Nitrifying Bacteria. Where Are You?

[MnFish1]

I don’t have an informed opinion on how long nitrifying bacteria can live without ammonia. I have seen a death rate estimate of 1% per day. There is nothing like first hand experience. So yeah, feed your starving nitrifyers.


You did not miss the control discussion but a good topic to cover.

Starting an aquarium without adding bottled bacteria is possible. The time frame for achieving high nitrification rates is on the order of weeks, possibly longer if starting with Instant Ocean with 0.5 ppm total ammonia. So the observed high activity after four days is most likely from the BioSpira. This seemed like a good enough reason to skip a control. But, the lack of control could weaken any statement that BioSpira settled here or there because maybe the observed activity was already there. We will keep in mind these weak points in the experiment design when it is time to draw conclusions.



Definitely would be a nice touch to bring in modern technology. Just like remembering the weak points of the experiment design when drawing conclusions, let’s discuss how AquaBiomics might untangle any mysteries we discover in the results.

Thanks for your support.

Lets say a person took biospira and added lets say sugar (no ammonia). Another had Sugar water only, And another vial had biospira and ammonia - and let them grow for 4 days- that would be one way (after sending the samples to aquabiomics) - to decide whether its heterotrophs as compared to nitrifying bacteria doing the 'work' on the film. I suppose another way would be to somehow 'culture' biospira - on a slide and merely send the sample to Aquabiomics. Pseudomonas, which is a rapid growing ammonia using bacteria - also grows in a biofilm (it can live in betadine solution as well).

For example in Sixty_reefer's experiment - he had a huge bacterial bloom after scrubbing (which to me suggested heterotrophs - I'm assuming you haven't had that.

 

[threebuoys]

Following the assumption that nitrifying bacteria must attach to surfaces for greatest effectiveness,@Dan_P Something you may want to consider at some point in the future is what amount of surface area is required. Perhaps, you might consider this for a future expansion of your experiment.

I found this info some time ago, and I have found 30 ppi foam to be very effective in both freshwater and saltwater sumps. I also have live rock in my saltwater tank used primarily for landscaping, so I can't conclusively say how much ammonia is nitrifyed by bacteria in the foam vs rock. However, based on this information, I believe the foam could be very effective. I also use foam in my QT tank filtatrion regularly and I've not encountered any problems with ammonia spikes.

Based on this info, a very small piece of foam with sufficient water flow should handle a relatively large population of fish.

7. Aquarium Filter Media

The media in an aquarium filter is the most important variable when setting up a filter.

aquariumscience.org

7.3. Aquarium Filter Media Volume

Calculating aquarium filter media volume requirements is relatively straight forward math problem.

aquariumscience.org

(assumes fish are feed 1.5% of body mass daily, juveniles or very active fish would likely be higher, Common assumptions are that fish will consume between1% and 4% of body mass daily, dependent upon age and activity.)

 

[MnFish1]

Following the assumption that nitrifying bacteria must attach to surfaces for greatest effectiveness,@Dan_P Something you may want to consider at some point in the future is what amount of surface area is required. Perhaps, you might consider this for a future expansion of your experiment.

I found this info some time ago, and I have found 30 ppi foam to be very effective in both freshwater and saltwater sumps. I also have live rock in my saltwater tank used primarily for landscaping, so I can't conclusively say how much ammonia is nitrifyed by bacteria in the foam vs rock. However, based on this information, I believe the foam could be very effective. I also use foam in my QT tank filtatrion regularly and I've not encountered any problems with ammonia spikes.

Based on this info, a very small piece of foam with sufficient water flow should handle a relatively large population of fish.

7. Aquarium Filter Media

The media in an aquarium filter is the most important variable when setting up a filter.

aquariumscience.org

7.3. Aquarium Filter Media Volume

Calculating aquarium filter media volume requirements is relatively straight forward math problem.

aquariumscience.org

(assumes fish are feed 1.5% of body mass daily, juveniles or very active fish would likely be higher, Common assumptions are that fish will consume between1% and 4% of body mass daily, dependent upon age and activity.)

This is fascinating - and - If Dan does not want to do it - I will be happy to do it in my tanks.

