How Ammonia is Introduced:

Ammonia is introduced into the aquarium in various biological processes, but it mainly comes down to four primary ways of breaking-down proteins:

• Uneaten food
  • Fish food contains proteins and nutrients meant for the fish, but many also contain processed additives unneeded by the fish’s systems. For food which sinks to the bottom and goes uneaten, it breaks down. Since it was not eaten, there is a higher level of proteins and nutrients in the food and more ammonia is added to the system. This is why it is vital to not overfeed.
• Decaying waste
  • Fish metabolisms break down proteins and nutrients from their food, what is unneeded is passed through as waste, which, as the waste breaks down it releases toxic ammonia as well as hydrogen ions. The less fillers in the food (also the less processed) the less waste there will be; a healthy diet can reduce the amount of ammonia going into the system via fish waste. Nitrogenous waste may be released as ammonia, or as urea, also known as carbamide.
• Decaying plants/dead organisms
  • When dead animals and plants decay, it is the first step to the nutrients being recycled (see the DECOMPOSITION section for detailed information). One byproduct: ammonia.
• Released through the gills of fish during respiration
  • >80% of nitrogenous waste produced by internal processes of fish is released via the gills in passive diffusion (transcellularly, through a cell, or paracellularly, passing through the space between cells; release of NH₃ into water), NH₃ Trapping, (Protons pumped out through the gills combine with NH₃ to produce NH₄+ which acidifies the small amount of water surrounding the gills and maintains the gradient of NH₃; high content in blood, low in water) and finally the conversion of CO₂ to HCO₃^- and H+ due to the carbonic anhydrase enzyme
  • In saltwater fish, the process is similar yet different; starting with passive diffusion of NH₃ into the water (transcellularly and paracellularly) as well as the passive diffusion of NH₄+ paracellularly via certain junctions. Since saltwater is well buffered due to the presence of salt and other compounds, trapping of the un-ionized ammonia is not possible, so passive diffusion is then followed by the active transport of ionized ammonium into the gill by the replacing of K+ in Na+-K+-ATPases, and finally the active transport of ionized ammonium into the water by replacing H+ in HNEs
  • Recently, a new form of symbiosis has been discovered to take place within the gills of zebra danios and carp, where the nitrification process takes place in the gills by nitrifying bacteria.

These organic sources break down into either un-ionized ammonia (NH3) or ionized ammonium (NH4+ ), with, of course, various byproducts (mainly organic compounds/nutrients to be recycled by various other processes).

Ammonium is harmless to fish, except in high concentrations where it may substitute K+ in ion transporters and disrupt electrochemical gradients (gradient of electrochemical potential) , Ammonia is very toxic, deadly even, as it increases the internal pH and may inhibit key enzymes necessary for the generation of energy by destabilizing proteins. When ammonia is dissolved in water, a small amount becomes ammonium- which is actually very important as a form of nitrogen for plants (see PLANTED TANK section for more details).

Production of Ammonia by Fish:

In ammonotelic species (those which release nitrogenous waste primarily as un-ionized ammonia) such as fish, 80-90% of the nitrogenous waste is released as ammonia, while the rest is released as urea. 95% of total ammonia in fish tissue exists as ionized ammonium, which cannot be diffused over the epithelia (covering/lining tissues). For fish, ammonia is mainly produced by two catabolic processes (catabolism is the set of pathways which break down molecules into smaller units which are used for energy or oxidized), though only one is related to ammonotelic species:

• Amino Acid Catabolism
  • 50-70% of ammonia produced by transdeamination is done so in the liver, up to 99% in goldfish, with the rest in the kidneys, muscle, gills, and intestines.
  • Mainly carried out by transdeamination:
    • Transamination → Deamination

In ureotelic species (those which release nitrogenous waste primarily as urea), such as air-breathing fish (i.e. african lungfish) which do not spent all their time in the water, there is 10x less water required for the excretion than in the process used by ammonotelic species. A short overview for ureotelic species, undescribed:

Minor passive diffusion of urea into water transcellularly → active transport to outside gills

Nitrification is the biological oxidation of ammonia and ammonium to nitrite, which is then followed by the oxidation of nitrite into nitrates. The oxidation of NH₃ is a common limiting factor in environments for the accumulation of NO₂- (Nitrogen Dioxide, the nitrite ion).

