Exchange W/ Dr Tim Re Nutrifying Bacteria

[TwoTankAmin]

This is the origin of this thread:

I figured the easiest solution was to contact Dr. Hovanec directly and ask him to clarify things. I will post both my email his reply in a new thread when I receive it. What is most interesting is he says 5 ppm can start to poison and then that 10 ppm will inhibit them in different places.

I also asked him to clarify the death of a bacteria vis-a-vis rupturing of the cell wall etc. He says that the bacteria in a bottle only remain usefull for about one year max. But he also states that these bacteria have survived on the planet for millions of years because they can shut down during periods of extended drought. So I asked if the can survive extended drought and continue the survival of the species, then why can't they do the came after a few years in a bottle.

He has replied to past emails so I am assuming I will get an answer but it may take time as I am sure he is a busy man.

From http/www.fishforum...385852-ammonia/


Here is the exchange- My questions are in black and his replies are in blue:

Hello - thanks for email, please see below for comments to your questions.

On Feb 1, 2012, at 6:37 PM, cwl........ wrote:

Hi- I have been a fan of your work and products for a number of years. I am a bit confused about certain facts regarding these bacteria and have read conflicting statements on your site and could use a little clarification.

In one place you state that levels of ammonia over 5 ppm can poison nitrite oxidizers.

"Nitrite does not spike until somewhere between days 14 and 20. You want to be careful adding more ammonia because you do not want the nitrite-nitrogen over 5 mg/L as this will start to poison the nitrite-oxidizing bacteria."

To me this means that these levels can and will kill the bacteria. However, In another part of the site you state:

"If the ammonia or nitrite concentration in the aquarium is above 10 ppm (as nitrogen) or 10 mg/L-N you should do a partial water change before adding One & Only. The reason is that these bacteria prefer low concentrations of ammonia/nitrite and can actually be inhibited by high levels of these chemicals."

This implies not killing but just slowing them down and that would imply lowering the levels would allow the bacteria to "revive". Can you give me a bit more precise explaination about the effects of elevated ammonia/nitrites on the bacteria.


As I wrote at a nitrite concentration of 5 mg/L of "starts to poison" the bacteria. This is just like if you were in a closed room and it was filled to a low level of carbon monoxide - you would feel sick and have headaches and you are slowly being poisoned. You probably won't die at this low level but you surely are not healthy.

Once the values get higher they become toxic and can kill the bacteria. It's most semantics the absolutely values depends on many environment conditions but the point is that you need to keep the nitrite low or the process will get stuck because the nitrite-oxidizing bacteria cannot function at these higher levels.

How it works varies by bacterial type for ammonia -oxidizers the nitrite effect the enzyme AMO. You can search for this article and read more should you wish the details
Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite LISA Y. STEIN AND DANIEL J. ARP* Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 Received 16 April 1998/Accepted 28 July

Also, in your articles on fishless cycling and bacteria living in bottle you state:

"The only way a bacterial cell truly 'dies' is if the cell wall breaks apart (called cell lyses) and the cell contents spill out or if the cell in poisoned by some substance that penetrates the cell wall and kills the cell."

However, you also indicate there is a maximum useful time that the bacteria can survivie in a bottle:

"They can live in a bottle but under optimal conditions the time period is about one year. The nitrifying bacteria don't die in the bottle, their activity level drops and eventually it becomes so low that there is little measurable positive effect when they are poured into the aquarium water."

I am again confused. Especially in light of your statement which says:

"Nitrifiers belong to a very old line of bacteria (milloins of years) and they have developed ways to cope with very long periods of "drought."

This would seem to indicate that they can "shut down" for extended periods and come back to "life" once better conditions are present. So why wouldn't the bacteria in the bottle also be able to "revive" after many years as well?

Again this is semantics. The cell may be alive but it is not useful to use in the sense that it cannot rapidly (in hours/days) convert ammonia to nitrite. Bacteria in a bottle can be revived and even an old bottle of bacteria is better than no bacteria (as long as they were the right bacteria in the first place). But people expect things in different time periods.

The reason for the "old wives tale" that bacteria in a bottle don't work is due more to the fact that the bacteria in the bottle were the WRONG bacteria in the first place so they didn't even work before placed in the bottle.

But for the cells to stay active they need to be fed and this cannot happen in a bottle. So once placed in the bottle the bacteria activity slowly fades - they are not dead but they are not real active hope that makes sense .

Thanks in advance for your reply. Chris L.

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From the above I conclude the following:

First- It is possible to slow down, stall or outright destroy the cycling process by over-dosing ammonia. However, the effect is a function of how much ammonia in combination with other water related issues such as pH, temp. etc. However, 5 ppm would be the advisable maximum limit as it is about here that inhibition starts to occur and by about 10 ppm it is killing the bacteria. It isn't the eleveated ammonia levels that do the killing, it is the elevated nitrite the ammonia eaters produce that does it. However, the cause is the overdosing of the ammonia and the elevated nitrate can kill the ammonia oxidizers as well the nitrite ones..

I wanted to look at the study Dr. Tim referenced, so I entered "Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite" into Google. I was amazed by what came back in terms of quantity and not just limited to that specific study. There is a lot of independant research which supports what Dr Tim is saying in this regard. You can reduce the 7500+ responses by using Google Scholar to limit them to just over 300.


Second- If a bottled bacteria additive contains the proper strains of bacteria which have been cultured on a hard surface and have formed an EPS coating, they can remain sufficiently active for up to a year after being bottled to assist in cycling a tank fairly rapidly (six months is a safer guidline). After that they are normally too dormant to revive in time to prevent rising ammonia and nitrite levels where fish are involved. However, the bacteria are not dead, the are just very, very dormant and reviving them would take more time. Plus to then get them back to being able to reproduce takes even much longer. However, if one wants to use them, they can become active again. (This all assumes that the bacteria have not been subjected to fatal conditions such as freezing or over heating.)


Third- A solid surface on which the bacteria can colonize and form a protective coating of EPS is crucial to establishing a fully and properly cycled tank. To that extent it is clear that bacteria not only colonize the filter media but other surfaces, especially, but not limited to, the substrate. If you doubt this, I am waiting to hear your explanation for how and why a UGF works.

Related to this one can do a Google Scholar search for "exopolymeric substances" and discover what Dr. Tim states about them is indeed true. You will find EPS mentioned/discussed/measured in countless studies in a very wide variety of bacteria, not just our tank ones. EPS play a central role in the ability of bacteria to live and to survive adverse conditions. It really is a fascinating topic.


Fourth- Once you get a tank fully cycled and well established to the extent the bacteria develop their EPS coating, you can remove all the fish and all other sources of ammonia and just keep the filter running and the bacteria will not die (just don't let them freeze or overheat), they will, at worst, begin to become inactive/dormant. However, you can revive them and restore the tank to functionally cycled by adding an ammonia source. How long it takes to revive them depends upon how long they have been inactively dormant. But surely at least 6 months as conditions in tank are much more hospitable than in a bottle. I would not be surprised if this were not also possible after twice that time. So much for folks who claim 10% of the bacteria dies each day etc. etc.


Fifth- Actually killing established tank bacteria takes active action such as freezing it, heating it to 104 degrees F for at least a day, less the higher you go. Killing it involves breaking the cell wall to damage the inside. This is how chlorine/chloramine kills our nitrifyers. The upshot is, we cant kill the established bacteria in our tanks merely by withdrawing food and/or oxygen. All we can do this way would be to make them become inactive/dormant.

Sixth- Helping others seed their tanks by shipping them your established bacteria is a snap. All you need to do is ship some of your substrate and make sure it stays moist and the temp stays clearly above freezing and below the hi 90sF/36.5C range. If it takes a week or a month it should arrive ready to use and alive. Of course some of your bio-media works even better.

I am curious what other folks think about all this.

 

[Prime Ordeal]

Lots of interesting stuff in his reply.

There's one instantly obvious anomaly though. He says "Once the values get higher they become toxic and can kill the bacteria. It's most semantics the absolutely values depends on many environment conditions but the point is that you need to keep the nitrite low or the process will get stuck because the nitrite-oxidizing bacteria cannot function at these higher levels."

The inhibitory effect of high nitrite levels is well known, which is why I always recommend water changes during cycling to keep them below 5ppm (i.e. within a measurable range). But to say that the NOB cannot function at higher levels is plainly wrong. If that were the case then following the cycling method on this forum, where nitrite is allowed to build to ridiculous levels (low 100's ppm sometimes) would permanently stall the cycle and we know anecdotally that many have cycled successfully using this method.

 

[TwoTankAmin]

But to say that the NOB cannot function at higher levels is plainly wrong. If that were the case then following the cycling method on this forum, where nitrite is allowed to build to ridiculous levels (low 100's ppm sometimes) would permanently stall the cycle and we know anecdotally that many have cycled successfully using this method.

Bah is all I can say. this is the scientific section.

When you that "But to say that the NOB cannot function at higher levels is plainly wrong." Unless you have published peer reviewed research proving this, than what you say is basically meaningless in a scientific forum. If you have published, please link us to your research, if not then provide links to the the appropriate peer reviewed research by anybody else who has published such work.

