Nitrogen Cycle

Understanding the nitrogen cycle and how it works is fundamental to being a successful aquarium hobbyist. Fish food added to the system is eventually consumed by animals or processed by macro and microorganisms. When fish consume food, they produce ammonia, which is released through their gills into the water column. Uneaten food will be processed by detritivores and microbes that release ammonia into the water column. Ammonia in the aquarium can reach toxic concentrations without bacteria and archaea processing it into less toxic nitrate.

In aquatic systems nitrifying bacteria (chemolithoautotrophs [chemo-litho-auto-trophs]) and archaea are required to keep your aquarium water safe for fish^[1].

Chemolithoautotrophs means chemical reactions (chemo), inorganic chemicals (litho), inorganic carbon (auto), eater (troph). These nitrifying bacteria and archaea live on all surface area within the system that is in contact with oxygen-rich water, including substrate, decor, plants, sides of the aquarium, and the parts of the filtration system. These beneficial bacteria and archaea are often referred to as biological filtration in the aquarium and pond hobbies.

Archaea (a group of micro-organisms that are similar to, but evolutionarily distinct from bacteria) have also been shown to aid in nitrification and denitrification. Studies of recirculation aquaculture systems have also shown over time that chemolithoautotrophic bacteria are dominant oxidizers of ammonia and nitrite.

Nitrifying bacteria and archaea oxidize ammonia to nitrite and then to nitrate. Nitrate is a nutrient for plants. In systems with a low bioload, it is common to have 0 ppm of nitrate. For most heavily stocked aquarium and pond systems, nitrate accumulates over time and is reduced with water changes.

The chemolithoautotrophs need a relatively clean surface to colonize. The slime-covered filters are not conducive to growing and harboring chemolithoautotrophs.

Freshwater chemolithoautotrophs are not the same species found in saltwater, and temperature and pH can also affect the dominant species responsible for the nitrogen cycle. Like fish, bacteria and archaea responsible for the nitrogen cycle have evolved to live in a limited range of water chemistry, pH, and temperature. The species of bacteria and archaea responsible for the nitrogen cycle can change based on environmental factors^[69].

The heterotrophic bacteria are also beneficial to the aquarium ecosystem. The slime-covered areas within a filtration system are heavily colonized by heterotrophic bacteria and archaea, which aid in decomposing organic material (fish waste, uneaten food, plant matter). Slime-covered heterotrophic areas prevent chemolithoautotrophs from accessing the ammonia and nitrite in the water column.

Heterotrophic bacteria and archaea break down detritus and create bioavailable nutrients (natural fertilizer) for plants. These bacteria colonize the substrate and filtration systems.

Scientific research shows that under the right conditions, anaerobic bacteria in the substrate also aid in nitrate reduction. The reduction of nitrate through either plants or bacteria is referred to as Natural Nitrate Reduction (NNR). The reduction of nitrate completes the nitrogen cycle. A system that does not accumulate nitrate is considered balanced.

Hobbyists that maintain balanced systems can still have good plant growth. Balanced systems still produce ammonium and nitrate. In balanced systems, the production of ammonium and nitrate is used quickly.

A mature nitrogen cycle will always have no ammonia and nitrite in the test results.

There are three popular methods for establishing a mature nitrogen cycle:

When starting a new aquarium, the tank is nearly sterile. The first 35 days (depending on temperature and water chemistry) after the aquarium was set up (with fish) is the most difficult time for fish to survive. Warm water of 84 to 85°F (29 to 30°C) will help speed up the reproduction of nitrifying bacteria. During this time, the slow-growing nitrifying bacteria population tries to catch up with the fish population load. Once the nitrogen cycle is established, set the temperature to the desired range. Hobbyists need to monitor the pH (Potential of Hydrogen) and three chemicals that make up the nitrogen cycle:

In nature, when measured with an aquarium or pond hobbyists test kit, unpolluted water's ammonia, nitrite, and nitrate will always measure 0 ppm (parts per million).

