixty_reefer]

PS for those interested in this topic here is an article - @sixty_reefer this might help explain some of what I was getting at in a more concise manner. https://www.sciencedirect.com/science/article/pii/S003807172200068

most people don’t realise that nitrifying and denitrifying bacteria don’t have a significant impact in a mature reef. Most of the filtration if not all in some cases is being done by heterotrophic bacteria, the excessive use of large protein skimmers is one of the reasons some people have so many issues in early stage of a tank maturity. As most of the beneficial bacteria is actually in the water column so are the nutrients.

 

[MnFish1]

You are right although we still on the first part of the experiment, the issue we been having so far is that many say that carbon dosing won’t affect phosphates, the theory I’ve wrote has been extremely criticised on those grounds that are near to be proven wrong. hopefully this will end up today, as for which bacteria is doing the work I think I would have to try and find a way, unfortunately I don’t have a facility over here like aquabiomics, wend I started last night it was a theory today it looks like it’s more evidence based and more work will need to be done

I think that people that think PO4 is not lowered by bacterial growth are 'incorrect' - in the experimental conditions you have set up - almost by default, PO4 will decrease if bacteria are growing.

But - IMHO - this is where there is a key difference. in your experiment - during 'a bloom' - during the logarithmic growth phase - PO4 will decrease - my GUESS is that in a tank - with all of the variables - additions of food, etc - the PO4 will not decline significantly.

I guess I look at it this way. Take a tank - that produces a stable amount of ammonia, nitrate, PO4/day. Start dosing xxx Carbon. At first - there will be a logarithmic increase in bacteria x,y,z......... Then - that extra 'carbon' is utilized. Then one of 2 things will happen - if you dont add more carbon the nutrients will be re-released into the tank - right? So - you repeatedly dose carbon - and eventually a new steady state will be accomplished - and in theory the nitrate after a month lets say - of carbon dosing will be lower than the start.

But - if you're merely maintaining a population of bacteria thats basically eating ammonia and or nitrate (heterotrophs - with carbon) - they are not going to be using as much PO4 once they are in the 'stable' phase - as compared to the active growth phase. Does that make sense?

By the way - if you read the article I provided - my GUESS is that when you increase heterotrophs using carbon, the reason nitrate falls is not that the bacteria are eating nitrate 'per se' - though some do - its because they are utilizing ammonia - thus less nitrate is produced. I.e. carbon increases heterotrophs --->. Heterotrophs utilize both carbon and ammonia ---> less nitrate.

Note these are merely my ramblings - perhaps @Lasse has a comment - as he would have much more knowledge than I

 

[MnFish1]

most people don’t realise that nitrifying and denitrifying bacteria don’t have a significant impact in a mature reef. Most of the filtration if not all in some cases is being done by heterotrophic bacteria, the excessive use of large protein skimmers is one of the reasons some people have so many issues in early stage of a tank maturity. As most of the beneficial bacteria is actually in the water column so are the nutrients.

I liked this comment - but I'm not sure it's totally correct. The reason being - that there are multiple experiments done with 100% water changes - and no change in ammonia, etc levels.

 

[sixty_reefer]

I liked this comment - but I'm not sure it's totally correct. The reason being - that there are multiple experiments done with 100% water changes - and no change in ammonia, etc levels.

If you remember from our testing in freshwater, every time we done a 100%water change it took a couple days for the ammonia to be fully processed in comparison to keep 10% of the old water

 

[sixty_reefer]

18 hours results are in:

phosphates are a clear zero

nitrates climbed back up to 20 as @taricha predicted

this is consistent with the nutrient limitations that tell us if a tank becomes limited in phosphates a increase in nitrates is expected I still got a few more hours to go so I’ve added 0.6 ppm of phosphates again to see the changes. This morning there was jus a small haze to the tank and now is almost milky white.

At 10 hours

At 18 hours and around 7 hours after being phosphates limited

 

[sixty_reefer]

I think that people that think PO4 is not lowered by bacterial growth are 'incorrect' - in the experimental conditions you have set up - almost by default, PO4 will decrease if bacteria are growing.

But - IMHO - this is where there is a key difference. in your experiment - during 'a bloom' - during the logarithmic growth phase - PO4 will decrease - my GUESS is that in a tank - with all of the variables - additions of food, etc - the PO4 will not decline significantly.

