Nitrifying Bacteria. Where Are You?

[MnFish1]

I take it that your system does not produce nitrite, just straight on to nitrate.

Admit - I was lazy this time - I did not measure nitrite. There is not much if any nitrite produced. I think it is 'over limit' on nitrite processors. I would be happy to send some samples to @AquaBiomics. I was hoping he was looking at these and would maybe give a discount code in the interest of science

 

[Azedenkae]

Admit - I was lazy this time - I did not measure nitrite. There is not much if any nitrite produced. I think it is 'over limit' on nitrite processors. I would be happy to send some samples to @AquaBiomics. I was hoping he was looking at these and would maybe give a discount code in the interest of science

If you do do this, just bare in mind the resolution of the test. I can't remember, but I am pretty sure their test only narrows down to the genus level, rather than species level. And we know different species (and indeed strains) of nitrifiers, even from the same genus, can exhibit wildly different nitrification rates.

So just because for example there is a relatively 'small' population of a specific type of nitrifier does not mean they are not performing a significant degree of nitrification, or vice versa. Though I suppose if we find a lack of other species, then that would still point towards the nitrifiers rather than non-nitrifying heterotrophs being predominantly responsible for ammonia consumption.

 

[MnFish1]

If you do do this, just bare in mind the resolution of the test. I can't remember, but I am pretty sure their test only narrows down to the genus level, rather than species level. And we know different species (and indeed strains) of nitrifiers, even from the same genus, can exhibit wildly different nitrification rates.

So just because for example there is a relatively 'small' population of a specific type of nitrifier does not mean they are not performing a significant degree of nitrification, or vice versa. Though I suppose if we find a lack of other species, then that would still point towards the nitrifiers rather than non-nitrifying heterotrophs being predominantly responsible for ammonia consumption.

My standard comment on any of this stuff is - Yes you're right. BUT - lets say there are 0 nitrifiers. that would mean something, IMHO. It at least gives some information. For example my rocks may be covered with pseudomonas - IDK. Thats the problem again IMHO - no one knows what are in these experimental tanks. Perhaps Aquabiomics is not perfect - but it IMHO does give an idea.

 

[Azedenkae]

My standard comment on any of this stuff is - Yes you're right. BUT - lets say there are 0 nitrifiers. that would mean something, IMHO. It at least gives some information. For example my rocks may be covered with pseudomonas - IDK. Thats the problem again IMHO - no one knows what are in these experimental tanks. Perhaps Aquabiomics is not perfect - but it IMHO does give an idea.

Oh for sure, I absolutely am rooting for you to do a test via AquaBiomics. I am super keen to see the results lol. Just wanted to raise a thought beforehand, but absolutely it would be interesting to see the results. ^_^

 

[Dan_P]

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. Another observation is that the presence of aragonite surfaces increase nitrification activity over just plastic surfaces, my bare bottom tank. Sand or rock slices enhance nitrification equally well. The near constant nitrification rate over the three tests suggests that only one dose of ammonia might be needed for a fish-less aquarium cycling. 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 multiple doses. And finally, insufficient surface area can limit nitrification rates but this may not be an issue for a hobby-size aquarium.

Enjoy!

 

[Lasse]

Very important findings that it is enough with 1 dose of NH3/NH4 when start.

Sincerely Lasse

 

[MnFish1]

Great job - very thorough and well thought out.

Question for you and @Lasse

This is a little different than the results in my experiment. In which over the first 3 experiments - the rate of ammonia consumption increased each time (and also in the experiments done by Coxey and Sixty reefer). Do you think its because when you do the water change that the bacteria were not completely adherent? Due to the short time?

When I look at graph 1 - the water section - it looks to me like the water nitrification decreases in every case between test 1 and test 3. suggesting over time - that there is more adherence and less bacteria going into the water.

I dont know if you tested for statistical significance - what PPM total ammonia did you start with? What did you use to measure free ammonia?

Could you do me a favor could you do a graph in which you add the 'water bare' +'Box bare' for all 3 tests? in theory - The total ammonia removal would be the sum of the 2, right?

Again great job

 

[J1a]

Great experiment!

I would like to find out, how is the surface area of the rocks determined?