Hobbyists can monitor ammonia, nitrite, and nitrate concentrations with aquarium test kits. Every aquarist should have an aquarium test kit for ammonia, nitrite, nitrate, and pH when establishing the nitrogen cycle in a new aquarium. Master test kits from many companies include tests for ammonia, nitrite, nitrate, and pH.

Hobbyists must keep ammonia below .5 ppm (not Total Ammonia Nitrogen [TAN]) and nitrite concentrations below 5 ppm to maximize the growth rate of chemolithoautotrophs in the aquarium.

The nitrogen cycle creates hydrogen ions that consume alkalinity (carbonate hardness, KH). KH is covered in more detail in the water chemistry section of this ebook. If the water in the system is low in KH (< 1 dKH), pH will need to be monitored and adjusted to keep it above 7.0.

Ammonia/ammonium in “most” systems with a mature nitrogen cycle will always remain at 0 ppm. In freshwater Nitrosomonas oligotropha^[2] (in saltwater Nitrosomonas marina) bacteria (a.k.a ammonia-oxidizing bateria [AOB]^[3])^[4]and ammonia-oxidizing archaea (AOA)^[5] are known to oxidize ammonia into nitrite in freshwater aquatic ecosystems. It is important to note that ammonia above .5 ppm (not Total Ammonia Nitrogen [TAN]) inhibits the growth of Nitrosomonas spp., and they are intolerant of nitrite above 5 ppm. AOB oxidize ammonia, not ammonium.

When establishing the nitrogen cycle, the ammonia/ammonium levels will peak around two to three weeks after adding fish to a new system. The population of AOB doubles every 30+ hours.

Ammonia is eliminated from the fish's blood through the gills (branchial ammonia excretion). Branchial ammonia excretion varies between different species and environments and primarily involves ammonia passive diffusion and ammonium (NH₄⁺)/sodium (Na⁺) exchange. Ammonia at high enough concentrations can damage the fish's organs (gills, liver, kidney, spleen, and other organ tissues) and make it difficult to take in oxygen^[7]. Heterotrophic bacteria also produce ammonia as they process organic detritus. Exposure to elevated levels of ammonia can have long-term physiological effects on fish^[8]. Exposure to elevated ammonia levels can affect the growth rate, feeding behavior, swimming activity, and fish reproduction.

When you test for ammonia with your aquarium or pond test kit, the reading you have is a combination of ammonium (NH₄⁺ or ionized ammonia) and ammonia (NH₃ or unionized ammonia), known as Total Ammonia Nitrogen (TAN). The result of the ammonia test kit only shows if the sample is positive (green) or not (yellow). Ammonium is not toxic to fish at concentrations we see in aquatic systems. High levels of ammonium ( > 10 ppm) can be toxic to plants. For fish, ammonia is the toxic part of the TAN. Understanding the difference between the two chemicals in the test result is crucial to determining the toxicity in a sample. Ammonia concentration of as little as .6 ppm is toxic to many fish species.

The amount of ammonia in a test result is affected by the pH and, to a lesser extent, temperature. At a pH of 7.0, almost all of the result is ammonium. The higher the pH, the greater the amount of the result is toxic ammonia. Higher temperatures also make the result more toxic, but its effect is minor.

For years, authors of aquarium literature advised limiting the feeding for a “fish in cycle.” We now know from scientific research that this advice may have done more harm than good. Research shows that fish are more resistant to ammonia exposure in the system when fed than starved^[9].

Often in freshwater aquariums, fish will survive the ammonia spike when a new tank is cycling because the pH is low enough that the ammonia concentration stays low.

In an established aquarium, the nitrite level should always be at 0 ppm. Nitrospira bacteria oxidize nitrite into nitrate^[10]. Nitrospira bacteria growth is inhibited by ammonia above .5 ppm. The population of nitrite-oxidizing bacteria (NOB) doubles every 40+ hours.

More recent scientific research has shown that comammox (COMplete AMMonia OXidation) can be performed by chemolithoautotrophic bacteria in the Nitrospira genus^[11]. Comammox is an organism that can convert ammonia into nitrite and then nitrate through nitrification.

