Nitrogen is an element essential to life. In its simplest form, it occurs as dinitrogen (N2), the major component of air. How it is converted to a chemical form usable to living organisms, the path it travels through biological processes, and how it is eventually returned to dinitrogen is the nitrogen cycle.
Nitrogen begins its journey into the realm of living organisms via a process known as nitrogen fixation. Fixation occurs mainly when microorganisms having the nitrogenase enzyme extract dinitrogen from the air and convert it to ammonia. These microorganisms may be free-living or may be symbiotic with plants. Regardless, the ammonia produced is quickly utilized by plants or other microbes and converted to proteins or other organic nitrogen compounds. This is known as nitrogen uptake. As a side note, high energy natural events, such as lightning, and industrial processes may also fixate nitrogen.
The organic compounds created are metabolized by living organisms to sustain life processes. The compounds travel through the food chain as critters eat plants and critters eat other critters. The end users that benefit from these organic nitrogen compounds are heterotrophic microorganisms that break them down to ammonia (NH3) and ammonium (NH4+), a process known as nitrogen mineralization. Simplified, this is decay.
Inorganic nitrogen may follow one of two paths at this point. It may be used by plants or free-living microorganisms and returned to an organic state through the nitrogen uptake process, or it may enter the nitrification process.
Nitrification is the conversion of ammonia to nitrate through biological oxidation. It is an aerobic process that involves several stages and numerous species of microorganisms. The two genera that contribute to nitrification the most are Nitrosomonas and Nitrobacter. Nitrosomonas sp. convert ammonia (NH3) to nitrite (NO2-) and Nitrobacter sp. convert nitrite (NO2-) to nitrate (NO3). The nitrate is then either used by plants or free-living microorganisms and returned to an organic state through the nitrogen uptake process, or is denitrified.
Denitrification is the process through which anaerobic microorganisms convert nitrate back to nitrite, and then to nitrous oxide, nitric oxide and dinitrogen. Dinitrogen escapes to the atmosphere to rejoin its friends and family, and to await fixation. Some nitric oxide and nitrous oxide may remain, but dinitrogen is the main product of denitrification.
It is the next to last phase of the nitrogen cycle, nitrification, which is of most interest to the aquarist.
In the Aquarium
Where does ammonia in the aquarium come from? Whereas mammals, birds, reptiles and most terrestrial amphibians excrete excess nitrogen in an organic form, teleost fish, aquatic invertebrates and aquatic amphibians are ammonotelic: they excrete excess nitrogen directly as ammonia. The ammonia is passed out through the gills during respiration. This is the first source of ammonia in an aquarium.
The second source is the result of heterotrophic microorganisms causing the decay of additional organic matter such as uneaten food, dead plant material, undigested fecal matter and that guppy under the driftwood that disappeared last week, but was never found.
At this point, the first stage of nitrification begins. Nitrification is the biological oxidation of ammonia to the final form of nitrate. Ammonia is highly soluble and is absorbed into water. Once absorbed, it circulates until coming in contact with Nitrosomonas bacteria. Nitrosomonas are substrate-dwelling, aerobic autotrophs. Autotrophs are microorganisms capable of synthesizing their own organic substances from inorganic compounds. Nitrosomonas are present in soils and water, and colonize any available surface, including plant leaves and stems. In the presence of dissolved oxygen (DO) and carbon, they convert ammonia (NH3) to nitrite (NO2-).
Left unchecked, ammonia is lethal to aquatic life at 1 part per million (ppm) and causes health problems as low as 0.25 ppm. Symptoms of ammonia poisoning include fish “gasping” at the surface listlessly, a milky appearance as the slime coat degenerates, and blood streaks in the fins and on the body in advanced stages. Even if action is taken and the fish survives, a shortened lifespan and permanent damage may occur.
Nitrite (NO2-) produced in the first stage is then converted to nitrate (NO3) by Nitrobacter bacteria. Like Nitrosomonas, Nitrobacter are substrate-dwelling, aerobic autotrophs and require dissolved oxygen and carbon to metabolize nitrites. In marine systems, there is evidence to support that the oxidation of nitrite to nitrate may be dominated by Nitrospira sp. rather than Nitrobacter.
Nitrite is dangerous to aquatic life at concentrations as low as 0.1 ppm, though this is somewhat species dependent and some species show greater tolerance than others. Symptoms include “gasping” at the surface listlessly and a brownish color to the gills. Nitrites react with hemoglobin, preventing oxygen uptake by blood in the gills, thus suffocating the fish.
