New Nitrifying Bacteria Experiment.

[MnFish1]

Today's results - End of Experiment 2.

My thoughts go something like this.

The rock biofilm begins to change the minute you move the rock to a new environment. Some life forms die, some leave the rock, others grow to fill the new space. The effects I would wonder about include, chemical and biological.

Chemical. Will the rock chemical emissions effect my experiment measurements? Will NH3 be produced by the rock irrespective of what is added?

Biological. Is the rock ammonia consumption bacteria or algae dominated? How does an algae dominated biofilm behave in the dark?

Interestingly enough - when the formerly 'Dark' Rock was 'Lit' and the formerly 'Lit' rock was left 'dark' - there was no real difference - except BOTH sets processed ammonia more quickly - than before - but the Dark (formerly lit) tank - did it one day faster. As @Lasse suggested - will try the rest of the experiments 'in the dark'

 

[MnFish1]

Experiment 2 Final results - Tank 1 - Lit (former dark sump rock) - Tank 2 - Dark (former lighted display tank)

(My) Conclusions:

1. Both tanks continued to process ammonia
2. Even though dark, Tank 2 continued to process more ammonia than Tank 1.
3. Both tanks processed ammonia more quickly - approximately 24 hours faster than Experiment 1.
4. I may have had the first incidence of a 'partly false API measurement - more on this later - or it could be that the ammonia in tank 1 was still not entirely processed. There were VERY slight differences in the Seachem alert badges but - only well seen in pictures - if I had read Tank 1 alone - I would say 'SAFE'. Comparing the 2 there was a slightly different color. I decided to just go to the next step - because - we will be using new water anyway - and these first few are just 'preparatory' to figuring out how to best do the future rinsing ones. The fact that the nitrate processed is more in Tank 2 than tank 1 - suggests also that the slight green was not 'error'

5. Unfortunately @Lasse - the Nitrite is just not sensitive enough - or none is being measured - will continue to try

 

[MnFish1]

Experiment 3, Day 1. Ammonia added to Tank 1 (Dry sump rock - DARK) and Tank 2 (Former Lighted Display tank rock - DARK)

 

[MnFish1]

Comments on the API 'false positive' ammonia test.

It seems to be common 'wisdom' that the API tests (esp for ammonia) are not 'accurate'.

I have used 2 different sets (bought new) - and done a couple tests - that might shed light on this. This was not a formal study - but mostly - just whats happened when I made various errors:

1. For the most part, with about 30-40 tests - when the ammonia is supposed to be 'zero' (I.e. fresh saltwater, A fully cycled tank, RODI, Tap water with no Chloramine, etc - the vial reads perfect 'zero'. WHEN THE TEST IS DONE CORRECTLY. There was one exception - see #, and 3e and f.
2. In the most recent experiment - the Seachem alert to me looked like 'zero/safe' - so I expected that the API test would also be '0' (it was in Tank 2). In Tank 1 - it was 0-0.25 ppm. On closer inspection, the Seachem alert in Tank one - though it looked 'SAFE' - was a slightly more 'green color' - so my guess is that the API test was in fact reading between 0 and 0.25.
3. Causes for error (I tested several of these out when I was practicing to do the experiments)
a. I find it extremely difficult to hold the bottle exactly vertical - without steadying my arm somehow. Varying drop size - can markedly change the results. (sometimes causing a very faint green - in water with 0 ammonia.
b. Not mixing vial 1 before adding vial 2 - can give variable results.
c. Not very througoughly shaking vial 2 - can give variable results. When they say 30 seconds at least - they mean it lol
d. Not properly timing the test. It is unclear from the instructions - whether you start timing immediately after finishing shaking vial 2 (which is what I think) - as compared to starting the timer after you put the #2 drops in the vial before starting to shake. If you don't do this step properly - you may get higher results (if too long) than expected - and lower results (than expected) - if timed too short.
e. According to the instructions - the way to read the test is to use the color CLOSEST to the one in the vial. So for example - I have seen a lot of people tempted to read a very minimally green test as '.25 ppm' - a false positive.
f. At pH 8 - .25 ppm is a safe ammonia level (free ammonia) in any case - So - if you (as many here say) have a tank that 'looks cycled' in every other way - your 0.25ppm is probably really '0'.