When establishing the nitrogen cycle, the nitrite levels will peak around three to four weeks after adding fish to a new system.

Nitrite is extremely toxic to fish. It only takes .6 ppm of nitrite to cause stress and 2 ppm to kill some species of fish exposed for 24 hours. The toxicity of nitrite is highly variable, depending on species, water chemistry, and temperature. Per Dr. Melanie Greeley, DVM, "The LC50 for most freshwater fish ranges from 0.6 to 200 mg/l^[12]."

Scientific research on nitrite toxicity for aquatic animals often uses 24 hours of exposure to a lethal concentration that causes 50% loss (abbreviated as LC50). If not 24 hours, the time will often be prefixed (96-hour LC50 or 96LC₅₀)^[13].

Nitrite is primarily absorbed through the gills and digestive tract and attaches to hemoglobin. Nitrite in the water will displace oxygen in the fish's blood, causing methemoglobinemia (MetHb) or “brown blood disease.” MetHb prevents oxygen from getting transported to an animal's cells.

Fish exposed to elevated nitrite levels will often initially go to the surface to find water with a higher oxygen concentration. This behavior is natural for many species of fish. Unfortunately, the issue in the system is not the lack of oxygen in the water but nitrite in the blood preventing oxygen from attaching to blood. After the fish become exhausted, they start resting on the bottom. An immediate remedy for nitrite poisoning is to perform a nearly 100% water change and add chloride.

Chloride (Cl^-) ions in the water are well documented as a way to block the uptake of nitrite in freshwater fish^[14]. The receptors in the fish's gills can be disrupted from taking up nitrite by adding calcium chloride (CaCl₂), magnesium chloride (MgCl₂), or sodium chloride (NaCl). Some studies have concluded that calcium chloride may be even more effective than sodium chloride. In planted aquariums, sodium chloride should be avoided as most plants do not tolerate high levels of sodium. Calcium chloride, magnesium chloride, and sodium chloride are covered in more detail in the water chemistry section of this ebook.

Nitrate is the final toxic chemical in the nitrogen cycle. In most freshwater aquariums, nitrate builds up over time. The rate of accumulation of nitrate is directly related to fish respiration, waste, decay of aquatic plant matter, and if there are any natural nitrate reduction (NNR) strategies employed within the aquarium ecosystem. Aquariums with a heavy fish load, fed often with high protein food, can accumulate nitrate very quickly, often well over 100 ppm in a month.

In unpolluted natural bodies of water, nitrate is rarely detectable (< 1 ppm) with an aquarium test kit.

For fish, keeping the nitrate close to 0 ppm is optimal. The nitrogen cycle will lower the system's pH and carbonate hardness (KH) over time. Low alkalinity (≤ 2 dKH or 35.72 ppm) can be a health problem for fish physiologically evolved in a higher pH range (Lake Tanganyika, Lake Malawi, Central America, Mexico, Florida Springs, Northern South America, and Gulf of Mexico American States). In freshwater aquariums, the general guidance is not to exceed 40 ppm of nitrate.

While nitrate is not as toxic as ammonia and nitrite, long-term exposure to nitrate can affect fish's growth rate, reproduction, and the immune system's ability to protect against disease. Fish exposed to very high nitrate levels for an extended period are known to develop a hole in the head (HITH) condition where the skin around the head recedes. The HITH condition is commonly seen in Oscars (Astronotus ocellatus) and other large Central and South American cichlids.

Discus hatcheries are well-known for doing nearly 100% water changes daily to keep nitrate as low as possible.

The oxidation of ammonia and nitrite and nitrite to nitrate can consume KH quickly in high-bioload systems. Nitrate is a weak acid, and in systems with a low alkalinity level (≤ 1 dKH or 18 ppm), it can lower the pH below 6.0. Systems with low KH and high nitrate (> 40 ppm) will often have a pH test result below 6.0. If pH is below 6.0, the ammonia test result will often be positive.

