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  1. #1

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    Default A closer look at the Nitrogen cycle


    7 Not allowed!
    I wrote this post prompted by my own experience cycling my first tank. I was surprised at the amount of contradictory information I found about the best way to cycle a tank, so I decided to get a deeper understanding of the nitrogen cycle to learn the reasons behind the guidelines used to cycle a tank.

    This is not meant to be a primer on cycling -- there already are excellent stickies in this forum on cycling with fish and on fishless cycling (If you haven't read the cycling stickies you should probably do that before reading this). Instead, I examine the available scientific data on the nitrogen cycle and the bacteria involved to get a more thorough understanding of the processes behind our biological filters and a better understanding of the guidelines commonly used to cycle an aquarium.

    As you already know, fish produce highly toxic ammonia as waste. The nitrogen cycle is the process that converts this ammonia to nitrites and then to nitrates by beneficial bacteria in our tanks. So, the nitrogen cycle consists of two separate steps: first the conversion of ammonia to nitrites, and then the conversion of nitrites to nitrates. Together these two steps are referred to as "nitrification".

    Nitrification
    Ammonia is a compound made of nitrogen and hydrogen with a chemical formula of NH3. We commonly hear there are two forms of ammonia in our tanks: "regular" ammonia and "less toxic" ammonium. Ammonium, which is NH4+, is simply an ammonia molecule that has gained a hydrogen proton. This can happen by "protonation" where, if the pH of the water is low enough, the ammonia can gain a proton from the water. It can also happen if we add a conditioner to the water to "bind" ammonia into ammonium. We often hear of "false ammonia readings" from ammonia tests -- this happens because some tests react to the presence of both ammonia and ammonium, giving us a "total" ammonia reading, while others read only the ammonia itself. Some tests also raise the pH of the sample, reversing protonation and converting some ammonium back into ammonia. This difference in testing is important because ammonia is highly toxic to fish while ammonium is much safer. Still, for purposes of the nitrogen cycle (and even more so if you are cycling without fish) the difference is less critical because both ammonia and ammonium can be converted into nitrites and nitrates through nitrification.

    The first step in nitrification is "nitritation": conversion of ammonia (or ammonium) into nitrite by oxidation. Nitritation takes the hydrogen in the ammonia or ammonium and replaces it with oxygen to create nitrite, which is NO2- . For the chemically inclined, the exact chemical reactions are:

    Nitritation of ammonia: NH3 + O2 → NO2- + 3H+ + 2e-
    Nitritation of ammonium: 2NH4+ + 3O2 → 2NO2- + 2H2O + 4H+

    The second step in nitrification is "nitratation": conversion of nitrites into nitrates by oxidation. Nitratation adds oxygen to each molecule of nitrite to convert it into nitrate, which is NO3-. The chemical reaction involved is:

    Nitratation: NO2- + H2O → NO3- + 2H+ + 2e-

    The chemical formulas are not really important. I only include them to show that in addition to producing nitrites and nitrates, nitrification also produces some by-products, including hydrogen protons (H+). These protons are important because their creation will acidify the water. We often hear that cycling can cause fluctuations in pH, and this is one reason for those fluctuations. pH is a measure of the concentration of hydrogen protons in a substance. The higher the hydrogen proton concentration, the lower the pH, so adding hydrogen protons to the water affects pH in a very direct way. In other words, if there are no other factors affecting pH, the conversion of ammonia and ammonium into nitrates will, over time, tend to lower the pH of our tank water. As you probably know, drops in pH can be buffered by high levels of carbonate hardness (KH) so if your tank has high KH you will only experience very mild drops in pH, which will be easily offset by your routine water changes. On the other hand, if your KH is low you should pay constant attention to your pH because it will tend to drop over time, and you may need more frequent water changes to maintain it at a desirable level.

    Note that high KH levels might not fully protect you against this drop in pH: While bicarbonate does not appear anywhere in the chemical reactions for nitrification, beneficial bacteria use bicarbonate as their source for carbon and utilize large amounts of it during nitrification. As a result KH levels can drop during nitrification, reducing the buffering capacity of tank water and increasing the impact of the pH drop. This creates the need to monitor pH levels even in tanks with higher KH.

