All plants, animals and micro-organisms require nitrogen containing compounds like proteins and nucleic acids. Though air has 79% nitrogen gas (N₂), most organisms cannot use nitrogen in this form. Plants use Nitrogenous compounds like ammonia (NH₃) , nitrate ions (NO₃⁻) , urea (NH₂)₂CO. Animals secure their nitrogen from plants or animals that have fed on plants.

Ammonia in a pond or a fish tank is formed from fish waste, uneaten food, decaying plant matter, bacteria and micro-organisms. In a properly filtered pond with nitrifying bacteria, ammonia levels are zero at all times. However, at times, due to pump failure, medications or drop in temperature, overstocking and overfeeding can lead to increased levels of ammonia. Biological filter can fail if chlorinated water is introduced without a dechlorinator or carbon filter.

When water is acidic or neutral (pH is 7.0 or less), majority of the nitrogen is in the ionic form of ammonia i.e., ammonium (NH₄⁺). When pH is over 8.0, the nitrogen is mostly ammonia (NH₃). Ammonia form is more toxic to fish than ammonium form. Ammonia can cause gill damage even at 0.1ppm! Fish may gasp (especially the large ones) or show hyperventilation (rapid movement of gills). When you suspect ammonia, always use a test kit that measures the concentration of ammonia form.

Ammonia can be taken up directly by plants — usually through their roots. However, most of the ammonia produced by decay is converted into nitrates.

Recent research shows that in fresh water aquaria Nitrosococcus may be the true ammonia-oxidizer.

The biological conversion of ammonia to nitrate is called nitrification. There are 2 types of bacteria that can nitrify ammonia.
a) Both soil and the ocean contain Crenarchaeota (archaeal microbes), that convert ammonia to nitrites. They are more abundant than the nitrifying bacteria and play an important role in the nitrogen cycle.

b) Nitrifying bacteria such as Nitrosomonas and Nitrobacter that are present in freshwater are also capable of nitrifying ammonia.

The C0₂ produced by organisms and plants in water, is converted into Carbonic acid. But Carbonic acid is unstable. It breaks down to form bicarbonate or carbonate and hydrogen ions.

H₂0 + C0₂  => H₂C0₃
Water + Carbon dioxide  => Carbonic acid

H₂C0₃ =>  HC0₃^- + H⁺  => C0₃ + 2H⁺
Carbonic acid  => Bicarbonate + Hydrogen ion  => Carbonate ion + Hydrogen ion

Ammonia oxidizing bacteria (AOB) like Nitrosomonas, Nitrospira, Nitrosococcus, Nitrosolobus and Nitrosovibrio oxidize ammonia to gain energy. The energy is used in metabolizing their food i.e. Carbonate.

In the first step, ammonia is converted into hydroxylamine with the help of an enzyme called mono-oxidase.

NH₃ + O₂ + 2 H⁺ + 2 e^- => NH₂OH + H₂O

In the second step, hydroxylamine is converted into nitrite
NH₂OH + H₂O => NO₂^- + 5H⁺ + 4e^-

In the third step, oxygen and hydrogen combine to form water.

½ O₂ + 2 H⁺ + 2 e^- => H₂O

The total reaction can be written as

NH₃+ 1.5 0₂  => H⁺ + H₂0 + N0₂^-

Since ammonia is just a source of energy, this reaction produces very little sludge. Conversion of Ammonia to Nitrite produces acid (H⁺).

In the second step, Nitrite oxidizing bacteria (NOB) like Nitrobacter, Nitrospina, Nitrococcus and Nitrospira bacteria convert nitrite to nitrate with the help of nitrite oxido-reductase.

N0₂- + 0.5 0₂ => N0₃^-

The total reaction of Nitritation and Nitratation can be written as

NH₃ + 20₂  => N0₃^- + H⁺ + H₂0

Research has shown that NH₃ is the substrate for the enzyme, not NH₄⁺. Both nitritation and nitratation run at different velocities. Therefore, if nitritation is faster than nitratation, there will be buildup of nitrite as well as nitrous acid (HN0₂^-). Nitrous acid is toxic to both bacterial species. If nitratation is faster, nitrite may be undetectable.

Nitrite formation produces 274.91 kJ/mol energy whereas nitrate formation produces 74.16kJ/mol. Due to the high energy available, ammonia oxidizing bacteria (AOB) can double in 7-8 hours where as nitrite oxidizing bacteria (NOB) takes 10-13 hours.