Anecdotal evidence has no place in a scientific discussion. Personally, I doubt the veracity of any such reports about cycling in the presence off 100+ppm of nitrite.

Unless one is trained on the graduate level in chemistry and microbiology it can be difficult to make sense of the scientific research one can find. A lot of the information can be hard to digest for us lay people. But if you look you will finds tons of references to nitrite/free nitrous acid as being inhibitor or toxic to nitrifying bacteria in many published research studies.

Fortunately, I managed to stumble across a good, easy to understand and credible source of info on this topic. It is titled "Ammonia and Nitrification Information Sheet" and is written by a company called ENVA Water Treatment which is based in Ireland. They appear to be a 20+ year old outfit which is involved in all forms of water treatment: "Our customer base is nationwide and covers the entire spectrum of engineering, pharmachem, food processing, paper, general industrial, municipal water and wastewater."

From reading on their site they appear to know what they are doing. You can decide that for yourself if you would like to surf their site ENVA Water Treatment

Here are some key point they make from the above mentioned "Ammonia and Nitrification Information Sheet":

"How does nitrification take place in an activated sludge system? It is important to note that it is the ammonium ion (NH4+) and not ammonia (NH3) that is oxidized during nitrification.

Nitrification is a bio-chemical reaction that occurs inside bacteria. Two species of bacteria are involved in the process - Nitrosomonas and Nitrobacter. These bacteria are collectively known as nitrifiers and are autotrophic, i.e. they get their carbon
source from inorganic carbon (carbonates, bicarbonates) or carbon dioxide. (The bacteria responsible for breaking down carbonaceous BOD in an activated sludge system are known as organotrophs or heterotrophs, i.e. they require an organic source of carbon). Approximately 90-97% of the bacteria in an activated sludge process are organotrophic, while the remaining 3-10% of
bacteria are autotrophic (nitrifiers). Nitrifiers possess cytomembranes, which are extensions of the cell membrane away from the cell wall and toward the cytoplasm. These are the active sites for oxidation of ammonium and nitrite ions. It is on the cytomembranes of Nitrosomonas and Nitrobacter, where ammonium ions and nitrite ions, respectively, come in contact with enzymes that add oxygen to each ion.

Nitrifiers are obligate aerobes, i.e. they require free molecular oxygen and are killed off by anaerobic conditions. Maximum nitrification occurs at a D.O. (Dissolved Oxygen) level of 3.0 mg/l. Significant nitrification occurs at a D.O. level of 2.0 to 2.9 mg/l. Nitrification
ceases at D.O. levels of <0.5 mg/l.

Nitrification is temperature sensitive. The optimum temperature for nitrification is generally considered to be 30°C. Temperature and Nitrification (after Gerardi, M.H., 2002)
Temperature-----------Effect upon Nitrification
>45°C-------------------Nitrification ceases
28-32°C---------------Optimal temperature range
16°C-------------------Approx. 50% of nitrification rate at 30°C
10°C-------------------Significant reduction in nitrification rate - 20% of rate at 30°C
< 5°C-------------------Nitrification ceases

Nitrous acid (HNO2) is also produced during the oxidation of ammonium ions.

e. Inhibition/Toxicity
Inhibition is temporary short-term or long-term loss of enzymatic activity. Toxicity is permanent loss of enzymatic activity or irreversible damage to cellular structure. Small increases in inhibitory wastes can cause a dramatic reduction in nitrification.

Nitrifiers grow slowly and only account for a small portion of the bacterial assemblage in an aeration system. Wastes toxic to nitrifiers include cyanide, heavy metals, mercaptans, phenols and thiourea.

Nitrifiers are excellent indicators of toxic shock in an effluent treatment plant – significant loss of nitrification will occur before loss in efficiency of carbonaceous BOD removal. This can give an operator time to take remedial action to prevent excess damage to the organotrophic population.

Nitrifying bacteria are also inhibited by relatively low concentrations of free ammonia (10 mg/l for Nitrosomonas; 0.1 mg/l for Nitrobacter) and free nitrous acid (1.0 mg/l for both Nitrosomonas and Nitrobacter). Free ammonia (NH3) is produced from ammonium ions under a high pH in the aeration tank. Free nitrous acid (NHO2) is produced from nitrite ions under a low pH in the aeration tank. This type of inhibition is known as substrate inhibition. Substrate inhibition usually occurs at a concentration of 400-500 mg/l ammonium ions or when ammonium ions are converted to nitrite ions at a faster rate than nitrite ions are converted to nitrate ions."
From http/www.google.co...S54W0Mg&cad=rja

I have highlighted two pieces of the info especially relavant to this discussion. The second time was to make sure it is clear that elevated nitrites via free nitrous acid at a very low level begins to inhibit nitrification and elevated ammonia does the same.

The first time was to insure folks understand that free nitrous acid is a natural by product of the ammonia oxidation phase of the nitrification cycle. Why?

The role of nitrite and free nitrous acid (FNA) in wastewater treatment plants.
Zhou Y, Oehmen A, Lim M, Vadivelu V, Ng WJ.
Source
Advanced Environmental Biotechnology Centre (AEBC), Nanyang Environment and Water Research Institute (NEWRI), Nanyang Technological University, School of Biological Science, Level N-B2-01, 60 Nanyang Avenue, Singapore 639798, Singapore. [email protected]

Abstract
Nitrite is known to accumulate in wastewater treatment plants (WWTPs) under certain environmental conditions. The protonated form of nitrite, free nitrous acid (FNA), has been found to cause severe inhibition to numerous bioprocesses at WWTPs. However, this inhibitory effect of FNA may possibly be gainfully exploited, such as repressing nitrite oxidizing bacteria (NOB) growth to achieve N removal via the nitrite shortcut. However, the inhibition threshold of FNA to repress NOB (∼0.02 mg HNO2-N/L) may also inhibit other bioprocesses. This paper reviews the inhibitory effects of FNA on nitrifiers, denitrifiers, anammox bacteria, phosphorus accumulating organisms (PAO), methanogens, and other microorganisms in populations used in WWTPs. The possible inhibition mechanisms of FNA on microorganisms are discussed and compared. It is concluded that a single inhibition mechanism is not sufficient to explain the negative impacts of FNA on microbial metabolisms and that multiple inhibitory effects can be generated from FNA. The review would suggest further research is necessary before the FNA inhibition mechanisms can be more effectively used to optimize WWTP bioprocesses. Perspectives on research directions, how the outcomes may be used to manipulate bioprocesses and the overall implications of FNA on WWTPs are also discussed.

Copyright © 2011 Elsevier Ltd. All rights reserved.

From http/www.ncbi.nlm....pubmed/21762944

Abstract
The effects of free ammonia (FA; NH[sub]3[/sub]) and free nitrous acid (FNA; HNO[sub]2[/sub]) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonaseuropaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO[sub]2[/sub], HCO[sub]3[/sub][sup]−[/sup], and CO[sub]3[/sub][sup]2−[/sup]). It was revealed that FA of up to 16.0 mgNH[sub]3[/sub]-N · L[sup]−1[/sup], which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO[sub]2[/sub]-N · L[sup]−1[/sup], and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO[sub]2[/sub]-N · L[sup]−1[/sup]. The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50–0.63 mgHNO[sub]2[/sub]-N · L[sup]−1[/sup]. The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH[sub]3[/sub]-N · L[sup]−1[/sup], independent of the presence or absence of inorganic carbon. © 2006 Wiley Periodicals, Inc.

From http/onlinelibrary...hly+maintenance

Rather than cluttering things up with full date as above here are the key sentence and the link to the abstract:

The much smaller values of K[sub]I[/sub] for nitrite oxidation reflected the susceptibility of that reaction to inhibition by FA and FNA, which could lead to accumulation of nitrite during nitrification. from http/www.sciencedi...359511309000506

MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, from http/onlinelibrary....22631/abstract

The inhibitory effect of free ammonia (NH[sub]3[/sub] or FA) and free nitrous acid (HNO[sub]2[/sub] or FNA) on the ammonia oxidizing bacteria (AOB) and the dependence of the AOB activity on the concentration of total inorganic carbon (TIC) are well-established. from http/www.sciencedi...960852410004591

I could keep listing, but it gets boring. The conclusions that one should draw from all of this is that elevated levels of free ammonia and even more so, free nitrous acid acts to inhibit nitrification at fairly low levels. At elevated levels it merely gets worse. And FNA is produced in our tanks by nitrite.

I will now wait for Prime Ordeal, or anybody else, to post the quotes and links to pulished research which supports his saying that Dr. Tim is wrong and further that neither elevated ammonia (really FA) nor nitrite (really FNA) don't prevent or inhibit the cycle nor even kill any of the bacteria.

 

[Prime Ordeal]

You're not telling me anything I don't already know TTA. Indeed my method of cycling (which is completely at odds with the methods suggested on this forum) is based directly on this information.

