I Found New Research On Tank Bacteria And Commercial Bacteria Starter

TwoTankAmin

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I have posted both in the scientific section and the other areas of the forum over the years regarding what bacteria handle ammonia and nitrite in tanks and which commercial bacterial starter products may or may not work. For the most part I relied on the work of Dr. Hovanec et. al. One reason it is hard to find research on other commercial bacterial starters is that science is usually not interested in the brands but in the specific bacteria involved. Moreover, science usually does not want to end up in a fight with manufacturers by naming their product as not working. So finding any real research into this subject is not easy.
 
But today i finally came across a great study. "Novel application of nitrifying bacterial consortia to ease ammonia toxicity in ornamental fish transport units: trials with zebrafish." It looked at two commercial starter products and a control group with nothing added. They were investigating whether the products would help control ammonia in fish bags during transport. The study refused to name the two products but they did identify the specific strains of bacteria in each. The two products contained different ammonia oxidizing bacteria (AOB) but the same nitrite ones (NOB).
 
The upshot of all this is that the study basically confirmed what I have been posting now for a number of years. Here is a summary of what the study indicated that is relevant to us.
 
1. The strain of AOB matters. Those AOB with a lower affinity for ammonia were more effective than ones with a high affinity.
2. Even when the starter product with the higher affinity AOB was used, they soon detected those lower affinity AOB present even though they were not in the product added.
3. Both products contained Nitrobacter spp as the NOB. This is an NOB with a high affinity for nitrite- they are typically found in waste treatment but not in aquariums. But they did not test for nitrite, so they did not comment on the effectiveness of these.
4. However, when they tested for NOB they discovered all the tanks (even the controls) contained Nitrospira. This is a lower affinity NOB and the same ones identified by Hovanec et. al. There is a patent involved with this and most starter products will not contain this strain. Neither of the commercial additives contained nitrospira.
 
So as I see things, here is a study with no connection to Dr Hovanec or his co researchers which has come to the same conclusion they did. And it is being confirmed by completely unrelated researchers
 
If you want to read it for yourselves it is a full study to be found here http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2011.05050.x/full
 

Ch4rlie

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Nice to fnd a separate study that supports what you have been saying on this forum for quite some time now.
 
Good little find.
 

daizeUK

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 Thanks for sharing this.
 
Does 'affinity for ammonia' basically mean the preferred concentration of ammonia that the bacteria will thrive in?  i.e. a strain that prefers the low concentration present in our fish tanks rather than the sort of bacteria used for waste treatment?
 
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TwoTankAmin

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Yes, daize. A lower affinity means it can thrive at lower levels. When it comes to ammonia, it is actually the Archaea which have the lowest affinity. They can thrive in levels too low for almost any of the bacteria.
 
The other term used relative to bacteria that can be a bit confusing is the term substrate. We use this term for our gravel/sand, and bacteria will also attach to the substrate. However, when used relative to the bacteria, substrate also refers to the basic nutrients on which they feed. Here is a decent one page explanation of this. http://www.biotecharticles.com/Applications-Article/Microbial-Growth-Substrates-657.html
 

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This thread is a little old now, but I thought I'd post as its an interesting paper - the DNA sequencing part went straight over my head though!

I was curious about a few of things. Firstly, the two samples of bacteria starter had bacteria in different substrates - one in a liquid, the other a solid medium. As bacteria grow at different rates in different media, doesn't this introduce bias into the experiment? How can we be sure that the observed differences aren't at least partly due to the medium rather than the actual bacteria.

I was also curious as to whether supersaturating the containers with oxygen could also be a confounding variable. Each species of bacteria must have an optimum level of oxygen where it functions at peak efficiency. Is it possible that the high level of oxygen could give a competitive advantage to Nitrospira if it is better suited to high oxygen environments? And if so, could it also be possible that Nitrobacter might outcompete Nitrospira at a lower oxygen saturation? I'm just wondering if there's any evidence that Nitrospira outperforms Nitrobacter at all oxygen saturations, or if there's a point where the results are reversed.

Finally, I was wondering how closely the experiment matches the environment in an uncycled tank. Firstly, I can't envision such high levels of oxygen in a cycling tank; and secondly, wouldn't there be much more flow in a tank, which would affect the bacteria's ability to capture ammonia in their surroundings?
 

