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The Cycling Experiment - Part 1: Water Chemistry


EMeyer

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Hi everyone. I've just completed a month-long experiment in my new lab at AquaBiomics at thought you might be interested in reading about it. 

The subject is a familiar one: cycling a tank. In other words, establishing a nitrifying community of bacteria (capable of converting toxic ammonia into nitrites, and then into the relatively non-toxic nitrates). Hobbyists have been doing this for decades, and in recent years have been measuring seawater chemistry to indirectly measure the development of these communities. I've repeated this, following  more or less standard practices, in order to directly measure the changes in microbial communities at each stage. Along the way, I've dosed with ammonia and measured water chemistry to monitor development of the tanks. These are the results I have to share. 

My study includes several features not often included in the "forum literature":

1. replication - I prepared all experimental tanks at the same time using identical materials, and set up duplicate identical tanks in each group

2. controls - normal people don't invest time and resources setting up tanks in a way they know won't work. I did this here, because controls are important if we want to draw confident conclusions about the effects of our practices. 

3. semi-sterile environment - I set up my experimental tanks in a way that minimizes contamination from environmental bacteria, and took great pains to avoid any contamination of the tanks during sampling and maintenance. 

 

What I did

Built experimental tanks

For this and other studies I set up an array of 12 experimental tanks. For these experiments I built custom all-in-one nano tanks (20 gal), designed to minimize cross contamination between systems. They're not designed to be pretty display tanks, but a couple months in I'm happy with their performance. 

Here are shots of one tank during testing, with a top view during construction to show how it works.

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Its an all-in-one design, with a two-chambered sump in the back. Water flows from display, then down through live rock chamber, then up into the return pump/heater chamber, then back into the display.

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The design reflects an effort to minimize costs so I could afford 12 independent systems, minimize contamination from the environment into the tank, and to fit the essential reef keeping functions in a small space. 

Here are the tanks on shelves (dry in the pictures but they've actually been wet almost 2 months). I fit 12 of these mini reefs into my little lab in Junction City. 

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Started tanks four different ways

To measure the effects of different practices for cycling a tank (here, I'll use this to mean "establishing a community of bacteria capable of metabolizing ammonia into nitrate"), I set up duplicate tanks in each of four different ways:

  • F -  maricultured rock from Fiji (artificial base rock)
  • T - maricultured branch rock, Tonga (real coral skeletons)
  • D - dry Pukani base rock (acid washed and neutralized then soaked in hydrogen peroxide)
  • B - dry rock (as D) with a popular "bacteria in a bottle" product 

I first sterilized each tank and all pumps. etc with alcohol and bleach, then filled each tank with sterile-filtered (0.2 µm) UV-sterilized artificial seawater. I added 4 lbs sand to each tank, which I sterilized using hydrogen peroxide prior to adding to the tank. I used the bacterial inoculant product (treatment B) according to the manufacturers instructions. I'm not gonna name the product here because my results are not terribly flattering, but it is advertised as establishing and maintaining a biofilter capable of converting ammonia into nitrate.

Here are images of the rock I used. I went to great pains to locate something as close as possible to real live rock (which is sadly very hard to come by). The Fiji rock had absolutely beautiful growth. The rock underneath is kind of garbage; its a mixture of cement, sand, and a little shells and rubble... with that ugly artificial purple color added. I'd rate this rock A for growth, F for the rock itself. Very pretty and very real in terms of the biology, but very fake in terms of the rock itself. All the visible growth died shortly after arrival (even though I maintained the rock at temperature with circulation and skimmer, with regular water changes to minimize dieoff). But presumably the bacteria remain.

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The Tonga rock had a mixture of coralline ranging from pale to bright purple. No other visible life, but I consider live coraline a good indicator of live bacteria. Its very, very porous stuff, real branch rock from old dead coral branches. Its also pretty small and brittle, would be challenging to aquascape with in a big tank. But great for my purposes. Really, I'd consider this ideal sump rock material: porous, and relatively fresh from the ocean. 