Nitrate and low pH have been linked to the lower oxygen-carrying capacity of the fish's blood from an increased level of methemoglobin^[15]. A low pH and high nitrate level are double trouble for the fish's ability to take up oxygen. Methemoglobinemia (MetHb), also known as brown blood disease, prevents oxygen from reaching an animal's cells.

Nitrate typically builds up in the aquarium over time, but it can be slowed down and kept from rising. Natural nitrate reduction (NNR) can be accomplished by implementing nitrate-reducing elements into your aquarium ecosystem and keeping fish populations (biomass) in check.

Fast-growing live plants under intense light will take up nutrients from the decomposition of fish waste, biodegraded uneaten food, and plant matter. The brighter the light, the faster the plants will take up ammonium and nitrate. An aquarium positioned in the home so that it will receive a couple of hours of natural sunlight can help reduce nitrate, as well as help bring out the natural colors of fish and plants. As fish release ammonia through their gills, most of it will be converted immediately to ammonium and thus available for plants as a nutrient (nitrogen). Plants in a brightly lit aquarium are one of the best ways to control nitrate accumulation in the freshwater system.

A hydroponics system can also be added to an aquarium ecosystem to control and reduce nitrates. The hydroponics method may be best for systems with a high bio load, like large cichlids, plant eaters, and grow-out tanks. Hydroponics systems use emergent plants for nitrate reduction. Emergent plants have access to the higher carbon dioxide (CO₂) concentration in the atmosphere and typically remove nutrients faster.

Many hobbyists use emergent plants at the top of the aquarium. Hobbyists can purchase many plants at garden centers that can be used as emergent plants for aquariums and, in some cases, submerged.

Heterotrophic and autotrophic^[16] anaerobic bacteria found in sediment also aid in denitrification^[17]. Heterotrophic bacteria known as Pseudomonas denitrificans can reduce nitrate. Some hobbyists use ethanol alcohol (vodka) as a carbon source to increase the population of nitrate-reducing heterotrophic bacteria. Autotrophic bacteria from the genera Paracoccus, Thiobacillus, Thiosphaera, and others can accomplish denitrification autotrophically using hydrogen or various reduced sulfur compounds.

Some hobbyists may be confused by the terms anaerobic and anoxic. In microbiology, anoxic is used to describe environments without molecular oxygen. Anaerobic refers to microorganisms that are able to live without molecular oxygen. The metabolism bacteria use is also called anaerobic. Easy distinction, anoxic refers to environments, and anaerobic refers to microorganisms and processes.

A substrate comprised of fine sand or gravel can also help with denitrification. Anaerobic bacteria live in anoxic areas devoid of oxygen. Anaerobic bacteria use the oxygen attached to the nitrate molecule freeing up nitrogen gas that will escape into the atmosphere.

Anaerobic bacteria first reduce nitrate to nitrite and then to nitrogen gas. If you use a denitrator filter on a system, this process can be observed by flowing water through too fast. The effluent will test positive for nitrite. Slowing the effluent will complete the reduction to nitrogen gas and will test negative for nitrate and nitrite.

Anaerobic bacteria will colonize the depths of a substrate. Using Malaysian live-bearing snails (a.k.a Malaysian trumpet snails, Melanoides tuberculata) to turn over the sand bed slowly will help exchange nitrate-rich water through the sand to the anaerobic bacteria. Catfish from the Corydoras genus and other sand-sifting fish can also assist with the transport of nitrate to the anaerobic bacteria.

It is recommended that the system have at least 1 ½ to 2 inches (3 to 5 cm) of fine sand. A large mesh substrate (3 mesh [grit] or larger pea gravel) allows the exchange of oxygenated water to move easily through the substrate and will require more depth to achieve an anaerobic layer. The finer the substrate, the shallower the depth must be for denitrification.

The amount of current in a system can also affect how deep the substrate must be to achieve denitrification. Gravel substrates can provide denitrification in low current systems at a much shallower depth.

The pH also affects the anaerobic bacteria population. The pH should be kept on the basic (alkaline) side to maintain a healthy anaerobic bacteria population. Some research suggests the ideal range for freshwater denitrification is between a pH of 7 to 8^[68].