Nitrate is the end product of nitrification. It is only removed from the system through nitrogen uptake by plants or microorganisms, by water changes or by denitrification. Denitrification is an anaerobic process by which microorganisms convert nitrate back to nitrite, then to nitrous oxide, nitric oxide and dinitrogen. Anaerobic conditions are the result of a lack of dissolved oxygen in the water, and should be avoided in an aquarium due to the potential of a build-up of hydrogen sulfide gas (H2S). If H2S is released from anaerobic areas, it may cause an unpleasant odor from the water and may be dangerous to the health of the tank’s critters.
“Anoxic” is another term occasionally encountered by aquarists. Simply put, an anoxic state is an anaerobic condition where H2S producing microgranisms are absent. Anoxic conditions result in denitrification in the same manner as other anaerobic states.
While ammonia and nitrite are very toxic to fish at low concentrations, fish can adapt to an amazingly high concentration of nitrate, dependent on pH, dissolved oxygen, temperature, and age, size and species of fish. Salmonids suffer ill-effects from nitrate at a concentration of 10 ppm. On the other hand, fish living at over 200 ppm of nitrate is not unheard of and actually occurs in some environments in nature. The ill effects of high nitrate concentrations is often exhibited long-term rather than having immediate consequences like ammonia poisoning or nitrite poisoning.
The final form of inorganic nitrogen common in aquariums is the ammonium ion. Ammonium is formed when ammonia picks up an extra hydrogen molecule, converting the NH3 (ammonia) to NH4+ (ammonium ion). Ammonium is non-toxic and is more prevalent at a low pH (this will be discussed in more detail in the Nitrification and Water Chemistry section). Ammonium will often catch another molecule to form an ammonium salt. Many hobbyist grade ammonia test kits do not distinguish between ammonia and ammonium, and may give a false high reading for ammonia. This is why a tank that has been in service for a long period of time without a water change may show an ammonia reading, but the fish are still alive. The water has become acidic due to the lack of buffering capacity and the resulting ammonium is falsely reading as ammonia.
Nitrification and Water Chemistry
Although many parameters affect nitrification in the aquarium, four factors play a greater role than others. These are dissolved oxygen (DO), temperature, pH and alkalinity. These will be explored in some detail and others will be briefly touched on in this section.
It is important to note that there are several species of both Nitrosomonas and Nitrobacter, and some species have multiple strains. Though most of the information below can be generalized for all, individual strains or species may exhibit variation from the parameters outlined in this section.
Dissolved Oxygen – As previously noted, nitrification is the biological oxidation of ammonia to nitrate. In the conversion of ammonia to nitrate, approximately 4 mg of oxygen are consumed. Both Nitrosomonas and Nitrobacter require dissolved oxygen to facilitate this process.
The amount of oxygen used in an aquarium can be stated as chemical oxygen demand (COD) and biological oxygen demand (BOD). COD is the amount of oxygen used when the decomposition of organic materials causes oxidization. BOD is the amount of oxygen required by living organisms to facilitate metabolic processes. Dissolved oxygen (DO) supplies these demands. Nitrification decreases rapidly under 3 ppm of DO and is inhibited below 2 ppm, excess COD or BOD can slow or inhibit nitrification. To minimize this potential, regular vacuuming the gravel to remove excess detritus (and subsequent COD from oxidization and excess BOD from heterotrophic bacteria) is important. Nitrobacter are affected more severely by low DO than Nitrosomonas, so a lack of DO may be seen as a spike in nitrites without a preceding spike in ammonia.
Temperature – Both Nitrosomonas and Nitrobacter reproduce through binary division: one bacterium divides into two individuals. Nitrifying activity remains constant from 72 to 96 degrees, but optimal division and growth is achieved from 78-86 F. Nitrification is reduced by 50% at 64F, by 75% at 50 F. Activity ceases completely at 40 F and nitrifying bacteria die at freezing. On the other end of the spectrum, nitrification ceases at 104 F and nitrifiers die at 120 F. Nitrobacter are less tolerant of colder temperatures than Nitrosomonas. It is convenient that the optimal temperature for division and growth falls into the range of temperatures that most tropical aquarium critters live in.
