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What is nitrogen?
Nitrogen is an element vital to all life processes on Earth. Nitrogen is very important in our biosphere, where nitrogen comprises 78% of the atmosphere, and is part of every living tissue. It is a component of amino acids, proteins and nucleic acids. With the exception of carbon, nitrogen is the most universal element of life. Life could not exist without nitrogen. Nitrogen is essential for organic development; nitrogenous compounds are also required by some organisms for metabolic functions and respiration. Unfortunately, free nitrogen in the atmosphere is not in a form that is usable by plants or animals. Because of its stable structural formula, it is relatively inert and does not combine readily with other elements.
All living organisms, from fish to plants, have great quantities of assimilated nitrogen in their tissues. Nitrogen is a fundamental ingredient for the formation of proteins and nucleic acids. Every organism you place in your aquarium adds nitrogen based compounds; from fish to coral, to live rock, to plants.
The introduction of food also adds nitrogen. Dead or alive, they are organic masses, and possess the same nitrogenous attributes as the fish, plants, invertebrates you added to your aquarium.
Inorganic nitrogen is added two ways: the atmosphere and new water. Atmospheric nitrogen (N2) is incorporated into our aquarium water by way of nitrogen fixing bacteria and by Cyanobacteria (bacteria that obtain their energy through photosynthesis) as ammonia (NH3). Some Cyanobacteria fix nitrogen gas, which cannot be used by plants, into ammonia (NH3), nitrites (NO2−) or nitrates (NO3−). Nitrates can then be utilized by plants and converted to nucleic acids and protein.
Inorganic nitrogenous compounds from our tap or well water also enter our aquarium, often as Nitrates or Nitrates. Reverse Osmosis can remove much of this. For more about tap water, please see this article: “What should I know about tap water for my aquarium? From Chlorine and Chloramines to Phosphates”
Four Types of Bacteria are important to the Nitrogen Cycle;
• One of the most important biological conversions involves the transformation of N2 into a form readily available for plants.
Specialized gram negative bacteria that live in soil (Rhizobium) have the capability of converting N2 into NH3 (ammonia) a chemical that plants can absorb through their roots. Ammonia can be used to synthesize amino acids (Rhizobia bacteria require a plant host as they cannot independently fix nitrogen). The process of converting N2 + 3H2 > 2NH3 is called Nitrogen fixation. The bacteria that perform this task are anaerobic (no oxygen required). They possess the enzyme nitrogenase, necessary to catalyze this reaction. Certain plants develop anaerobic nodules on their roots that contain the nitrogenase-producing bacteria such as soybeans.
As well Blue-Green Algae (actually Cyanobacteria) can “fix” nitrogen and often live symbiotically with some plants; such is the case with the tiny aquatic Azolla fern found throughout the tropical & temperate regions of the world.
• Nitrifying bacteria change ammonium (NH4+) to nitrite (NO2-) then to nitrate (NO3-). This is the most important aspect of the nitrogen cycle in an aquarium & pond and will be discussed in depth as this article progresses.
• Denitrifying bacteria convert nitrate to nitrogen (N2) gas or Hydrogen sulfide under certain conditions in aquariums or ponds. Denitrifying bacteria are anaerobic, meaning they are active where oxygen is absent. This process is also important to aquariums & ponds (especially marine reef aquariums) and will be discussed in depth as the article progresses.
• Another large group of bacteria are Actinomycetes that grow branching filamentous cells (hyphae) like fungi. These Heterotrophic bacteria are active at high pH levels, are generally gram positive and aerobic. Actinomycetes are common in soil and are responsible for the characteristic smell of freshly turned healthy soil. Actinomycetes decompose large organic substrates/compounds that are hard-to-decompose such as cellulose or Chitin found in exoskeletons of crustaceans.
Fungi/Saprolegnia are more important in degrading these compounds at low pH.
OTHER ORGANIC WASTE
I think it is noteworthy to explain that not all organic waste contributes to the nitrogen cycle, (hence high ammonia, etc.). The importance of knowing this is that certain organics may have low amounts or NO nitrogen containing compounds, so controlling the addition of high content nitrogen containing compounds can be important in keeping lower nitrates in established aquariums or lower ammonia/nitrites in less established aquariums (or aquariums that have had their bio cycling process interrupted).
All plant and animals contain proteins (amino acids) and lipids, of which varying amounts of nitrogen atoms are contained depending on the molecule in question’
HOWEVER sugars, carbohydrates (starches) and most fats (fats are a form of lipids, generally without nitrogen atoms though) do not contain nitrogen atoms and therefore CANNOT directly contribute to the nitrogen cycle of your aquarium or pond (bacteria that feed on these oils can multiply and indirectly affect ammonia/nitrites/nitrates).
As well different animal and plant matter have varying amounts of nitrogen containing molecules (with plants in general containing less than animals). The few lipids that contain nitrogen atoms generally have few, and are often less of a factor as well.
With this in mind, more fish equals more nitrogen compounds released into an aquarium/pond, as well higher protein foods such as shrimp will also add more nitrogen compounds. While plants, even decomposing plants generally release fewer nitrogen bearing compounds than a decomposing fish or fish waste. So controlling these molecules via fish numbers vs. plant numbers and diet is important even for an established aquarium (so as to control nitrates).
Here are a few chemical formulas for example:
*Glycerol; a basic sugar and a central component of many lipids: C3-H5-(OH)3
*Glucose; a common sugar: C6-H12-O6
*alpha Linolenic Acid, a fat; Short-chain omega-3 fatty acid: CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH
* Psychosine, a lipid intermediate in the biosynthesis: C24-H47-NO7
* Leucine, an essential amino acid (protein): C6H13NO2
Further reference: http://www.newton.dep.anl.gov/askasci/mole00/mole00027.htm
NITRIFICATION:
When an organism dies, nitrogen is moved from plant or animal into the inorganic chemical ammonia by the process of bacterial decay. Ammonia is also produced by bacteria in the breakdown of protein. This process is called Mineralization and is the end result of the metabolism of food. However, ammonia is produced from both metabolism and mineralization. The decomposition (mineralization) process produces large quantities of ammonia (NH3) through the process of ammoniafication. Heterotrophic microbes (organisms that require organic substrates to get its carbon for their growth and development) utilize the organic compounds of decomposing matter as their carbon source. Ammonia (NH3) is the byproduct of this consumption.