Secondly, if we assume that the bacteria mainlu grow on surfaces, then perhaps the bacteria in the water column are adhered to to particulate matters in the water? If this is the case, then filtered water will have much less ability to nitrify.

 

[Rick Mathew]

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. Another observation is that the presence of aragonite surfaces increase nitrification activity over just plastic surfaces, my bare bottom tank. Sand or rock slices enhance nitrification equally well. The near constant nitrification rate over the three tests suggests that only one dose of ammonia might be needed for a fish-less aquarium cycling. 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 multiple doses. And finally, insufficient surface area can limit nitrification rates but this may not be an issue for a hobby-size aquarium.

Enjoy!

Dan, as usual outstanding work!! You are an experimental machine!! Well done!

Rick

 

[Dan_P]

Very important findings that it is enough with 1 dose of NH3/NH4 when start.

Sincerely Lasse

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.

 

[Lasse]

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.

Its a good idea but I do not think it will work in the long run - let us say - to store it for 10 days - can you figure out why?

Sincerely Lasse

 

[threebuoys]

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.

Seems like this should work. Not much different than keeping "charged" media in the sump for future use.

 

[Dan_P]

Great experiment!

I would like to find out, how is the surface area of the rocks determined?

I traced the outline of the rock slice on paper, cut out 5he paper, and weighed the paper. For edges, I ran string along edges and estimated the average thickness of the slice with a ruler

Secondly, if we assume that the bacteria mainlu grow on surfaces, then perhaps the bacteria in the water column are adhered to to particulate matters in the water? If this is the case, then filtered water will have much less ability to nitrify.

My thoughts exactly! It would be fun to see the effect on activity after filtering the water with different porosity filters, after removing particulates and then after using a 0.20 micron filter

 

[Dan_P]

Its a good idea but I do not think it will work in the long run - let us say - to store it for 10 days - can you figure out why?

Sincerely Lasse

I can’t figure it out?

My thinking is if bacteria stay wet, fed, oxygenated and waste removed, all should be well. What is the test question answer?

 

[Lasse]

Seems like this should work. Not much different than keeping "charged" media in the sump for future use.

Yes it is different - guess why?

sincerely Lasse

 

[Lasse]

oxygenated

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

 

[J1a]

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

Actually. I suppose a thin film of water exposed to air will likely stay more oxygenated than submerged in a tub of water. As long as the film don't dry out.

I traced the outline of the rock slice on paper, cut out 5he paper, and weighed the paper. For edges, I ran string along edges and estimated the average thickness of the slice with a ruler

This would be the external area. How about internal though? It's probably hard to quantify that. But I would say that's something important.

 

[Lasse]

tually. I suppose a thin film of water exposed to air will likely stay more oxygenated than submerged in a tub of water. As long as the film don't dry out.

Exactly - but how do you hinder a surface with unlimited contact with air (with lesser RH than 100 %) not to dry out - you can´t - it will dry out. If you hinder free contact - oxygen in the surrounding air will disappear and you get anaerobic - example wrapt in plastic environment

Sincerely Lasse

 

[J1a]

Exactly - but how do you hinder a surface with unlimited contact with air (with lesser RH than 100 %) not to dry out - you can´t - it will dry out. If you hinder free contact - oxygen in the surrounding air will disappear and you get anaerobic - example wrapt in plastic environment

Sincerely Lasse

Probably going to have to keep spraying water at intervals.

 

[Dan_P]

Great job - very thorough and well thought out.

Question for you and @Lasse

This is a little different than the results in my experiment. In which over the first 3 experiments - the rate of ammonia consumption increased each time (and also in the experiments done by Coxey and Sixty reefer). Do you think its because when you do the water change that the bacteria were not completely adherent? Due to the short time?

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.

When I look at graph 1 - the water section - it looks to me like the water nitrification decreases in every case between test 1 and test 3. suggesting over time - that there is more adherence and less bacteria going into the water.

I think you are right. The maturing biofilm seems to be shedding less and less. And this is consistent with little or no ammonia oxidation occurring in the water of older aquaria.

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

I dont know if you tested for statistical significance - what PPM total ammonia did you start with? What did you use to measure free ammonia?

@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.

Could you do me a favor could you do a graph in which you add the 'water bare' +'Box bare' for all 3 tests? in theory - The total ammonia removal would be the sum of the 2, right?

Again great job

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.