However, to suggest that the many people who have cycled a tank according to this forum's method are effectively making it all up is ridiculous. Even Bignose has an article somewhere on this forum where he doses every day for 20 odd days and still the tank cycles. In that time the tank would have accrued in excess of 100ppm nitrite. If the information about the degree of inhibition this would cause is true then it would never have cycled nor would any of the many other successfully cycled tanks. I can't explain the incongruity, but there it is.

I really think we believe the same science but I will always believe experiment before I will believe theory, that's the scientific method.

 

[snazy]

Sixth- Helping others seed their tanks by shipping them your established bacteria is a snap. All you need to do is ship some of your substrate and make sure it stays moist and the temp stays clearly above freezing and below the hi 90sF/36.5C range. If it takes a week or a month it should arrive ready to use and alive. Of course some of your bio-media works even better.

Just to add my piece of opinion on this. The water and the oxygen/ammonia/nitrite it carries needs to be in very close contact or literally flow through the surface where you want to grow bacteria, whether this is a sponge or substrate. That's why undergravel filters work as well. But in a tank without an undergravel filter there probably is little nitrifying bacteria established on the substrate.
If it was the case that a mature sponge or gravel with no flow through grows bacteria and cycles a tank, then why waste energy by turning on the filter?

 

[TwoTankAmin]

snazy based on what you wrote above would you be surprised to know that you can find many forums and fish related sites which say the exact opposite. None of them would qualify as being scientific evidence any more than what you stated above or most of what Prime Ordeal has either. In fact, you state its your opinion. If I undersatnd what your post is meant to say is that there is virtually no meaningful amount of bacteria on/in the substrate. Since this is the scentific forum and you have stated " But in a tank without an undergravel filter there probably is little nitrifying bacteria established on the substrate." I challenge you to offer scientific proof of what you say.

1. The substrate for a fish only aquarium serves the purpose of providing a surface for the beneficial bacteria to build colonies.
from http/www.algone.co...arium-substrate

2. The lions share of nitrifying bacteria resides in the substrate and filter media.
from http/freshaquarium.../f/seedtank.htm

3. The greatest population of bacteria in a healthy balanced aquarium occurs in the substrate, not the filter. The floc or humic compost that collects in the substrate is the host for the biofilms; this is why the substrate in planted tanks should never be disturbed, and many aquarists apply this to non-planted tanks as well. from http/www.tropicalf...aquarium-74891/

4. The substrate layer acts as a medium harboring beneficial bacteria. These beneficial bacteria are responsible for the breakdown of organics in the aquarium. Known as the nitrogen cycle. These bacteria will colonize on any surface in the aquarium. Having substrate in an aquarium adds much surface area for the bacteria to colonize.from http/mraqua.net/th...ting-substrate/

5. The bacteria responsible for oxidizing ammonia to nitrite, and nitrite to nitrate are called lithotrophic bacteria, or lithotrophs. This name means "rock-lover" or "rock-grower", because they are found firmly attached to solid, usually quite firm (to them if not necessarily so to us), substrates. To these bacteria, plastic foams (sponges) are hard, as are most synthetic fibers. These bacteria do not occur as functional metabolizing and multiplying forms in the water column, only when attached. They can and will occupy any solid surface in the tank - gravel, rocks, glass, filter sponges or floss of fiber mats, ceramic or glass beads, plastics, etc. from http/theaquariumwi...:_Nitrification

6. The slow exchange of water through the sand bed allows two important and distinct ecological layers to develop. The top layer of the bed which is in contact with oxygen rich water is heavily colonized by the nitrifying bacteria from the Nitrosopira and Nitrospira genus. from http/www.aquaworld...lent_killer.htm

7. A goldfish aquarium doesn't need a gravel substrate but the goldfish in that goldfish aquarium surely would appreciate it. Not only is a gravel substrate aesthetically pleasing, it creates a place for beneficial nitrifying bacteria to grow. from http/howtotakecare...avel-substrate/

BTW- I have never said there is no bacteria in a filter, only that there is a decent amount of it on the substrate and other hard surfaces of a tank. My point for the list above is that one can find many fish sites that say many things, most of the above is stated just the way folks on this forum also make statements of fact without being able to provide scientific support. I am reminded of that old story attributed to Abe Lincoln:

Abraham Lincoln faced with some thorny issue that could be settled by a twist of language, or a slight abuse of power, asks his questioner how many legs would a dog have, if we called the dog's tail, a leg. "Five," the questioner responds confident in his mathematical ability to do simple addition. "No," Lincoln says. "Calling a dog's tail a leg, doesn't make it a leg."

Prime- I still can not find any reference to 100ppm nitrite levels in cycling anywhere on this site. Some of that is because the search engine here isn't real sophisticated so it is hard to find certain things. Please link me. I did find the fishless cycling article in the Beginner's Resource Center and it makes absolutely no mention of nitrite levels exceeding 100ppm. In fact the only thing it mentioned about nitrites was that they would exceed the maximum level shown on the FW API FW kit which is 5 ppm.

And I am still waiting for you to post one single shred of scientific proof for anything you have said in our back and forths. Despite this being the Scientific Forum you have not posted anything scientific in nature- not one link to research, not one quote from any scientific authority. I have provided numerous links and quotes from independent sources and you have replied with anecdotes and opinions. You have failed to refute any of the evidence I have presented. A reasonable person might conclude that you are unable to do so.

I have also come across an article by Dr. Hovanec in which he explains, in more lay friendly terms, the methods and results of his research. Since at the end it says: "© Timothy Hovanec. May not be reproduced without permission", I will only provide the link to it rather than any quotes: http/www.alltropic...t/view/119/101/

 

[snazy]

4. The substrate layer acts as a medium harboring beneficial bacteria. These beneficial bacteria are responsible for the breakdown of organics in the aquarium. Known as the nitrogen cycle. These bacteria will colonize on any surface in the aquarium. Having substrate in an aquarium adds much surface area for the bacteria to colonize.from http/mraqua.net/th...ting-substrate/

The substrate for a fish only aquarium serves the purpose of providing a surface for the beneficial bacteria to build colonies.
from http/www.algone.co...arium-substrate

There are many types of beneficial bacteria in an aquarium. But not all of it is responsible for converting ammonia and nitrite. Which ones are we talking about?

There is a type of bacteria that lives deep in the substrate which is heterotrophic facultative.
It means that in anaerobic conditions when there is no oxygen, it converts organic matter(dead plants, fish waste, etc) to ammonia. Which in turn gets converted by the autotrophic bacteria like ammonia/nitrite oxydizers to nitrite. However, this bacteria can also be facultative, meaning it will perform different functions when it is exposed to oxygen(when stirring the substrate regularly for example). One of this functions is converting ammonia to nitrite at a very slow level compared to the "true" nitrifiers like nitrosomonas and nitrospira in fresh water tanks(some articles state the rate is as slow as 1 : 1 000 000 compared to the autotrophic nitrifiers).
Another function this facultative heterotrophs perform is the denitrification process(nitrate to nitrogen gas conversion).

Point 1, 2 and 4 from your statement above seem to be talking about the beneficial bacteria, but not specifically the ammonia/nitrite oxydizers in fresh water tanks.

Lithotroph means rock eaters, or in other words inorganic matter eaters, and it has nothing to do with the bacteria loving rocks or gravel to live on. The oposite of lithotrophs is organotrophs.


So in this sense nitrifying bacteria is lithotrophs to which I agree. But I think you are understanding the term lithotrophs incorrectly.
Ammonia/nitrite oxydizers are lithotrophs/chemoautotrophs that use inorganic matter like ammonia/nitrite and oxygen to perform their nitrifying function

5. The bacteria responsible for oxidizing ammonia to nitrite, and nitrite to nitrate are called lithotrophic bacteria, or lithotrophs. This name means "rock-lover" or "rock-grower", because they are found firmly attached to solid, usually quite firm (to them if not necessarily so to us), substrates. To these bacteria, plastic foams (sponges) are hard, as are most synthetic fibers. These bacteria do not occur as functional metabolizing and multiplying forms in the water column, only when attached. They can and will occupy any solid surface in the tank - gravel, rocks, glass, filter sponges or floss of fiber mats, ceramic or glass beads, plastics, etc. from http/theaquariumwi...:_Nitrification

The article given in point 5 in your statement above actually says the following:

"but the most favorable dwelling for them is in the filter. That is the case whether it is gravel for a UG or RFUG, sponges, floss, beads, spheres, or whatever, in canisters or HOT/HOB power filters, or in fluidized bed filters. This is the most favorable location for them because the constant flow of water is providing the bugs with the two things they need most after an attachment site - oxygen and an energy source (ammonia or nitrite) along with an assortment of other nutrients."

Which I was trying to explain in my previous post about the water flow and access to oxygen/ammonia/nitrite

I would agree that in the surface layers in the substrate in a tank there is a bit of nitrifiers established. But this is only because there is oxygen in the upper layers of the substrate. In the lower layers, there is different beneficial bacteria , but they are not the ammonia/nitrite oxydizing bacteria which I thought we were talking about.

