Nitrifying Bacteria. Where Are You?

[Dan_P]

You figured it out . If you treat the media for not drying out - you will also limit the oxygen that the film can get. But as long as it is some moist and contact with fresh air (read new oxygen) it will work. It can be difficult to uppfill both demands - moist and free access to rather dry air

Sincerely Lasse

I think you make a good case for what to investigate first in developing this approach.
Dan

 

[Lasse]

Probably going to have to keep spraying water at intervals.

It has a name - wet and dry filter.

Sincerely Lasse

 

[Lasse]

I think you make a good case for what to investigate first in developing this approach.

Don´t mention H2O2

Sincerely Lasse

 

[taricha]

Multiple ammonia additions in this experiment did not enhance nitrification capability, suggesting that fish-less cycling may not benefit from multiple doses.

Did we use the same concentration of ammonia? If my water change protocol removed bacteria, wouldn’t ammonia consumption become higher as the biofilm increased in size experiment to experiment?

Awesome work. The whole bacterial-shedding-into-the-water thing is fascinating and unexpected.
Never a majority, but significant and persistent under these conditions. Suggests that at least in the early stages of biofilm formation, the films are putting live nitrifying bacteria into the water in a way that would be important for colonization of new surfaces.

Agree that your data illustrates multiple 0.5ppm ammonia additions spaced a few days apart did not scale up initial biospira bacterial nitrification capability of surfaces.
(If you add up all the activity in the repeatedly discarded water, then you would come up with an increase, perhaps a doubling.)
My less-thorough looks at biospira and my sandbed, I saw large increases (from a couple of tenths ppm ammonia/day to 2+ppm/day) in nitification ability over the course of ~10 days when ammonia was persistently fed 1-2ppm. I didn't probe low portions of ammonia very much, but it seems plausible and the little data I have is consistent with the idea that the nitrifier population in my system didn't increase much at all during the portion when the total ammonia dropped below 0.5ppm.

I feel pretty confident that if you had fed 2ppm ammonia under the same conditions you would have seen a big scale-up.

 

[taricha]

I feel pretty confident that if you had fed 2ppm ammonia under the same conditions you would have seen a big scale-up.

...and Dan knows when I say I'm "confident" about how one of his nitrifying biofilm experiments would go, I'm mostly wrong.

 

[MnFish1]

Maybe it will be difficult to compare experiments because we don’t know the number of viable bacteria we each started with. Did we use the same concentration of ammonia? If my water change protocol removed bacteria, wouldn’t ammonia consumption become higher as the biofilm increased in size experiment to experiment?

There are so many questions generated by this data that if I am not careful, I will spend all my time studying these bacteria.

Yes - Exactly. It might be interesting to try the experiment with a higher initial ppm Ammonia? and/or a different bacteria (fritz?). The other difference - is that your system was oversaturated with bacteria as compared to mine which was oversaturated with ammonia.

 

[Dan_P]

I feel pretty confident that if you had fed 2ppm ammonia under the same conditions you would have seen a big scale-up.

That experiment is in progress. I will run up the ammonia concentration until nitrification stops increasing. I am using the bare boxes and boxes with sand from this experiment.

 

[MnFish1]

As to the no nitrite or nitrate startup - I don't understand how this would work - in your system. Unless there are multiple anaerobic areas.

The way you calculated surface area is interesting. What is impossible to know - is the microscopic surface area - lets say comparing sand to rock slices and clean plastic - really as similar as you think? (My guess is even a polished roc surface (take an agate) vs slices of a brick - would be different)

 

[Jon_W79]

One more idea for you to consider. I call it the no nitrite/no nitrate start up for a new aquarium. It is an idea with no data to say whether it would work.

What if you establish a nitrifying biofilm by wetting new rock and sand with saltwater containing ammonia and bottled bacteria, keeping it covered to prevent trying. You might need to keep rewetting the sand and rocks with fresh saltwater and ammonia, maybe more bacteria. Then after X days, rinse the sand and rock with fresh saltwater and assemble the aquarium. In principle you will have your nitrifying biofilm but no need for water changes, no nitrite or no nitrate in the water, and no more posts about ammonia in a new aquarium.

You could make a wet and dry filter using new rock, and connect it to a small water reservoir that you dose ammonium and phosphate into with a dosing pump many times a day.