Ammonia, in its neutral state, exists as ammonium (NH4+). Ammonium (NH4) is formed by the protonation (the addition of a Proton (H+) to the molecule. During this process of protonation NH3 (which is a base) converts into a weak acid (an acid which has the tendency to lose, or "donate" a hydrogen ion, also known as a “Brønsted-Lowry acid”. This tendency to “donate” a hydrogen ion is how NH4 converts back to NH3 as pH rises. Products such as Prime block this process, maintaining the extra hydrogen ion
Ammonia is assimilated in more than one way. Plants (such as Hornwort) and algae can assimilate ammonia and ammonium directly for the biosynthesis. The remaining bulk of decomposed byproducts are utilized by bacteria in a process called nitrification. Fortunately Ammonia does not last long in a healthy aquarium environment. Nitrifying bacteria such as Nitrosomonas quickly break down ammonia into less toxic Nitrite (NO2). During this process, specific species of nitrifying bacteria strip the ammonium of its hydrogen molecules as an energy source. Oxygen molecules are then affixed to the stripped nitrogen, forming the oxide nitrite (NO2).
Another group of bacteria (Nitrobacter ) utilize the enzyme nitrite oxidase that is then responsible for converting nitrite into nitrate (NO3). This nitrate can either be used by plants as a nutrient source, or can be further broken down into nitrogen gas (N2) through the activity of anaerobic bacteria such as Pseudomonas .
It should be noted, that without oxygen (nitrification is an oxidative process), none of this process can take place.
What are nitrifying bacteria?
There's a lot of confusion among aquarists about nitrifying bacteria. This is due in large part to the recent emergence of a wide variety of bacterial products claiming to be nitrifying aids. Most of these products actually contain species of Heterotrophic bacteria from the genera Bacillus, Pseudomonas, Escherichia, and others (as these bacteria are much easier packaged with a reasonable shelf life under normal conditions/room temperatures).
 True nitrifying bacteria are Autotrophic and considered to be those belonging to the family Nitrobacteraceae whose energy sources are derived from the chemical conversion of ammonia to nitrite, or, nitrite to nitrate (Autotrophic bacteria are organisms that produce complex organic compounds from simple inorganic molecules). They require oxygen, utilize mostly inorganic (without carbon) compounds as their energy source, and require carbon dioxide (CO2) for their source of carbon. In the case of the Nitrobacteraceae these energy sources are derived from the chemical conversion of ammonia to nitrite, or, nitrite to nitrate.
The desired Autotrophic aerobic bacteria of nitrifying bacteria are present everywhere (e.g., in the air), oxygen and at least some moisture is present (not in areas void of oxygen) (reference: 3). It is important to note that although the desired nitrifying species of bacteria are “all around us”, they do not readily store in sealed oxygen free containers (dying rapidly without oxygen), and it takes some time for the sparse air born nitrifying bacteria to populate an aquarium or pond, so do not expect these bacteria to “magically” populate your aquarium overnight, even a re-started aquarium will need to be re-populated (please see cycling methods further down in the article).
These bacteria are primarily gram POSITIVE, which is why you need to be careful when treating with medications that are primarily gram positive such as Erythromycin (Maracyn), Ampicillin, or Penicillin (I have also found Tetracycline Hydrochloride to be hard on nitrifying bacteria as well). Another point of misinformation about these bacteria since they are gram positive is that most aquarium diseases are gram negative (especially marine) such as Vibrio, Columnaris, Pseudomonas, Aeromonas and treatment with antibiotics such as Kanamycin or Nitrofurazone which are primarily gram negative will NOT interfere with the nitrifying cycling process.
Another commonly used product for aquarium Ich infestation is Malachite Green, despite some common anecdotal comments Malachite Green, as well as Copper Sulfate DO NOT affect nitrifying bacteria (please see references below)
Five genera are generally accepted as ammonia-oxidizers and four genera as nitrite-oxidizers. Of these, Nitrosomonas (FW), Nitrosococcus (SW), and Nitrospira (ammonia-oxidizers); Nitrococcus (SW), Nitrobacter (FW), and Nitrospina (nitrite-oxidizers) are the most important. Species of marine nitrifying bacteria are different from those that prefer fresh water, and yet, are very closely related.
Heterotrophic Bacteria (which are generally found in most over the counter aquarium cycling products due to their portability) are usually aerobic in these products, they can be either gram-positive (ex: Bacillus) or gram-negative (ex: Pseudomonas), heterotrophs (an organism that requires organic substrates to get its carbon for growth and development). Some are strictly aerobic, but many are facultative anaerobes (they can survive in either the presence or absence of oxygen).
Another point is growth (which is why Heterotrophic bacteria are favored for cycling products); nitrifying (Autotrophic) bacteria will double in population every 15-24 hours under optimal growth conditions. Heterotrophic bacteria, on the other hand, can reproduce in as little as 15 minutes to 1 hour.
Unfortunately research has shown that up to one million times more of these heterotrophic bacteria are required to perform a comparable level of ammonia conversion that is attained by true autotrophic nitrifying bacteria, in part due to the fact of Heterotrophic Bacteria to convert many organics into food.
The use of only Heterotrophic Bacteria to cycle an aquarium (or pond) can result in a bio environment that does not contain the necessary Autotrophic nitrifying bacteria to rapidly adapt to changes in bio load either from added fish, wastes, or similar; thus often resulting in sudden spikes in ammonia or nitrites when these Heterotrophic bacteria cycling products are not added in a timely or regular schedule!
Low pH and Nitrification Important;
It is also noteworthy that the primary nitrifying bacteria are affected by pH.
PH levels of 7.5 to 8.5 are considered optimal for healthy nitrification of ammonia, and nitrites, as nitrification rates are rapidly depressed as the pH is reduced below 7.0.
During the nitrification process carbonates are used by the aquarium or pond to counter acids produced, however without an adequate KH (even for Amazon River Fish such as Discus or German Rams), this can become critical in crowded aquariums with low pH, thus allowing for a rise in ammonia and nitrites, sometimes to toxic levels. However the flip side of low pH is that ammonia is converted to non toxic ammonium at lower pH, but keeping a low pH/KH that suppresses nitrifying bacteria can be a double edged sword that allows the growth of pathogenic Fungi/Saprolegnia.
This is often misunderstood by some aquarists keeping such fish, resulting in the fish keeping adding cycling products, changing water, or anything else in a desperate attempt to lower ammonia/nitrites when the problem is a low pH/KH in a crowded aquarium that does not allow for adequate nitrification by chemolithotrophic nitrifying bacteria.