The question is how much of ammonia/nitrite oxydizers are established on the upper layer of the substrate compared to the filter media?
Well in my opinion that depends on the fish tank itself.
You can have many ammonia/nitrifiers in the substrate in your tank I may have none.
In some tanks, there maybe none due to lack of maintanace, waste build up in the substrate depriving it from oxygen, or if the gravel is very compact and doesn't allow oxygen flow, etc...

Diana Walstad's method is based on using many routed plants in the substrate and also using substrate that doesn't get compacted easily. This way oxygen is reaching more levels and the nitrifiers get established there.

 

[snazy]

Prime- I still can not find any reference to 100ppm nitrite levels in cycling anywhere on this site. Some of that is because the search engine here isn't real sophisticated so it is hard to find certain things.

Not addressed to me, but I think he bases that on the ratio of molecular weights of ammonia/nitrite/nitrate.
According to this article(http/www.coloradokoi.com/nitrific.htm), the ratio is 17/46/62 respectively.
Which means that 1ppm ammonia can convert to 2.7ppm nitrIte which in respect converts to 3.65 nitrAte.

In simple maths, 100ppm nitrite can accumulate in the absence of nitrite converting bacteria when 4ppm ammonia is dosed for 9 days in a row(4ppm ammonia multiplied by 2.7 equals 10.7ppm nitrIte. Multiply that by 9 days of ammonia dosing and you are going to come up to a figure of nearly 100ppm nitrIte.)
Which of course we can't measure with the Api test kit as it only measures up to 5ppm. Normally when that't the case, the Api nitrite test turns purple in the matter of seconds, or turns completely clear colour not available on the chart.

 

[Prime Ordeal]

Prime- I still can not find any reference to 100ppm nitrite levels in cycling anywhere on this site. Some of that is because the search engine here isn't real sophisticated so it is hard to find certain things.

Not addressed to me, but I think he bases that on the ratio of molecular weights of ammonia/nitrite/nitrate.
According to this article(http/www.coloradokoi.com/nitrific.htm), the ratio is 17/46/62 respectively.
Which means that 1ppm ammonia can convert to 2.7ppm nitrIte which in respect converts to 3.65 nitrAte.

In simple maths, 100ppm nitrite can accumulate in the absence of nitrite converting bacteria when 4ppm ammonia is dosed for 9 days in a row(4ppm ammonia multiplied by 2.7 equals 10.7ppm nitrIte. Multiply that by 9 days of ammonia dosing and you are going to come up to a figure of nearly 100ppm nitrIte.)
Which of course we can't measure with the Api test kit as it only measures up to 5ppm. Normally when that't the case, the Api nitrite test turns purple in the matter of seconds, or turns completely clear colour not available on the chart.

I'm glad somebody in this thread has the common sense and basic maths skills to not require a citation for even the most mundane of facts. And imagine it's one of those long cycles that goes on for three,four, five weeks or more of dosing!

 

[raptorrex]

This is the origin of this thread:

I figured the easiest solution was to contact Dr. Hovanec directly and ask him to clarify things. I will post both my email his reply in a new thread when I receive it. What is most interesting is he says 5 ppm can start to poison and then that 10 ppm will inhibit them in different places.

I also asked him to clarify the death of a bacteria vis-a-vis rupturing of the cell wall etc. He says that the bacteria in a bottle only remain usefull for about one year max. But he also states that these bacteria have survived on the planet for millions of years because they can shut down during periods of extended drought. So I asked if the can survive extended drought and continue the survival of the species, then why can't they do the came after a few years in a bottle.

He has replied to past emails so I am assuming I will get an answer but it may take time as I am sure he is a busy man.

From http/www.fishforum...385852-ammonia/


Here is the exchange- My questions are in black and his replies are in blue:

Hello - thanks for email, please see below for comments to your questions.

On Feb 1, 2012, at 6:37 PM, cwl........ wrote:

Hi- I have been a fan of your work and products for a number of years. I am a bit confused about certain facts regarding these bacteria and have read conflicting statements on your site and could use a little clarification.

In one place you state that levels of ammonia over 5 ppm can poison nitrite oxidizers.

"Nitrite does not spike until somewhere between days 14 and 20. You want to be careful adding more ammonia because you do not want the nitrite-nitrogen over 5 mg/L as this will start to poison the nitrite-oxidizing bacteria."

To me this means that these levels can and will kill the bacteria. However, In another part of the site you state:

"If the ammonia or nitrite concentration in the aquarium is above 10 ppm (as nitrogen) or 10 mg/L-N you should do a partial water change before adding One & Only. The reason is that these bacteria prefer low concentrations of ammonia/nitrite and can actually be inhibited by high levels of these chemicals."

This implies not killing but just slowing them down and that would imply lowering the levels would allow the bacteria to "revive". Can you give me a bit more precise explaination about the effects of elevated ammonia/nitrites on the bacteria.


As I wrote at a nitrite concentration of 5 mg/L of "starts to poison" the bacteria. This is just like if you were in a closed room and it was filled to a low level of carbon monoxide - you would feel sick and have headaches and you are slowly being poisoned. You probably won't die at this low level but you surely are not healthy.

Once the values get higher they become toxic and can kill the bacteria. It's most semantics the absolutely values depends on many environment conditions but the point is that you need to keep the nitrite low or the process will get stuck because the nitrite-oxidizing bacteria cannot function at these higher levels.

How it works varies by bacterial type for ammonia -oxidizers the nitrite effect the enzyme AMO. You can search for this article and read more should you wish the details
Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite LISA Y. STEIN AND DANIEL J. ARP* Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 Received 16 April 1998/Accepted 28 July Also, in your articles on fishless cycling and bacteria living in bottle you state:

"The only way a bacterial cell truly 'dies' is if the cell wall breaks apart (called cell lyses) and the cell contents spill out or if the cell in poisoned by some substance that penetrates the cell wall and kills the cell."

However, you also indicate there is a maximum useful time that the bacteria can survivie in a bottle:

"They can live in a bottle but under optimal conditions the time period is about one year. The nitrifying bacteria don't die in the bottle, their activity level drops and eventually it becomes so low that there is little measurable positive effect when they are poured into the aquarium water."

I am again confused. Especially in light of your statement which says:

"Nitrifiers belong to a very old line of bacteria (milloins of years) and they have developed ways to cope with very long periods of "drought."

This would seem to indicate that they can "shut down" for extended periods and come back to "life" once better conditions are present. So why wouldn't the bacteria in the bottle also be able to "revive" after many years as well?

Again this is semantics. The cell may be alive but it is not useful to use in the sense that it cannot rapidly (in hours/days) convert ammonia to nitrite. Bacteria in a bottle can be revived and even an old bottle of bacteria is better than no bacteria (as long as they were the right bacteria in the first place). But people expect things in different time periods.

The reason for the "old wives tale" that bacteria in a bottle don't work is due more to the fact that the bacteria in the bottle were the WRONG bacteria in the first place so they didn't even work before placed in the bottle.

But for the cells to stay active they need to be fed and this cannot happen in a bottle. So once placed in the bottle the bacteria activity slowly fades - they are not dead but they are not real active hope that makes sense .

Thanks in advance for your reply. Chris L.

-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --


From the above I conclude the following:

First- It is possible to slow down, stall or outright destroy the cycling process by over-dosing ammonia. However, the effect is a function of how much ammonia in combination with other water related issues such as pH, temp. etc. However, 5 ppm would be the advisable maximum limit as it is about here that inhibition starts to occur and by about 10 ppm it is killing the bacteria. It isn't the eleveated ammonia levels that do the killing, it is the elevated nitrite the ammonia eaters produce that does it. However, the cause is the overdosing of the ammonia and the elevated nitrate can kill the ammonia oxidizers as well the nitrite ones..

I wanted to look at the study Dr. Tim referenced, so I entered "Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite" into Google. I was amazed by what came back in terms of quantity and not just limited to that specific study. There is a lot of independant research which supports what Dr Tim is saying in this regard. You can reduce the 7500+ responses by using Google Scholar to limit them to just over 300.


Second- If a bottled bacteria additive contains the proper strains of bacteria which have been cultured on a hard surface and have formed an EPS coating, they can remain sufficiently active for up to a year after being bottled to assist in cycling a tank fairly rapidly (six months is a safer guidline). After that they are normally too dormant to revive in time to prevent rising ammonia and nitrite levels where fish are involved. However, the bacteria are not dead, the are just very, very dormant and reviving them would take more time. Plus to then get them back to being able to reproduce takes even much longer. However, if one wants to use them, they can become active again. (This all assumes that the bacteria have not been subjected to fatal conditions such as freezing or over heating.)


Third- A solid surface on which the bacteria can colonize and form a protective coating of EPS is crucial to establishing a fully and properly cycled tank. To that extent it is clear that bacteria not only colonize the filter media but other surfaces, especially, but not limited to, the substrate. If you doubt this, I am waiting to hear your explanation for how and why a UGF works.

Related to this one can do a Google Scholar search for "exopolymeric substances" and discover what Dr. Tim states about them is indeed true. You will find EPS mentioned/discussed/measured in countless studies in a very wide variety of bacteria, not just our tank ones. EPS play a central role in the ability of bacteria to live and to survive adverse conditions. It really is a fascinating topic.


