Nitrifying Bacteria. Where Are You?

[Dan_P]

Nitrifying Bacteria. Where Are You?

Introduction. I am studying the formation and development of photosynthetic (algal) biofilms to understand why some biofims become dominated by nuisance organisms, such as, diatoms, dinoflagellates and cyanobacteria. Bacteria are an integral part of these biofilms, potentially playing a role in supporting nuisance organism growth. One group of bacteria that may occupy an algal biofilm are the nitrifying bacteria. As an information gathering exercise, I have several experiments in mind to study these bacteria. The first exercise might be of broadest interest. When I add bottled nitrifying bacteria, where do they go?

Method. I have already conducting screening experiments and settled on a way to follow the bacterial journey. I will post pictures of the experiment with the results. Here is experiment.

BioSpira bacteria are dosed to very small acrylic aquaria (~200 mL party favor cubes) containing nothing, a thin layer of aragonite sand or thin slices of aragonite rock. The water is 100 mL of Instant Ocean (35 ppt) containing ammonium chloride (0.5 ppm total ammonia) and phosphate (0.2 ppm). The covered cubes are placed on an orbital shaker in the dark. The shaker is set to gently swirl the water.The bacteria will be located by there ability to convert ammonia to nitrite and nitrite to nitrate.

After the ammonia is consumed (4 days), the water is decanted and saved. Fresh IO and ammonia is added to each box and the decanted water is dosed with ammonia and phosphate and placed in clean capped 50 mL centrifuge tubes. All the boxes and centrifuge tubes are returned to the orbital shaker. Samples are removed at 1, 2 and 4 days to assess the consumption of ammonia in all containers. This represents one activity assessment.

I plan on a total of at least three activity assessments. I am also considering a study of these matured nitrifying biofilms to see how long it takes for them to lose ammonia oxidizing capability with ammonia minimal feeding. Activity assessments will start to be reported soon.

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.

Dan

EDIT: Final results on post #85

 

[LRT]

@Dan_P this is going to be interesting! 1 obviously being to see how much ammonia the water can continue to process once all other sources are removed.
In my recent upgrade I set up 2 identical tanks equipment wise.
My shroom lagoon, tank 1 was cycled using controlled measured feedings to build nh3 processing over the course of 24hrs and then stocked with current inhabitants. Used around 20-30% high nutrient water from old system to new salt mix water on start up. Nutrients barely recordable at around 72hrs. Slow dosed nutrients to keep recorded and manageable during first 2-3 months until regular feeding caught back up and stabilized itself. Basically 100% control to best of my ability all the way through!
Tank 2 went a little different at about 70-80% high nutrient old system water to new salt mix. Tank 2 inhabitant consisted of 5-6" pufferfish that ate probably 2oz food on average. Did not interrupt nh3 monitoring after initial cycled described above.
Nitrates at 40ppm at 72hrs to off the charts high and not recordable with red sea kit around week 2 and stayed that way until I tore it down last month getting ready for a move.
Judging from what ive seen I feel like you would see a huge difference in nuisance algae by overloading bioload combined with heavy feeding and raising nitrates up front. Dinos was a big issue for me as I couldn't keep Phosphates balanced and recordable on a stable level with such high Nitrates.
Could you do one of the tanks with higher ammonia processing with higher Nitrates?
Also would it be possible to substitute ammonia dosing with equal measured feedings with maybe small mysis shrimp? Only reason for that request is ive never dosed ammonia and would like to see if actual food helps fuel nuisance algae opposed to dosed tanks.