 

[Dan_P]

Experiment 2 Final results - Tank 1 - Lit (former dark sump rock) - Tank 2 - Dark (former lighted display tank)

(My) Conclusions:

1. Both tanks continued to process ammonia
2. Even though dark, Tank 2 continued to process more ammonia than Tank 1.
3. Both tanks processed ammonia more quickly - approximately 24 hours faster than Experiment 1.
4. I may have had the first incidence of a 'partly false API measurement - more on this later - or it could be that the ammonia in tank 1 was still not entirely processed. There were VERY slight differences in the Seachem alert badges but - only well seen in pictures - if I had read Tank 1 alone - I would say 'SAFE'. Comparing the 2 there was a slightly different color. I decided to just go to the next step - because - we will be using new water anyway - and these first few are just 'preparatory' to figuring out how to best do the future rinsing ones. The fact that the nitrate processed is more in Tank 2 than tank 1 - suggests also that the slight green was not 'error'

5. Unfortunately @Lasse - the Nitrite is just not sensitive enough - or none is being measured - will continue to try

Does the nitrate nitrogen produced equal the ammonia nitrogen added?

 

[DrZoidburg]

closer to 4 ppm

3.7823 lol I duno yours seems a little darker too. Is hard to get spot on. What I wonder is if tank 1 will be lower in nitrate in all tests.

 

[MnFish1]

Does the nitrate nitrogen produced equal the ammonia nitrogen added?

One problem - as you know - the API tests are not that 'sensitive'. a reading of lets say 40 - can be 30 or 50. This is one of the problems with using 'scientific' conclusions - with hobby grade experiments. - I wondered the same thing

 

[MnFish1]

3.7823 lol I duno yours seems a little darker too. Is hard to get spot on. What I wonder is if tank 1 will be lower in nitrate in all tests.

we will see.... but - going forward - the highest (per my eyes) the Ammonia will be is 2 ppm. Frankly - to me - the next experiment - will be the start of the 'repeat' of the others. But it will be 2 ppm - according to API

 

[MnFish1]

I decided to post the results a little differently to make day-day changes easier to read. I will Reply to the first post in every experiment and then keep replying so a person can see all of the results in one place. Starting now - One problem is that I don't believe all of the images transfer:

Experiment 3, Day 1. Ammonia added to Tank 1 (Dry sump rock - DARK) and Tank 2 (Former Lighted Display tank rock - DARK)

Experiment 3 Day 2 (24 hours). Tank 1 and Tank 2 Dark:
There was a much more rapid reduction in ammonia today - Tank 2 still way ahead - but .25-.5 ppm - Tank 1 is already 1 ppm

 

[DrZoidburg]

Just noticed asterina starfish made it through all that E3 day 1 photo. It deserves a name now.

 

[sixty_reefer]

Looking really good so far @MnFish1, wend you intend to start the freshwater trials?

 

[brandon429]

Post #144 vastly helpful on api procedure consistency 3c is affecting me in chat as we speak + the other confounds that we never bothered to ensure ahead of time. I ask them for .5 max, they show up having dosed to dark green, 2 ish, solid find causative combo.

Fine discoveries being posted here

 

[MnFish1]

Looking really good so far @MnFish1, wend you intend to start the freshwater trials?

So - After this experiment (3) is done (which should replicate the one from the day before), the plan is:

1. I'm going to use an accurate syringe - and get as close to 2 ppm as possible - and then use that in each tank every day (Note - I have marked the volume on the edges of both tanks - so I know how much saltwater to add every day. For all of the rest of the experiments.
2. I want to TRY to get the tanks to the point where it processes 2 ppm/day. Going forward - both tanks will be in complete darkness as @Lasse recommended.
3. So - My HOPE is since Tank 2 is almost there - my guess is that tomorrow - Experiment 3 will be over and will start Experiment 4a and 4b, c, d, etc as needed (maybe I should use covid variant numbers lol) to get the tanks to process 2 ppm ammonia in 24 hours.
4. 4a, b, etc are only designed to repeat the first experiment with as close to 2 ppm as possible As I said before - but for new people who might be reading - I think I was putting closer to 3-4 ppm ammonia in the tank on each day 1.
5. Experiment 5 will be to verify this (this is similar to @Coxey81 and @sixty_reefer 's 'step 1'.
6. Then since many people wondered about whether glass, filters, heaters, and the water itself were participating. There will be another sub-experiment 6 - which will keep the same rock (non-rinsed - BUT - the tank, HOB (they have no media - just for oxygen) - and heater will be cleaned - to see whether they contributed to the ammonia reduction.
7. Then - since you (@sixty_reefer) wondered about the water column itself - will repeat experiment 6 - cleaning the tank, HOB, etc then replace the old water into the tank. This - hopefully - will answer the question about 'the water' and what its contribution to nitrification is in this system. (Up to this one - all of the water has been 100% changed)
8. THEN LOL - will start again - to replicate the experiments you and @Coxey81 did. 2 times - processing ammonia of 2 ppm in 24 hours - then various types of rinsing.