Fourth- Once you get a tank fully cycled and well established to the extent the bacteria develop their EPS coating, you can remove all the fish and all other sources of ammonia and just keep the filter running and the bacteria will not die (just don't let them freeze or overheat), they will, at worst, begin to become inactive/dormant. However, you can revive them and restore the tank to functionally cycled by adding an ammonia source. How long it takes to revive them depends upon how long they have been inactively dormant. But surely at least 6 months as conditions in tank are much more hospitable than in a bottle. I would not be surprised if this were not also possible after twice that time. So much for folks who claim 10% of the bacteria dies each day etc. etc.


Fifth- Actually killing established tank bacteria takes active action such as freezing it, heating it to 104 degrees F for at least a day, less the higher you go. Killing it involves breaking the cell wall to damage the inside. This is how chlorine/chloramine kills our nitrifyers. The upshot is, we cant kill the established bacteria in our tanks merely by withdrawing food and/or oxygen. All we can do this way would be to make them become inactive/dormant.

Sixth- Helping others seed their tanks by shipping them your established bacteria is a snap. All you need to do is ship some of your substrate and make sure it stays moist and the temp stays clearly above freezing and below the hi 90sF/36.5C range. If it takes a week or a month it should arrive ready to use and alive. Of course some of your bio-media works even better.

I am curious what other folks think about all this.

oh dear, conned by more sales speak.

this tosh is not peer reviewed. its got some words from his business partner. not to mention the fact it would work, reliably. if it were half what its claimed.
still you seem to be enjoying yourself and nobody with knowledge is will be fooled. so go ahead, fill your boots.

 

[snazy]

If someone is curious how long the ammonia oxydizer bacteria can survive without any ammonia source, I found an interesting article: http/aem.asm.org/content/71/3/1276.full.pdf
According to which AOB species can recover quite rapidly even if deprived from ammonia source for weeks. Wouldn't that suggest they can be preserved in a bottle?

Here is a quote from the above article based on depriving the AOBs from ammonia for periods of time:

In summary, we demonstrated that N. briensis is able to
recover rapidly after periods of starvation up to 2 weeks. The
culture responded within minutes to the addition of fresh
NH4

and the maximum potential ammonia-oxidizing activity
was reached after 30 to 60 min (Fig. 4). A fast recovery after
starvation within the first weeks has been observed for other
AOB, e.g., Nitrosomonas europaea, Nitrosomonas cryotolerans,
and the culture G5-7, closely related to Nitrosomonas oligotropha
(8, 17, 23, 31, 46). Recovery after longer starvation periods
revealed differences between the AOB strains; Nitrosomonas
europaea and Nitrosomonas cryotolerans were recovering very
fast after 10 weeks of starvation, whereas the culture G5-7
and N. briensis showed a longer lag time before they regained
their activity (8, 23, 32). Despite this recovery after longer
starvation periods of N. briensis that was slower than with other
ammonia-oxidizing bacteria, N. briensis showed recovery patterns
similar to other AOB with respect to short-term starvation.
The delay in reaching the maximum potential ammoniaoxidizing
activity after starvation (Fig. 4) could be explained by
the need for key molecules necessary for the metabolism. The
addition of the intermediate NH2OH to a starved culture of
Nitrosomonas europaea reduced the time delay before reaching
the maximum activity (17).

 

[Prime Ordeal]

This is the origin of this thread:

I figured the easiest solution was to contact Dr. Hovanec directly and ask him to clarify things. I will post both my email his reply in a new thread when I receive it. What is most interesting is he says 5 ppm can start to poison and then that 10 ppm will inhibit them in different places.

I also asked him to clarify the death of a bacteria vis-a-vis rupturing of the cell wall etc. He says that the bacteria in a bottle only remain usefull for about one year max. But he also states that these bacteria have survived on the planet for millions of years because they can shut down during periods of extended drought. So I asked if the can survive extended drought and continue the survival of the species, then why can't they do the came after a few years in a bottle.

He has replied to past emails so I am assuming I will get an answer but it may take time as I am sure he is a busy man.

From http/www.fishforum...385852-ammonia/


Here is the exchange- My questions are in black and his replies are in blue:

Hello - thanks for email, please see below for comments to your questions.

On Feb 1, 2012, at 6:37 PM, cwl........ wrote:

Hi- I have been a fan of your work and products for a number of years. I am a bit confused about certain facts regarding these bacteria and have read conflicting statements on your site and could use a little clarification.

In one place you state that levels of ammonia over 5 ppm can poison nitrite oxidizers.

"Nitrite does not spike until somewhere between days 14 and 20. You want to be careful adding more ammonia because you do not want the nitrite-nitrogen over 5 mg/L as this will start to poison the nitrite-oxidizing bacteria."

To me this means that these levels can and will kill the bacteria. However, In another part of the site you state:

"If the ammonia or nitrite concentration in the aquarium is above 10 ppm (as nitrogen) or 10 mg/L-N you should do a partial water change before adding One & Only. The reason is that these bacteria prefer low concentrations of ammonia/nitrite and can actually be inhibited by high levels of these chemicals."

This implies not killing but just slowing them down and that would imply lowering the levels would allow the bacteria to "revive". Can you give me a bit more precise explaination about the effects of elevated ammonia/nitrites on the bacteria.


As I wrote at a nitrite concentration of 5 mg/L of "starts to poison" the bacteria. This is just like if you were in a closed room and it was filled to a low level of carbon monoxide - you would feel sick and have headaches and you are slowly being poisoned. You probably won't die at this low level but you surely are not healthy.

Once the values get higher they become toxic and can kill the bacteria. It's most semantics the absolutely values depends on many environment conditions but the point is that you need to keep the nitrite low or the process will get stuck because the nitrite-oxidizing bacteria cannot function at these higher levels.

How it works varies by bacterial type for ammonia -oxidizers the nitrite effect the enzyme AMO. You can search for this article and read more should you wish the details
Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite LISA Y. STEIN AND DANIEL J. ARP* Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 Received 16 April 1998/Accepted 28 July Also, in your articles on fishless cycling and bacteria living in bottle you state:

"The only way a bacterial cell truly 'dies' is if the cell wall breaks apart (called cell lyses) and the cell contents spill out or if the cell in poisoned by some substance that penetrates the cell wall and kills the cell."

However, you also indicate there is a maximum useful time that the bacteria can survivie in a bottle:

"They can live in a bottle but under optimal conditions the time period is about one year. The nitrifying bacteria don't die in the bottle, their activity level drops and eventually it becomes so low that there is little measurable positive effect when they are poured into the aquarium water."

I am again confused. Especially in light of your statement which says:

"Nitrifiers belong to a very old line of bacteria (milloins of years) and they have developed ways to cope with very long periods of "drought."

This would seem to indicate that they can "shut down" for extended periods and come back to "life" once better conditions are present. So why wouldn't the bacteria in the bottle also be able to "revive" after many years as well?

Again this is semantics. The cell may be alive but it is not useful to use in the sense that it cannot rapidly (in hours/days) convert ammonia to nitrite. Bacteria in a bottle can be revived and even an old bottle of bacteria is better than no bacteria (as long as they were the right bacteria in the first place). But people expect things in different time periods.

The reason for the "old wives tale" that bacteria in a bottle don't work is due more to the fact that the bacteria in the bottle were the WRONG bacteria in the first place so they didn't even work before placed in the bottle.

But for the cells to stay active they need to be fed and this cannot happen in a bottle. So once placed in the bottle the bacteria activity slowly fades - they are not dead but they are not real active hope that makes sense .

Thanks in advance for your reply. Chris L.

-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --


From the above I conclude the following:

First- It is possible to slow down, stall or outright destroy the cycling process by over-dosing ammonia. However, the effect is a function of how much ammonia in combination with other water related issues such as pH, temp. etc. However, 5 ppm would be the advisable maximum limit as it is about here that inhibition starts to occur and by about 10 ppm it is killing the bacteria. It isn't the eleveated ammonia levels that do the killing, it is the elevated nitrite the ammonia eaters produce that does it. However, the cause is the overdosing of the ammonia and the elevated nitrate can kill the ammonia oxidizers as well the nitrite ones..

I wanted to look at the study Dr. Tim referenced, so I entered "Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite" into Google. I was amazed by what came back in terms of quantity and not just limited to that specific study. There is a lot of independant research which supports what Dr Tim is saying in this regard. You can reduce the 7500+ responses by using Google Scholar to limit them to just over 300.


Second- If a bottled bacteria additive contains the proper strains of bacteria which have been cultured on a hard surface and have formed an EPS coating, they can remain sufficiently active for up to a year after being bottled to assist in cycling a tank fairly rapidly (six months is a safer guidline). After that they are normally too dormant to revive in time to prevent rising ammonia and nitrite levels where fish are involved. However, the bacteria are not dead, the are just very, very dormant and reviving them would take more time. Plus to then get them back to being able to reproduce takes even much longer. However, if one wants to use them, they can become active again. (This all assumes that the bacteria have not been subjected to fatal conditions such as freezing or over heating.)