References:
Affects of chemicals on the biofilter, autotroph , Cloudiness in the Water, & What are bacteria?
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=525248
http://www.environmentalleverage.com/Total%20Nitrification%20Balance.htm
A few key points about ammonia:
• Ammonia in the form of toxic NH3 commonly found in aquariums from wastes
(Animal and plant) will convert to NH4 (which is much less toxic) in a pH of around 6.4 or less.
• The symptoms of ammonia poisoning can often appear as a disease or actually allow a disease such as Aeromonas infections to establish in the fish under stress from ammonia poisoning.
The most common symptom of ammonia poisoning is rapid respiration (often near the surface), followed by flared or abnormally red gills.
Prolonged exposure to even moderate levels of ammonia (or nitrites) can result in fin deterioration and loss of color. This is noteworthy, as many aquarists will treat for fin rot or other diseases when the problem is either directly ammonia poisoning or a result of ammonia poisoning. Such treatments will usually fail and often make the situation worse as dumping in medications to solve an ammonia poison issue will often exasperate the problem further (the use of products such as Prime are OK as a temporary solution to the problem of high ammonia)
• Surface agitation can cause some ammonia ions to evaporate, but not quickly enough to be significant to a healthy aquarium. The ammonia commonly found in aquariums should not be confused with ammonia carbonate used in cooking which more readily evaporates nor should an aquarist confuse the process of evaporation in a cooling system commonly used in RV refrigerators which heats the ammonia for evaporation and cooling (this system basically works by heating anhydrous ammonia to evaporate it then the ammonia condenses and cools and the process starts over again).
• Toxic NH3 ammonia can be remove or detoxified by products such as Prime , Ammo Lock, or Amquel but this is a short term solution to proper bio filtration.
DENITRIFICATION:
Denitrification is the process by which microorganisms convert nitrate (NO3) to nitrogen gas (N2). In terms of the global nitrogen cycle, denitrification serves to balance nitrogen fixation by removing fixed nitrogen (rather than supplying it) to the biosphere. Most denitrifying bacteria are heterotrophic (such as Paracoccus denitrificans and various pseudomonads), utilizing organic carbon, hydrogen or hydrogen sulfide as electron donor and nitrate as electron acceptor. The electron donor is oxidized (to CO2, water or sulfate) and nitrate is contemporaneously reduced to dinitrogen gas (N2). Denitrifying bacteria require a source of reductant (energy) and a source of oxidant (nitrate).
This process can take place in an environment without oxygen by anaerobic bacteria. This process is more common in Marine aquaria and takes place in fine #00 sand, live rock, or “aquarium mud”. In freshwater aquariums this process often produces deadly Hydrogen Sulfide, but by maintaining an oxygen level above 1 ppm, this can be avoided. Plants roots are great for maintaining this balance of oxygen in the gravel for proper Nitrate removal.
It is also noteworthy that many premium aquarium/pond cycling aids or waste digesters such as SeaChem Stability or Natural Environmental Pond Keeper contain anaerobic heterotrophic bacteria and can be useful to add during spikes in the bio load of an aquarium or pond to aid in nitrate reduction and lower incidence of Hydrogen Sulfide production from decaying organic wastes (which can also affect water clarity and algae blooms).
 The production of Hydrogen Sulfide in aquariums (both salt and even more so freshwater) is a controversial subject, often with unclear answers as to whether anaerobic de-nitrification is beneficial in freshwater due to the POSSIBLE production of Hydrogen Sulfide.
With the most current research (although admittedly not conclusive in my view), you CAN have anaerobic de--nitrification and NOT have dangerous levels of Hydrogen Sulfide produced.
A tell-tale sign of Hydro Sulfide production is black areas in the deep areas of sand or substrate, whether freshwater, marine, or especially ponds. The rotten egg odor is another sign although as Hydrogen Sulfide levels in the air increase, research has shown that human’s olfactory senses tend to block out the smell.
Please see the picture to the left of what the "black" Hydrogen Sulfide producing layers look like in sand (click picture to enlarge).
One key to allow de-nitrification without production of Hydrogen Sulfide is to allow some oxygen penetration of the substrate and as well, the occasional mixing of substrate via vacuuming or simply mixing of the substrate by hand. In freshwater, plant roots achieve this well and also remove raw ammonia as well as nitrates.
I have a new article dealing with the subject of Hydrogen Sulfides in De-Nitrification:
*Hydrogen Sulfide production in anaerobic De-Nitrification for Aquarium/Pond Nitrate Removal
As well as this article more specifically dealing with the subject of Nitrates:
* “Aquarium Answers; Nitrates”
BIOFILTRATION:
Nature can pack a lot of bacteria into small places, which is to the advantage of the aquarist. For bacterial growth, all that is required is ammonia and oxygenated water. This is the beginning of the nitrogen process and the growth of bacterial colonies.
Remember you need oxygen and a lot of surface area for bacterial colonies. Wet/dry filters, sponge filters, ceramic media, and loosely packed upper layers of gravel are all sources for bacterial accumulation. If there is not adequate surface area in oxygen areas of the filter media or gravel, nitrification will be poor. Relatively new scientific evidence shows nitrifying bacteria to be sticky and adheres to the surfaces like glue, so agitation of filter media when rinsing (no tap water), or vacuuming of gravel will not destroy these colonies. This is why water exchanges with established aquariums usually are ineffective for cycling aquariums and why the myth of UV Sterilizers killing beneficial bacteria is just that, a myth.
Water will follow the path of least resistance, so if your filter or gravel has a build-up of non-nitrifying bacterial slime or is packed to tightly, nitrification will not be achieved. Sponge Filters or Pre-Filters are an inexpensive filter medium for colonies of nitrifying bacteria. Wet/dry filters and live rock (in Marine Aquariums) are excellent sources of bio filtration in larger aquaria. Bio-Wheels are very popular, but in my tests in my maintenance business they are VASTLY overrated. They tend to accumulate hard water deposits and stop and even when they are working my tests have shown little difference in aquariums when they are removed as compared to sponge filters or wet/dry filters. Canister filters are also good sources for nitrification as long as the proper media is used (such as ceramic rings) and the media is not packed too tight and is rinsed regularly.
Bio Filtration during use of medications in aquariums:
During the use of medications, in particular gram positive antibiotics/antimicrobials your aquarium (or pond’s) important aerobic nitrifying filter bed may be damaged or outright destroyed. However, the majority of aquarium fish diseases are gram negative, so using medications such as Kanamycin are much less likely to damage your aquariums bio filter bed when used properly, including water changes prior to each treatment.