    Incidentally, the form of ammonia many of us use to cycle our tanks is 10% ammonium hydroxide, which is simply a solution of ammonia in water. The pH of 10% ammonium hydroxide is 12.4. This means that regular additions of ammonia to our tanks during cycling will tend to increase the pH of our tank water. Thus, during the first few days of cycling, when the nitrogen cycle hasn't started yet, your tank's pH will probably tend to increase (again, the increase will depend on your KH level). Once nitrification starts, however, the drop in pH caused by the oxidation of ammonia (or ammonium) will somewhat offset this increase. Predicting the exact net effect on pH is impossible because each tank is different, so it's important to keep a close eye on your pH levels. Preventing excessive drops in pH while cycling is crucial because nitrification is carried out by live bacteria sensitive to pH -- which leads into the second part of this article.

    Beneficial bacteria
    Nitrification in our aquariums is carried out by two different types of bacteria: ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). Unfortunately, a quick internet search will reveal a lot of inconsistent and even contradictory anecdotal information about these bacteria.

    Bacteria carry out these oxidation processes to obtain energy for reproduction. So, more efficient nitrification not only removes waste from the water, it also allows the colonies to grow faster -- but "faster" is a relative term. The oxidation processes of ammonia and nitrite produce very little energy, so large amounts of oxidation must occur before bacteria can reproduce. As a result, these colonies grow very slowly when compared to other types of bacteria: AOB colony size doubles every 7-8 hours, and NOB colony size doubles every 10-13 hours. The slower growth rate of NOB colonies is one possible reason why the first half of our cycles happens faster than the second half and we get a buildup of nitrites until the NOB colony size can catch up.

    Until recently it was thought that nitritation and nitratation in fish tanks were carried out by Nitrosomonas (also called “Nitrosomas”) and Nitrobacter bacteria, respectively. Recent studies suggest, however, that other organisms may be at work instead. For example, studies have found that nitratation in aquariums is primarily carried out by Nitrospira bacteria, with little or no involvement of Nitrobacter species. In reality there are different types of AOB and NOB bacteria and there are probably different bacteria responsible for the nitrogen cycle in any given fish tank. Bacteria evolve and adapt to their environment and each tank is slightly different, so bacteria colonies in one tank are likely to be somewhat different from colonies in any other tank. Still, regardless of the specific types of AOB and NOB colonies in a tank, the same basic factors affect the health and growth of beneficial bacteria.

    Temperature: Like all microbes, nitrifying bacteria are sensitive to temperature. The optimum temperature range for growth of both AOB and NOB colonies has been reported as 77 to 86 °F, with peak growth taking place at 86°F for AOB and 82°F for NOB. This is why we often hear that the ideal cycling temperature is 82°F. Since AOB colonies can grow faster than NOB, it makes sense to set the temperature for NOB peak growth and decrease the gap in growth rates between both colonies. So, this is one area where "conventional wisdom" is correct: for faster cycling you should set your tank temperature at 82°F.

    It is important to differentiate between ideal temperatures to promote faster colony growth, and temperatures under which nitrification can occur. Nitrification has been observed to occur at temperatures as low as 45°F. Nitrifying bacteria will die at 32°F and 120°F, but these levels are well beyond the temperatures normally encountered by aquariums. Thus, once your colonies are established you can safely lower the temperature of your tank to normal aquarium levels and nitrification will continue.

    pH: Nitrifying bacteria are quite sensitive to pH. The ideal pH range for colony growth is between 7.5 to 8.0 for most AOB and between 7.6 to 7.8 for most NOB. Nitrification and colony growth can continue outside of these ranges, but at pH levels of 6.5 or lower the rate of activity and growth cease almost completely for both AOB and NOB, severely impacting the ability to cycle a tank. The good news is that this doesn't necessarily mean nitrification cannot occur at all with pH levels of 6.5 or below. It has been observed that once a colony is established, the slime layer where bacteria live in our fish tanks can protect them from unfavorable conditions, creating the possibility for nitrification to occur even in pH levels below 6.5. Over the long term the colonies might be able to adapt to the low pH and survive. Still, low pH levels can make cycling a tank extremely difficult, and a pH of 6.5 or lower will almost certainly stall your cycle. For fast cycling you should aim to maintain pH levels in your tank between 7.6 and 7.8, and never allow it to fall to 6.5 or below.