pH and Alkalinity – These two parameters are so intricately linked, they will be discussed together. Please note, basic/basicity will be used to note measurement of pH to avoid confusion of pH with discussion pertaining to alkalinity, which will be the term used to signify measurement of the buffering capacity of the water to increasing hydrogen ions.
pH is a measure of the acidity or basicity of a liquid, in this case water. The most simplistic definition is that it is a measure of the concentration of dissolved hydrogen ions (H+). Acidic water (water with a pH less than 7) contains a high concentration of hydrogen ions. On the other end of the scale, basic water (water with a pH greater than 7) contains few hydrogen ions and a high concentration of hydroxide (OH-). The higher the concentration of hydrogen ions in the water, the more acidic the water is. A pH of 7 is neutral. The pH scale is logarithmic; a change of one point on the pH scale indicates a change in the hydrogen ion concentration of a magnitude of ten. For example a pH of 5 is ten times more acidic than a ph of 6, and a pH of 4 is ten times more acidic than a pH of 5 and 100 times more acidic than a pH of 6. Conversely, a pH of 9 is ten times more basic than a pH of 8, and a pH of 10 is ten times more basic than a pH of 9 and 100 times more basic than a pH of 8. Hydrogen ions are a natural by-product of the metabolic functions of living organisms, thus over time the pH of a closed aquatic system will decrease as the hydrogen ion concentration increases.
Alkalinity is the measurement of water’s ability to counteract this natural increase of hydrogen ions and the amount of alkalinity is often referred to as buffering capacity. Although many substances commonly occurring in water contribute to alkalinity (hydroxide and phosphates, for example), the measurement of most concern to hobbyists is carbonate alkalinity, or carbonate hardness. This is a measure of the concentration of carbonate (CO3 2-) and bicarbonate (HCO3-) ions in the water. These ions react with hydrogen ions (H+), effectively preventing acidification, preventing suppression of nitrification by bacteria, and maintaining ammonia in an aqueous solution accessible to nitrifiers rather than allowing the promotion of ammonia to unavailable ammonium (see next paragraph). Please note, carbon dioxide (CO2) does not contribute to alkalinity and in fact destroys buffering capacity due to a tendency to form carbonic acid in aqueous solution. Calcium carbonate and sodium bicarbonate are two substances often driving carbonate hardness.
At a low pH, hydrogen ions (H+) react with ammonia (NH3) to create the non-toxic ammonium ion (NH4+). At a constant low pH, the ammonium itself does not present a hazard to aquatic life. The threat occurs when the pH is raised, alkalinity is increased and the ammonium rapidly reverts to ammonia. The sudden release of ammonia may overload the nitrification capacity of the established colony of nitrifying bacteria, creating a toxic environment.
The high concentrations of hydrogen ions in acidic water react with aqueous carbonates or bicarbonates, removing the carbon from the pool of carbon available to nitrifying bacteria and reducing the buffering capacity of the water to avoid low pH conditions. Carbonates and bicarbonates are the primary source of carbon for nitrifying bacteria. Carbonate hardness has to be replenished on a regular basis to ensure nitrifying bacteria in an aquarium remain healthy and active. Although these bacteria will utilize carbon dioxide as a carbon source to some extent, the nitrification rate is greatly reduced. The optimal means to replenish carbonate alkalinity is through frequent water changes, as this also removes accumulated hydrogen ions that have not been buffered.
It should be noted that nitrifying bacteria are inhibited at a pH below 5.5 and above a pH of 10.5, regardless of other water parameters or conditions. The exact mechanism of this inhibition has not been determined at either end of the scale. Though the upper end of this range is seldom encountered or encouraged by aquarists, the lower end is often seen when a hobbyist recreates a “black water” environment high in organic material. In such cases frequent water changes and an alternate method of ammonia removal (such as a filter containing zeolite) is necessary. Although the ammonia concentration may be low, the toxicity tends to be acute.
Trace Elements – Iron, magnesium, calcium, molybdenum and copper are all required at trace levels for either growth or activity in Nitrosomonas, Nitrobacter , or both. At high concentrations all of these as well as chromium, nickel and most other metals have a negative impact to either growth or activity in one or both species.
Phosphate – Phosphate is required by both species for nitrification. However, phosphate addition is generally not required as phophate is released as a by-product in fish waste and is often present in water supplies. Excess phosphates will contribute to algae and cyanobacteria growth.