The rate of nitrification reaction is highly dependent on a number of environmental factors.  In most cases the rate is limited by the first step, the oxidation of the ammonia.  Nitrification reaction is so fast that the amount of NO₂^- present is usually small. A nitrite level of 0.1ppm can be harmful to fish if exposure is prolonged. Fish may gasp, gulp or show rapid gill movements. Since nitrite binds to hemoglobin (the part of red blood cell that is responsible for carrying oxygen), fish may not be able to breathe in the presence of nitrite. This is called “brown blood syndrome”. The damage caused by nitrite is irreversible. It was originally thought to be Nitrobacter that is responsible for this reaction but recent research by Dr. Timothy Hovanec indicates that Nitrospira is responsible.

Factors influencing Nitrification:
i) Amount of ammonia or nitrite/nitrous acid: Low amounts of ammonia or nitrite can slow the nitrification because bacteria can not multiply due to low amount of energy available to them.

ii) pH: Nitrification occurs between pH 6.8-8.5 but optimum pH is 8.2-8.3 because at this pH ammonium ions are less than ammonia. Since nitrification process produces acid, it can lower the pH if the water is not buffered properly. Continuous aeration strips C0₂ and increases pH. In nitrification plants, 1% C0₂ - air mixture are introduced during aeration. But in ponds, koi and other living animals, plants introduce C0₂. Reduced pH can reduce the growth of nitrifying bacteria.

On the other hand, High concentration of ammonia can increase the pH. Nitrosomonas is more sensitive to high pH.

If large amount of ammonia is present, the bacteria can kill themselves by producing lots of acid while converting it into nitrite.

iii) Retention time: Nitrification requires a long retention time. The time required for nitrification is directly proportional to the amount of nitrifying bacteria present because the rate of oxidation of ammonia is linear. Minimum retention time is 3.9 hours at 71*F - 75*F (22-24*C). So, if you are feeding your fish frequently, you must wait for 4 hours in between feedings.

iv) Oxygen: Nitrifying bacteria need oxygen. So, the nitrification is “aerobic”. Even at low levels of dissolved oxygen like 1mg/litre, nitrification is active but not very efficient.

v) Temperature: Nitrification proceeds at a slower rate, at temperatures below 75*F (20*C) and continues to 50*F (10*C) or less. But if for some reason it stops, then it will not resume until the temperatures increase well over 50*F (10*C). Nitrification rate is maximum between 86*F – 95*F (30*C – 35*C). However, at these temperatures, water can not hold much oxygen. So, aeration should be vigorous. Rapid changes in temperature do not produce rapid changes in growth rates.  Rather a slow adaptation period with a lower than expected rate follows such change. But by 95*F (35*C), the growth rate begins to rapidly fall off toward zero.

vi)The conversion of Nitrite to Nitrate is inhibited in the presence of ammonia gas, even at low concentrations of 1.4 mg/L. So, presence of ammonia gas can be very dangerous because Ammonia oxidizing bacteria are not inhibited by ammonia gas and will continue to produce nitrite.

vii) Inhibiting substances: Many substances can potentially inhibit the nitrification.  Metals (example, copper) are particularly strong inhibitors of the reactions.  When exposed to more than one inhibitor, the extent of inhibition increases greatly.

viii) Alkalinity: Since nitrification itself produces acid, water must be buffered in order for the nitrification to continue. 

For every Ppm (mg/litre) of ammonia converted into nitrate, 7.1 ppm (mg/litre) of alkalinity as CaCO₃ is used. This is why an alkalinity of 50-100mg/litre must be maintained.

NH₄+ 1.83 0₂ + 1.98 HC0₃-  0.021 => C₅H₇0₂N + 0.98 N0₃^- + 1.041 H₂0 + 1.88 H₂C0₃^-

NH₄⁺ + 1.9 0₂ + 2HC0₃-  => 1.9 C0₂ + 2.9 H₂0 + 0.1 CH₂

For every pound of ammonia oxidized to nitrate

• 4.18 pounds of oxygen
• 7.14 pounds of alkalinity as Calcium carbonate or 12 pounds of alkalinity as Sodium bicarbonate

are consumed.
In other words, to process 1ppm ammonia (3.785mg/gallon), one must provide 45.42mg/gallon of sodium bicarbonate (baking soda) or 27mg/gallon calcium carbonate. Alkalinity should be at least eight times the concentration of ammonia and ideally over 100 mg/L. Since nitrification is a continuous process, replenishing alkalinity must also be done frequently, either through water changes or by passing water through calcium carbonate (coral shells, lime rock or plaster of paris) or by addition of baking soda (sodium bicarbonate).

Between 50 - 63*F (10-17*C), nitrate formation is slower than nitrite disappearance. So nitrite build up can be seen until the temperature goes above 63*F. This is why ponds suffer from “new pond syndrome” in spring.