    As explained in the first part of this article, nitrification tends to cause KH to drop over time. If this tendency is left unchecked it could then lead to a drop in pH, causing a well-established biological filter to stop working. This drop in KH and pH is one possible reason for what is known as "old tank syndrome" where a tank that has been established for a long time suddenly starts experiencing high levels of ammonia and nitrites.

    Light: One factor seldom discussed by aquarists is sensitivity of beneficial bacteria to light. Both AOB and NOB are sensitive to light and oxidation can be inhibited by even short exposures to sunlight, UV light, or even artificial light. Note that exposure to light doesn't harm the bacteria, it simply slows down the rate at which it can oxidize ammonia or nitrite (and therefore the rate at which it can grow). As a result inhibition is temporary, and bacteria return to normal activity some time after light exposure ends. NOB appear to be more sensitive to light than AOB.

    How much inhibition takes place or how quickly it happens depends on the frequency and intensity of the light, but inhibition has been observed with as little as 10 minutes of exposure to ambient light under certain conditions. The time for the bacteria to recover depends on a number of factors, including the amount, duration and frequency of light, as well as the levels of oxygen, ammonia and nitrites in the water. In general, both AOB and NOB are back to normal activity a few hours after short exposures to light.

    For aquariums, this means that colonies are more likely to develop in areas of the tank not usually exposed to light -- such as inside filters or deep inside sponges. It's also a reason to leave filters undisturbed during cycling, to avoid exposing your biomedia to light while you are trying to establish your bacteria colonies.

    Oxygen: It is clear from the chemical reactions above that bacteria need oxygen for nitrification. AOB are inhibited when oxygen levels in the water drop below 1 mg of oxygen per liter and NOB are inhibited below 2-4 mg/L. In a correctly set up tank this should not pose any problems because oxygen levels will typically exceed 5 mg/L. While the ideal cycling temperature of 82°F will reduce the oxygen content in the water, oxygen content should still remain well above the required 4mg/L, particularly in a tank undergoing fishless cycling where there are no fish to compete for oxygen. Thus, in a properly set up tank with enough water flow and surface agitation to promote good gas exchange, oxygen content should not be a limiting factor in cycling a tank.

    While oxygen content itself should not be a problem in a properly set up tank, water flow around the bacteria colonies could be an issue. Because bacteria tend to live inside our filters, maintaining proper water flow is important to keep oxygen at adequate levels around filter media. Stagnant water or poor flow can lead to depletion of oxygen, inhibiting nitrification and, in prolonged cases, result in bacteria die offs. While this is normally not a problem in adequately maintained filters, it can become an issue when filters stop operating for prolonged periods (as in the case of power outages). Still, beneficial bacteria are hardier than what most aquarists think. It has been observed that AOB and NOB bacteria can survive undamaged without oxygen for periods of up to 70 hours. This means that colonies should be able to recover even after 3 days without proper water circulation, and even longer if oxygen content in the water is high enough when water flow stops.

    Ammonia: It is well known by aquarists that excessive levels of ammonia can interfere with a cycle. The reason is that oxidation of nitrite by NOB is inhibited by high levels of ammonia in the water, delaying the establishment of the NOB colony. The point where inhibition begins varies widely depending the specific type of NOB involved. Studies have found, for example, that Nitrobacter are inhibited at ammonia concentrations above 30 ppm ammonia, while Nitrospira are inhibited with concentrations as low as 0.1 ppm ammonia and below. Fortunately, inhibition by ammonia is fully reversible, and nitratation resumes once ammonia drops below the inhibition threshold.

    We now know that nitratation in aquariums is carried out primarily by Nitrospira bacteria. As a result, even the relatively low ammonia levels used to cycle a tank can inhibit establishment of the NOB colony. On the other hand, maintaining an adequate level of ammonia is important to avoid starving the AOB colony. So, how can we achieve a balance between feeding the AOB colony, while maintaining low enough levels of ammonia to allow faster development of the NOB colony?