 

[Dan_P]

@Dan_P this is going to be interesting! 1 obviously being to see how much ammonia the water can continue to process once all other sources are removed.
In my recent upgrade I set up 2 identical tanks equipment wise.
My shroom lagoon, tank 1 was cycled using controlled measured feedings to build nh3 processing over the course of 24hrs and then stocked with current inhabitants. Used around 20-30% high nutrient water from old system to new salt mix water on start up. Nutrients barely recordable at around 72hrs. Slow dosed nutrients to keep recorded and manageable during first 2-3 months until regular feeding caught back up and stabilized itself. Basically 100% control to best of my ability all the way through!
Tank 2 went a little different at about 70-80% high nutrient old system water to new salt mix. Tank 2 inhabitant consisted of 5-6" pufferfish that ate probably 2oz food on average. Did not interrupt nh3 monitoring after initial cycled described above.
Nitrates at 40ppm at 72hrs to off the charts high and not recordable with red sea kit around week 2 and stayed that way until I tore it down last month getting ready for a move.
Judging from what ive seen I feel like you would see a huge difference in nuisance algae by overloading bioload combined with heavy feeding and raising nitrates up front. Dinos was a big issue for me as I couldn't keep Phosphates balanced and recordable on a stable level with such high Nitrates.
Could you do one of the tanks with higher ammonia processing with higher Nitrates?
Also would it be possible to substitute ammonia dosing with equal measured feedings with maybe small mysis shrimp? Only reason for that request is ive never dosed ammonia and would like to see if actual food helps fuel nuisance algae opposed to dosed tanks.

Interesting observations. I will definitely have you run your eye over future experiment designs as I look at different nutrient combinations.

The role of organic material in supporting nuisance organism is on my list to investigate though its a complicated thing to study, but studied it will be.

Also on the list is looking at mixed nitrogen sources and their role in nudging different organisms to grow like crazy. Nitrate and ammonia are likely to be present together and will be studied. Too bad my current study protocol takes two weeks. More tests in parallel I guess.

I have had poor luck growing dinoflagellates, go figure. The only time I had success, turning one of my small aquarium into a brown mess, was the rare time I did get a dinoflagellate bloom and I increased the nitrate concentration a bit.

 

[Dan_P]

Nitrifying Bacteria. Where Are You?

Activity Assessment #1. Recall that I am using little aquaria (200 mL) for this experiment.

After the BioSpira was dosed to the aquaria, the ammonia was consumed over four days. The water in each box was decanted and fresh Instant Ocean with 0.5 ppm total ammonia was added to each box. The water decanted from each box was dosed with ammonia to 0.5 ppm and added to clean 50 mL screw cap test tubes. The amount of ammonia consumed and concentration of nitrite were determined on days 1,2 and 4.

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?

Discovering the linkage between activity on surfaces and that in water will require a separate experiment. Even if someone has a good explanation, I will want to confirm it in the lab.

This experiment will continue with at least 2 more activity assessments being made which will include looking at the decanted water for activity. This data will give some indication of how quickly activity ramps up. I will then use these ramped up biofilms to test how long they maintain activity under depleted ammonia conditions.

My list of questions so far

1) How long does nitrification in the water last in an aquarium?
2) Do nitrifying bacteria in the water play some role in determining the severity of the “nasties”?
3) What is the role of aragonite surfaces in seemingly ramping up activity both in water and on surfaces?

Don’t forget, comments, ideas, questions, and theories welcomed.

 

[Rick Mathew]

Nitrifying Bacteria. Where Are You?

Activity Assessment #1. Recall that I am using little aquaria (200 mL) for this experiment.

After the BioSpira was dosed to the aquaria, the ammonia was consumed over four days. The water in each box was decanted and fresh Instant Ocean with 0.5 ppm total ammonia was added to each box. The water decanted from each box was dosed with ammonia to 0.5 ppm and added to clean 50 mL screw cap test tubes. The amount of ammonia consumed and concentration of nitrite were determined on days 1,2 and 4.

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?

Discovering the linkage between activity on surfaces and that in water will require a separate experiment. Even if someone has a good explanation, I will want to confirm it in the lab.

This experiment will continue with at least 2 more activity assessments being made which will include looking at the decanted water for activity. This data will give some indication of how quickly activity ramps up. I will then use these ramped up biofilms to test how long they maintain activity under depleted ammonia conditions.

My list of questions so far

1) How long does nitrification in the water last in an aquarium?
2) Do nitrifying bacteria in the water play some role in determining the severity of the “nasties”?
3) What is the role of aragonite surfaces in seemingly ramping up activity both in water and on surfaces?