Though there are many strains of nitrifying bacteria, the rate of formation for Nitrosomonas is typically 1000 to 30,000 mgN/day/g dry weight cells and for Nitrobacter is 5000 to 70000 mgN/day/g dry weight cells, which is much higher than the formation rates of the other strains capable of nitrification, That is the reason why these two are the most useful strains. Recently, it has been discovered that certain sulfide oxidizing, denitrifying bacteria, such compared to Paracoccus pantotrophus and Bacillus mojavensi AMH 118, which are also capable of heterotrophic ammonia oxidation to nitrite. These bacteria do NOT replace true Nitrosomonas and Nitrobacter, but can augment performance nitrifiers in low oxygen levels, acidic pH range or low temperatures in spring and fall etc.

For example, Nitrospina, and Nitrococcus are also known to convert nitrite into nitrate. They are mesophiles, with a temperature optimum of 83*F (28*C) and a pH range of 5.8-8.5, with an optimum pH range between 7.6 and 7.8.

Often, Koi breeders/owners release water from their pond into nearby rivers, streams or ponds while doing the 10% weekly change or daily change in case of a continuous flow-through system. This water may contain high amounts of nitrates. Nitrates in drinking water can cause Blue Baby syndrome. Increases in levels of nitrates can also make the water more acidic and increase solubility of toxic metals like mercury. This can kill organisms and fish living in the receiving water. Moreover, Nitrates will stimulate algae growth and reduce dissolved oxygen. Removing nitrates before releasing water is good practice.

 In a pond, nitrates must be maintained below 25-50ppm. One can remove nitrate by converting it into nitrogen gas. Conversion of nitrate to nitrogen gas is a reduction reaction (opposite of ammonia to nitrite) and is called Denitrification.

The denitrification process involves heterotrophic bacteria like Aerobacter, Alcaligenes, Bacillus, Brevibacterium, Flavobacterium, Lactobacillus, Micrococcus, Proteus, Pseudomonas and Spirillum. These bacteria can get their oxygen from either dissolved oxygen or nitrate. They need a carbon source like Methanol, sugar or vinegar to conduct this reaction. If dissolved oxygen is not available, they use nitrate as source of oxygen. They use nitrate only when dissolved oxygen levels drop below 0.5mg/litre. When bacteria breakdown nitrate to gain oxygen, nitrate is reduced to nitrous oxide and in turn nitrogen gas. Nitrogen gas has low solubility and so escapes into atmosphere as gas bubbles.

N0₃^-  => N0₂^- => N0  => N₂0  => N₂

6N0₃- + 5CH₃0H  => 3N₂ + 5C0₂ + 7H₂0 + 60H-

In the above equation, CH₃0H (Methanol) is the carbon source required for denitrification to occur. In a pond, carbon containing compounds present in the sludge can supply carbon source. Optimum pH value for denitrification is 7.0 - 8.5. Denitrification is an alkalinity producing process. About 3.0 – 3.6 pounds of alkalinity (as CaC0₃) is produced per pound of nitrate, thus partially mitigating the lowering of pH caused by nitrification in water.

Though carbon source can also be sludge or organic matter, Methanol, Acetic acid (vinegar) and sugar provide the highest rate of denitrification. Denitrification occurs between 41*F – 86*F (5*C – 30*C).

5 parts of chloride ions (Cl-) are used up for every part of NO₂^- oxidized to NO₃^-

Regular tap water contains chloramines. Chloramines can be nitrified in water systems if the water is stored after disinfection. The problem is greatest when temperatures are warm and water usage is low. For example, a number of water systems in Texas observed nitrification in their water storage during the rainy summer of 2007. E. coli or Azospirillum convert the nitrates into Nitrogen gas. Tubifex worms can also denitrify with the enzymes present in their gut.

Experiments:

1. After feeding koi, test ammonia and nitrite in the moving bed every 15 minutes and find the optimum feeding intervals.

2. Set up a large column and fill up with media. Trickle unfiltered water  directly on the media (to avoid aeration). Measure nitrates in the effluent and determine the time taken for denitrification. The media must develop sludge in order to denitrify. So, do not clean the media too often.

Bibliography:

Anthonisen A.C, Loehr R. C., Prakasam T.B.S, et al,“Inhibition of Nitrification by Ammonia and Nitrous Acid”, Journal of Water Pollution Control Federation, 48(5) pp. 835-852 (1976)

 Buday J, Drtil M, Hutnan M, et al. “Substrate and product inhibition of nitrification”, Chemical Papers-Chemicke Zvesti, 53(6) pp 379-383 (1999)

Philips S, Wyffels S, Sprengers R, et al. “Oxygen-limited autotrophic nitrification/denitrification by ammonia oxidisers enables upward motion towards more favourable conditions”,Applied Microbiology and Biotechnology, 59(4-5) pp. 557-566 (2002)

Lakeram Chatarpaul, John B. Robinson, Narinder K. Kaushik, Canadian Journal of Fisheries and Aquatic Sciences, 1980, 37(4): 656-663, 10.1139/f80-082