I had an aqua clear HOB I think a 1000? - that came with a large sponge. Every year - the Koi come into a 72 gallon tank (I know - Koi Police) - and I always start it up fresh except for the aqua clear that was running over the summer on another tank. Long story short - the 1000 broke - and I had a smaller one. Well. needless to say - the water was cloudy constantly. I decided to take the block of foam - as well as the ceramic disks - and just drop them in the tank. It is now crystal clear. Note. there is no flow going through the foam - or the ceramic -they are just sitting on the bottom. (There is no rock or other surface - except for the glass - and the small aqua clear 400) in the tank. This is 6 x 6in fish in a 72 gallon tank.

I actually considered buying the foam blocks (replacements from aqua clear) - and lining the bottom an area of my sump with them.

Thanks for that information @threebuoys

 

[Malcontent]

Something you may want to consider at some point in the future is what amount of surface area is required.

7. Aquarium Filter Media

The media in an aquarium filter is the most important variable when setting up a filter.

aquariumscience.org

I would be hesitant to cite aquariumscience. He's been caught making stuff up before. I'm not sure he actually performs his experiments.

There are two surface area experiments that I'm aware of. One done on plantedtank.net and one by MarinePure.

A good summary of the planted tank experiment.

MarinePure's famously self-owned where their own product cames out on top but if you read between the lines only 1 L of the worst performing bio media could support 12-24 lbs of fish at normal feeding rates (0.5-1% BW/day).

So it seems bacteria aren't surface area limited at all and the surface area marketing wars can simply be ignored.

 

[Dan_P]

Following the assumption that nitrifying bacteria must attach to surfaces for greatest effectiveness,@Dan_P Something you may want to consider at some point in the future is what amount of surface area is required. Perhaps, you might consider this for a future expansion of your experiment.

I found this info some time ago, and I have found 30 ppi foam to be very effective in both freshwater and saltwater sumps. I also have live rock in my saltwater tank used primarily for landscaping, so I can't conclusively say how much ammonia is nitrifyed by bacteria in the foam vs rock. However, based on this information, I believe the foam could be very effective. I also use foam in my QT tank filtatrion regularly and I've not encountered any problems with ammonia spikes.

Based on this info, a very small piece of foam with sufficient water flow should handle a relatively large population of fish.

7. Aquarium Filter Media

The media in an aquarium filter is the most important variable when setting up a filter.

aquariumscience.org

7.3. Aquarium Filter Media Volume

Calculating aquarium filter media volume requirements is relatively straight forward math problem.

aquariumscience.org

(assumes fish are feed 1.5% of body mass daily, juveniles or very active fish would likely be higher, Common assumptions are that fish will consume between1% and 4% of body mass daily, dependent upon age and activity.)

Yes, of course, foam! Brilliant.Thanks.

This is the type of material I could use to dial in a specific surface area and not have to slice rocks.

 

[Dan_P]

I would be hesitant to cite aquariumscience. He's been caught making stuff up before. I'm not sure he actually performs his experiments.

There are two surface area experiments that I'm aware of. One done on plantedtank.net and one by MarinePure.

A good summary of the planted tank experiment.

MarinePure's famously self-owned where their own product cames out on top but if you read between the lines only 1 L of the worst performing bio media could support 12-24 lbs of fish at normal feeding rates (0.5-1% BW/day).

So it seems bacteria aren't surface area limited at all and the surface area marketing wars can simply be ignored.

I read aquariumscience and there seemed to be some axe grinding, but I found the information useful to make a list to check up on as I read professional papers. Thanks for the heads up.

As for surface area, I find the subject very interesting but maybe not important at this time. Still, I have these reactors available…

 

[Dan_P]

Lets say a person took biospira and added lets say sugar (no ammonia). Another had Sugar water only, And another vial had biospira and ammonia - and let them grow for 4 days- that would be one way (after sending the samples to aquabiomics) - to decide whether its heterotrophs as compared to nitrifying bacteria doing the 'work' on the film. I suppose another way would be to somehow 'culture' biospira - on a slide and merely send the sample to Aquabiomics. Pseudomonas, which is a rapid growing ammonia using bacteria - also grows in a biofilm (it can live in betadine solution as well).

For example in Sixty_reefer's experiment - he had a huge bacterial bloom after scrubbing (which to me suggested heterotrophs - I'm assuming you haven't had that.

I am assuming nitrite production and consumption to mean nitrifying and nitrafying bacteria are at work in my experiments. The other thing is that Instant Ocean by itself has little or no oxygen demand. I take that to mean heterotrophic bacteria don’t do well in freshly prepared IO, which is what I am using in the activity test.

