Post your test data

[Caliban07]

I haven't been much in the mood to read scientific papers today so I haven't made any research advances. Tomorrow maybe...
Let me know Caliban if you read something good!

Also where is Sixtyfou? Did he miss a couple days and now is too overwhelmed with the 100 new posts

Ok I have more reading to do. Another note. Higher temperatures increases the metabolism of nitrosomonas thus increasing activity. This uses more saturated oxygen. Nitrospira does not perform as well under lower o2 levels as nitrosomonas.

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[Delapool]

Hi all, quickly skimmed - has anything been seen on what will slow down the nitrate conversion part of the cycle? All I say was a reference to 20ppm (link below). Also that certain metals (at low ph) may be an issue (which prime or such would take care of anyway).

Total Nitrification balance

Edit - brain is fried, off to the fish shop lol.

Another edit - what was meant by phosphate blocker and water changes fixing?

 

[threnjen]

Delapool this is the phase of research I'm at too and I haven't gotten to do all the reading- the nitrate conversion part. And arguably that's the most important part to solve, because that's where people get hung up. I've just had a busy couple days and haven't had a chance to work on it more.

That looks like a good link! Although they name Nitrobacter as the stuff that converts to Nitrate, where we now know that in aquaria there may be more Nitrospira as the conversion bacteria. This whole topic just requires some additional study.

Re: Phosphates
One of the bacterias (or is it all?) actually requires phosphates for the energy for their conversion, in very low amounts. Generally there is enough in the water since the amount needed is very trace. But Caliban's argument is that sometimes a WC is necessary because the nitrite stage has stalled because all available phosphates in the water are used up, and a WC will restore those. I also propose that adding some flake food at the start of the cycle could add enough phosphate to take the cycle to the end.

As a note: On the crazy cycle I am running right now I did NOT add any phosphates

 

[threnjen]

Ok I have more reading to do. Another note. Higher temperatures increases the metabolism of nitrosomonas thus increasing activity. This uses more saturated oxygen. Nitrospira does not perform as well under lower o2 levels as nitrosomonas.

One thing I also saw somewhere is that Nitrospira prefers low nitrites and high temps, where Nitrobacter prefers high nitrites and low temps.
It's very, very possible that we are building different bacterial colonies from each other when we choose different cycling techniques, that all accomplish the same thing in the end. It's interesting!

 

[threnjen]

We need more people to run fishless cycles for data gathering

I made this post, I hope some people will sign up

On an unrelated note, @Caliban, I am traditionally a member of the "fishless cycling 4eva!" brigade, but even I have to admit that some of the stuff we have learned makes me thing that fish-in cycling may not be so bad in the hands of an informed keeper.

 

[threnjen]

2 signups already for testing!

So I'm proposing 3 dosing plans-
Conservative method aka Caliban's plan
Standard method aka the 4ppm method
Aggressive method aka "my" method (I am not the inventor of the method, for that I give credit to jrman83 of aquariumforum.com)

So I'm hoping we get at least 6 signups min, so there can be at least 2 tests per method.

I'm also thinking I'll get one more filter and heater so that I can run all three methods as well, rather than just two.

 

[Caliban07]

A whole new spin on this experiment. Do chlorine/chloramines really affect the bacteria? We could run another test that does not dechlorinate water beforehand. Would this new condition breed another bacteria strain that is sensitive to parameters that nirospira are not? Would the cycle start slower and start once the chlorine has been dispersed from the water?

Dum dum duuum! Or am I just complicating matters?

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[threnjen]

lol!! too many variables! Save that one for a later experiment

I'm bummed what you noted about the filters and tank sizes for others running the cycle though! I mean you're totally right. We just don't have that much control.

 

[threnjen]

My husband just asked me how my "thesis" is coming. He has noticed I haven't been reading the science papers today. Such a slacker I am.

It's only 12:45AM, sounds like a great time to start some heavy reading.

 

[threnjen]

A task for those more scientifically inclined:

From the Havonec report about Nitrospira as the nitrite oxidizing bacteria.

"Three all-glass aquaria were established as described above. A 34-liter sample of city tap water, which was passed through activated carbon, was added to each aquarium, which contained 4.53 kg of gravel. Initially, 0.71 mmol of filter-sterilized (0.2-mm-pore-size filter) ammonium chlo-ride was added to each tank, followed by an additional dosing of 5.0 mmol of NH4 Cl on the fourth day. On days 10, 15, 18, 23, and 30, further ammonia additions of 8.9 mmol were made to each aquarium. During the test, a total of 50.4 mmol of ammonia was added to each aquarium. Water samples werecollected daily"

Question: What is mmol of ammonium chloride as a ppm in their 34 liter water sample?