Sometimes it is necessary to treat with wide spectrum (mixed antibiotic treatments) or strong gram positive antibiotics such as Erythromycin for unknown problems or diseases such as Streptococcus or Eye Infections. In this case I strongly recommend having several “seeded” (seasoned/aged) bio filters/media such as sponge filters, grids, foam inserts, even gravel in high flow areas to add to the aquarium under treatment every 3-4 days (all the while monitoring ammonia levels), with one final “plant” of this seasoned filter media 2 days after the last treatment.
Although systemic treatment of a display aquarium is often necessary, when possible, treatment in a quarantine tank will avoid the above problem in the first place.
Please see this article for further information about medications:
“Aquarium Medications/Treatments”
SUMMARY OF LEVELS;
In healthy aquarium ammonia and nitrites should be at 0 ppm
In a healthy freshwater aquarium Nitrates should be 15-50 ppm (below 15 ppm is not healthy for planted freshwater aquariums).
In a healthy Saltwater fish aquarium nitrates should be below 40 ppm.
In a healthy Marine Reef aquarium nitrates should be below 20 ppm (or even less, many reef keepers aim for less than 10 ppm).
METHODS OF AQUARIUM CYCLING:
All the methods I will describe below are versions of “FISHLESS CYCLING”. Many associate fishless cycling with the pure ammonia method, however fishless cycling is ANY method that does not introduce fish until the aquarium has gone thru the nitrogen cycle, in other words, the ammonia and nitrites have gone up then back down which can take from 10 to as long as 45 days depending on method tank size and temperature. I will note that method one (if done properly) rarely results in an ammonia and nitrite spike, this is why it is my preferred method WHEN possible (which often it is not).
I do NOT recommend adding “starter” or cycling fish to start your nitrogen cycle for either freshwater or saltwater.
Finally, before I jump into different cycling methods, I would like to note that testing your and keeping a journal of your water parameters during the cycling process (as well later on, but not at as frequent of intervals) is important in my opinion and will help you note subtle changes. The frequency is a mater of preference/experience, but I would recommend at least every other day for beginners (generally more advanced aquarists can recognize signs and perform cycling as a routine that requires less testing).
These tests include the more obvious ammonia/nitrite & nitrate, but almost as important is the testing of KH/pH as carbonates are used up during the cycling process and this can result in pH drops that can seriously slow the growth of nitrifying bacteria
 [1] Seasoned Filter Media:
My preferred cycling method is to transfer filter media (sponges work well as Autotrophic nitrifying bacteria tend to cling to sponge media in high quantities and sponge media is easily transferred), although floss, ceramic media, volcanic rock, etc. are also fine from an established aquarium and possibly along with some gravel, then introduce the fish SLOWLY after 3-7 days. The method of adding “aged” or "seasoned" media is much faster (you still have to take it slow, but this with this method some fish can and should be introduced immediately), and provides all the necessary bacteria, the only negative is adding disease pathogens to your aquarium, but I have rarely encountered this problem. To prevent this transfer of disease pathogens and parasites, only use a media source where no new fish have been added in 30 days, ALL water parameters are good, and if possible has UV Sterilization (although not necessary).
It is also important to note with this method that you do not rinse filter media/gravel prior to addition to the new tank (especially not with tap water which can kill the nitrifying bacteria). It is important to NOT rinse so as retain some of the organics necessary to “feed” the bacteria, especially while fish are not present. It should also be noted that true Autotrophic nitrifying bacteria secrete a glue like substance (as noted elsewhere in this article) that allow the bacteria to cling to filter media/surfaces, so merely squeezing sponges or similar will NOT add many of these bacteria to your aquarium.
If fish are not added before 3-7 days, I suggest adding a small amount of fish food to feed your growing bacterial colonies. I will also note that the 3-7 days seems a bit vague, however this can vary due to temperature, amount of bacterial seed, and other factors, so I would not be overly concerned about the exact timing as I have added fish as soon as 2 days and as long as over a week with no problems.
For marine tanks the use of seasoned or “cured” live rock serve this purpose quite well.
I recommend this method even more with Marine tanks using seasoned (cured) live rock and/or live sand as well as filter media. In Marine tanks I still prefer to added aged media (not essential, but still better) along with 1-2 lbs (2.2 -4.4 kg) CURED live rock per gallon (approx. 4 liters).
This method (added media) will give you more instant bio bacterial colonies and this is method that is used by far the most by the professional aquarium maintenance community (which needs faster more sure results for their clients), despite the internet popularity of the next two cycling methods.
Also keep in mind that many pathogens such as pseudomonas are usually present in a healthy aquarium, but when fish are stressed, the fish are in poor health due to poor feeding and lack of proper minerals, and/or water conditions are less than desirable- these pathogens will be opportunistic and cause a disease in the fish.
We used this method for our Aquarium Maintenance route for years and never lost a fish to Ammonia or nitrite poisoning, and disease transfer was minimal.
It should also be noted that when done properly, you will rarely see ammonia levels rise past .50 ppm in the aged media method.
Another product that does not necessarily help speed up the cycling process itself, but de-toxifies the ammonia and nitrites during this process, allowing for less stress on fish is "Prime"
As a negative to the aged bio-filter media, this simply may not be possible for a new aquarium owner who has no friends to obtain aged filter media or if one does not trust a Fish Stores (or even a friend) aquarium health for obtaining aged/seasoned filter media. In this case, I would recommend one of the next two methods.
Further seasoned media cycling method tips
An ammonia spike is not uncommon with the use of seasoned media for there to be an ammonia spike when more fish are added, often this spike may take a several days depending on the amount of fish added as well as the bio load that the seasoned media has grown to handle.
Generally this is not too much of a concern, but the use of Prime, cutting back on feeding, and definitely placing the addition of any other fish "on hold" until the nitrifying bacteria "catch up" with the new bio load are good practices.
As an example of what happens (and this is NOT scientific, just an analogy):
If say you added a sponge filter from a 20 gallon tank that is fully seeded to another 20 gallon tank that is brand new or restarted after bleaching.
This seasoned Sponge filter for the sake of argument carried 50% of the bio load, in theory you could only stock this new tank to 50%.
If the seasoned filter only carried 25% of the bio load from the tank it was removed from, then you could only initially stock the new tank to 25% initially.