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Its a good topic as there are so many misconceptions where, when, what & why questions still have not been fully tested and understood.
The ones that stump me is that bacteria lives everywhere media, water, ornaments, substrates even glass! 
The one thing I wanted to see is data on the natural selection process of bacteria, as they all compete for Ammonia, Oxygen Nitrogen and other primary, secondary chemicals that make bacteria either survive or thrive? A spanner in the works is soft water or hardwater, what breeds Brackish, Alkaline or Acidic?
A recent misconception that has been spinning around is that when you move over to a new fish tank most people in the old days will say move as much of the old tank equipment over including water.
An example of this on you tube most advocate that bacteria does not live in water and there is no point to move the water over aswell. But regardless of the bacteria or chemicals in the water it would be an essential source of food for bacteria? Like I said if bacteria thrive they will compete for food source? 
Another interesting debate is the natural waste produced by plants or fish which is a source of neutrient food for plants and bacteria and some will say these free radiacals cause more swings in water conditions than actually having a chemical induced stability the question is can this be maintained to that level constantly? 
In normal everydays terms we do water changes to reduce the risk of these swings, but some breeders will advocate messing with water parameters is not a good thing, but these types have a fix formular that works for them.
From what I can tell over the years water changes takes out the science out of the hobby and people feel comfortable with safe, tried and tested ideas!
 
New one for me is the Nitrate levels?
Is Nitrate really bad or is it the build up of other chemicals that have a symptom of hight levels of Nitrate, as most would change the water to lower Nitrate but essentially are you improving the whole water quality? Pure Nitrate is used for plants and its in substrates and pure forms makes you think?
 

Byron

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I will comment on some of the issues you raise, and TTA (who has researched this much more than I have) can expand where needed.
 
The one thing I wanted to see is data on the natural selection process of bacteria, as they all compete for Ammonia, Oxygen Nitrogen and other primary, secondary chemicals that make bacteria either survive or thrive? 
 
 
I'm not sure, but maybe you have a misconception here.  Not all bacteria use/need ammonia, nor oxygen, nor nitrogen, in the sense that they specifically use these, so in that sense there is no competing.
 
A spanner in the works is soft water or hardwater, what breeds Brackish, Alkaline or Acidic?
 
 
In a sense.  In very acidic water, nitrifying bacteria (meaning, the bacteria that are AOB and NOB or ammonia/nitrite oxidizing bacteria, but not necessarily other bacteria species) do see a slower reproduction and will even cease when the pH is very low.  Temperature plays a similar role.  As for brackish, there are marine strains of AOB and NOB that are related to but different than freshwater strains.
 
I'm not aware of soft or hard water having much impact, but TTA can correct this if in error.
 
An example of this on you tube most advocate that bacteria does not live in water and there is no point to move the water over aswell. But regardless of the bacteria or chemicals in the water it would be an essential source of food for bacteria? Like I said if bacteria thrive they will compete for food source?
 
 
Bacteria form on surfaces, in the biofilm.  Using "old" water can benefit (up to a point) when fish are being moved, to avoid severe shock from differing parameters, but there is no real benefit re bacteria because the ammonia/nitrite does not remain very long in the water.  The bacteria (or plants) would snap it up fairly quickly.  If this were not the case, our fish in established tanks would be suffering from ammonia or nitrite poisoning, and clearly they do not, yet they produce ammonia continually via respiration.
 
The nitrifying bacteria appear and colonize at a level needed to take up the ammonia/nitrite.  They do not exceed this level unless ammonia/nitrite increases, at which point the bacteria would multiply by binary division.  When/if ammonia or nitrite should lower, the bacteria are able to go into a state of hibernation in a sense.  So there is no real competition here either.
 
 
Another interesting debate is the natural waste produced by plants or fish which is a source of neutrient food for plants and bacteria and some will say these free radiacals cause more swings in water conditions than actually having a chemical induced stability the question is can this be maintained to that level constantly? 
In normal everydays terms we do water changes to reduce the risk of these swings, but some breeders will advocate messing with water parameters is not a good thing, but these types have a fix formular that works for them.
From what I can tell over the years water changes takes out the science out of the hobby and people feel comfortable with safe, tried and tested ideas!
 
 
Water stability is actually very common when nature is doing the work.  It is more when we start dumping in chemicals and concoctions that things start acting up.  Live plants with a balanced fish load in an aquartium receiving regular substantial water changes will be about as stable as you can hope to achieve.  Breeders know that fry need substantive water changes in order to develop at their best and remain healthy.  There is (so far) no replacement for water changes.  No filter or filtration media on the market can do what a simple water change achieves.  There is "stuff" in the water that can only be removed with a water change, or it simply builds up.
 
New one for me is the Nitrate levels?
Is Nitrate really bad or is it the build up of other chemicals that have a symptom of hight levels of Nitrate, as most would change the water to lower Nitrate but essentially are you improving the whole water quality? Pure Nitrate is used for plants and its in substrates and pure forms makes you think?
 