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The dry Pukani was also hard to come by. I ordered all in stock that I could find online, then a little more from local stores. I sterilized this stuff pretty rigorously with first and acid wash (neutralized with baking soda), then soaking for 2 weeks in hydrogen peroxide, with new H2O2 added several times. I doubt it was surgical-level sterile when I added it to the tanks, but it was as close as I could make it without an autoclave. 

Added ammonia to fuel bacterial growth

I dosed ammonia to 1 ppm in each tank using ammonia sulfate. I measured the initial levels after dosing, then measured ammonia levels weekly and dosed additional ammonia to maintain 1 ppm in each tank throughout the experiment. 

Monitored changes in water chemistry

I measured ammonia, nitrite, and nitrate levels throughout the experiment using hobbyist kits, and read the results using a spectrophotometer. This is a subject I'll write up in a separate post, because it deserves the space. For now, I'll keep it brief. The spec works as an electronic eye, and reads the kits in the same way as a human. Except it can tell the difference between fine shades of green or pink that are invisible to the human eye. 

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I ran these tests according to the kit instructions, then transferred the solution into a cuvette for reading in a spectrophotometer. I used Red Sea kits for all three. [After using these for a month or so, I can say I trust the data from these kits but am exploring other tests currently. The ammonia test has more cloudiness than I would like, and there are cheaper alternatives for NO2/NO3. ]

In a subsequent post I'll describe the details of these measurements for those who are into that sort of thing.  

Collected samples for microbiome analysis

I collected samples from each tank weekly throughout the experiment, using the same process and supplies included in the AquaBiomics sampling kit

I haven't analyzed these yet; that'll be part 2. 

 

What I found

Falling Ammonia levels are an unreliable indicator of nitrification

This is a minor point, but maybe worth noting. During the first few weeks of the experiment, ammonia was consumed in all tanks. Once a week I measured ammonia levels in all tanks and dosed additional ammonia to maintain levels at 1 ppm. Based on these data, we can estimate ammonia consumption rates for each tank in each week, revealing that a large fraction of the ammonia was consumed in each tank (43-92%). For example, the data for week 2 are shown in Figure 1. 

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Despite the consumption of ammonia during the early weeks, there was no evidence of nitrite or nitrate production in most tanks (Figure 2-3).

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Although initially confusing, this actually makes sense if we remember that ammonia is a valuable nutrient and is rapidly taken up by lots of organisms in the ocean. Its not all oxidized into nitrite and nitrate by bacteria. 

Some of the ammonia went into algal biomass, because during the first week I foolishly left the lights on a 12:12 cycle. This promoted a little bit of microalgal growth in the LR tanks, and algae consume a lot of ammonia. And some of it went into bacterial biomass - I observed visible bacterial film growth in most tanks. 

In the final week of the experiment, I measured NH4 every 2 days after dosing the tanks at ~1 ppm. Figure 4 illustrates how rapidly ammonia is consumed in tanks with nitrifying bacterial communites (F, T) and how slowly it's consumed in dry rock tanks (D, B).

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It may also be useful to view the complete time series for NO2 and NO3 (Fig 5 and 6).

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Live rock rapidly establishes a nitrifying community

My results demonstrate what we already knew -- but I like having numbers and controls supporting this conclusion. The tanks started with live rock clearly established nitrifying communities within the first 2 weeks, based on the production of NO2 and NO3 along with consumption of NH4 (Figs 2, 3). 

I'm not adding livestock to anything yet, but these tanks would be ready to stock within 2 weeks of starting the tank (and likely within the first week). 

I see no convincing evidence of a difference between these live rock sources at the level of nitrification. This surprises me considering the difference in quality of the base rock. I anticipate their microbiomes will be very different based on their sources; it will be interesting to see what they have in common. 