I should note that with the use of seasoned filter media, generally the nitrifying filter media will quickly colonize the rest of the tank, so waiting 6-8 weeks to add more fish is rarely necessary (at least I have never observed this in many trials using seasoned filter media).
 [2] Ammonia Method;
Another method is fishless cycling where un-scented pure ammonia is poured into the aquarium (3-5 drops per gallon pure ammonia or 1-2 teaspoons per 10 gallons) so as to bring your ammonia level to 3-4 ppm. Then it takes about 2-6 weeks (usually about 3 weeks) for the aquarium to cycle (when your ammonia and nitrites have dropped to 0).
However, please check your ammonia purchase by shaking the bottle at the store, if it foams or bubbles, it has detergents and should not be used.
This method is growing in popularity, however it is not without a few drawbacks. Here are some pluses and minuses to this method;
*This method does not add actual bacteria (and is not really any quicker than the method below: adding fish food to a fishless aquarium) and because human nature is to want to add fish sooner than the 2-6 weeks it takes for this method.
*It is still not as quick as my preferred method above (seeded media, gravel, sand, live rock).
*This method is especially dangerous when used with live rock and/or sand that have already been added as the ammonia will kill me organisms that reside in live rock adding even more ammonia and pollution to your tank thus defeating the reason for this method.
*As a positive, if patience can be observed, this method is very safe when one considers the possibility of disease pathogen introduction from the aged media method (no matter how remote the risk).
*The pure ammonia method also has a positive over the fish food method in that there is no risk of Saprolegnia (mold) introduction to the new aquarium (although the “raw shrimp method” is of vastly higher risk for this than the fish food method)
 [3] Fish Food Method;
Another method is the gradual addition of fish food to an otherwise empty aquarium (no fish). This can be a very effective means of cycling that is preferred by many experienced aquarists. This method takes about the same time as the pure ammonia method (2-6 weeks, usually about 3).
This is my preferred method when aged bio media is not available (not everyone has a friend or helpful local fish store to give them some aged media).
The only risk of the fish food method is the possibility of Saprolegnia (mold) growing on rotting fish food which can become pathogenic to new fish that will be introduced later.
This is easily avoided with a fish flake food by powdering it between fingers before introduction to the aquarium (shaking fish flake food in a cup of water can also accomplish this). This risk is relatively small and basically non-existent when you use an easily “liquefied” fish flake food. This unfortunately is NOT the case with the raw shrimp method (recommended by a few poorly researched sites).
4 ppm is a typical fishless cycling target whether using the fish food method or ammonia. Higher (7 ppm) or lower (3 ppm) is also fine for healthy bacterial colony growth (based on mine and others in the maintenance community’s experience).
Regardless of fishless cycling method chosen, the bio load is always going to be in flux (higher or lower). When higher is needed, nitrifying bacteria double in population in 18 to 24 hours. When less are needed, they die back and are consumed by each other.
[4] Other fishless cycling methods;
There are other methods of fishless cycling being recommended or used however one method being pushed on the internet is the use of Raw Shrimp; however this is a recycled idea (which included the use of silversides, frozen shrimp, and even dead feeder fish) and has reappeared on the internet even though it was debunked in the early 1990s.
I do not recommend this method, not because it does not work for cycling, but because it may also allow a Saprolegnia infection to get started in your new aquarium. Saprolegnia is a mold (often called a fungus) that easily gets a foot hold in decaying nitrogenous matter such as raw shrimp and I have seen this many times in my experiments. Even after the source of Saprolegnia growth is removed, the secondary zoospores which are the primary mode of pathogenic transmission can remain, even after large water changes/vacuumings.
A new tank is the worst time to have a Saprolegnia infection get started as this is when fish are often much less resistant to disease due to the stressor of a new tank environment.
I should note back when this method was making its “rounds” in popularity that it worked fine in many instances and with a 100% water change and vacuuming of gravel can reduce this risk even further, however some risk still remains as per my many tests of pathogens and as per the often misunderstood lifecycle of Saprolegnia, please see this article for further information about this subject (as well as more resources/references):
“Saprolegnia/Columnaris”
Another note/point is that even the fish food method (as noted earlier) can allow for Saprolegnia to get a foothold in an aquarium if food is simply dumped into an aquarium, making this method not any better than the “Raw Shrimp or Silversides” method as its decay will also attract Saprolegnia, so make sure to liquefy fish food prior to addition to your aquarium when used for cycling.
As a final point, this article has a section dealing with water changes and their affect on pathogens or similar: “Aquarium Disease Prevention; Section 1, Cleanliness”
[5] Cycling Products;
There are many products for cycling available too, but most in my opinion do not work well although properly cared for BioSpira (continuous refrigeration is a must, as well shelf life is still short) or the newer Fritz-Zyme TURBO START #700 can be effective (the key is properly cared for, the shelf life is short and it should be stored between 34 F and 40 F).
I will note however that many I know in the aquatic community such as others in aquarium maintenance profession as well as aquatic forums have not had all that good of results in tests (my own experience with this product is limited to tests with no scientific controls in place).
This is in part (IMO) due to the fact that aerobic nitrifying bacteria cling to media and gravel (this has been PROVEN scientifically) and do not work while suspended in water. Also poor storage and shelf life undoubtedly play a role as well, and proper since storage and handling cannot often be well verified, this explains the often mixed results especially with BioSpira (FritzZyme ships directly, so this may be the better choice of these two products).
It should also be noted that many freshwater products generally cannot be used in saltwater or vice versa.
Some companies claim to make products from the proper Autotrophic bacteria that contain the correct freshwater (Nitrosomonas and Nitrobacter) or saltwater bacteria (these must be refrigerated). Fritz-Zyme # 7 or #9 are good examples.
Liquid Cycle and Stress-Zyme are just preserved bacteria (mostly Heterotrophs) that are more useful for over feeding or other bio over loads in an established aquarium (as aerobic bacteria needed for nitrification do not store well in liquid form at room temperature without oxygen). Even Fritz-Zyme which contains the proper nitrifying autotrophic bacteria tends to use it’s in bottle food supply (which supposedly includes encapsulated oxygen, based on a trade rep., but I have no proof of this statement) and be less effective based on my test/use.
Honestly in my tests Cycle or Stress Zyme are really only useful to aid in breakdown of excess wastes from over feeding, poor filtration, etc. (Cycle can also be used as an aid to organic breakdown while waiting for your aquarium Nitrogen Cycle to get started from other means when fish are present). The Heterotrophic Bacteria within these products can aid in the decomposing of excess organic waste however they are basically useless for actually seeding an aquarium and this is a FACT.