Third- A solid surface on which the bacteria can colonize and form a protective coating of EPS is crucial to establishing a fully and properly cycled tank. To that extent it is clear that bacteria not only colonize the filter media but other surfaces, especially, but not limited to, the substrate. If you doubt this, I am waiting to hear your explanation for how and why a UGF works.

Related to this one can do a Google Scholar search for "exopolymeric substances" and discover what Dr. Tim states about them is indeed true. You will find EPS mentioned/discussed/measured in countless studies in a very wide variety of bacteria, not just our tank ones. EPS play a central role in the ability of bacteria to live and to survive adverse conditions. It really is a fascinating topic.


Fourth- Once you get a tank fully cycled and well established to the extent the bacteria develop their EPS coating, you can remove all the fish and all other sources of ammonia and just keep the filter running and the bacteria will not die (just don't let them freeze or overheat), they will, at worst, begin to become inactive/dormant. However, you can revive them and restore the tank to functionally cycled by adding an ammonia source. How long it takes to revive them depends upon how long they have been inactively dormant. But surely at least 6 months as conditions in tank are much more hospitable than in a bottle. I would not be surprised if this were not also possible after twice that time. So much for folks who claim 10% of the bacteria dies each day etc. etc.


Fifth- Actually killing established tank bacteria takes active action such as freezing it, heating it to 104 degrees F for at least a day, less the higher you go. Killing it involves breaking the cell wall to damage the inside. This is how chlorine/chloramine kills our nitrifyers. The upshot is, we cant kill the established bacteria in our tanks merely by withdrawing food and/or oxygen. All we can do this way would be to make them become inactive/dormant.

Sixth- Helping others seed their tanks by shipping them your established bacteria is a snap. All you need to do is ship some of your substrate and make sure it stays moist and the temp stays clearly above freezing and below the hi 90sF/36.5C range. If it takes a week or a month it should arrive ready to use and alive. Of course some of your bio-media works even better.

I am curious what other folks think about all this.

oh dear, conned by more sales speak.

this tosh is not peer reviewed. its got some words from his business partner. not to mention the fact it would work, reliably. if it were half what its claimed.
still you seem to be enjoying yourself and nobody with knowledge is will be fooled. so go ahead, fill your boots.

This is why I didn't bother responding.

 

[TwoTankAmin]

Boy- between snazy and prime they have totally managed to misdirect this thread. Lets back up for a few seconds and look at how it started. The debate was about whether elevated ammonia and more importantly elevated levels of nitrites can stall the nitrogen cycle,

I provided a ton of evidence that it they do and Prime responded with anecdotes and insinuations that selling a product invalidates science. Prime- I believe you can not provide one shred of scientific evidence to support your contentions so you resort to trying to change the discussion. I still challenge folks here to link to one single scientific study that shows elevated nitrite levels do not trash the nitrogen cycle by inhibiting/stopping it and perhaps even killing some of the nitrifiers.

I said that article was a lay version of Dr. Hovanec's peer reviewed research, you respond by saying the lay version is not peer reviewed. You know full well the actual research is both valid and accepted by the scientific community today and is peer reviewed because you still can not provide support for what you say about high nitrite levels from any respected and accepted researchers any where in the world.

snazy- you have replied to this discussion by trying to change the topic as well, you have tried to bring all sorts of other bacteria into the discussion and unfortunately you have gotten a bit of the info wrong. This thread is about lithotrophic nitrifying bacteria and whether or not ammonia and nitrite levels can and do inhibit or kill them.

I will respond to the other bacteria issues in a separate thread which I will start in this section soon which will look at the denitrification process the deeper substrate anaerobic bacteria as well as some of the others you mentioned.

However let me respond to a couple of your comments. Lithotroph ltterally does mean rock eaters. But what is meant by that is the fact they can take energy from inorganic sources. Here is a quick overview:

Lithotrophic Bacteria - Rock Eaters

©2000 Timothy Paustian, University of Wisconsin-Madison
So far in catabolism we have spent our time looking at microbes that use organic compounds as their source of food (energy and building blocks for making cells). Interest in these species is understandable since our metabolism is very similar, but this ignores a large group of microbes that are capable of using inorganic substances as their source of energy. They are termed lithotrophs, literally meaning rock eaters.


General Concepts

• Common habitats of lithotrophs include waste water, volcanoes, deep sea ocean vents, the atmosphere, mines, seawater, fresh water. Basically they can be found everywhere.
• Energy is generated from reduced inorganic molecules. These molecules have high potential electrons that can be used to drive ETS. Typical energy substrates include, H[sub]2[/sub], CH[sub]4[/sub], CO, S, H[sub]2[/sub]S, NH[sub]4[/sub], NO[sub]2[/sub], N[sub]2[/sub]O, Fe[sup]+2[/sup] and Mn[sup]+2[/sup].
• Oxygen is often, though not always, the terminal electron accepter. Due to its willingness to accept electrons, oxygen gives the largest energy gain and is often employed by lithotrophs when available.
• Cell carbon is often from CO[sub]2[/sub] frequently using the Calvin Cycle. There are also microbes that can grow heterotrophically.
• Energy yields from lithotrophy are low per substrate oxidized and a large amount of substrate has to be metabolized per cell.
• Their appetite for substrate makes lithotrophs important in the global cycling of elements that they attack. Lithotrophs have a major impact on the movement of nitrogen, sulfur and carbon though the biosphere.

To help the reader get a general idea of what these microbes are like, we look at two interesting lithotrophs. These are just two examples in a sea (pun intended) of many aquatic and terrestrial lithotrophs.

from http/dwb.unl.edu/T...ithotrophs.html

The author proceeds from the above points with a discussion of nitrifying bacteria. Click through to see it.

The thing about the nitrifying bacteria is that they are not free swimming in the water. They need to attach to a solid surface, not for food, but to provide them a place to which they can anchor and then, in concert with a host of other bacteria to create the EPS shield which protects them. I can easily provide links to research showing the "bio-film" actually houses a diverse number of bacteria which all are contributing to making the bio-film and happily living together in it. These bacteria do not actively seek out food, they wait for it to come to them. The need to attach is why fish keepers can share gravel or much of the other decor from a healthy cycled tank to act as seed material for new tanks. If there were no signiificant bacteria present in surface substrate, then how much help in reducing cycling times can this process be? The EPS coating is why the bacteria can survive during times of adverse conditions, so it also a survival mechanism.

Moreover, this is why if you have ever used a bottle of Dr Tome's One and Only you will discover after shaking the bottle as per the directions, what pours out is a milky substance- the bacteria have been cultered on a solid medium which is then put into the bottle. The bacteria arrive attached and encapsulated in bio-film (EPS). Since I have never used any of the other barns , I can not say how they are packaged.

Now there has never been any doubt about one fact. The nitrifying bacteria need food and oxygen to thrive and reproduce. So the largest concentration of nitrifying bacteria in any tank will be found where the best sources of food and oxygen are. Clearly this should be in the filter if it is properly maintained. Dirty filters that do not have their mech media changed regularly and bio-media rinsed to keep it from clogging will always have lower bacterial counts than they would if properly maintained. And the fewer bacteria supported in the filter, the more that must live elsewhere in a cycled tank system.

Even when the filter is well maintained, this doesn't mean that there will not be significant portions of the tank's total beneficial bacteria in other parts of a tank. (significant does not imply the majority). In a healthy tank the circulation throughout the tank should be good. If you have a healthy tank with any kind of substrate, be it sand, gravel or some other choice, there should be adequate circulation to encourage lots of bacteria to colonize that as well as the filter(s). It may not be the majority, but it will be a meaningful amount and if one was to remove all of the substrate from a healthy cycled tank, they will get ammonia and nitririte spikes soon after.

I would go a step further. Any tank with substrate that does not have adequate circulation to keep a decent supply of ammonia and oxygen moving across the surface of the substrate will also have a lot of problems with fish health and likely not be able to keep bottom dwellers like corys, plecos, loaches and catfish in general alive very long. And maybe other fish will suffer as well.

But again all of this digresses from the original issue of whether or not elevated levels of nitrite can interfere with the the nitrifying bacteria and the nitrogen cycle I have provided ample scientific evidence that elevated/high nitrites do inhibit the nitrogen cycle. In fact waste treatment facilities count on using high nitrite levels to stall the cycle so they can then denitrify the system at a more opportune point. Please, read some of the research to understand the I am not positing this, it is scientists and engineers who are doing so. This is not just theory, but is being applied daily around the world in terms of the waste treatment industry. I guess nobody told them what they are doing every day doesn't really work, that their sophisticated instruments are giving them bad readings.

So far you and Prime are the only two sources I can find who claim all the peer reviewed research and science and the actual use it is being put to are untrue and not valid. So again, I challenge you guys to prove what you are saying. Start linking us to research papers which contradict what I have presented. Find even one single peer reviewed research study that claims to prove that nitrite (likely in the form of nitrous acid) at elevated levels does nothing to inhibit the nitrogen cycle.

Come on Prime, I am offering you the chance to make me look like a fool for saying the things I have in these exchanges. Come on, prove to everybody on TFF that I don't have a clue and that you are indeed the person with the right information.