Without environmental contamination, dry rock cycles very slowly

This one was a little more surprising. Many of us have started tanks with live rock and successfully cycled tanks started with dry rock. I've always seeded it with a little live rock, but I know many have started them without any live rock source. And eventually (weeks to months) these tanks are expected to cycle. 

In contrast, I find no evidence of nitrification in my dry rock tanks. With the lights off, ammonia levels remain nearly constant in these tanks, and after a month of maintaining 1 ppm ammonia there is no evidence of NO2 or NO3 in the tanks (Fig 5, 6). 

How can we explain this contrast? I believe the explanation is that I took great care to avoid environmental contamination in these tanks. I sterilized the tanks, the water, the sand, and the rocks prior to starting each tank. The tanks are covered to minimize dust and aerosols. And I only handle the tanks with bleach-sterilized gloves or tubes. No sensible hobbyist starts a tank like that! But it provides an important control for my experiments, since it demonstrates that bacterial communities in my other treatments were produced by the treatments themselves rather than environmental contamination.

The implication for the hobby is - its important to get a new tank dirty. You really can make them too clean - I've just done it.

Some bottled bacterial products provide no measurable benefit

This one will seem obvious to some and complete nonsense to others. But the data in hand are pretty convincing for me. Although I used this product as directed by the manufacturer, and set these up side by side with dry-rock controls, the tanks started with a bacterial product also show no evidence of nitrification at all, after 1 month (Fig 4, 5, 6).

Just like the dry rock tanks, the bacteria in a bottle tanks did not deplete ammonia in my time series, and show no evidence of NO2 or NO3 after a month with NH4 at 1 ppm. I don't want to over-generalize. But I have to conclude that in this case, the product appeared to have no measurable benefit for establishing a nitrifying community.

Why did it have no benefit for me, while many have successfully started tanks with these products? I would gently suggest that many peoples' experiences with these products were based in non-sterile environments, and lacked replication or controls (otherwise identical tanks started without the product). I suspect that the bacterial communities established in standard hobbyist environments (like my own tanks in my living room) are seeded from environmental sources, whether bottled bacteria are added or not... 

It will be interesting to see what the microbial analysis of these tanks over the past month reveals! And I'm starting additional experiments now to follow up on these results. 

I'll be curious to hear how these results compare with your own experiences.

 

Edited by EMeyer
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Unfortunately some of your content isn't appearing properly.  You might want to edit your post and just take screen shots of those Google Docs graphs and paste them in instead:

Your content:
<iframe width="458" height="371" seamless frameborder="0" scrolling="no" src="https://docs.google.com/spreadsheets/d/e/2PACX-1vTlOabLtYnMati6AA45D0LCt47ZU6eCWUFAHtt36Phu6TWAkMiiNlW-dy5HabjdK6E0Od9lFC-7Z3qk/pubchart?oid=1315943485&amp;format=interactive
"></iframe>

Screenshot:
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It won't be interactive, but at least it's visible.

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Very cool experiment.  Thanks for writing this up!

I started my tank with 60 lbs of dry Pukani rock, and used Dr. Tim's bacteria.  I did not acid wash the rock, but did soak it in heated saltwater for 2 months before putting it into the tank.  It then took 37 days until I finally measured zero nitrite.

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Thanks, you bring up another endpoint I should measure - returning to zero nitrites. One of my 4 live rock tanks has returned to zero... I'll go ahead and quantify that difference too. Perhaps that endpoint will show a difference between LR sources. (The timing of NO2 and NO3 appearance was pretty much identical in both, but I'm skeptical they're really gonna behave the same).

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Pretty intense experiment and write up. Great job.

I'd add that Randy Holmes Farley has explained some interesting stuff about how nitrite toxicity is not a real threat for saltwater fish (at least at the levels we experience) so you can technically be safe once ammonia levels drop. Nitrite is outcompeted by chloride for the same uptake mechanisms in saltwater fish. I.e. The process that intakes nitrite into the fish is too busy being inundated by chloride. Your experiment reminded of that article, as I had kind of forgotten about the nitrite deal.