There are several types of bacteria which are in the aquatic setting, however the primary processors of ammonia consist of Nitrosomonas, Nitrobacter and Nitrospira.

(Note: The aerobic oxidation of ammonium is also referred to as anammox)

Quick summary of the bacterial processing:

Nitrosomonas oxidize* the ammonia as a metabolic process, essentially eliminating it. This produces the byproduct of Nitrites, which is just as toxic.

Nitrobacter will process these nitrites into nitrates, which are not as toxic to inhabitants as nitrites or ammonia.

Nitrospira are aerobic* chemolithoautotrophic* nitrite-oxidizing bacteria, which both convert ammonia to nitrites and nitrogen dioxide to nitrates.

*oxidation: the loss of electrons during a reaction by a molecule, atom, or ion, and

occurs when the oxidation state is increased. The oxidation state refers to the

level of oxidation of a molecule, atom, or ion.

*aerobic: living, active, or occurring only in the presence of oxygen; requiring the presence of oxygen to live.

*chemolithoautotrophic bacteria are autotrophic microorganisms that gains energy through the process of oxidizing inorganic compounds. Most chemolithotrophs (an organism that is able to use inorganic reduced compounds to produce/gain energy) are autotrophs (primary producer, an organism that can produce its own food using light, water, CO2, or other chemicals).

Ammonia-oxidizing archaea were discovered to have the amoA gene in Crenarchaeota in 2004, proving their capabilities as ammonia oxidizers. Until then ammonia-oxidizing bacteria were the only known ammonia oxidizers. While they are known to be ammonia-oxidizers, it is unclear whether or not they take the same metabolic processes as AOB; although it has been suggested that they may generate nitroxyl hydride (HNO) which can subsequently be converted/oxidised into NO₂- via nitroxyl oxidoreductase (NxOR)

Chemical formulas for the oxidation processes:

Ammonia Oxidizing Bacteria (NH3 to NO2-):

NH₃ + O2 + 2H⁺ + 2e^- → NH₂OH + H₂O → NO₂^- + 5H⁺ + 4e^-

Nitrite Oxidizing Bacteria (convert NO2- to NO3-):

NO₂^- + H₂O → NO₃^- + 2H⁺ + 2e^-

Aerobic Oxidation of Nitrite (Stoichiometry):

NO₂^- + 0.5O₂ → NO₃^-

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Quick Overview of Nitrites and Nitrates, Involved in the Bacterial Processing and Conversion:

Nitrites can be toxic in levels as low as .10mg/liter in freshwater, they bind with hemoglobin and can prevent erythrocytes from delivering oxygen to cells (so, it can cause brown blood disease, aka methemoglobinemia). Chloride cells in fish gills cannot tell the difference of nitrite and chloride ions, the rate of nitrite uptake into the fish’s system depends on the nitrite:chloride ratio in the system. Since the uptake of nitrite by the gills can be inhibited with sodium chloride, nitrite is not as heavy of a threat to saltwater settings, though it is still dangerous.

Note: Nitrite oxidation in soil is catalyzed by nitrite oxidoreductase, and nitrite oxidoreductase (NOR/NXR) is the enzyme involved with the last step of aerobic ammonia oxidation

Nitrates are much less toxic than ammonia and nitrites, but are still toxic in high enough amounts. Nitrate is a polyatomic ion with the molecular formula of NO-3

These are the byproduct of nitrite oxidation, and is one of the most common groundwater contaminants in rural areas.

High levels of either nitrites or nitrates in an aquatic setting can result in ill and susceptible stock to diseases, just as high levels of ammonia can do the same. Note that almost every aquatic disease is caused by poor water quality, referring to the TAN and nitrite/nitrate levels, as well as the pH relative to the species of fish in question.

