Does my decade old sand bed actually nitrify? Who eats Ammonia in our tanks?

MnFish1

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@taricha - here are all of the results - I'll figure out what came from where in a bit - but - except for some control samples - all came from the same tank - just different places:

1000121, 1000122. These samples are relatively low in diversity and completely unlike any reef tank samples I've seen. (They are also very different from each other). I will set these aside for now and return to the mnext in an effort to deduce their origins. It's an interesting puzzle.



1000123.

  • This sample has a relatively low diversity, but within the range observed in my samples.
  • The balance score is quite low, but if you examine the family abundance barplot you see most of the core microbiome families are present. The low score appears to result from differences in the levels of these families, primarily the deficiency of this sample in Pelagibacteraceae and Flavobacteracieae, and excess in Thiotrichaceae and Oceanospirillaceae.
  • This sample has moderate to high levels of nitrifying microbes, since both ammonia-oxidizing and nitrite oxidizing microbes were detected.
  • No evidence of cyanobacteria or bacterial pathogens of fish or corals.
  • I would guess this sample represents a different aquarium or a different part of the aquarium than the samples below.


1000124

  • This sample has established a new record as the most diverse sample I've ever tested, with over 800 different types of microbes detected.
  • The balance score is quite low, indicating a different community composition than the typical water sample. If you examine the family abundance barplot you see that this sample has higher than usual levels of Flammeovirgaceae. This group is associated with marine sediments.
  • While the levels of ammonia-oxidizing microbes are on the lower end of the typical range, the diversity is unusually high, with three different groups represented. Nitrite oxidizing bacteria are present at high levels.
  • Cyanobacteria are present at high levels and very high diversity relative to their typical abundance in water + biofilm samples.
  • No evidence of bacterial pathogens of fish or corals.
  • I would guess this sample was taken from the sediments, rocks, or a similar part of the aquarium.


1000125, 1000126, 1000127.

  • These 3 samples gave nearly identical results, so I will discuss them together, interpreting them as replicate sample from the same aquarium.
  • These samples had high and very similar diversity scores (410-469), showing a more diverse community than about 3/4 of the tanks I've sampled.
  • These samples showed very low and similar balance scores (0.03 - 0.08), indicating a community that is very different than the typical reef tank. I think it's important to recognize that different doesn't necessarily mean unhealthy; this score indicates "atypical" rather than "unhealthy".
  • The composition of the communities in all three samples is nearly identical (part 2).
  • All show elevated levels of Flammeovirgaceae, a group associated with marine sediments. And they show lower than usual levels of Pelagibacteraceae, a free-living group that is abundant in the open ocean. They also show lower than usual levels of Alteromonadaceae, Flavobacteriaceae, and Vibrionaceae. Together, these differences suggest a very different nutrient environment in your tank than many other reef tanks. You can read more about these groups here.
  • All three samples contain detectable ammonia-oxidizing microbes, although the types of AOB detected differ among samples. 2 out of 3 samples contain detected nitrite oxidizing bacteria, while they were not detected in the third. This suggests these groups were present at very low levels, highlighting the challenges of detecting rare bacterial types. This is why I always caution people that a lack of detection is not the same thing as absence.
  • Two out of three samples showed no cyanobacteria, while the their showed moderate levels of a common type. I would not interpret any of these results as a cause for concern.
  • All three samples were free of known bacterial pathogens of fish or corals. This is far from a universal result; perhaps 1 in 10 tanks has a detectable pathogen. So to have 3 clear tests seems to be convincing evidence these are not present in your system.


1000128. This sample did not amplify in any of the different conditions I tried. In all cases, this sample behaved similarly to sterile water samples (Iwhich showed no amplification in any conditions tested). I would not trust any sequence data from this sample. If this was a negative control it was a successful one! If it was a sample you hoped to gather data from, we should discuss a replacement or refund. (But I hope it was a negative control, since it behaved just like my negative controls).
 
AS

Dan_P

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Let me guess . the glass surface consume more ammonia?

Sincerely Lasse
Biofilms on aragonite consumed ammonia but produced no nitrite or nitrate. Biofilms on glass produced ammonia. I assume the ammonia production meant that I harmed the biofilms. I am now trying to grow a nitrifying biofilm on glass with Bio-Spira.
 

Brew12

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Awesome. I knew you'd have a good way to think of this. By subtraction systems that pulled surfaces or algae can see ammonia spikes, implying these systems have water that can't keep up with the ammonia processing rate for the tank.
Thank you.

I think the only way you could convince me that water played a significant role in nitrification is if a large water change could cause an ammonia spike. I haven't come across anyone reasonably making this claim.
 

Lasse

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Biofilms on aragonite consumed ammonia but produced no nitrite or nitrate. Biofilms on glass produced ammonia. I assume the ammonia production meant that I harmed the biofilms.
Interesting - however it contradict the most I know about nitrification. May I ask for your test set up and what did you use in order to measure a biofilms production and/or uptake of ammonia? Ion sensitive microelectrodes?

Sincerely Lasse
 

Dan_P

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Interesting - however it contradict the most I know about nitrification. May I ask for your test set up and what did you use in order to measure a biofilms production and/or uptake of ammonia? Ion sensitive microelectrodes?

Sincerely Lasse
Lasse, the glass slides were stored vertical in the aquarium sump in the dark. Periodically a 2 were removed and placed in a capped centrifuge tube containing the test medium. The tubes were placed on an orbital shaker for three days. At the end of this time, the medium was tested for PO4, total ammonia, NO2 and NO3 when there was no or little NO2.

Initially, I was using freshly prepared Instant Ocean dosed with PO4 and NH4Cl, just several tenths of ppm each. Glass slides consistently increased the ammonia in the medium. In a discussion with @flampton, it was concluded that switching the medium from tank water to Instant Ocean could have somehow stressed the biofilm community and it started to disperse and digest the film. I then switched to boiled aquarium water as the test medium and the ammonia production was almost elimated.

Growing a biofilm on aragonite involved spreading a thin film of super glue on a glass slide and attaching a layer of washed fine grain aragonite sand. This was done on both sides. The biofilm that grew on aragonite consumed ammonia from the test medium but produced no NO3 or NO3. By day 21, the biofilm started to produce ammonia in the test solution.

Work is in progress growing a nitrifying biofilm on glass with the aid of Bio-Spira.

Lasse, I would appreciate any and all ideas and comments.

Dan
 
AS

Dan_P

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What did you measure with?

Any duplicate?

Sincerely Lasse
I always test at least two slides for each time point. @taricha and I modified the API test so the color intensity can be determined in a spectrometer or Hanna checker. It detects total ammonia down to 0.02-0.05 ppm, quantitates above 0.05 ppm.
 

Dan_P

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I´ll think that nitrification rate is very difficult to measure that way you do, It is very oxygen sensitive - the second step normally demand between 4 - 5 mg/l free O2.

Sincerely Lasse
Thanks Lasse. I will start checking O2 levels in the medium to make sure the biofilms are not oxygen starved.
 

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