 
We got into a nitrate discussion recently in another thread.  There is little scientific study in this area, because most have wrongly assumed nitrate to be harmless, but this just is not the case.  Ammonia, nitrite and nitrate all poison fish, no one can reasonably debate that.  The differing views involve how much nitrate and for what time period, and which fish species.  Studies have proven that the higher the nitrate, and/or the longer the fish is exposed, the more likely it will be seriously affected and to the point of death.  Some fish can tolerate nitrate better than other species.  But nitrtate is still a toxic substance no matter what.
 
As for plants, they only resort to nitrate when ammonia/ammonium is not sufficient to balance light and other nutrients.  In a planted tank, plants actually out-compete the nitrifying bacteria for ammonia, and plants can take up quite a bit, though this has its limits as ammonia is toxic to plants as well as fish, and to some bacteria.  Plants taking up nitrate have to internally change it back into ammonium, which is more energy from the plant, so they only resort to this when necessary.
 
Byron.
 
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TwoTankAmin

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The topic of microorganisms in a tank or in nature is quite complex. The real key to a lot of it is in the biofilms. These holed a myriad of varying bacteria. There is ample research on the make of of such biofilms. Science know how the various microorganism inside the biofim are layer. They actually work in concert.
 
When it comes to which bacteria thrive in a given system, it is very much ammonia dependent. That is why there are different bacteria dominant in and aquarium vs a waste water treatment plant. You will find that aquariums have much more in common with drinking water plants and aquaculture.
 
What is at issue here is which types/strains of nitrifiers do most of the nitrification in tanks. As Byron pointed out, the ammonia oxidizers in fw and sw are not the same. Recent research (since 1005) has discovered that another group or microorganisms called Archaea also appear to do ammonia oxidation. They are clearly doing this in salt water and they are found in most soil and freshwater habitats as well. However, there is still more research needed in terms of their potential role in fw tanks and other fw environs. Some believe they are key players here while others still are unconvinced. I am in the camp of the unconvinced. the one thing that is know about these Archaea, they have the lowest affinity for ammonia which means they do best at the lowest levels. As levels rise they are usually  by bacteria.
 
Moreover, no matter which strains dominate in an given system, there are normally some amount of other strains present. These few seem to hang around because nothing is stable and when things change, i.e. ammonia levels drop or spike up, the bacterial mix will change in response.
 
There is an amazing amount of research out there on these topics. Most of it is over my head in many of the specifics, but I do get the gist of these things. So when a paper talks about the specific gene testing protocols, I am lost until they get to the line that states as a reult of doing them they found X to be the case. I understand the X.
 
My reason for posting this thread was due to a long discussion/argument  on this site that went on for quite some time on the consept of bottled bacteria. I argued that it was viable, did work but was dependent on the strain of bacteria involved, how it was grown and packaged. the other side, with a a number of folks arguing for it claimed this was hokum and that Dr. Hovanec's research was not valid because it ended up years later with his selling bottled bacteria based on it. It took me months of quoting research studies etc. before some folks finally saw the light. But this whi refused to accept the science always came back with the argument that it was not showing research done using specific products. And when I did find a study all could read w/o institutional access, this was it and here I posted it.
 
There is actually a lot of research on this topic out there. For example, while it is true that the normal bacteria we have in or tanks will stop processing ammonia when the pH drops below about 6.0, the reason for this is at those pH levels the ammonia all exists in the for of NH4 (ammonium) and none in the toxic NH3 form (ammoia gas).  So when the NH3 vanishes, the NH3 using bacteria don't function. But there are other bacteria that do. The have receptors for NH4 and can and do use it, They process less effectivly than those found in the higher pH levels, but nitrification continues.
 
Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance
http://aem.asm.org/content/72/6/4283.full.pdf+html
 
Changes in ammonia oxidiser population during transition to low pH in a biofilm reactor starting with Nitrosomonas europaea
http://gwri-ic.technion.ac.il/pdf/Professors/Michal_Green/11.pdf
 
And I can confirm that there is nitrification in acid waters. I brought a number of wild caught altum angels into and uncycled tank at pH 4.2. I have a second tank i was working to have the media cycled using fishless methods to produce cycled media at 6.0. The plan was to raise the tanks to 6.0 gradually and when it reached that level the bacteria being developed in the biofarm tank would be ready and functioning at 6.0. But something went wrong with that plant. The angel tank cycled itself during that time and there was no need for the cycled filters. trace ammonia levels between .25 and .5 ppm which had been in the angel tank early on (but not harmful at the pH) completely vanished and there was never an issue. So I know, anecdotally, from my own experience that nitrification takes place at well under 6.0.
 