 

[Caliban07]

lol!! too many variables! Save that one for a later experiment

I'm bummed what you noted about the filters and tank sizes for others running the cycle though! I mean you're totally right. We just don't have that much control.

Yes its a shame, we just dont know if available surface area either by larger types of the same media or different media such as ceramics increases reproduction time.

Heres an interesting phrase

Ammonia oxidation is an acidogenic reaction. In the presence of excess ammonium, the pH of the medium decreases during ammonia oxidation until it is sufficiently low that further ammonia oxidation is prevented. If the starting pH is lowered, the amount of ammonia which must be oxidized to reach this limiting pH is decreased. Thus, at lower pH values, more ammonium remained in the medium of incubations containing 15 or 50 mM initial ammonium (as measured by a less nitrite production, data not shown).

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[Caliban07]

Yes its a shame, we just dont know if available surface area either by larger types of the same media or different media such as ceramics increases reproduction time.

Heres an interesting phrase

Ammonia oxidation is an acidogenic reaction. In the presence of excess ammonium, the pH of the medium decreases during ammonia oxidation until it is sufficiently low that further ammonia oxidation is prevented. If the starting pH is lowered, the amount of ammonia which must be oxidized to reach this limiting pH is decreased. Thus, at lower pH values, more ammonium remained in the medium of incubations containing 15 or 50 mM initial ammonium (as measured by a less nitrite production, data not shown).

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I think its time to start adding buffers or increasing pH. If you want to limit water changes then we best get the baking soda out.

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[threnjen]

I just found the more simplified thing I read about the pH crash:

"The nitrification process produces acid. This acid formation lowers the pH of the biological
population in the aeration tank and can cause a reduction of the growth rate of nitrifying bacteria.
The optimum pH for Nitrosomonas and Nitrobacter is between 7.5 and 8.5; most treatment plants
are able to effectively nitrify with a pH of 6.5 to 7.0. Nitrification stops at a pH below 6.0. The
nitrification reaction (that is, the conversion of ammonia to nitrate) consumes 7.1 mg/L
of alkalinity as CaCO3 for each mg/L of ammonia nitrogen oxidized. An alkalinity of no less than 50-100 mg/L is required to insure adequate buffering."

So basically, the nitrogen cycle literally eats pH. It eats alkalinity.

This paper is about waste treatment so talks about nitrobacter, but the thing about the ph crash is the important part
Source

I think just using baking soda is fine. While in a "permanent" system (aka a set up aquarium where we don't want to have to add chemicals) a permanent buffer like coral would be better, while we are just trying to keep the cycling pH at some level then adding chemicals isn't a big deal. We're not worried about shocking any fish.

 

[threnjen]

OK I found lots of good tidbits tonight in http://ftp.mpi-bremen.de/pub/moleco.../Gieseke_EM2003.pdf?origin=publication_detail

It's not all new but at least it's good to have confirmation of things sometimes.
Question: When something says inhibit, does that mean SLOW or STOP?

“results indicated inhibition of nitrite-oxidizing bacteria (NOB), but not of ammonia-oxidizing bacteria (AOB) by free ammonia under HA(high ammonium) conditions. “
Too high FREE ammonia (NH3) does not inhibit ammonia-oxidizing bacteria and will not stall your cycle at the ammonia stage.
Too high NH3 can inhibit nitrite-oxidizing bacteria. This could stall your cycle if too high at the nitrite stage.

“Nitrite-oxidizing bacteria of the genera Nitrobacter and Nitrospira(with at least two dis-tinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached“
Nitrospira can tolerate low oxygen better than Nitrobacter.

“In both experiments, A and B, production of nitrite occurred in the oxic part of the biofilm, and was highest near the surface. This also held true for experiment D. Nitrite production occurred near the surface”
Oxygen increases the growth. We know this. More airstones!

“Nitrate production in general was reduced with decreased oxygen concentration. “
Again, yup

“Among the NOB, one population was related to Nitrobacter spp., as hybridized with the probe NIT3. Further-more, we could detect cells related to Nitrospiras pp. “
Both Nitrospira and Nitrobacter grew in their experiment.
“Popula-tions of NSR1253-positive and NSR1253-negative Nitro-spira spp. were often found in close proximity (Fig“
I'm just curious about this one - does this mean there was both gram-positive and gram-negative nitrospira, or does this mean something else?