Sorry if this seems complicated - its actually just trying to answer a lot of questions brought up from the last thread. #8 - is going to be really the 'start' of the replication you guys so nicely did. But - I guess I'm happy to say - that so far - your results are replicated - - the aiptasia

 

[MnFish1]

Post #144 vastly helpful on api procedure consistency 3c is affecting me in chat as we speak + the other confounds that we never bothered to ensure ahead of time. I ask them for .5 max, they show up having dosed to dark green, 2 ish, solid find causative combo.

Fine discoveries being posted here

Thanks @brandon429

 

[Dan_P]

So - After this experiment (3) is done (which should replicate the one from the day before), the plan is:

1. I'm going to use an accurate syringe - and get as close to 2 ppm as possible - and then use that in each tank every day (Note - I have marked the volume on the edges of both tanks - so I know how much saltwater to add every day. For all of the rest of the experiments.
2. I want to TRY to get the tanks to the point where it processes 2 ppm/day. Going forward - both tanks will be in complete darkness as @Lasse recommended.
3. So - My HOPE is since Tank 2 is almost there - my guess is that tomorrow - Experiment 3 will be over and will start Experiment 4a and 4b, c, d, etc as needed (maybe I should use covid variant numbers lol) to get the tanks to process 2 ppm ammonia in 24 hours.
4. 4a, b, etc are only designed to repeat the first experiment with as close to 2 ppm as possible As I said before - but for new people who might be reading - I think I was putting closer to 3-4 ppm ammonia in the tank on each day 1.
5. Experiment 5 will be to verify this (this is similar to @Coxey81 and @sixty_reefer 's 'step 1'.
6. Then since many people wondered about whether glass, filters, heaters, and the water itself were participating. There will be another sub-experiment 6 - which will keep the same rock (non-rinsed - BUT - the tank, HOB (they have no media - just for oxygen) - and heater will be cleaned - to see whether they contributed to the ammonia reduction.
7. Then - since you (@sixty_reefer) wondered about the water column itself - will repeat experiment 6 - cleaning the tank, HOB, etc then replace the old water into the tank. This - hopefully - will answer the question about 'the water' and what its contribution to nitrification is in this system. (Up to this one - all of the water has been 100% changed)
8. THEN LOL - will start again - to replicate the experiments you and @Coxey81 did. 2 times - processing ammonia of 2 ppm in 24 hours - then various types of rinsing.

Sorry if this seems complicated - its actually just trying to answer a lot of questions brought up from the last thread. #8 - is going to be really the 'start' of the replication you guys so nicely did. But - I guess I'm happy to say - that so far - your results are replicated - - the aiptasia

I was able to grow a sustainable Bio-Spira biofilm on glass microscope slides. Will be interesting to see what you observe.

I observed the water from one Bio-Spira experiment, the only time I looked, oxidized ammonia as well as the acrylic container surfaces it was cultured in. I wonder if eventually this activity settles out.

 

[sixty_reefer]

So - After this experiment (3) is done (which should replicate the one from the day before), the plan is:

1. I'm going to use an accurate syringe - and get as close to 2 ppm as possible - and then use that in each tank every day (Note - I have marked the volume on the edges of both tanks - so I know how much saltwater to add every day. For all of the rest of the experiments.
2. I want to TRY to get the tanks to the point where it processes 2 ppm/day. Going forward - both tanks will be in complete darkness as @Lasse recommended.
3. So - My HOPE is since Tank 2 is almost there - my guess is that tomorrow - Experiment 3 will be over and will start Experiment 4a and 4b, c, d, etc as needed (maybe I should use covid variant numbers lol) to get the tanks to process 2 ppm ammonia in 24 hours.
4. 4a, b, etc are only designed to repeat the first experiment with as close to 2 ppm as possible As I said before - but for new people who might be reading - I think I was putting closer to 3-4 ppm ammonia in the tank on each day 1.
5. Experiment 5 will be to verify this (this is similar to @Coxey81 and @sixty_reefer 's 'step 1'.
6. Then since many people wondered about whether glass, filters, heaters, and the water itself were participating. There will be another sub-experiment 6 - which will keep the same rock (non-rinsed - BUT - the tank, HOB (they have no media - just for oxygen) - and heater will be cleaned - to see whether they contributed to the ammonia reduction.
7. Then - since you (@sixty_reefer) wondered about the water column itself - will repeat experiment 6 - cleaning the tank, HOB, etc then replace the old water into the tank. This - hopefully - will answer the question about 'the water' and what its contribution to nitrification is in this system. (Up to this one - all of the water has been 100% changed)
8. THEN LOL - will start again - to replicate the experiments you and @Coxey81 did. 2 times - processing ammonia of 2 ppm in 24 hours - then various types of rinsing.