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Nitrosomonas are ammonia oxidizing bacteria/biomass (AOB) which oxidize ammonia and ammonium (NH₃/NH₄+) into Hydroxylamine (NH₂OH), which requires a molecular dioxygen (O₂) and a pair of electrons (currently thought to be provided through Coenzyme Q10, also known as Ubiquinol)* for the AMO, ammonia monooxygenase*. Though the exact process of conversion is not fully known, it can be assumed the conversion from ammonia and ammonium into nitrites is through the process of ubiquinone reduction*, assuming that the source of the pair of electrons is the ubiquinone. This leads us to believe the process is related to the oxidation of Hydroxylamine (NH₂OH) to Nitrogen Dioxide (NO₂-) occurring in the bacterial periplasm*. The conversion to nitrogen dioxide is believed to be a reaction with four electrons, which pass on to cytochrome C₅₅₄ . There are approximately three theorized pathways for these electrons to take in this conversion, but in this specific path, in order for the conversion of ammonia to take place and complete, half of the ubiquinol molecules must be oxidized by the AMO. From there, the remainder of the electron path is not exactly known.

*Ubiquinol is the active form of CoQ10, with the inactive form being ubiquinone

*Ammonia monooxygenase: a metalloenzyme (an enzyme protein containing one or more metal ion as a part of its active structure) that catalyzes (speeds up the reaction without being consumed by it) the oxidation of ammonia into Hydroxylamine

*Reduction: the chemical reaction involving the gaining of electrons by one of the atoms involved between the two elements or compounds.

*Periplasm: space between the inner and outer membrane of Gram-negative bacteria.

Nitrobacter are nitrite-oxidizing bacteria/biomass (NOB) that are chemolithoautotrophs. They derive energy from nitrite oxidation and carbon dioxide fixation; when there are no nitrites they will use solely carbon sources with the use of the Calvin cycle as a chemoorganoheterotroph (use organic compounds for energy). When oxygen is present, they will oxidize nitrites into nitrates. Nitrobacter interact with nitrosomonas, which oxidize ammonia into nitrites, which the Nitrobacter then proceed to oxidize into nitrates. The exact process is not fully documented or yet understood.

*Carbon dioxide fixation: the conversion process of inorganic carbon (CO2) to

organic compounds by biotic organisms.

Nitrospira are aerobic chemolithoautotrophic nitrite-oxidizing bacterium important in marine and freshwater environments. Not only do these bacteria oxidize the nitrites and convert them to nitrates as a byproduct of the metabolic process, but they are also able to feed the ammonia-oxidizing Nitrobacter by feeding them with ammonia released through the urea (carbamide, an organic compound, CO(NH2)2) or or cyanate (an anion, [OCN]- or [NCO]-, that acts as a base to form isocyanic acid, HNCO, in aqueous solutions). Nitrospira has even been studied being capable of catalyzing both steps of nitrification, both ammonia-oxidation and nitrite-oxidation, and are also considered to be complete comammox organisms (complete ammonia-oxidizing). Some strains of Nitrospira are capable of using different substrates, H₂ and formate (HCOO-/CHOO-, etc.. IUPAC name: methanoate, the anion derived from formic acid, the simplest carboxylic acid) for example, and using oxygen or nitrates as a terminal electron acceptor*

*electron acceptors: a chemical entity that accepts electrons transferred to it from

another compound, it is an oxidizing agent which is reduced in the process of oxidation.

• Terminal electron acceptors are compounds which receive or accept an electron during cellular respiration or photosynthesis. Again, the acceptor itself is during the process.

Nitrospina are a lesser known nitrite-oxidizing aerobic chemolithoautotrophic bacteria which is a major (and exclusive) marine processor which uses the reductive tricarboxylic acid pathway for CO₂ fixation. It is the only species of NOB bacteria growing in above 40 g NaCl l–1, possibly related to the low net energy obtained from their metabolism (ΔG0′=–74 kJ mol–1 NO2–) that is not only lacking for proper growth, but also osmoregulation (active regulation of the osmotic pressure of an organism’s body fluids, internal balance between water and dissolved material- such as salt in marine environments. This is what saltwater fish use to process the salt of their marine environments).