 

[Prime Ordeal]

The author proceeds from the above points with a discussion of nitrifying bacteria. Click through to see it.

The thing about the nitrifying bacteria is that they are not free swimming in the water.

All nitrifying bacteria possess flagella. And that's why you will always find them in the water column. You really need to be careful about the 'peer reviewed' citations you quote TTA.

 

[TwoTankAmin]

Preface- I am not a microbiologist, a chemist nor even an environmental engineer. However, I am a pretty adept researcher in terms of being able to track down reputable and reliable information on most any subject. Since I have no scientific credentials, I hesitate to state facts which I can not back up from personal experience. Instead I choose to site other sources which do have the credentials. This is why I so often quote research papers or other published sources.

I used to have a lot of misconceptions about the bacteria in our tanks. It took me years and a lot of work to learn what the facts are vs the urban myths that are so often espoused on most fish forums.

So I apologize in advance to providing so many references, but it is because I am not asking folks to believe what I might say but rather to believe what the people who have the education, experience and credentials to state the facts say.

* * * * * * * * * * * *​

In post # 12 in the thread here http/www.fishforums.net/index.php?/topic/385852-ammonia/page__p__3243748#entry3243748 , Prime Ordeal made the following claim:

Furthermore, he may be the best known researcher in this field but he is far from the only one. And those other researchers publish copious peer reviewed papers where you can find equally good but often contradictory information relating to nitrifying bacteria and their optimal parameters for survival. It may even surprise you to learn that not everyone agrees with Tim Hovanec's paper on nitrospira, but that's another subject.

That particular quote about the longevity of bacteria and drying out relates to bacteria capable of sporulation and is unquestionably wrong in relation to nitrifying bacteria. It raised my eyebrow to astounding heights when I read it, particularly coming from a person who should know better. But it helps to fool you (if left unquestioned) into believing his claims as to the long term efficacy of his nitrifier products.

Lets start with the issue of “not everyone agrees with Tim Hovanec's paper on nitrospira”. I have asked Prime in multiple threads to please provide just one link to any peer reviewed research which refutes either the paper published in 1998 re nitrite oxidizing bacteria and authored by Timothy A. Hovanec, Lance T. Taylor, Andrew Blakis, and Edward F. Delong (all 3 co-authors were associated with the Monterey Bay Aquarium Research Institute, Moss Landing, CA at the time); or the paper published in 2001 re ammonia oxidizers by Paul C. Burrell, Carol M. Phalen, and Timothy A. Hovanec. So its not just Dr. Hovanec, but the other five researchers that Prime believes also are wrong. It takes a bit of work to find info on Ms. Phalen- she is at least an MS, but Paul C. Burrell has a long career in the field and has published numerous papers (almost none involving Dr. Hovanec. Google him.). And I think the reputation of the Monteray Bay Aquarium is internationally known and speaks for itself.

Now Prime so far has been unable to link to anything that refutes the work of these six folks. What I can add to this is that you can find 126 research papers and books which all cite the work on nitrite oxidizers and another 61 which site the paper on ammonia oxidizers which Prime Ordeal claims that not everyone agrees with. You can find the list and information regarding these 126 here http/scholar.google.com/scholar?cites=12645801936650989137&as_sdt=5,33&sciodt=1,33&hl=en and the 61 here http/scholar.google.com/scholar?cites=13880933799637190045&as_sdt=5,33&sciodt=1,33&hl=en

So no identified sources of published disagreement with these six researchers as claimed by Prime, but 126 published research papers or books that do seem to agree. So far its Hovanec et al 187 and Prime Ordeal 0 on these topics as far as I can tell.


As to optimal conditions “relating to nitrifying bacteria and their optimal parameters for survival” that is not the topic of either of Dr. Hovanec’s papers. However, there are a diversity of nitrifying bacteria, most of which have nothing to to do with aquaria, and these are known to have somewhat different needs and survival indications. It is known that the levels of ammonia present will determine which bacteria tend to colonize and be active- the ones we end up with in our tanks prefer a lower ammonia environment than those found in waste water treatment for example. Which is another reason not to overdose ammonia.



Now on to the issues regarding the survival of non-sporulating, nitrifying bacteria. Many bacteria produce spores. In the process of spore formation the mother cell dies. When a spore comes back to life it a small daughter cell that does so long after the mother cell is dead. Nitrifying bacteria do not form spores, yet they can survive adverse conditions for years. They can even survive drought conditions and no food for years despite Prime’s claim this is not possible.



Since the the early 1900s, long before any of us were born, science has known about the ability of nitrifying bacteria using EPS to form a biofilm in which they live and which they can use to survive drought conditions. This 1962 study refers to research done from the early 1900s on.

Now there is an impressive list of names in the above. I am not going to do all the legwork for readers on all of the names but here is a taste of the people that Prime claims have no clue because he knows more than not only any of them, but all of them.


Sergio Winogradsky - "is best known for discovering chemoautotrophy, which soon became popularly known as chemosynthesis, the process by which organisms derive energy from a number of different inorganic compoundshttp://en.wikipedia.org/wiki/Inorganic_compound and obtain carbonhttp://en.wikipedia.org/wiki/Carbon in the form of carbon dioxidehttp://en.wikipedia.org/wiki/Carbon_dioxide. In 1888, he relocated toZyrichhttp://en.wikipedia.org/wiki/Zurich, where he began investigation into the process of nitrification, identifying the genera Nitrosomonas and Nitrosococcus, which oxidizes ammoniumhttp://en.wikipedia.org/wiki/Ammonium to nitritehttp://en.wikipedia.org/wiki/Nitrite, and Nitrobacterhttp://en.wikipedia.org/wiki/Nitrobacter, which oxidizes nitrite to nitratehttp://en.wikipedia.org/wiki/Nitrate."

WILLIAM MERRIOT GIBBS, Ph.D., Professor of Bacteriology and Bacteriologist, Idaho Experiment Station BS(Agr.), University of Missouri, 1915; MS, University of Wisconsin, 1916; Ph.D., 1919; Scholar, University of Wisconsin, 1915-16

George Stronach Fraps- He attended North Carolina College of Agriculture and Mechanical Arts (now North Carolina State University) and graduated with a B.S. in 1896. After serving as an assistant chemist, Fraps attended Johns Hopkins University, where he received a Ph.D. in 1899. In 1913 Fraps wrote Principles of Agricultural Chemistry. His most important writings appeared as bulletins of the Texas Agricultural Experiment Station and articles in scientific journals.

I won’t do all the legwork for the curious, readers can check into the credentials and research work by the others named on their own if they want.

Or how about this study

Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment

• Ian M. Clarkhttp://www.sciencedirect.com/science/article/pii/S0038071707004683#cor1
• Penny R. Hirsch

• Plant Pathology and Microbiology Department, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

• Received 20 June 2007. Revised 27 November 2007. Accepted 30 November 2007

“Abstract
Dried soil samples from many sources have been stored in archives world-wide over the years, but there has been little research on their value for studying microbial populations. Samples collected since 1843 from the Broadbalk field experiment on crop nutrition at Rothamsted have been used to document changes in the structure and composition of soils as agricultural practices evolve, also offering an invaluable record of environmental changes from the pre- to post-industrial era in the UK. To date, the microbial communities of these soils have not been studied, in part due to the well-documented drop in bacterial culturability in dried soils. However, modern molecular methods based on PCR amplification of DNA extracted directly from soil do not require bacterial cells to be viable or intact and may allow investigations into the legacy of bacteria that were present at the time of sample collection.

In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from α- and β-proteobacteria as well as firmicutes. PCR using primers specific for ammonia oxidizing bacteria showed similar band profiles across the two treatments in recently collected samples, however older samples from the NPK plot showed greater divergence. Primers specific for the genus Pseudomonas were designed and used in real-time quantitative PCR to indicate that archived soil collected in 1868 contained 10-fold less pseudomonad DNA than fresh soil, representing around 10[sup]5[/sup] genomes g[sup]−1[/sup] soil. Prior to milling, dramatically less pseudomonad DNA was extracted from recently collected air-dried soil from the NPK compared to the FYM plot; otherwise, the two plots followed similar trends. Overall bacterial abundance, diversity and survival during the archiving process differed in the two soils, possibly due to differences in clay and soil organic matter content. Nevertheless, the results demonstrate that air-dried soils can protect microbial DNA for more than 150 years and offer an invaluable resource for future research.” (Note- the red is added by me for emphasis.)



So even though Prime has told us, unequivocally, that this is not possible, here are a whole host of people that seem to say that Prime is the one who is incorrect.

One of the greatest problems in doing research into aquarium bacteria specifically is that there is little incentive for it in economic terms. Agriculture and waste treatment are much more pressing, relevent and economically important areas for research. But often the conclusions can have some application across related areas.



But lets put most of the science aside for a moment and I will offer the following challenge for Prime or anybody else to explain.