 

[Dan_P]

You could make a wet and dry filter using new rock, and connect it to a small water reservoir that you dose ammonium and phosphate into with a dosing pump many times a day.

I think you’ve got it.

I will give this a try with sand, though I‘ll use a fluidized bed.

 

[Jon_W79]

I think you’ve got it.

I will give this a try with sand, though I‘ll use a fluidized bed.

I think that you may want to find out if very high nitrate can be harmful to nitrifying bacteria(maybe like 500 ppm plus). I read something that made me think that at least 500 ppm plus may be harmful, but I'm not sure.

 

[Dan_P]

As to the no nitrite or nitrate startup - I don't understand how this would work - in your system. Unless there are multiple anaerobic areas.

I was thinking to lay down a nitrifying biofilm, I just need to scale up the process that I used in the little reactors. As long as there is just enough seawater to keep the surfaces wet, a constant supply of ammonia and oxygen, the rock or sand will be quickly populated with a dose of BioSpira and minimum water. Then with a little fresh seawater to rinse off the conditioning medium, an aquarium can be assembled with a nitrifying biofilm and nitrogen free water.

I need to add sand to my aquarium and withll give this idea a try. I will report the results in a month or so. I need to work out the plumbing for this large system and experimental controls.

The way you calculated surface area is interesting. What is impossible to know - is the microscopic surface area - lets say comparing sand to rock slices and clean plastic - really as similar as you think? (My guess is even a polished roc surface (take an agate) vs slices of a brick - would be different)

Surface area was definitely an estimate in this experiment and wasn’t even something that I planned to investigate. The fact that the nitrification rates correlated with my surface area estimates gave me some confidence that I might be on the right trail to understanding the differences in activity of my little aquaria. It is a working hypothesis at this time. Mine was but a first step towards understanding.

 

[Dan_P]

I think that you may want to find out if very high nitrate can be harmful to nitrifying bacteria(maybe like 500 ppm plus). I read something that made me think that at least 500 ppm plus may be harmful, but I'm not sure.

Thanks for the heads up.

I was thinking of refreshing the medium to prevent the build up of nitrite and nitrate concentrations for the very reason you raise.

 

[LRT]

Excellent work Dan!

 

[LRT]

Nitrifying Bacteria. Where Are You?

FINAL RESULTS

You will recall that I am interested in observing where nitrifying bacteria settle in a new aquarium. For practical reasons I am using very small, 0.3 L, aquaria to perform replicate tests with different surfaces. The experiment is simple. Grow BioSpira in these little aquaria, then do a 100% water change but save the water. Then test the water and the aquaria separately for their ability to consume ammonia and nitrite. The water is tested by adding ammonia to it and observing how fast the ammonia and nitrite are consumed. The aquarium surfaces are tested for nitrification by adding fresh Instant Ocean and ammonia and observing ammonia and nitrite consumption. After four days, when the ammonia is depleted and little nitrite is left, I repeat the test. Perform a 100% water change, etc. After a total of three tests were performed, the data was analyzed. Here is the data and what seems to tell us.

The bar charts below show how much ammonia and nitrite are consumed after one day by the aquarium surfaces and the water removed in the 100% water change. Ammonia and nitrite consumption charts tell similar stories. There is substantial nitrification occurring in the water, but the activity is higher for the aquarium surfaces. The presence of nitrification in the water suggests that nitrifying bacteria are being shed or dispersed from the surfaces during the tests. No further enhancement in capability is gained by repeated ammonia doses.

How is aragonite sand or rock slices enhancing ammonia and possibly nitrite consumption rates over the plastic surfaces of the aquarium? The simplest explanation is increased surface area. Larger surface areas accommodate larger bacteria populations (see plots below).

A comparison of ammonia and nitrite consumption to the total wetted surface area for the bare aquarium and rock slice containing aquarium (see table below) demonstrates that increasing surface area increases nitrification activity. If there is sufficient surface area, adding more does not increase the nitrification rate (compare rock and sand containing aquaria nitrification rates above)

This surface area effect can be also observed in the same aquarium. When the nitrification activity for the rock slices and aquarium (“Box-Rocks”) are assessed separately, the correlation between surface area and nitrification is seen once again.