“The NOB populations related to Nitro-spira spp. and Nitrobacter spp., respectively, showed a very inhomogeneous spatial distribution. On polystyrene, we exclusively detected Nitrospira spp., which occurred distributed throughout the biofilm. Numerous large microcolonies were also found close to the substratum (Fig. 2A). In most sections from biofilm grown on Kaldnes substratum, Nitrospira spp. dominated the NOB guild whereas cells of Nitrobacter spp. were only rarely detected. In a few sections, however, Nitrobacter spp. represented the dominant NOB population whereas numbers of Nitrospira -like cells were low (Fig. 3). These results indicate a large scale heterogeneity. Because of the optical density of the biofilm material, however, no additional evidence could be provided by analysis of clay bead material by CLSM. In homogenized biofilm material, however, Nitrospira spp.-related cells were detected predominantly.“
I thought this was very interesting. Different filter media grew different bacteria.

“High concentrations of free ammonia (FA) and free nitrous acid (FNA) can hamper activity and growth of nitrifying bacteria. Concentrations of 10–150 mg FA were reported to be inhibitory to Nitrosomonas spp. whereas 0.1–1.0 mg FA was found to inhibit Nitrobacter spp. “
Here's the numbers again about the free ammonia level that inhibits. Worth noting that even in my MADCAP dosing scheme my free ammonia isn't over .5 (and obviously my cycle is not inhibited).
Again though still only studying ammonia, not nitrites as an inhibitor

“Whether FA inhibits NOB of the genus Nitrospirais not known. “
Interesting.

“The two phylogenetically unrelated groups of NOB affiliated to the genera Nitrobacter and Nitrospira did not exclude each other from the system. Earlier studies suggest Nitrobacter spp. to be adapted to high substrate concentrations, in contrast to Nitrospira spp., which were found predominantly in a zone of lower substrate and oxygen concentrations in a membrane-grown biofilm. is possible that the ‘clone clade 2’-related Nitrospira spp. are adapted to features common for the SBR design. “
From another study I recall that when they refer to "substrate" they basically mean food, suggesting this DOES refer to the nitrite levels. Therefore it is suggesting that Nitrobacter does well in high nitrites, and Nitrospira does well in lower nitrites. But it also suggests that the specific species of Nitrospira that have been discovered in freshwater systems seem to be better adapted to high nitrites.
I will be finding that study at a later time and posting from that too of course.


“cells inactive under anaerobic conditions, can easily be reactivated (Fig. 1, experiment D) and take up inorganic carbon when brought to aerobic conditions (Daims et al ., 2001b). The strategy of the Nitrospira strains might therefore not only consist of a high affinity towards oxygen as suggested earlier (Okabe et al.,1999; Schramm et al., 1999; 2000), but the cells, while being inactive, maintain their ribosome content for a long period of inactivity (Morgenroth et al., 2000). Thus, they could act as a seed population that immediately recovers its activity in other places when detached“
[FONT=&quot]Inactive bacteria can become active again. They don't just die.




I hope this was a good contribution for tonight
Goodnight from me!!
[/FONT]

 

[Caliban07]

I just found the more simplified thing I read about the pH crash:

"The nitrification process produces acid. This acid formation lowers the pH of the biological
population in the aeration tank and can cause a reduction of the growth rate of nitrifying bacteria.
The optimum pH for Nitrosomonas and Nitrobacter is between 7.5 and 8.5; most treatment plants
are able to effectively nitrify with a pH of 6.5 to 7.0. Nitrification stops at a pH below 6.0. The
nitrification reaction (that is, the conversion of ammonia to nitrate) consumes 7.1 mg/L
of alkalinity as CaCO3 for each mg/L of ammonia nitrogen oxidized. An alkalinity of no less than 50-100 mg/L is required to insure adequate buffering."

So basically, the nitrogen cycle literally eats pH. It eats alkalinity.

This paper is about waste treatment so talks about nitrobacter, but the thing about the ph crash is the important part
Source

I think just using baking soda is fine. While in a "permanent" system (aka a set up aquarium where we don't want to have to add chemicals) a permanent buffer like coral would be better, while we are just trying to keep the cycling pH at some level then adding chemicals isn't a big deal. We're not worried about shocking any fish.

Yes ive read that one. Good article.

I need help. This is going a bit far but im trying to convert 15mM in to ppm.

15mM seems to be the concentrations of ammonium these scientists are using. This is really tough. We will be using pure ammonia not ammonium.