Sodium – Sodium is not required for either species. The effect it has on Nitrosomonas is of note, however. At lower concentrations sodium inhibits growth but enhances nitrifying activity, at higher concentrations it enhances growth but inhibits nitrifying activity. Approximately 1.5 mg/l is the threshold where this change takes place.
Amino Acids – High concentrations of amino acids tend to repress or inhibit nitrification. These may be removed with water changes.
Chlorine/Chloramines – Both will kill nitrifying bacteria at high enough concentrations. Before adding water to an aquarium, use a commercial chlorine/chloramine remover available from a pet store.
Light – Light has a minor inhibitory effect. The effect is negligible within the confines of an aquarium.
Medications – Medications may or may not have an affect on these bacteria. The best practice is to have a quarantine tank to move fish to for treatment to avoid threatening the stability of the nitrifying colony.
Other Factors – There are many other factors not mentioned that may have an affect on nitrification. The ones above are discussed as they are either important to the process or are frequently asked about by new aquarists.
Establishing Nitrifying Bacteria in an Aquarium: Cycling the Tank
Establishing nitrifying bacteria in the aquarium, starting with very few bacteria to having a colony of bacteria that will reduce ammonia and nitrite to zero, is called cycling a tank. After adding water, initially the population of bacteria in the aquarium is small and incapable of handling the ammonia that will be excreted by livestock. In order to establish these bacteria, a source of ammonia must be provided. There are several methods used to cycle an aquarium.
For a fishless cycle, add ammonia and test the water. Use clear ammonia that does not contain surfactants, soaps, coloring or scents, as these may inhibit establishment of a nitrifying colony and will have negative health consequences for livestock added later. Add ammonia until a reading of approximately 5 ppm (5 mg/l) is obtained. Note the amount of ammonia that was added (in drops, mg, teaspoons or whatever measurement is used) to achieve that concentration. Add the same amount of ammonia daily. The ammonia concentration will remain steady or increase a bit, but at this time no nitrites will be detectable. Measure the ammonia and nitrites daily. Once nitrites appear, reduce the addition of ammonia to half of the amount added initially (for example (6 ml would be reduced to 3 ml). Nitrites will rise to a concentration similar to those of the ammonia. At some point the concentrations of both the ammonia and nitrite will begin to decrease. Continue adding the half dose of ammonia daily until no ammonia or nitrites are detectable. At this point, cycling is complete. A water change of 90% to 100% is recommended to remove the nitrates that have built up in the aquarium during the cycling process, and then livestock may be added.
To cycle with fish, add one or two hardy fish and test the nitrite and ammonia daily as above. The same increases and spikes as described for a fishless cycle will be seen in both followed by an eventual decrease to no ammonia and nitrites. When cycling with fish, a daily water change of 50% should be conducted, to make the process easier on the critters.
A third method used is to add fish food to an empty tank, allowing the decomposition of the food to be the ammonia source. A small amount of food is added daily and testing proceeds the same way as in the previous methods until no ammonia or nitrites are detectable.
To speed up the process, gravel, decorations or other objects from an established aquarium can be added to seed the new aquarium by introducing a large number of bacteria immediately. This can speed up the cycling time immensely. A sponge filter or a filter pad from a hang on back filter that has been used in an established could also be used to seed the tank. Another method of speeding the cycle is to use an aquarium product that adds bacteria to the tank. Usually these are sold in a liquid form and are available from most pet stores.
Live plants may be added to an aquarium immediately, prior to stocking. This may also speed the cycle.
The length of time to fully cycle a tank varies but without seeding may take 4-8 weeks. Seeding may shorten the process to a week or even less. Regardless, once the ammonia and nitrite read 0 ppm, the fun part begins. Ya can add critters!
This may have been far more than many hobbyists care to know. It attempts to provide background on the nitrification cycle, how it works, and how it applies in an aquarium, as well as answer many of the frequent questions I have asked or have been asked through the years. Please feel free to take what ya need and ignore the rest.
One final note. The list of papers, individuals, and other resources that provided information used in this article is extensive and a bibliography would likely run a dozen pages or more. I understand that there are inconsistencies within the literature, and some of what has been stated can be supported in one paper and contradicted in another. The point of this article, however, is not to nitpick whether Nitrosomonas reproduces faster at 78 or 80 degrees, but to provide a baseline understanding of the topic for the aquarium hobbyist.
May all yer tanks nitrify!