Don’t forget, comments, ideas, questions, and theories welcomed.

Well done!

 

[brandon429]

Dan I still need to reread that excellent data to absorb it and reflect but coming from our chats on the matter I was wondering above if you feel surface area plays as much or more of a role in the reduction of free ammonia vs quantity or locus of bacteria

meaning what if the bare tank is retaining in its bioslicks as much bacteria per unit of surface area/ but the zones with more surface area are able to move the sample much quicker due to more housing for residents? hope I'm asking the question like I'm thinking/my best attempt

 

[Rick Mathew]

Nitrifying Bacteria. Where Are You?

Activity Assessment #1. Recall that I am using little aquaria (200 mL) for this experiment.

After the BioSpira was dosed to the aquaria, the ammonia was consumed over four days. The water in each box was decanted and fresh Instant Ocean with 0.5 ppm total ammonia was added to each box. The water decanted from each box was dosed with ammonia to 0.5 ppm and added to clean 50 mL screw cap test tubes. The amount of ammonia consumed and concentration of nitrite were determined on days 1,2 and 4.

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?

Discovering the linkage between activity on surfaces and that in water will require a separate experiment. Even if someone has a good explanation, I will want to confirm it in the lab.

This experiment will continue with at least 2 more activity assessments being made which will include looking at the decanted water for activity. This data will give some indication of how quickly activity ramps up. I will then use these ramped up biofilms to test how long they maintain activity under depleted ammonia conditions.

My list of questions so far

1) How long does nitrification in the water last in an aquarium?
2) Do nitrifying bacteria in the water play some role in determining the severity of the “nasties”?
3) What is the role of aragonite surfaces in seemingly ramping up activity both in water and on surfaces?

Don’t forget, comments, ideas, questions, and theories welcomed.

Is it the type of rock? or is it some other property of placing a object in that some how increases "available" surfaces for bacteria to grow...even over sand which could be compacting to not provide nice "homes" for the bacteria.....Would placing something like "Bio-Balls" or some other type of "free" space material give a similar response to the aragonite...or is something about its "chemistry"...Just a random thought...could be totally out to lunch

 

[brandon429]

Dan one thing I may have fully backwards: you are only testing the decanted water but not actually the systems it was poured from?

I'm so used to doing 100% water changes in cycles/then re testing the known clean system for oxidation ability I may have imprinted that here. I need to wrap my head around the poured off water had varying degrees of control based on where they originated

 

[Dan_P]

Well done!

I bet the next time you see one of those cubes filled with candy you will think about nitrifying bacteria!

 

[Dan_P]

Dan one thing I may have fully backwards: you are only testing the decanted water but not actually the systems it was poured from?

I'm so used to doing 100% water changes in cycles/then re testing the known clean system for oxidation ability I may have imprinted that here. I need to wrap my head around the poured off water had varying degrees of control based on where they originated

Brandon, I am testing both. I am doing a 100% water change and checking the activity of the refilled box AND the water that was poured off I am dosing with ammonia and looking at its activity.

I am thinking about the imprinted activity myself. It is probably not quantum entanglement, but who knows, but I wonder if it is related to biofilms sloughing off bacteria in overcrowded areas. Could it be particulates in the water carrying the very active bacteria? Both rocks and sand would have very small particulates associated with them.

 

[Dan_P]

Is it the type of rock? or is it some other property of placing a object in that some how increases "available" surfaces for bacteria to grow...even over sand which could be compacting to not provide nice "homes" for the bacteria.....Would placing something like "Bio-Balls" or some other type of "free" space material give a similar response to the aragonite...or is something about its "chemistry"...Just a random thought...could be totally out to lunch

Racking my brains on this oo. I could try granite slices of the same size to separate material of construction from surface area. The bio-ball is a good idea because it is an aquarium relevant material. Ceramic chips maybe would be easiest.