This is done by multplying concentration by molecular weight.
Molecular weight of ammonia is 17g/mol
1mM = 0.001mol/L
So 17 x 0.001 = 0.017g = 17mg since mg/L = ppm 17mg/L =17ppm of ammonium x 15 = 255ppm ammonium

That cant be right surely.

That means at a ph of 7.5, 12.75ppm would be free ammonia and this would inhibit oxidising rates according to some studies. If this is correct 4ppm total ammonia would be pretty insignificant so why does it kill fish?

Ahhhhh

We need to write what we have learned about the cycle so far.

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[threnjen]

The conversion is out of my scope. I'm absolutely terrible at math. Hopefully someone else can check your work if you are unsure.
I agree, that looks too high though.

How is the inhibition of oxidation related to the fish though? It just kills the fish because it's poisonous. The filter would still take it, the fish will just be dead

I agree we need a more simplified write up. But I feel like I'm still missing a key link. We still haven't seen anywhere where it talks about high nitrites stalling the cycle.

I still have gobs more to read though, I only got through a few papers tonight.

 

[threnjen]

Oh I forgot to post my other tidbits, because most of them I got from that one great paper.

Again not all necessarily new. I just think it's important to have multiple citations to back up our claims if we reach a point where we want to challenge what everyone knows to be true.

FromAn Error Occurred Setting Your User Cookie
" Nitrospira moscoviensis were used for the pure culture trials. The results from this study suggest that free ammonia (NH3-N) concentrations of up to 10 mg/L were not inhibitory to Nitrospira either in situ or in pure culture
"
Studying if Nitrospira are inhibited by NH3. Study says NOPE. So now we have it that none of the three primary bacterias are inhibited until very high levels of NH3 that we would never reach.

From http://www.drtimhovanec.com/Publications/ScientificPapers/files/page8_2.pdf
"Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter. the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina"Nothing new here. Just a tidbit from the paper first asserting the dominance of Nitrospira.


"The daily con-centrations of ammonia, nitrite, and nitrate over the first 33days after setup of a new aquarium are presented in Fig. 6. The trends were as expected, with ammonia peaking about day 12.Nitrite values increased starting at day 12, peaked at day 21,and decreased to below detection limits by day 26. Nitrate values steadily increased from about day 15 onwards"

From the Nitrospira paper, logging their cycle length

 

[Caliban07]

Oh I forgot to post my other tidbits, because most of them I got from that one great paper.

Again not all necessarily new. I just think it's important to have multiple citations to back up our claims if we reach a point where we want to challenge what everyone knows to be true.

FromAn Error Occurred Setting Your User Cookie
" Nitrospira moscoviensis were used for the pure culture trials. The results from this study suggest that free ammonia (NH3-N) concentrations of up to 10 mg/L were not inhibitory to Nitrospira either in situ or in pure culture
"
Studying if Nitrospira are inhibited by NH3. Study says NOPE. So now we have it that none of the three primary bacterias are inhibited until very high levels of NH3 that we would never reach.

From http://www.drtimhovanec.com/Publications/ScientificPapers/files/page8_2.pdf
"Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter. the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina"Nothing new here. Just a tidbit from the paper first asserting the dominance of Nitrospira.


"The daily con-centrations of ammonia, nitrite, and nitrate over the first 33days after setup of a new aquarium are presented in Fig. 6. The trends were as expected, with ammonia peaking about day 12.Nitrite values increased starting at day 12, peaked at day 21,and decreased to below detection limits by day 26. Nitrate values steadily increased from about day 15 onwards"

From the Nitrospira paper, logging their cycle length

Ok excellent stuff. Are you keeping a jounal with this info too?

Check this abstract

ABSTRACT

Ammonia-starved cells of*Nitrosomonas europaea*are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of*N. europaea*starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.

Does this suggest high nitrites do not effect nitrosomonas? If so we need to see if high nitrites effect nitrospira.

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[threnjen]

ABSTRACT

Ammonia-starved cells of*Nitrosomonas europaea*are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of*N. europaea*starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.

Does this suggest high nitrites do not effect nitrosomonas? If so we need to see if high nitrites effect nitrospira.

Looks like it to me. Nice find!

What paper was that from?

 

[threnjen]

Ok excellent stuff. Are you keeping a jounal with this info too?

Yeah I'm journaling all my finds with citations. Do you have yours backed up too? I need to add yours to my journal too. Hopefully at the end we can organize it into something more "chronological" about the cycling process... or something. I'm not sure how to organize all of this information dump into something readable to the layman. It's been MANY years since I've written a proper paper, lol.