The bacteria responsible for the nitrogen cycle rely on the presence of oxygen and carbon dioxide in order to undergo the processes that drive the cycle or aid in it’s progression, such as carbon dioxide fixation serving to produce energy for some of the chemolithoautotrophs (i.e. Nitrobacter require oxygen to be present in order to oxidize nitrites into nitrates, but will use carbon sources as energy if they are unable to nitrify the nitrites), not to mention to just exist in general. The kinetics of nitrification is mainly limited by the concentration of carbon in the system, as the heterotroph/autotroph populations of the system rely heavily on the organic carbon/nitrogen ratio (C/N); theoretically, with higher levels of each there will be a decreased conversion of ammonium due to the heterotrophs dominating the autotrophic nitrifying bacteria-- in other words, an increased presence of organic carbon can, in theory, inhibit the activity of nitrifying bacteria by supporting the growth of the heterotrophs.

From Fowler’s Zoo and Wild Animal Medicine, Chapter 25 - Advanced Water Quality Evaluation for Zoo Veterinarians, page 1:

“CO₂ reacts with water to form carbonic acid (H₂CO₃), which dissociates to

bicarbonate (HCO₃−) and then carbonate (CO₃²− ) and hydrogen ions (H+ ),

chemically proportioned by temperature, pressure, and salinity equilibria²⁴:

CO₂ + H₂O ⇆ H₂CO₃ ⇆ H⁺ +HCO₃- ⇆ 2H⁺ + CO₃²- ”

From Effect of Carbon Dioxide on Nitrification Rates:

“Stoichiometrically, the required C/N [Carbon Dioxide/Nitrogen] ratio is 0.0236 for

Nitrobacter spp. and 0.0863

(µmol bicarbonate per µmol ammonium or nitrite oxidized) for Nitrosomonas and Nitrospira spp.” (1).

Short Overview of Blackwater Systems and Acidity, effects on the Nitrogen Cycle

Blackwater aquariums are those which use tannins from wood and leaf litter, as well as other organic plant matter, to tint the water a light yellowish-brown tea color to a deep red similar to the dark red of South African redbush tea (which, evidently, is not a tea, but is also brewed and used to obtain various levels of blackwater in a tank). They usually are filled with an abundant amount of decaying leaf litter from various trees (such as catappa/indian almond leaves and oak) and wood (manzanita, oak, grape vine, malaysian driftwood, etc.) as they release tannins into the water. Blackwater tanks are known to have a more acidic pH than clear tanks lacking in tannins, generally around 6.8ppm. As discussed above, this affects the process of ammonification by allowing for an increased conversion/production of ammonium rather than ammonia.

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Some Side Info about Tannins:

Tannins, or humic acid, is a type of polyphenol* produced by plants, and mostly present in the surface wax, buds, leaf tissues, root tissues, seed tissues, stem tissues and vacuoles* where they do not interact or interfere with the metabolism. They are classified as phenolics, which are aromatic benzene ring (compounds with 1+ hydroxyl groups-- basically made as protection against stress factors.

They bind and precipitate proteins, and are composed of a diverse group of oligomers* and polymers*.

*Vacuoles are membrane-bound, fluid-filled organelles in plant and animal cells with many functions.

*Polyphenols are a micronutrient, as well as secondary metabolites of plants generally involved in defence against UV radiation of aggression by pathogens.

*Oligomers are low molecular weight polymers consisting of small repeating units.

Polymers are macromolecules composed of many repeated subunits.

As described by Hovarth in 1981, tannins are: “Any phenolic compound of sufficiently high molecular weight containing sufficient hydroxyls and other suitable groups (i.e. carboxyls) to form effectively strong complexes with protein and other macromolecules under the particular environmental conditions being studied”

For literally everything you didn’t need to know about tannins, go here: http://poisonousplants.ansci.cornell.edu/toxicagents/tannin.html

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