Another product on the market by a VERY reputable company; SeaChem, is “Stability”.
Although I am a big fan of most SeaChem products, this product has a few claims that do not wash with known facts about nitrifying bacteria. SeaChem claims this to be a synergistic blend of aerobic (including encapsulated oxygen), anaerobic, and facultative bacteria; however facultative bacteria are NOT the primary bacteria of nitrification and are mostly Heterotrophs of which I discussed the differences earlier in the article.
This said, the bacteria found in Stability, albeit not the primary bacteria of nitrification is the closest bottled product to date that I have found to be a reliably useful product.
However my recommendation as it pertains to Stability is to use it as an aid in cycling of new aquariums or (better) as a boost when the bio load of an aquarium suddenly “jumps’ for whatever reason. If any cycling product is to be used in an aquarium, Stability would be the one I would recommend, especially for an aquarium that already has fish and is encountering cycling problems (whether an established aquarium that had a spike in bio load for whatever reason or a new tank that had a set back for whatever reason).
Quote from a fish forum about the use of Stability, Cycle, Stress-Zyme
I can't say enough about Seachem's Stability. It is a miracle worker. I started a 45 liter Eclipse tank just after Christmas, and it never stabilized. In fact, after 7 weeks, the ammonia had risen to well past the 8.0 ppm on the test card. The guys at the local fish store couldn't believe anything was still alive in there, the fish guys told me to use heavy duty doses of Cycle to clear up the ammonia, but two weeks later with no change, I decided to scrap the whole setup and start over. I waited a week to put the fish back in. Within 24 hours, the ammonia level had climbed two colors on the chart, and I hadn't even fed the fish! Exasperated, I went online to find a solution. i found lots of recommendations for Bio-Spira, and I would have probably gotten it if the store had not been out of it. There was only one bottle of Stability, I took it home and added it to my quickly clouding water, and within 24 hours, 80% of the cloudiness was gone. Within 36 hours the ammonia level had dropped to almost normal. I couldn't believe my eyes! After using a half a bottle of Cycle over 2 weeks to no avail, (ditto with the Stresszyme) this Stability came along and fixed what no one else seemed to be able to! I am one happy camper, and I would recommend Stability to everyone!
Maire S
PLEASE reference this article for more information to back up what I am saying: http://www.ucmp.berkeley.edu/bacteria/bacterialh.html.
[6] Marine Aquariums;
 Seasoned Live Rock is an excellent way to jump start your marine tank nitrogen cycle.
By seasoned I mean live rock that has been in a healthy established marine/saltwater aquarium at least 6 weeks, and more preferably 8 plus weeks.
Be careful of buying so-called live rock for a new aquarium that just arrived at your local retailer or online.
This live rock should likely also have some “food” for the nitrifying bacteria as well as healthy colonies of Nitrosococcus, Nitrospira, & Nitrococcus nitrifying bacteria in the outer areas where oxygenated water circulation can reach these bacteria. It is important to keep good circulation around your live rock with a power head or propeller pump once introduced into your new aquarium.
I would point out that live sand does not add nitrifying bacteria in significant amounts to help jump start your nitrogen cycle nearly as well as live rock due to the fact of oxygen depth penetration. Also it should be noted that these high priced bags of live sand commonly sold primarily contain Heterotrophic Bacteria which are NOT the primary bacteria of nitrification. True nitrifying Autotrophic Bacteria will NOT live long in a sealed bag so the best you will get out of these pre-packaged live sand bags is some de-nitrifying anaerobic bacteria which in my opinion is not worth the price and one is better off just buying dry #00 oolite sand for much less.
This is not to advocate against a deep sand bed, far from it, but only to state that the primary objective for this is for anaerobic bacteria which results in Nitrate control (not ammonia/nitrites).
*Also the use of “live saltwater” is NOT based in any real scientific evidence since nitrifying bacteria secrete glue like substance to adhere to substances such as live rock and are generally not found in real quantity in the water column.
*Finally I will note as to different cycling methods; I have used these different methods in my Aquarium maintenance and research business in controlled tests and still found that the added media/sand/gravel/seasoned live rock method works fastest and with the best results, although I have also had good results with this method combined with the fish food method (do not combine with the pure ammonia method)
*Effect of plants on Aquarium Cycling:
As mentioned earlier, many plants also remove nitrogenous waste such as Hornwort. I usually do not add all the plants I desire until the aquarium is fully cycled (regardless of method used), which is usually 2-3 weeks. The plants will help carry the waste load vs. an aquarium without plants, but I like to see at least some bacteria establish it self before a full load of plants are added. Having plants in the beginning does help keep the dangers of ammonia (NH3) or Nitrites (NO2) from building to toxic levels, which allows for a quicker addition of fish to your aquarium.
Summary:
Please note that whatever method you choose to cycle your aquarium from aged media, to pure ammonia, to Bio Spira; that if your fish are exposed to high ammonia and nitrites for any prolonged period (over 24 hours in my opinion), these fish can and will suffer permanent gill damage that will cause future problems with disease resistance and even overall aquarium health as these fish may become a starting point for opportunistic infections, which is another reason that either adding generous amounts of “seasoned” filter media while fish are present or simply waiting until the tank/pond is cycled is best for the health of fish that may be subjected to high ammonia/nitrites.
Another important point as to the use of cycling products, whether it be Cycle or the highly touted Bio Spira; often aquarists (even so called experts, and I have to admit to being guilty to this at times myself), will base an opinion on whether a product or method works are not on non-controlled observations.
Let me give an example; If you added fish to a non cycled aquarium, then started adding Cycle to this aquarium and noticed improved fish survival and lower ammonia/nitrites you might assume that this product is cycling your aquarium, this would be a wrong assumption!
Here is my explanation, the Heterotrophic Bacteria contained in Cycle will decompose some of the organics, thus lowering initial ammonia output, but this is not actually “seeding” your aquarium because as soon as you cease using this product your ammonia will go right up due to the lack of Autotrophic nitrifying bacteria. This can also even be said about Bio Spira as it is highly possible for the Autotrophic bacteria to die off leaving the Heterotrophic bacteria behind (the Heterotrophic bacteria survive much longer in a sealed container) giving the casual observer the impression that the product is performing an action it is not. Even the use of products such as Prime or Amquel Plus (which I highly recommend) will give a similar impression by limiting the toxic side effects of ammonia/nitrite while being used, but these products do not actually cycle your aquarium either!