Real interesting that you were not able to generate a cycled tank with dry rock. I would think that even if you completely sanitized those tanks, bacteria would cross contaminate from the other tanks via the air.

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Yeah , it surprised me too. I think its because I took such pains to avoid contamination. The tanks are covered, and I only work on them with sterilized gloves. Hard to argue with 4 tanks that completely lack nitrification after a month, though!

I was equally surprised that bacteria in a bottle did absolutely nothing. My expectation was that these would promote nitrification but do little for bacterial diversity. SO far all the evidence in hand points to a placebo effect...

And thanks for your comments on NO2 toxicity or the lack thereof, that matches my understanding. Seems to me thats an argument in support of considering NO3 appearance as the endpoint rather than NO2=0. 

Edited by EMeyer
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This is so interesting! I've always wanted to do something similar so I'm glad somebody else did it in such a careful way so it's much easier to trust the results. I've cycled probably 150 tanks (mostly freshwater) over the years using everything out there and have had the best fastest results using live rock with saltwater tanks. I've only cycled one tank using dry base rock and nitrifying bacteria and I do remember it taking quite a bit longer than others but I guess I didn't think too much of it until now. It's nice you made this simple to understand for someone like me! I'm very interested now in seeing your future posts about this subject and what you found with the

spectrophotometer

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This is fantastic, @EMeyer. I can't wait to see part 2!

Your results suggest using extreme caution when using bottled bacteria products. This is a bummer, since I am about to start up a quarantine system with one of these products. I know now to make sure to start with some live rock or a pre-cycled sponge filter.

I wonder if the bacteria product you used might have overheated in transit to you before you used it. Can you use your microbiome analysis to see whether your bottle does contain viable bacteria as a way validating your treatment?

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This is fantastic, Eli. I can't wait to see part 2!
Your results suggest using extreme caution when using bottled bacteria products. This is a bummer, since I am about to start up a quarantine system with one of these products. I know now to make sure to start with some live rock or a pre-cycled sponge filter.
I wonder if the bacteria product you used might have overheated in transit to you before you used it. Can you use your microbiome analysis to see whether your bottle does contain viable bacteria as a way validating your treatment?
Oh that would be interesting to find out, how often you get a bad bottle would be something I'm sure a lot of people would like to know.

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25 minutes ago, TaylorW said:

Oh that would be interesting to find out, how often you get a bad bottle would be something I'm sure a lot of people would like to know.

Sent from my BLU R1 HD using Tapatalk
 

I was just wondering this, because I just cycled my tank, which was dead rock, with a mix of Dr Tim's and Microbacter7. I used a lot more than recommended and multiple times during the cycle. I didn't add any live rock to the system before the cycle finished. After adding the products I observed visually what I assumed was bacterial blooms in the tank, usually the next day.

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I'm skeptical enough about the negative result on the bacterial product that I went ahead and dosed those tanks again with bacteria and ammonia. I'll run another time course with this fresh dose. 

I will absolutely be including both these tanks and the bottle itself in my first batch of samples (preparing this batch now).

I can say for sure the bottle and cold pack were cold on arrival and I've kept them cold since arrival... but cant vouch for its history beyond that. If I get another round of negatives I will buy another bottle to triple check.

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I have an update that totally changes my conclusions about the bacterial product. 

I couldn't  believe the total lack of an effect the first time. So I added an additional dose of bottled bacteria to both of the B tanks, and sampled the microbiome again in each of the B tanks and the D tanks (dry rock controls). Then I measured water chemistry as usual. I only have ~4 days of data in hand but its clear that it worked this time, very convincingly. 