Where things become muddled is after the initial growth of BioSira, that is, after day 4 and the beginning of the activity assessment. Nitrifiers make organic matter. Between their exudates and lysis, we cannot overlook the possibility that the surfaces I am testing over and over again are developing a significant heterotrophic bacteria population living off the nitrifiers. AquaBiomics data would certainly help identify the constituents of the maturing biofilm. The influence of heterotrophic bacteria on the results would be a good thing to remember come conclusion time. So far though, I have only see the telltale sign of surface slime growth but crystal clear water.

 

[MnFish1]

I think the sand in most of our tanks is quite porous, especially aragonite. It makes sense that sand would be a pretty heavy lifter in the nitrification process.

But it doesnt explain the issue that - all of the rocks in our tank is made from the same stuff. BUT - like I said with my sponge comment - maybe there is not such a requirement for oxygenated water flowing through.... I will phone a friend @Lasse

 

[damsels are not mean]

But it doesnt explain the issue that - all of the rocks in our tank is made from the same stuff. BUT - like I said with my sponge comment - maybe there is not such a requirement for oxygenated water flowing through.... I will phone a friend @Lasse

yes but only the surface of the rocks is in contact with enough oxygenated water so 10 lb of rock is a lot les surface area than 10 lb of sand. Even if both are made of the same stuff it's the way it contacts the water.

 

[Lasse]

Why is the water activity so similar to the surface activity?

Could it be particulates in the water carrying the very active bacteria?

My answer to this is that with a higher amount of bacteria film on surface - higher amount of particles that can carry nitrification bacteria. More mini-particles will detach from the biofilm and form small particles in the water column - with optimal conditions for nitrification bacteria

I can’t get off the starting block with the question how is a biofilm on plastic different from one on aragonite.

area, area and area. A rock or a sand gravel have a lot of cavities and ridges that can act as an surface for biofilm and maybe it is more difficult to attach om plastic (or glass) that have very few micro-micro cavities in the surface - compared with rocks and sand gravel

Where things become muddled is after the initial growth of BioSira, that is, after day 4 and the beginning of the activity assessment. Nitrifiers make organic matter. Between their exudates and lysis, we cannot overlook the possibility that the surfaces I am testing over and over again are developing a significant heterotrophic bacteria population living off the nitrifiers.

Very important

The main objection I have is that you do not measure oxygen. For a good nitrification rate - there is IMO four main requirements thats need to be meet.
1) Oxygen (higher amount - higher penetration into the biofilm)
2) Area (indirect amount of nitrifier)
3) Lack of competitors (i.e. heterotrophs that use both area and oxygen but have a propagation rate more than thousand times higher)
4) at least micro levels of nutrients

Sincerely Lasse

 

[J1a]

How about the inner surface of the aquarium? Perhaps should sterilize it in between experiments. Probably it's not a significant factor, but doesn't hurt to rule it out.

 

[Dan_P]

My answer to this is that with a higher amount of bacteria film on surface - higher amount of particles that can carry nitrification bacteria. More mini-particles will detach from the biofilm and form small particles in the water column - with optimal conditions for nitrification bacteria



area, area and area. A rock or a sand gravel have a lot of cavities and ridges that can act as an surface for biofilm and maybe it is more difficult to attach om plastic (or glass) that have very few micro-micro cavities in the surface - compared with rocks and sand gravel


Very important

The main objection I have is that you do not measure oxygen. For a good nitrification rate - there is IMO four main requirements thats need to be meet.
1) Oxygen (higher amount - higher penetration into the biofilm)
2) Area (indirect amount of nitrifier)
3) Lack of competitors (i.e. heterotrophs that use both area and oxygen but have a propagation rate more than thousand times higher)
4) at least micro levels of nutrients

Sincerely Lasse

Insightful comments as always. Thanks Lasse!

@Lasse, would you expand on your point that oxygen is not being measured in the experiment? Are you thinking along the line that differences in oxygen concentration in the water could account for differences in observed ammonia and nitrite oxidation rates?

Dan

 

[Lasse]

Are you thinking along the line that differences in oxygen concentration in the water could account for differences in observed ammonia and nitrite oxidation rates?