 

[Dan_P]

Dan I still need to reread that excellent data to absorb it and reflect but coming from our chats on the matter I was wondering above if you feel surface area plays as much or more of a role in the reduction of free ammonia vs quantity or locus of bacteria

meaning what if the bare tank is retaining in its bioslicks as much bacteria per unit of surface area/ but the zones with more surface area are able to move the sample much quicker due to more housing for residents? hope I'm asking the question like I'm thinking/my best attempt

My reading on biofilms has not helped with the observed surface differences. I can’t get off the starting block with the question how is a biofilm on plastic different from one on aragonite.

As for surface area, I feel dumber about the subject the more data I collect

 

[MnFish1]

Excellent job. I still have the aquaria that I set up for my experiments - I was planning on re-introducing ammonia (2 ppm) again after 1 month - with none) - that will give at least some information as to whether there is (I think there will be) - nitrification capability after a month - and how much is still there. Here are a couple comments:

1. It looks like a great model.
2. It would be nice to have a control - with no biospira (maybe I missed it) - since at least a couple people here have suggested that tanks will cycle on their own without any bacteria.
3. I might ask @AquaBiomics - whether it would be possible to verify that the bacteria on your films are the same as whats in 'biospira' - as compared to heterotrophs.

Thanks alot for doing this great work.

 

[MnFish1]

My reading on biofilms has not helped with the observed surface differences. I can’t get off the starting block with the question how is a biofilm on plastic different from one on aragonite.

As for surface area, I feel dumber about the subject the more data I collect

One interesting observation on my experiments - is that there was little - or no activity comparing - a bare tank with a filter, heater, powerhead (i.e. no ammonia was removed - after removing rock). Many people had a theory that additional nitrification ability came from the glass, filter (though empty), etc. But - it did not seem to have any activity.

Seems to me that compared to plastic, sand has exponentially more surface area. I have to admit I haven't looked at high magnification pictures of various types of plastic and aragonite.

 

[MnFish1]

PS. the water experiment is extremely interesting - am I understanding correctly - you're taking water from your system - then adding ammonia (with nothing else) - and it still processes it in nearly the same way? If so - it goes along with 2 hypotheses (and probably more) IMHO - 1. There are heterotrophs in the Sample (less likely) - or 2. as has been reported - various nitrifiers are motile - and could very well be in the water (but I would expect a delay).

I have another idea - would you consider repeating the experiments - with Fritz 9000 as well? Only because Its unclear what Biospira contains?

 

[damsels are not mean]

One interesting observation on my experiments - is that there was little - or no activity comparing - a bare tank with a filter, heater, powerhead (i.e. no ammonia was removed - after removing rock). Many people had a theory that additional nitrification ability came from the glass, filter (though empty), etc. But - it did not seem to have any activity.

Seems to me that compared to plastic, sand has exponentially more surface area. I have to admit I haven't looked at high magnification pictures of various types of plastic and aragonite.

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.

 

[Amhojjat]

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.

Damsels ARE MEANA!

 

[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.

Yes - but many people have no sand in their tanks. Much of the rock in our tanks is also aragonite

 

[damsels are not mean]

Yes - but many people have no sand in their tanks. Much of the rock in our tanks is also aragonite

Oh for sure. The bacteria will be wherever it can and in a system with sand I bet a lot of it is in there especially if it's deeper bed of larger pieces. I don't think too much space is needed for nitrification TBH but not sure how much research has been done on that.

 

[Dan_P]

Excellent job. I still have the aquaria that I set up for my experiments - I was planning on re-introducing ammonia (2 ppm) again after 1 month - with none) - that will give at least some information as to whether there is (I think there will be) - nitrification capability after a month - and how much is still there.

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.

Here are a couple comments:

1. It looks like a great model.
2. It would be nice to have a control - with no biospira
(maybe I missed it) - since at least a couple people here have suggested that tanks will cycle on their own without any bacteria.

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.

3. I might ask @AquaBiomics - whether it would be possible to verify that the bacteria on your films are the same as whats in 'biospira' - as compared to heterotrophs.

Thanks alot for doing this great work.

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.