Sorry if this seems complicated - its actually just trying to answer a lot of questions brought up from the last thread. #8 - is going to be really the 'start' of the replication you guys so nicely did. But - I guess I'm happy to say - that so far - your results are replicated - - the aiptasia

It’s nice of you to look at all possible angles, it’s good too follow the test as it happens also, will you be doing a separate write up with all the data in one place, once you get all the results?

 

[MnFish1]

So an update. 1. I messed up - I left the lights on on both tanks a day ago... (no update that day). But - as predicted - the ammonia was safe safe and 0 0 in both tanks. Yesterday was a mess and I missed the measuring time. I left the tanks dark. I thought - I'll use this little break - to check the nitrate. I'm just going to say the results - and post the pictures after:

1. The ammonia - Seachem - and API were both 0.
2. The nitrate - was still lower in Tank 1 than Tank 2. Despite having the same ammonia level added at the start.

This suggests - either a 1) test error, 2) the sump rock has different nitrate processing capability. 3) there was an ammonia dosing error (there was not) - any other ideas?

In any case - I'm working on the dose of ammonia to get exactly 2 ppm ammonia today - the experiments will resume tomorrow. . Sorry for no good updates.

PS - @brandon429 brought up an excellent idea in another thread - and it got me thinking.

As everyone (?) knows - the 'weight' of the rock was the same in each tank. The water displacement in the tank when put in was the same. BUT - in the 'display tank rock' - the rock was 'less dense' - due to there being some 'coral skeletons' present - even though I tried to control for this - by matching the size of the rocks put in each tank. SO - rather than photosynthesis it could very well be - that there is more surface area in the 'display rock tank' - than the sump rock tank - thus easier to process ammonia. IN any case - both tanks have markedly improved their ammonia processing capability.

 

[Coxey81]

Eta: nevermind

 

[Rmckoy]

So an update. 1. I messed up - I left the lights on on both tanks a day ago... (no update that day). But - as predicted - the ammonia was safe safe and 0 0 in both tanks. Yesterday was a mess and I missed the measuring time. I left the tanks dark. I thought - I'll use this little break - to check the nitrate. I'm just going to say the results - and post the pictures after:

1. The ammonia - Seachem - and API were both 0.
2. The nitrate - was still lower in Tank 1 than Tank 2. Despite having the same ammonia level added at the start.

This suggests - either a 1) test error, 2) the sump rock has different nitrate processing capability. 3) there was an ammonia dosing error (there was not) - any other ideas?

In any case - I'm working on the dose of ammonia to get exactly 2 ppm ammonia today - the experiments will resume tomorrow. . Sorry for no good updates.

PS - @brandon429 brought up an excellent idea in another thread - and it got me thinking.

As everyone (?) knows - the 'weight' of the rock was the same in each tank. The water displacement in the tank when put in was the same. BUT - in the 'display tank rock' - the rock was 'less dense' - due to there being some 'coral skeletons' present - even though I tried to control for this - by matching the size of the rocks put in each tank. SO - rather than photosynthesis it could very well be - that there is more surface area in the 'display rock tank' - than the sump rock tank - thus easier to process ammonia. IN any case - both tanks have markedly improved their ammonia processing capability.

Lbs / gallon was always only a number as surface area of the rocks is what has always mattered

 

[MnFish1]

Correct - I tried to control for the surface area by measuring the 'displacement' of the water - and piecing out the rock individually before I selected it for the experiment. For example 1 piece that was an inch - of each type, etc - and then weighed the pairs - and added the same 'weight' to each tank.

But its my impression - that the tank (2) - with display tank rock - has a larger surface area. This - this (as compared to photosynthesis) - is the reason tank 2 is doing better.

Second - after several tests today - using .78 cc of Dr. Tim's results in 2 PPM - I dosed today to about 3 ppm in each tank - once that is processed - will use .78 cc in each tank - for the rest of the experiments

Lbs / gallon was always only a number as surface area of the rocks is what has always mattered