Absolute. From fresh water I know that the second step (nitrite-nitrate) needs a higher oxygen pressure than the first step. Below 5 mg/L - the second step slow down and even the first step will slow down. With the fact that 25 degree saltwater have an 100% saturation at somewhere around 6.5 mg/L - 80 % saturation will be rather close to 5 mg/L in 35 PSU saltwater

Sincerely Lasse

 

[Dan_P]

Absolute. From fresh water I know that the second step (nitrite-nitrate) needs a higher oxygen pressure than the first step. Below 5 mg/L - the second step slow down and even the first step will slow down. With the fact that 25 degree saltwater have an 100% saturation at somewhere around 6.5 mg/L - 80 % saturation will be rather close to 5 mg/L in 53 PSU saltwater

Sincerely Lasse

Thanks Lasse. I fully understand your concern.

I had some odd ammonia oxidation results using 50 mL centrifuge tubes, but these disappeared upon scaling up the reactor size and increasing the head space volume. I might have been starving the bactetia of oxygen.

 

[taricha]

I know it's a pain, but a final NO3 measurement for the samples would help me decide which inevitably wrong hypothesis I wish to put forward as an explanation for this mess...

Here are the results for the boxes. The boxes containing sand and rocks showed greater activity than the bare box. It seems that more nitrifying capacity “settled” in the presence of rock and sand than on acrylic surfaces. This data does not tell us whether the greater activity is a result of more bacteria, faster reproduction, more active bacteria or all of the above. In the future I should subtract the data of the bare box from the boxes containing sand or rocks. Also, @brandon429 will want to know about activity on a square inch basis.

The nitrite concentration profile also follows a similar trend as ammonia consumption: greater capacity “settled” on rock and sand.

Did nitrifying activity remain in the water? Yes, quite a bit. And since the trend in the intensity of activity in the water mirrors that of the boxes, does this mean that the presence of aragonite somehow increased nitrification capacity both on the aragonite surfaces and in the water above the surfaces?

What about the nitrite concentration profile? Once again the concentration profile for the decanted water mirrors that of the boxes. Why is the water activity so similar to the surface activity?

 

[Dan_P]

I know it's a pain, but a final NO3 measurement for the samples would help me decide which inevitably wrong hypothesis I wish to put forward as an explanation for this mess...

HaHa, I know, it would be really cool. Chalk it up to pure laziness on my part.

I have a method to get rid of the nitrite which does not interfere with the nitrate measurement. Just cook the sample in urea and HCl.

Don’t start hypothesizing just yet. I have two more data sets like the one above and need to generate two additional ones to answer some questions the above data and Lasse generated. I need another two weeks (stupid biology experiments take so long ) to complete the new experiments. I will write a lab report for you and Rick before going live on R2R with the results.

 

[Aqua Man]

I would be interested in hearing about questions you might have about nitrifying bacteria or questions you heard someone else wonder about. And of course, comments and recommendations welcome.

Curious why the 0.2 ppm phosphate addition?

 

[Dan_P]

Curious why the 0.2 ppm phosphate addition?

0.2 ppm was my best guess. Here are some reasons.

Bacteria need PO4 to grow. Since I don’t know how much they need, how fast they use it up, and the reactors get fresh medium every four days, I gave them a generous aquarium size concentration.

Also, Hanna Checker shows 0.00 ppm PO4 for Instant Ocean. BioSpira label (I think that is where I read it) says water should be above 0 ppm PO4. In previous experiments, there seemed to be a small PO4 concentration effect on BioSpira activity.

 

[J1a]

I

0.2 ppm was my best guess. Here are some reasons.

Bacteria need PO4 to grow. Since I don’t know how much they need, how fast they use it up, and the reactors get fresh medium every four days, I gave them a generous aquarium size concentration.

Also, Hanna Checker shows 0.00 ppm PO4 for Instant Ocean. BioSpira label (I think that is where I read it) says water should be above 0 ppm PO4. In previous experiments, there seemed to be a small PO4 concentration effect on BioSpira activity.

I would suppose having po4 slightly in excess can exclude it as the limiting reagent. It will also unlikely to interfere with ammonium oxidation. Make sense.

What puzzled me though (slightly tangential) is that fishless cycle does not call for addition of phosphate sources. Any thoughts?

 

[MnFish1]

I have a method to get rid of the nitrite which does not interfere with the nitrate measurement. Just cook the sample in urea and HCl.

Do you just add prime? JUST KIDDING