Lets assume we have two brand new fish hobbyists- lets call them Countryboy and Citygirl. They have never had a tank before but have read about fishless cycling and decide to try it. Unfortunately, they have no access to either seed material in any form nor any bottled bacteria products, so they are forced to do a traditional fishless cycle. Lets further assume (so as not to complicate things), that they both have access to the same pure ammonia and that they both decide never to dose to a level above 3-4 ppm of ammonia. I assume that readers would agree that they both should be able to end up with cycled tanks even it it takes a couple of months.



Countryboy lives on a 20 acre property in the woods and his water comes from his own private well. For our purposes we will also assume its fairly decent water with no agricultural or industrial run offs etc. Citygirl lives in a larger city and gets her water provided by a municipal water system which treats its water with chloramines. So she uses a top quality dechlor while Countryboy does not need to do so. The other thing these two new fish keepers have in common is they are not using live plants nor driftwood but do both use a substrate. They use a packaged aquarium substrate that is coated (such as Estes gravel) and for sure it is purchased very dry. The point is there is nothing in their tanks on which nitrifyers might use to hitchhike in.



So how can they possibly get their tanks to cycle? Exactly where will the first bacteria come from which will start their colonies? According to Prime the nitrifyers can not survive drying, so they could not be found floating around in the air and I would concur that they are not airborne. Bear in mind the tank nitrifyers do not produce spores, so that they can not get in as either air or water borne spores.




So where do the bacteria come from to start their cycling processes? The answer is very simple, they come in with the tap water used by both of them. There is no other way. But how can this be if these bacteria die off in the absence of food or in the presence of chloramine. Countryboy has no ammonia in his well water and likely low disolved oxygen levels, so how can there possibly be viable bacteria in his well water to start his cycle. And what about Countrygirl’s water which has been treated with chloramine. Should that not have killed the nitrifying bacteria before they could arrive in her tap?





According to Prime: “Even the comment about how difficult it is to kill nitrifying bacteria is incorrect, as a moment's thought would tell you. Ever heard of chlorine and chloramine?”



So if one agrees with what Prime has stated, I guess our two budding fishkeepers would never be able to get their tanks cycled. But the fact is almost any member on this forum, will tell you both Cityboy and Countrygirl will get their tanks cycled, and I agree. And there are some very simple explanations for it. In the case of Countryboy, the bacteria are alive and well in his well water, but due to low oxygen levels and little or no food source, they are mostly dormant, not dead/absent, and not in huge numbers. But once they are in the tank and given food and oxygen, they revive and they reproduce and hence his cycle starts. But what about all those cloramines in Citygirl’s water, how can there be any living nitrifyers in that?




Unfortunately, I am going to have to revert to science and research for this. I must turn to a study entitled:




Development and Use of Microelectrodes to Evaluate Nitrification within Chloraminated Drinking Water System Biofilms, and the Effects of Phosphate as a Corrosion Inhibitor on Nitrifying Biofilm

(This is the 200 page PhD thesis of Woo Hyoung Lee dated Nov. 2, 2009, University of Cincinnati, Engineering : Environmental Engineering. This work and its defense were approved by: Committee Chair: Paul Bishop, PhD, William Heineman, PhD, George Sorial, PhD, Margaret Kupferle, PhD, PE and Jonathan Pressman, PhD. )



“The primary objective of this research was to develop, fabricate and evaluate microelectrodes to evaluate nitrification within chloraminated drinking water system biofilm, and to determine the effects of phosphate on nitrifying bacteria biofilm.”

In this study, microelectrodes were used as important experimental tools to measure directly monochloramine biofilm penetration and nitrifying microbial activities. Microbial viability upon the administration of chloramine disinfectant was investigated by observing confocal laser scanning microscopy (CLSM) with LIVE/DEAD® BacLight™. LIVE/DEAD® BacLight™ is a culture-independent membrane integrity based technique. A cell was considered viable or nonviable based on the ability of propidium iodide (PI) to penetrate its membrane and subsequent processing according to the method of LIVE/DEAD® BacLight™. The results from this study will provide answers for the following primary questions; 1) Does monochloramine fully penetrate into the biofilm? 2) How fast and how deep does monochloramine penetrate? 3) Are bacteria really dead or simply inactive when monochloramine fully penetrates? 4) If there is excess ammonia in the chloraminated water system, what is the response from the bacteria? 5) Do the bacteria which are deactivated recover? With these questions, the monochloramine biofilm penetration was investigated and will be discussed.”




Rather than quote and quote and quote I will summarize the results and if you want to read for yourself I will provide a link to the pdf full text of the research. It was observed that nitrifying activity completely stopped, however cell walls were not penetrated. And when the monochloramine was removed, the bacteria recovered.




“When monochloramine and DO full penetration into the biofilm was observed from the microprofiling, we investigated the possibility of microbial activity recovery achieved by feeding ammonia under the proper environmental conditions for nitrifier (i.e. pH 8.0, 23°C, 8.3 mg/L DO, and 4.2 mg N/L ammonia). Figure 5.6 shows the DO microprofile changes with time when fed ammonia. Biofilm aerobic microbial activity increased over time and 40% of DO consumption was recovered after 163 hrs (6.8 days). It appears that the nitrifiers recovered their activity over time in the absence of chloramines, even though they once had been exposed to relatively high concentrations of monochloramine.“

(Note- the red is added by me for emphasis.)

Full Thesis text: http/etd.ohiolink.edu/send-pdf.cgi/Lee%20Woo%20Hyoung.pdf?ucin1258489526



Interesting read especially since Prime has told us how easily nytrifyers can be killed with chloramine. So I guess again we have a PhD thesis reviewed by five other PhDs which clearly must be totally invalid. Poor fragile bacteria. Fortunately, they have never read Prime Ordeal’s statements.





Now on to Raptorex and his statement in post #10 above:



“oh dear, conned by more sales speak.

this tosh is not peer reviewed. its got some words from his business partner. not to mention the fact it would work, reliably. if it were half what its claimed.
still you seem to be enjoying yourself and nobody with knowledge is will be fooled. so go ahead, fill your boots.”



First he clearly did not read the post since it never stated it was even research let alone peer reviewed. It was an email exchange between me and Dr. Hovanec and then I stated what I had concluded. This is exactly the way I presented it. Second, what partner? There were exactly two folks involved, Dr. Hovanec and me. I have no connections to Dr. Hovanec, his company nor any of his products. So who is this fictitious partner Raptorex has invented?



There is no sales speak anywhere. I have and can continue to provide independent research studies which support all of what Dr. H explained in lay terms for me. And these studies are are totally unconnected to Dr. Hovanec in any way save proving most of what he says on his site.



Raptorex, just because you were apparently unable to follow the directions and get your tank cycled using One and Only, or one of the other few similar products, does not mean they do not work. All it means is you can’t get them to work for you. But I guess you would also argue that several major public aquariums which have successfully used this product to cycle immense tanks were also conned. The Georgia Aquarium managed to cycle two million gallons and Raptorex apparently failed to cycle what, 100 or 200 gallons? Of course, it is possible that Raptorex failed because the bottled bacteria he tried was not properly handled before or after it arrived in his possession and that poor handling killed all the bacteriabefore he poured them into his tank. Maybe they were either frozen or allowed to get over about 120F (although enough hours at 105F may also kill them if I remember right).



Raptorex, I assume you also believe that the 13 patents Dr. Hovanec et al hold for ammonia and nitrite oxidizing bacteria and methods for using and/or dectecting them which have been issued in the USA, Europe, Mexico and Australia should never been issued for such “snake oil”? And what about countless folks on forums all over the net who have posted that they did cycle their tanks exactly as the legit products they used, including Dr. Tim’s, said they would? All conned as well?



Finally, snazy I have spent way too much time on the above to want to have to do it again regarding all the other bacteria in our tanks. So I will point you and other members to one of the better pieces I have read that isnt all science speak and which covers the aerobic nitrifying part of this topic admirably. There are actually 2 articles- the first deals with the autotrophic nitrifyers we all want and the 2[sup]nd[/sup] deals with the heterotrophic bacteria snazy mentioned. Please note the author did get it wrong about chloramine killing off the bacteria completely, but he actually is more correct when chlorine is used as it is a much more effective as a killer of the nitrifying autotrophs. This fact is discussed on page 122/123 in the 200 page thesis referenced above:



“Monochloramine penetrated into the biofilm surface layer 49 times faster than chlorine within the nitrifying biofilm and 39 times faster in the multi-species biofilm than did chlorine. On the other hand, free chlorine seems to be consumed by oxidizing the organic matter in path of the biofilm, then diffuse further, indicating very slow chlorine penetration. However, CLSM image of membrane compromised cells in biofilm after chlorination confirmed that free chlorine is much stronger than monochloramine in disinfection.”



Part 1 http/www.oscarfish.com/article-home/water/71-autotrophic-bacteria-manifesto.html

Part 2 http/www.oscarfish.com/article-home/water/72-heterotrophic-bacteria.html



Eventually I will do a thread on the denitrifying bacteria snazy mentioned.



Until Prime Ordeal and Raptorex can actually produce some science on these topics, I am done responding to anecdotes, invented information and plain mistatements lacking any credible support.