I have been reading on these subject for a few years now. Unfortunately, I lack a lot of the background needed to understand the nuts and bolt part of the research, but I do understand the basic processes at work and generally what makes them tick. My current interest is with the process of denitrification which I am still learning about.
 
There are a few doctorate level practicing microbiologists in this hobby and I have learned a lot by paying attention to them. I have been fortunate enough to correspond with one and to meet and chat with the other. And I am nutty enough to have a running disagreement with the latter on the role of archaea vs bacteria in fw aquariums and fw in general. We have reached a point where he is willing to concede that the archaea might not be there in any numbers at the start, especially when one has to cycle using fish or ammonia dosing rather than lots of plants, but would appear and then dominate later once a tank is cycled. His belief is the Archaea come to dominate after this period when ammonia levels are  lower. I have come to accept that archaea may be present, but do not agree they would necessarily dominate. The science in general says more research is needed here to be more certain about what is going on in fw.
 
A lot of the problems with research is it is rarely aquarium specific. Most of it looks at more commercial considerations- waste water, drinking water, aquaculture and natural environments such as oceans, lakes, rivers soils etc. etc. Given the cost of such research, there are very few who will underwrite these costs when the research specifically involves our hobby.
 
I would suggest this, however. One might think that if you asked the two lifelong fish keeping practicing microbiologists mentioned above about how they filter their tanks, we might learn some interesting things. I am in the process of replacing a bunch of my filters with this approach as a result: http://www.swisstropicals.com/library/mattenfilter/ I just inserted the second one recently and have the first tank almost totally relying on one. It takes about 6-10 week to make the switchover gradually. So far so good.
 
As for nitrate, there is research, but a lot of it is not hugely helpful. It is almost universally related to natural environments which have a host of things living in it from crustaceans to fish and everything in between. What levels are safe for 100% of these is not representive of what night be safe for most fish we keep in tanks. I can find you research on channel catfish which shows they can be Ok in nitrate levels that would make us freak ou if we saw them in a tank. but how many of us keep daphnia or channel cats in out tanks? There is also research on farmed fish to some extend and for the salmonids- another thing we never keep in tanks. I have seen a bit of research on a few fish.
 
Recirculating systems for zebrafish
 
It is important to test total ammonia (NH4) and nitrite (NO2-), about once a week using a water quality test kit. The water sample should be taken from the wastewater sump tank, before the water has been processed by the biofilter.
Ammonia and nitrite levels should be below 0.3 ppm. Nitrate (NO3-) is relatively harmless to aquatic animals in low levels. There is no data that indicates any water quality problems with nitrate levels < 200 ppm. Weekly testing of nitrates is suggested, to maintain levels < 150 ppm. If ammonia, nitrites or nitrates reach the upper limits, a water exchange of 10% of the water volume at a time is recommended.
from http://integrated-aqua.com/wordpress/wp-content/uploads/2011/04/Lab-Animal-Zfish_scourtland.pdf
 
I can show you a paper where they looked at sw/brackish Juvenile turbot. The used 3 levels of nitrate and a control over a 42 days. They considerd low level nitrate to be "125 mg/L NO3–N (low nitrate LN)." because they measured the nitrat as nitrogen ions as opposed to the total ions or hobby kits measure, the number on our kits would have been 550 ppm. Now when we talk about nirtrate levels in tanks we consider 80-100 to be pretty high. the high level in that study was 500 mg/L NO3–N (2,200 ppm on an API kit). So rather that state it as Byron has done, I would say that above specified levels, nitrate can become toxic to fish. But for even the most sensitive of species i have never seen anything to support the idea that 0 is the required level as it usually is with ammonia or nitrite. For even the most sensitive of species i have never seen it suggested that nitrate needs to be below about 8.8 ppm NO3 (aka 2 mg/L-N).
 
Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates
 

from http://www.researchgate.net/profile/Alvaro_Alonso3/publication/8063535_Nitrate_toxicity_to_aquatic_animals_a_review_with_new_data_for_freshwater_invertebrates/links/543f9e150cf2f3e82851e42d.pdf
 
What is clear from the few studies i have been able to find is that is is not possible to use any single number for nitrate which would apply to everything. Different fish do have different tolerances. But this is the case for ammonia and nitrite as well. What we can conclude here is that for ammonia and nitrite undetectible is the only acceptable number and, with nitrate, that lower is always better but that undetectible is not necessary.
 