Here are the ammonia consumption data for the new trials, plotted along with the previous live rock data for comparison. I calculate nearly identical rates of ammonia consumption for bottled bacteria vs live rock (~45% per day over the first 4 days of data). Like before, all data shown here represent the average from duplicate identical tanks in each group. 

pubchart?oid=436020997&format=image

And there is convincing evidence this consumption results from nitrification. Here are the data for nitrite and nitrate accumulation. The bottled bacteria produce even more than the live rock tanks (again, I've plotted the new B and D trials alongside the older live rock data)

pubchart?oid=2085312070&format=image

pubchart?oid=1201184636&format=image

Its always frustrating when you try to repeat an experiment and get different results. In my research life we used to say "thats why you never repeat an experiment!" [This is a scientist joke; reproducibility is obviously important but theres this annoying tendency for things to go differently the second time... ]

But I am relieved. So many people are using these products, they couldnt be totally snake oil. They obviously worked great in my second trial.  So this is good, I can focus on the original question for those treatments instead of having to work hard to prove the product didnt work :)

So why did the first inoculation fail while second inoculations from the same bottle worked? I think there may have been residual chlorine in the tanks from bleach sterilization. I neutralized with thiosulfate and tested to confirm the absence of chlorine, but using a fairly insensitive kit. My sensitive kit arrived 2 days into the experiment and also showed zero. So I thought all was OK, but I cant rule out the presence of tiny amounts of chlorine for the first 2 days... it would make sense that this would affect suspended bacteria more than biofilms, and the product instructions do emphasize the sensitivity to chlorine. 

In conclusion - I now have three groups of functional nitrifying communities to analyze, in addition to the control. Next steps are to finally analyze these samples I've been accumulating!

-Eli

 

Edited by EMeyer
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I have an update that totally changes my conclusions about the bacterial product.  I couldn't  believe the total lack of an effect the first time. So I added an additional dose of bottled bacteria to both of the B tanks, and sampled the microbiome again in each of the B tanks and the D tanks (dry rock controls). Then I measured water chemistry as usual. I only have ~4 days of data in hand but its clear that it worked this time, very convincingly.  Here are the ammonia consumption data for the new trials, plotted along with the previous live rock data for comparison. I calculate nearly identical rates of ammonia consumption for bottled bacteria vs live rock (~45% per day over the first 4 days of data). Like before, all data shown here represent the average from duplicate identical tanks in each group. pubchart?oid=436020997&format=image

And there is convincing evidence this consumption results from nitrification. Here are the data for nitrite and nitrate accumulation. The bottled bacteria produce even more than the live rock tanks (again, I've plotted the new B and D trials alongside the older live rock data)

pubchart?oid=2085312070&format=image

pubchart?oid=1201184636&format=image

Its always frustrating when you try to repeat an experiment and get different results. In my research life we used to say "thats why you never repeat an experiment!" [This is a scientist joke; reproducibility is obviously important but theres this annoying tendency for things to go differently the second time... ]

But I am relieved. So many people are using these products, they couldnt be totally snake oil. They obviously worked great in my second trial.  So this is good, I can focus on the original question for those treatments instead of having to work hard to prove the product didnt work [emoji4]

So why did the first inoculation fail while second inoculations from the same bottle worked? I think there may have been residual chlorine in the tanks from bleach sterilization. I neutralized with thiosulfate and tested to confirm the absence of chlorine, but using a fairly insensitive kit. My sensitive kit arrived 2 days into the experiment and also showed zero. So I thought all was OK, but I cant rule out the presence of tiny amounts of chlorine for the first 2 days... it would make sense that this would affect suspended bacteria more than biofilms, and the product instructions do emphasize the sensitivity to chlorine. 

In conclusion - I now have three groups of functional nitrifying communities to analyze, in addition to the control. Next steps are to finally analyze these samples I've been accumulating!