    The answer may lie with the resistance of AOB to starvation. Many aquarists believe that AOB cannot survive long without ammonia. As a result, when cycling an aquarium most aquarists re-dose their tanks with ammonia as soon as they register a reading of zero to avoid starving their AOB colony. This could explain why NOB typically take much longer than AOB to become established: the constant redosing of ammonia means that there are very short periods of time where ammonia levels in the tank are low enough for NOB to grow uninhibited. In fact, this constant redosing is unnecessary and even counterproductive. Many studies have shown that under certain conditions most AOB can survive up to several weeks without any ammonia. While some types of AOB experience slow recovery after a long period of starvation, all types are able to recover almost instantly if starvation lasts just a few days. This suggests that when daily testing shows zero ammonia levels in a tank, waiting 1 or 2 days before dosing again could accelerate the establishment of the NOB colony by providing a longer ammonia-free period for the NOB to grow, without any adverse effects on the AOB colony.

    Guidelines for faster cycling
    While the rules and guidelines commonly used for cycling work well, the information presented above suggests that with minor modifications we might be able to shorten the time it takes to cycle a tank without fish.

    • Many of the usual principles still apply: water temperature should be set at 82°F, you must have proper oxygenation and water flow, and pH should be between 7.6 and 7.8. It is also usually said that you should not clean your filter during cycling. I will go one step further and say you should not disturb your filter in any way during cycling.
    • You should also minimize exposure of filter media to strong light or direct sunlight while cycling. If you have no plants in your tank, leave tank lights off during cycling. While light will not harm your bacteria, repeated exposures to light might slow down the establishment of your colony.
    • Monitoring pH levels during cycling is very important to ensure pH remains above 6.5 -- even if your tank has adequate KH levels. If your water source has a pH above 6.5, drops in pH can be easily corrected by simply doing a water change. Incidentally, since Nitrification can impact KH levels, a drop in pH might also indicate that your KH levels are dropping.
    • Perhaps the most important change to the common cycling procedure concerns the amount and timing of ammonia additions. Common practice is to redose ammonia the same day your tests register zero ammonia. As mentioned above, however, this is unnecessary and counterproductive. Instead, any time your tests register zero ammonia, wait 24 hours before redosing.
    • If you are not adding fish immediately, you can successfully maintain your cycle by redosing ammonia every other day, while you continue to monitor pH.



  2. #2

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    2 Not allowed!
    great post. very well written.
    Fishes go "pook pook"
    my spell check went on vaction.
    my Mts storie:http://www.aquaticcommunity.com/aqua...d.php?t=117355

  3. #3

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    2 Not allowed!
    Thank you :-) It has been sitting on my computer for a while, waiting for "the final edit".

    One reason for the delay is that I also wanted to take a look at how much ammonia, exactly, we need to cycle. How much ammonia do our filters really need to be able to handle in 24 hours, even under heavy stocking plans? If we could cycle with only, say, 1 ppm ammonia or lower, cycling would be much faster. Unfortunately (and surprisingly) there is precious little data on the bioload of different fish, and on how much bioload is typically produced by an average "fully" stocked aquarium. Perhaps that is a good topic for a follow up post :-)

  4. #4

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    2 Not allowed!
    I'll read the whole thing later, but I'm betting it's sticky-worthy

    I'll make sure Cliff sees it.
    Last edited by Slaphppy7; 02-27-2015 at 11:08 PM.
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  5. #5

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    3 Not allowed!
    I think it's a pretty decent breakdown of the techno-babble behind why cycling works, in a way that most people can understand.

  6. #6

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    1 Not allowed!
    Thank you all. I wrote it mostly for myself, but quickly realized it needed to be posted :-)

  7. #7

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    2 Not allowed!
    Quote Originally Posted by cajunqn81 View Post
    I think it's a pretty decent breakdown of the techno-babble behind why cycling works, in a way that most people can understand.
    "Laymen's terms", I think they call it
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  8. #8

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  9. #9

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    0 Not allowed!
    K, don't get me started...this is a family friendly forum
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  10. #10

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    1 Not allowed!
    Quote Originally Posted by Slaphppy7 View Post
    You've got that right.

    I can't believe--or maybe you can--how many times I've had to take some of the simplest terms and common phrases that 99% of native English-speaking people should know and break them down even further. Or explain something that's generally considered common knowledge. Ah, well... You've got to pick your battles, I suppose.

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