I guess I look at it this way. Take a tank - that produces a stable amount of ammonia, nitrate, PO4/day. Start dosing xxx Carbon. At first - there will be a logarithmic increase in bacteria x,y,z......... Then - that extra 'carbon' is utilized. Then one of 2 things will happen - if you dont add more carbon the nutrients will be re-released into the tank - right? So - you repeatedly dose carbon - and eventually a new steady state will be accomplished - and in theory the nitrate after a month lets say - of carbon dosing will be lower than the start.

But - if you're merely maintaining a population of bacteria thats basically eating ammonia and or nitrate (heterotrophs - with carbon) - they are not going to be using as much PO4 once they are in the 'stable' phase - as compared to the active growth phase. Does that make sense?

By the way - if you read the article I provided - my GUESS is that when you increase heterotrophs using carbon, the reason nitrate falls is not that the bacteria are eating nitrate 'per se' - though some do - its because they are utilizing ammonia - thus less nitrate is produced. I.e. carbon increases heterotrophs --->. Heterotrophs utilize both carbon and ammonia ---> less nitrate.

Note these are merely my ramblings - perhaps @Lasse has a comment - as he would have much more knowledge than I

The process is called filtration by assimilation and it’s similar to how macro algae exports nutrients, bacteria utilise C N P and then is exported via skimmer or ends up as marine detritus in our sump or substrate. It’s not a new thing.

 

[taricha]

nitrates climbed back up to 20 as @taricha predicted

Why?

I say that NO3 measurement "rebound" after an initial drop is possible for the O2 limiting reasons that others mentioned earlier.

Because 1mL vodka = .32 g ethanol = .17g Carbon, so 1mL vodka in 10L = 17mg/L Carbon. If half (ballpark guess) of that Carbon were oxidized to CO2, that would take 44 mg/L O2. Saltwater will have ~7 mg/L O2 max. So it's enough Carbon to deplete all the O2 like 6 times over. I'm guessing that the small pump may not keep up with the oxygen consumption once the bacterial bloom gets rolling.
If that happens, and O2 is gone in some part of the water while organic C remains, then NO3 could be reduced to NO2, and if that happens, then the NO3 test (Nyos?) may be very sensitive to the NO2 produced and generate a false high NO3. It was made even more likely since Sixty bumped the vodka up to 5mL.

(I've confused myself doing this sort of thing in a bottle of tank water before.)

this is one of the reasons the tank is bare, there’s no way anyone can say that denitrifying bacteria is removing the nutrients or interfering with the results

But that is exactly what I do say. Tank water bacteria under high Carbon and low O2 de-nitrified NO3->NO2 and gave a false NO3 "increase"

Pull out an NO2 test kit to see if nitrite was in fact generated. Test for ammonia too, just for kicks.

 

[sixty_reefer]

I say that NO3 measurement "rebound" after an initial drop is possible for the O2 limiting reasons that others mentioned earlier.

Because 1mL vodka = .32 g ethanol = .17g Carbon, so 1mL vodka in 10L = 17mg/L Carbon. If half (ballpark guess) of that Carbon were oxidized to CO2, that would take 44 mg/L O2. Saltwater will have ~7 mg/L O2 max. So it's enough Carbon to deplete all the O2 like 6 times over. I'm guessing that the small pump may not keep up with the oxygen consumption once the bacterial bloom gets rolling.
If that happens, and O2 is gone in some part of the water while organic C remains, then NO3 could be reduced to NO2, and if that happens, then the NO3 test (Nyos?) may be very sensitive to the NO2 produced and generate a false high NO3. It was made even more likely since Sixty bumped the vodka up to 5mL.

(I've confused myself doing this sort of thing in a bottle of tank water before.)




But that is exactly what I do say. Tank water bacteria under high Carbon and low O2 de-nitrified NO3->NO2 and gave a false NO3 "increase"

Pull out an NO2 test kit to see if nitrite was in fact generated. Test for ammonia too, just for kicks.

I see we’re it went wrong, I didn’t account for the o2 depleting and the hole system become anoxic.
we’re do I go from here? Repeat the experience? The first 10 hours were still anaerobic.

no2 is between 0.6 and 0.8 ppm
Ammonia is at zero ppm
Phosphates are still at zero even after a additional 0.9 ppm after the 18 hour mark, we’re the phosphates going?