-Eli

 

 

 

 

[emoji44] wow that's great! I'm glad you went ahead and tried it again though, I would bet that odds are not all the bottles contain live bacteria, some people do indeed get a bad bottle. From the attached thread above on Reef2Reef that lexiverts posted Dr. Tim posted that it's not the heat you need to worry about as much as freezing it. I'm wondering myself if I got a bad batch when I tried it, I used it as directed and remember it taking longer to cycle a tank with dry rock than just using live rock like I've done before and since. I used the Tetra Safe Start and that's the only kind I've ever tried myself, I bought it from Walmart and remember it was pretty close to the expiration date so maybe that had something to do with it....  Sent from my BLU R1 HD using Tapatalk 

 

 

 

 

 

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On 6/27/2019 at 4:41 PM, EMeyer said:

Some bottled bacterial products provide no measurable benefit

This one will seem obvious to some and complete nonsense to others. But the data in hand are pretty convincing for me. Although I used this product as directed by the manufacturer, and set these up side by side with dry-rock controls, the tanks started with a bacterial product also show no evidence of nitrification at all, after 1 month (Fig 4, 5, 6).

I didn't get a chance to read through this originally but it is pretty interesting... especially since the concept should work and, frankly, not be overly complicated to implement.  Wonder if they just have an issue stabilizing the bacteria (assuming there were some there to start with).

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1 hour ago, albertareef said:

I didn't get a chance to read through this originally but it is pretty interesting... especially since the concept should work and, frankly, not be overly complicated to implement.  Wonder if they just have an issue stabilizing the bacteria (assuming there were some there to start with).

You might want to check out the update - a second dose produced clear evidence of nitrification. The first dose did absolutely nothing but the second worked great. I speculate the first failed due to undetectable residual chlorine from bleach sterilization. 

Frankly I've always been skeptical of these products on the basis that

1. most (>99%; basically all) marine bacteria cannot be cultured

2. bacteria arent immortal, and the number of viable cells inoculated makes a difference

but I cant argue with the round 2 results. It definitely established a nitrifying community. I am curious now to know how that community compares to live rock communities. Extracted DNA today to start finding out...

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On 6/27/2019 at 10:37 PM, Higher Thinking said:

Pretty intense experiment and write up. Great job.

I'd add that Randy Holmes Farley has explained some interesting stuff about how nitrite toxicity is not a real threat for saltwater fish (at least at the levels we experience) so you can technically be safe once ammonia levels drop. Nitrite is outcompeted by chloride for the same uptake mechanisms in saltwater fish. I.e. The process that intakes nitrite into the fish is too busy being inundated by chloride. Your experiment reminded of that article, as I had kind of forgotten about the nitrite deal.

Real interesting that you were not able to generate a cycled tank with dry rock. I would think that even if you completely sanitized those tanks, bacteria would cross contaminate from the other tanks via the air.

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This is an excellent point that I discuss when I'm lecturing water chemistry with Fish and Invert Health Mgmt. students. However, while chlorides do out-compete nitrites in uptake across gill tissue, this varies from one marine fish species to another. In any case, nitrites inhibit the oxygen-carrying efficiency of fish blood by oxidizing hemoglobin into methemoglobin (Once again, major props and thanks to Dr. Noga.) If the animal is subject to multiple stressors(a very likely scenario if nitrites are present in any measurable concentration,) the "brown blood disease" of nitrite poisoning will likely result in anemia and push the animal past the point of no return. 

As for this WQ experiment, much respect and many thanks for spending the time and resources to set it up, document the results and sharing it with the collective. While my experiences with bacteria in a bottle are anecdotal rather than scientific, I regard these products with suspicion. In many cases over the last several years, I have observed stubbornly high and sustained NO2 levels in 20-gallon systems kick-started with these products, which were purchased new and very much before their expiration date. This is supposition, not science, but I believe that the genera of bacteria that oxidize NH3 into NO2 out-compete the genera that oxidize NO2 into NO3 for carbonates and other resources and actually retard the cycle. The NO2 concentrations persist even when mitigated by daily and aggressive water changes.

Could be that in addition to out-competing Nitrobacter, et.al., for carbonates, etc., the elevated NO2 levels inhibit respiration in these genera, as well. Again, this is experiential and supposition but I've seen it happen often enough that there might be a kernel of truth in there somewhere. 

Great thread!

 

 

 

 

 

 

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