Conclusion. The experimental data confirm that nitrification occurs on surfaces. Activity was also observed in the water, at least initially. Multiple ammonia additions in this experiment did not enhance nitrification capability, suggesting that fish-less cycling may not benefit from multipAnd finally, insufficient surface area can limit nitrification rates but this may not be an issue for a hobby-size aquarium.

Enjoy!

One of the greatest if not the most significant posts I've ever read especially in regards to cycling.

One more idea for you to consider. I call it the no nitrite/no nitrate start up for a new aquarium. It is an idea with no data to say whether it would work.
....In principle you will have your nitrifying biofilm but no need for water changes, no nitrite or no nitrate in the water, and no more posts about ammonia in a new aquarium.

Randys old post about why not just cycle with ammonia really shines out to me here especially considering your work with seneye compared to calibrated lab grade equipment. Your experiments here have proven there are much more efficient and precise instruments available to measure what's happening in our systems.
Some vehemently disagree but I ask why(especially after reading posts by certain members bashing results) if those disagreements aren't business driven on a competitive level at this point.

.. if I am not careful, I will spend all my time studying these bacteria.

Why and who cares? Whats significant is whats actually happening.

This phenomenon is an especially interesting to me. When does shedding stop? What causes it to stop?

Fascinating questioning.


@taricha and I modified the API ammonia test to detect total ammonia down to around 0.02 ppm and to quantitate it down to about 0.05 ppm. We used the Hanna low range silica Checker to measure the color intensity. The Checker was calibrated with a total ammonia reference standard. For this experiment, differences in readings of 10% were definitely different.

The experiments were performed with 0.5 ppm total ammonia.

I did not measure free ammonia.


Yes, this was my assumption, but it was continually proven wrong It would been so satisfying for the two activities to sum to the total of the unseparated samples. Here is why I think they don’t.

First, there is variability in the data. I did not replicate decanted water assessments (ran out of room on the orbital shaker) and cannot say for sure whether the sum is really different than what I expected. But there are cases where we can agree that the differences are big enough to say that the activity of the box and the water are bigger than the activity measured together. I think this arises because of the way I assessed activity.

Bacterial metabolism and growth are affected by nutrient concentration. Even though I used the same concentration of ammonia for assessing the water and box activities, the ratio of bacteria number to total nutrient content in the assessment is higher. This means the nutrient is depleted more slowly in twenty four hours which in turn means the bacteria can grow at a faster rate for a longer time. When the assessment activity of the box and water are summed, it would be larger than the activity of the previous box-water combination. This is my current thinking. I can be easily swayed.

I thought about ways around this and it involves observing ammonia consumption closer to time zero, before nutrient depletion can affect the growth rate. Could be fun.

Please don't stop doing what yall are doing.

 

[Lasse]

That experiment is in progress. I will run up the ammonia concentration until nitrification stops increasing. I am using the bare boxes and boxes with sand from this experiment.

In this case - I would suggest an experiment with different amount of sand (not the thickness but how much glass area it cover - just a thin layer of sand for 1/3, 2/3 and 3/3 of the available glass area covered with sand

I will give this a try with sand, though I‘ll use a fluidized bed

In this case - I would also test the "waters" ability to nitrify - but when you test that - be sure that the water is in movement with help of rather much air bubbling

Sincerely Lasse

 

[J1a]

I will give this a try with sand, though I‘ll use a fluidized bed.

Would fluidization cause even more find particles due to abrasion?

 

[Lasse]

Would fluidization cause even more find particles due to abrasion?

Exactly - google activated sludge nitrification

Sincerely Lasse

 

[Dan_P]

Would fluidization cause even more find particles due to abrasion?

Yes, abrasion would be a concern. Shear is one mechanism by which biofilm bacteria are dispersed.

There might be a sweet spot between too much abrasion and not enough oxygen. If we go much further thinking about this, we will be designing the next miracle product for starting nitrite/nitrate free new aquaria

 

[Dan_P]

Would fluidization cause even more find particles due to abrasion?

I did not read this correctly on the first read. A couple thoughts.

Newly washed sand under the microscope has very small particles attached to larger sand grains. I would guess these could free up in a fluidized sand bed. As for wearing down large particles by abrasion, I would guess not much happens when sand is just fluidized for a few days until the bacteria become attached.