The Nitrogen Cycle: Nitrification

Table N3. Rates of nitrification by some heterotrophic and autotrophic nitrifiers.

Nitrification is a Two Step Process

1. First Step: Ammonium Oxidation

The microorganisms involved are called the ammonia oxidizers. There are four recognized genera, with several species in each (see Table N4). Nitrosomonas is the most extensively studied and usually the most numerous in soil (see Table N5). Nitrosospira is an aquatic nitrifier.

• Nitrosomonas
• Nitrosospira
• Nitrosococcus
• Nitrosolobus

Biochemical Oxidation:

NH₄⁺ + 1/2 O₂ -------> NH₂OH + H⁺
Enzyme: Ammonia Monooxygenase

NH₂OH + O₂ ---------> NO₂^- + HOH + H⁺

• Overall Reaction: NH₄⁺ + 1 1/2 O₂ ----> NO₂^- + HOH + 2H⁺. Sixty-six kcal of energy are liberated per gram atom of ammonia oxidized.
• Under Anaerobic Conditions: NH₂OH --> NOH --> N₂O

Ammonia-Oxidizing Bacteria:

These bacteria are rod-shaped, spherical , spirillar, or lobular Gram-negative cells. Flagellation of motile cells is polar to subpolar or peritrichous. All species are aerobic but can grow at reduced oxygen partial pressure. The organisms are chemoautotrophs, growing with ammonia as the energy substrate and CO₂ as the main carbon source. Generally, the optimum growth temperature is 30^oC and the optimum pH is between 7.5 and 8.0. In enrichment cultures, but not in pure cultures, cells often form aggregations called zoogloeae or cysts. Species are distributed in a great variety of soils, oceans, brackish environments, rivers, lakes, and sewage disposal systems.

Table N4. Differential characteristics of the genera of the ammonia-oxidizing bacteria.

Table N5. Differential characteristics of the species of the genus Nitrosomonas.

2. Second step: Nitrite Oxidation

Microorganism involved: Nitrobacter

Biochemical Oxidation: NO₂^- + 1/2 O₂ -----> NO₃^-. Eighteen kcal of energy is liberated per gram atom of nitrite oxidized.

Nitrite-Oxidizing Bacteria (see Table N6)

These bacteria comprise a diverse group of rod, ellipsoidal, spherical, and spiral-shaped cells. All are Gram negative. Cells are motile or non-motile. When motile, flagella are polar or lateral. Nitrite-oxidizers are aerobes. As an exception, at least one strain of Nitrobacter has been described that can grow by anaerobic respiration (denitrification). NO and N₂O are formed if organic substances and nitrate are available. Investigations have shown that one strain of Nitrobacter was able to oxidize NO to nitrate. These chemoautotrophs obtain energy for cell growth from the oxidation of nitrite to nitrate. CO₂ is fixed via the Calvin cycle. Members of the genus Nitrobacter possess more than one type of the CO₂- fixing enzyme ribulose-1,5-biphosphate carboxylase. They are mesophiles, with a temperature optimum of 28^oC and a pH range of 5.8-8.5, with an optimum between 7.6 and 7.8. Unicellular, with the ability to form microcolonies that consist of loosely associated cells embedded in a polysaccharide layer or closely packed cells surrounded by a firm slime layer. The biofilm is referred to as zoogloeae or cysts. Nitrite-oxidizing bacteria are found in aerobic, but occasionally also in anaerobic, environments where organic matter is mineralized. They are widely distributed in soils, fresh water, brackish water, seawater, mudlayers, sewage disposal systems, and inside stones of historical buildings and rocks. They are also found inside corroded bricks and on concrete surfaces such as in cooling towers and highway-automobile tunnels.

Table N6. Differential characteristics of the genera of the nitrite-oxidizing bacteria.

Environmental Impact

(i) Nitrification aids in the decomposition of nitrogenous material and thus in the recycling of nitrogen atoms since the deamination of organic nitrogen produces ammonia that is subsequently oxidized to nitrate by nitrification.

(ii) The microbes that perform nitrification are inefficient. Most of them are autotrophs that use the energy gained from oxidizing ammonia to fix carbon. Thus these bacteria have a dual ecological role - they are involved in recycling nitrogen and in fixing carbon into organics. Carbon fixation by this method is not very efficient. Therefore a lot of nitrogenous oxidation is required to acquire enough energy to fix carbon. The fixation of one mole of carbon requires the oxidation of 35 moles of ammonia to nitrite and of 100 moles of nitrite to nitrate.

(iii) The microbes that perform nitrification are fragile. These organisms are acid-sensitive even though they produce acid! If a large source of nitrogen is dumped into the environment, these organisms can potentially kill themselves by metabolizing it to nitric acid. Since they are also strict aerobes, they can be killed if introduction of wastes leads to excessive growth of other species that deplete oxygen (i.e. eutrophication).

Agriculture

Nitrification is a nuisance to the agricultural industry. Under optimal conditions, the conversion of ammonia to nitrate can be very rapid. Studies from the Midwest have shown that one-half of applied fertilizer ammonia can be converted to nitrate within 30 days after application (see FATE OF NITRATE). Plant fertilizers contain ammonia in the form of the ammonium ion (NH₄⁺). This is provided to supplement soils, which are often deficient in nitrogen sources that plants can utilize. However, there is a competition for ammonium between plants, who want to incorporate it into organics (R-NH₂), and the microbes who want to oxidize it to nitrate. Although plants can use both ammonium and nitrate as nitrogen sources, ammonium is retained in soils by sticking to negatively-charged clay particles by electrostatic interactions. Nitrate has no such affinity for clay - it is repulsed by having the same charge. Nitrogen in fertilizer is thus wasted if it is converted to nitrate that simply leaches out of the soil when it rains. A strategy employed to combat this problem is to add nitrapyrin to the fertilizer. This chemical inhibits growth of nitrifiers by poisoning ammonia monooxygenase.

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