Of coarse my example also shows where these products can be useful as well, just do not depend on them for cycling (Bio Spira may help more than Cycle though). Also I might make the point that by either employing fishless cycling or aged filter media, this should never come down to the use of such products (unless your bio filter has been disrupted by the use of gram positive medications).
A FEW DOS AND DONTS FOR MAINTAINING A HEALTHY NITROGEN CYCLE:
*DO Rinse filter media in used tank water or de-chlorinated tap water so as to not kill nitrifying bacteria.
*Do NOT rinse your filter media in tap water or turn filters on and off, this will hamper the establishment of healthy bio colonies of nitrifying bacteria. Filters should run 24/7 except in rare instances.
*Do NOT wash gravel or totally change water, again this will destroy or hamper establishment of bio bacterial colonies.
*DO maintain carbonate hardness (KH), especially when cycling a new tank, as the process of cycling will use carbonates and without adequate carbonates (KH) your pH can crash which will slow the establishment of nitrifying bacteria
*DO rotate filter media changes so as to always have older media with bacterial colonies. In a HOB filter with a cartridge, place a second cartridge in 7-10 days before changing the old one of install a sponge pre-filter on your HOB filter such as a Filter-Max Pre-Filter
*Do add de-chlorinators when changing water over 10%
*Do NOT vacuum all ‘dead’ spots under live rock in the live sand of a “Marine Tank ONLY”. (This can destroy anaerobic bacterial colonies in Marine Tanks needed for de-nitrification). Even then try and vacuum only the top layer in open areas of live sand in marine tanks, with only occasional “dips” into deeper sand (the use of a ½ inch layer of #3 crushed coral makes this process easier and quite bluntly makes for better aerobic and anaerobic filtrations and as well trapping of detritus)
WHAT TO DO FOR HIGH AMMONIA OR NITRITES (when Fish are already present):
*Change anywhere from 20%- 50% water every other day (or even every day in severe cases) until you at least reach an ammonia or nitrite level of 1 ppm or less (0 is what you want eventually). The use of Prime or Amquel Plus immediately after each water change is strongly suggested. Also keep in mind that at for example 2.0 ppm ammonia, followed by a 50% water change, you are still at 1.0 ppm (which is why the Prime/Amquel + suggestion)
Here are a few suggested water change amounts based on ammonia reading (please note these are suggestions, not facts):
-4.0 or higher; up to 50% per day
-2.0- 4.0; up to 50% every other day or 25%- 50% per day
-1.0- 2.0; 25% every other day
-under 1.0; 20-25% every other day
Steps to temporarily improve high ammonia problems (emergency)
Followed by steps for a more permanent solution
*Use Prime (removes chlorine/chloramines, detoxifies ammonia & nitrite) or Amquel Plus or to a lesser extent many other older generation Ammonia/Nitrite de-toxifiers such Ammo Lock, or Amquel. These products do not remove ammonia they change the ammonia from highly toxic NH3 to less toxic Ammonium (NH4). Please note the ammonia will still test after using these products as the test kits do not differentiate between ammonia and ammonium (ionized ammonia). Prime or Amquel Plus will keep the ammonium safely “bound” even if the pH climbs (which would otherwise convert NH4 to NH3). The ammonium is still available for nitrifying bacteria to consume and therefore Prime will not interrupt the growth of healthy bio-bacterial colonies as the ionized ammonia and nitrites are still available for nitrifying bacteria. It should be also noted that most test kit will still show the ammonia/nitrites at the SAME level as before the addition of Prime/Amquel +, however rest assured this is in a non-toxic ionized form that is still bio available to Nitrobacteraceae bacteria that forming in your filter media/gravel.
Here is more information about Prime: “The detoxification of nitrite and nitrate by Prime (when used at elevated levels) is not well understood from a mechanistic standpoint. The most likely explanation is that the nitrite and nitrate is removed in a manner similar to the way ammonia is removed; i.e. it is bound and held in an inert state until such time that bacteria in the biological filter are able to take a hold of it, break it apart and use it. Two other possible scenarios are reduction to nitrogen (N2) gas or conversion into a benign organic nitrogen compound”.
See SeaChem Support AND
"Aquarium Answers: Aquatic Water Conditioners" for more information.
*Change water, at least 25%, (use a gravel vacuum so as to remove as much decomposing organic mulm as possible)
*Add a cycling aid, such as SeaChem Stability which interferes the least with establishment of a healthy bio filter in your substrate or filters as it provides both Autotrophic and Heterotrophic bacteria, as well unlike competing products, the bacteria employed by Stability are non-sulfur fixing and will not produce toxic hydrogen sulfide.
*Add salt (NaCl); this is a popular method for “nitrite poisoning” and should be added at 1 tablespoon per 5 gallons to 1 teaspoon per gallon depending upon fish sensitivities. The addition of salt will prevent methemoglobin that forms in the blood due to nitrite exposure from building up.
Salt can be combined with Prime or Amquel Plus, however from my experience the salt method is not nearly as effective as Prime when used by itself.
*For high ammonia or nitrite exposure (common in fish shipped from long distances), the use of Methylene Blue in 30 minute baths is very effective and also counters the effects of methemoglobin in the blood by increasing the hemoglobin oxygen carrying abilities.
Steps for a long term solution to lower ammonia levels
*Add "seasoned" by bio media such as sponge or filter fiber form another healthy tank to kick start your bio filtration.
If at all possible, this can do MUCH at lowering your ammonia/nitrites, especially if large amounts of seasoned bio filter media from other tanks are added (6 weeks plus in age is best).
*Add filter media with high abilities to maintain nitrifying bacteria such as Volcanic Rock, Ceramic Bio Media, and especially SeaChem Matrix
*Consider zeolite in the filters to absorb some of the ammonia (FRESHWATER ONLY!).
*Use of chemical absorbents such as SeaChem Purigen placed in a filter or other high flow areas
*Cut back on feeding and do not use fish foods high in non aquatic proteins, which are mostly un-digestible by fish and add to your ammonia.
For more about proper fish food ingredients, please see this article: Quality Fish Food; what ingredients are needed for proper fish nutrition, growth and health.
To expand on this point; ammonia has nitrogen in it (hence the “N” in NH3) and so do ALL proteins (where as carbohydrates/fats do not), so feeding fish foods high in proteins or indigestible proteins during an episode of high ammonia can be a MAJOR factor in high ammonia levels.