Once last comment. I do not consider that plants and bacteria compete for ammonia. The plants can not use NH3 only NH4. The bacteria need NH3. So the amount of bacteria in any tank will be what is required to handle the available NH3. When plants are included, that level stays lower. But here is what ZI see as the main difference between plants and bacteria in relation to total ammonia. No matter how many plants are in a tank, there is always some amount of NH3 and hence some bacteria present even if it is not all that much. It is especially present in the planted tank substrate. In fact, the plants have nitrifying bacteria living on them and their roots. Plants host bacteria but no bacteria hosts plants . So it is really not possible to find an established tank, no matter how well planted, without some level of nitrifying bacteria. On the other hand, it is commonplace to have tanks with no plants at all that rely 100% on bacteria (and archaea?) to remove 100% of the NH3/NH4.
 
I see the role of the bacteria/archaea as being one of clearing the ammonia as NH3 that may be available in any aquatic system. It doesn't matter if that is a very low or a higher level, they size in number to meet that need. While the plants may reduce the need, they are not really in direct competition with the bacteria. No matter how fast or how much plants can take up ammonium, they can never do so rapidly enough to eliminate any NH3 from being present even if its only there long enough to support a small colony of microorganisms.
 

Byron

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Once last comment. I do not consider that plants and bacteria compete for ammonia. The plants can not use NH3 only NH4. The bacteria need NH3. So the amount of bacteria in any tank will be what is required to handle the available NH3. When plants are included, that level stays lower. But here is what ZI see as the main difference between plants and bacteria in relation to total ammonia. No matter how many plants are in a tank, there is always some amount of NH3 and hence some bacteria present even if it is not all that much. It is especially present in the planted tank substrate. In fact, the plants have nitrifying bacteria living on them and their roots. Plants host bacteria but no bacteria hosts plants . So it is really not possible to find an established tank, no matter how well planted, without some level of nitrifying bacteria. On the other hand, it is commonplace to have tanks with no plants at all that rely 100% on bacteria (and archaea?) to remove 100% of the NH3/NH4.
 
 
The majority [and there are so few exceptions it is hardly worth mentioning] of aquatic plants will take up ammonium and/or ammonia, which ever is present, as their preferred source of nitrogen, and studies have shown that it is only when the ammonia/ammonium is exhausted that they turn to nitrate.  "Exhausted" is a bit misleading, as obviously ammonia/ammonium is continually being produced if fish are present, but if everything else including light is sufficient to drive photosynthesis, and ammonia/ammonium is the limiting factor, plants will then turn to nitrate.  I'll return to this momentarily.
 
Ammonium is prevalent in acidic water as we know, so it is readily taken up.  In basic water, plants will take up the ammonia just as quickly, and use it in two ways.  As ammonia enters the plant cells by simple diffusion across the membrane, it does one of two things.  It can combine with a hydrogen ion and convert to ammonium, which can be immediately used or stored in cell vacuoles.  Or it can immediately be used in its toxic state to synthesize proteins, by combining with stored carbohydrates to form amino acids.  This is why plants that grow fast and well can remove so much ammonia; they have the stored carbohydrates.
 
This is why I am so frequently recommending live plants, esp fast growing, in new tanks.  I agree that some of the ammonia will get past the plants and be grabbed by bacteria, and I agree with everything else in this cited paragraph about bacteria on plant leaves.  But this is a slower process than the uptake by plants, and I suppose that is why some authors call it out-competing.
 
Back to the ammonia/nitrate issue.  Aquatic plants take up ammonium much faster than nitrate.  One study with Pistia stratiotes (common Water Lettuce) showed ammonium uptake to be four hours (at 0.4 ppm N) compared to 20 hours for nitrate.  Ammonium also inhibits the uptake of nitrate by many organisms, including algae.  Other studies have shown that plants grow faster and better with ammonium than nitrate, though evolution has a part in this this; the faster uptake seems related to the plant's source where it evolved.
 
Plants including algae and all photosynthesizing organisms use the N of ammonium, not nitrate, to produce their proteins.  When plants need to turn to nitrate, nitrate reduction is required, whereby the plants convert the nitrate back into ammonium; this requires energy, and appears to be the mirror image of nitrification.  Plants use approximately the same amount of energy to change nitrate back into ammonium as bacteria do for the reverse.  This is why planted tanks should not "encourage" nitrification; the plants must spend twice the energy just to get the ammonium needed to synthesize their proteins.
 
Most of the above data is from Walstad.  However, similar info is available (without all the science) in Hiscock's book, and elsewhere.  I've also had discussions with Tom Barr on this.  Point is, I haven't the inherent knowledge to argue the science.

 
Byron.
 
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