*Increase pH slowly and more importantly KH, as a pH below 7.0 slows the growth of nitrifying bacteria (as stated earlier in this article). This is often a problem with keepers of Discus and German Rams, sometimes unbeknown to the fish keeper that is losing their valuable stock.
*Although as explained earlier that products such as “Cycle”, or “Stress Zyme” are not good choices for “kick starting” your aquarium or pond nitrogen cycle (in other words used as a biological cycling product), they DO contain Heterotrophic Bacteria than can at least consume some of the organic waster while your nitrogen cycle is still in the process of establishing itself or re-establishing itself if the bio filter has been interrupted by gram positive medications (assuming fish are present, otherwise I would not use them). The best choice here would be SeaChem Stability as it seems to do the best job and has the least interference on replacing true Autotrophic nitrifying bacteria.
The negative of using these types of products during this time, is that unlike Prime or Amquel Plus, these products WILL remove nutrients from the water column necessary for a your true Autotrophic nitrifying bacteria to establish themselves, thus delaying the cycling processes.
WHAT TO DO FOR HIGH NITRATES:
Although not generally toxic high nitrates can stunt fish growth and lead to health issues if fish are exposed to high nitrates for an extended time in freshwater. In saltwater the same holds true for fish, but with many invertebrates, especially coral and cephalopods nitrates above 20 ppm can be deadly.
*Prime as well as Amquel Plus will neutralize the Nitrate but is not a long term solution to high nitrate problems.
*Perform a water change using a gravel vacuum to remove not just dirty water, but “mulm” that will decompose and enter the nitrogen cycle and eventually become nitrates.
This can be a particular problem with Under Gravel Filters (UGF), decomposing organic debris will tend to build up under the filter plate. For these filters I recommend occasionally removing the lift tubes and placing a siphon into the opening and removing as much organic debris (mulm) as possible.
Even without UGF, poor vacuuming procedures (or none at all) can contribute to high to high nitrates. Make sure and vacuum around and under ornaments as well, although be careful around live plants. I think the Aquarium Cleaning Machine (Aquarium power siphon and re-circulating micron filter is a better idea for larger aquariums.
*Proper filtration and maintenance; Make sure and regularly rinse in de-chlorinated or used aquarium water (never tap water) your bio filter media. This includes bio rings and balls commonly found in wet dry filters and canister filters, sponge filters, bio wheels, or any other media that is not changed in the filter. This is especially important with filters that tend to become “nitrate factories”, which include in my experience (AND tests) to be; Canister Filters, Wet-Dry filters, and Emperor Filters. This is not to say these filters are bad, it is just important to not ignore properly cleaning these filters even though their large capacity makes it very easy to do.
I will however add that for reef aquariums I do not recommend the before mentioned filters unless one substitutes live rock crumbles (or volcanic rock) for bio balls.
The reason behind this is that bio balls although excellent for aerobic nitrification only contain bacteria on the outer surfaces and thus are NOT capable of sustaining anaerobic de-nitrifying bacteria necessary for nitrate removal as these bacteria live on surfaces where little oxygen penetrates. Live Rock crumbles/scrap are capable of containing these bacteria necessary deep inside their pores (as is volcanic rock, although not quite to the same level, but close)
I have seen nitrates in freshwater go down by simply changing filter systems (the over all health of the aquarium improved as well). The last time I did this was by removing an Emperor 400 and replacing it with a combinations Sponge filter/ Internal filter/ Fluidized Filter System.
*Add aquatic plants to freshwater especially hornwort.
For saltwater some green algae will perform this task as well (this is why I do not remove all my green algae in the marine aquariums I maintain). Caulerpa Algae (which looks like a plant performs nitrate removal reasonably well).
*For saltwater aquariums consider a Refugium or a Protein Skimmer. A Refugium uses plants in a sump with strong lighting to remove nutrients including nitrates. Protein skimmers remove nitrates before they enter the nitrogen cycle via foam refraction (a similar idea to vacuuming the “mulm”). Personally I have used many a Protein Skimmer and find many over rated considering the constant adjustments in many of the Skimmers available, rate of refraction compared to water volume. However this is not to say a protein skimmer does not work, these are useful tools in marine aquariums for nitrate control, and I just do not recommend “throwing all your eggs in one basket” with these devices. Also keep in mind that not all Protein Skimmers are created equal, so purchasing a true quality unit is worth the investment such as the V-2 Skim Professional Skimmer along with the purchase of a Ozone Generator to further improve the effectiveness of your protein skimmer.
A Mud Filter or Mud Filter/Refugium combination works well too.
 *Besides the Refugium, a DSB (Deep Sand Bed) in a bucket works well. Put 8" to 12" of fresh, clean sand in a 5 gallon bucket; Oolitic sugar-fine aragonite works best in my opinion. Drill a bulkhead about an inch or two higher than the top of the substrate for the water to flow out from back into your tank/sump. A flow rate of 150-200 gph, just enough flow over the sand to keep solids in suspension, I recommend pre-filtering the water first to prevent detritus build up. This is a very effective method (similar to Mud Filters) that can bring Nitrates down to near 0.
Please see this link for a DIY Deep Sand Bucket/Tank picture and more information about this idea: “DIY Deep Sand Bucket”
*The use of premium “over the counter” waste digesters or cycling aids (such as SeaChem Stability) which contain heterotrophic bacteria for waste digestion and nitrate removal (these products should be used as an aid and not depended upon for a long term solution to nitrate and organic decay problems)
Please see these excellent articles for more about nitrates and nitrate removal:
* “Aquarium Answers; Nitrates”
* “Hydrogen Sulfide production in anaerobic De-Nitrification for Aquarium/Pond Nitrate Removal”
FURTHER REFERENCES:
(1)Chemolithoautotrophic Nitrite-Oxidizing Bacterium Nitrobacter
(2)Removal of Nitrogenous Waste Products from Water
(3)http://www.cs.duke.edu/~narten/faq/cycling.html
(4)http://alpha.marsci.uga.edu/FacultyPages/Joye/research/belize.htm
(5)http://www.carteretcountyschools.org/chs/fptwebs/JBUTLER/organic/Nitrogen%20Cycle%20AP%20Biology.htm
(6)http://www.dls.ym.edu.tw/ol_biology2/ultranet/Eubacteria.html
(7) http://www.college.ucla.edu/webproject/micro7/studentprojects7/Rader/asludge2.htm
(8) http://soils.usda.gov/sqi/concepts/soil_biology/bacteria.html
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