Nitrification is the biological oxidation of ionized ammonia to nitrite (Equation 10.2) and/or the biological oxidation of nitrite to nitrate (Equation 10.3). Nitro-somonas and Nitrosospira oxidize ionized ammonia to nitrite, while Nitrobacter and Nitrospira oxidize nitrite to nitrate.

NH4+ + 1.5O2 Nitrosomonas & Nitrosospira , NO2- + 2H+ + energy (10.2) NO2- + 0.502 Nitrobacter & Nitrospira > NO3- + energy (10.3)

Nitrifying bacteria oxidize ionized ammonia and nitrite in order to obtain energy for cellular activity including reproduction. In the activated sludge process, this reproduction results in an increase in sludge. Carbon needed by nitrifying bacteria is obtained from bicarbonate alkalinity (HCO3-). Because nitrifying bacteria obtain very little energy from the oxidation of ionized ammonia and nitrite, bacterial growth or sludge production is relatively small. Approximately 0.06 pound of nitrifying bacteria or sludge is produced for every pound of ionized ammonia oxidized to nitrate.

Nitrifying bacteria are chemolithoautotrophs. As chemolithoautotrophic bacteria, they obtain their cellular energy by oxidizing a mineral such as nitrogen, and they obtain their carbon for cellular synthesis by consuming inorganic carbon. Inorganic carbon does not contain hydrogen (H). The inorganic carbon source for autotrophic nitrifying bacteria is carbon dioxide (CO2). Carbon dioxide is consumed in the form of bicarbonate alkalinity (HCO3-)—that is, carbon dioxide dissolved in water to form carbonic acid (H2CO3) that dissociates to form bicarbonate alkalinity (Equation 10.4).

Nitrifying bacteria are very efficient in oxidizing ionized ammonia and nitrite because they possess (1) unique nitrifying enzyme systems and (2) cytomembranes (Figure 10.4). Cytomembranes are an in-folding of the cellular membrane that

FIGURE 10.4 Cytomembranes in nitrifying bacteria.

provide an increase in the surface area of the cell membrane upon which nitrification can occur.

Nitrifying bacteria are free-living organisms and are found in the soil and water. They enter wastewater treatment plants through inflow and infiltration. Because nitrifying bacteria are strict aerobes, they live in the top 1-2 inches of soil.

Due to the relatively small quantity of energy obtained from the oxidation of ionized ammonia and nitrite, nitrifying bacteria reproduce very slowly. Under optimal conditions, the generation time for nitrifying bacteria is approximately 8-10 hours. Under the harsh conditions of an activated sludge process, the generation time for nitrifying bacteria is approximately 2-3 days. Therefore, in activated sludge processes, relatively high mean cell residence times (MCRT) are required to establish a population of nitrifying bacteria that are capable of effective nitrification.

The activity and generation time of nitrifying bacteria are temperature-dependent.With increasing temperature nitrifying bacteria become more active and reproduce more quickly. Nitrifying bacteria are active and reproduce over a range of temperature values from 5°C to 40°C. However, the maximum temperature for nitrification in the activated sludge process is considered to be 30°C. This temperature produces the maximum activity and shortest generation time for Nitrosomonas, one of the major nitrifying bacteria.

Nitrifying bacteria are poor floc-forming organisms. Their incorporation into ftoc particles is achieved largely through compatible charges between the nitrifying bacteria and the floc particle and their adsorption to the floc particles through coating action of higher life forms. Ciliated protozoa, rotifers, and free-living nematodes, several of the higher life forms in the activated sludge process, release secretions that coat the surface of nitrifying bacteria and renders the surface compatible for adsorption to the floc particles.

Due to the long generation time of nitrifying bacteria and their small population growth (sludge yield) from the oxidation of energy substrates (NH4+ and NO2-), nitrifying bacteria usually represent <10% of the bacterial population in the activated sludge process. Because nitrifying bacteria are strict aerobes, they are found mostly on the perimeter of the floc particles.

Although the activity and maximum population size of nitrifying bacteria are dependent upon the quantity of energy substrates that are available, there are several operational factors that influence the activity and population size of nitrifying bacteria and the ability of the activated sludge process to successfully nitrify

TABLE 10.3 Operational Factors that Influence Nitrification


Dissolved oxygen concentration Mean cell residence time (MCRT) pH

Temperature Toxicity

TABLE 10.4

Temperature and MCRT Recommended for



(°C) MCRT (days)











TABLE 10.5

Temperature and Nitrification


(°C) Effect on Nitrification


Optimum temperature for nitrification


Approximately 50% of optimum nitrification


Approximately 20% of optimum nitrification


Nitrification ceases

(Table 10.3). These factors include alkalinity and pH, dissolved oxygen concentration, MCRT and temperature, and toxicity. The most important factors are MCRT and temperature.

The most critical factors affecting the activity and population size of hitrifying bacteria and the success of nitrification in the activated sludge process are MCRT and temperature. Because there is an indirect relationship between temperature and activity of nitrifying bacteria, increasing MCRT is required with decreasing temperature (Table 10.4). With decreasing wastewater temperature, nitrifying bacteria become less active, and nitrification efficiency decreases. To compensate for the lost of activity and to improve nitrification efficiency, the number of nitrifying bacteria must be increased. An increase in the number of nitrifying bacteria requires an increase in the mixed liquor volatile suspended solids (MLVSS) and MCRT.

The most critical temperature value with respect to process control of nitrification is 15°C (Table 10.5). With decreasing wastewater temperature, approximately 50% of the ability of the activated sludge process to nitrify is lost at 15°C, unless appropriate operational measures are implemented to maintain effective nitrification (Table 10.6).

Adequate alkalinity is essential for successful nitrification. At least 50mg/liter of alkalinity should be present in the mixed liquor effluent after complete nitrification to ensure the presence of adequate alkalinity. After complete nitrification, the mixed

TABLE 10.6 Operational Measures Available for Maintaining Effective Nitrification During Cold Wastewater Temperature

Operational Measure Effect

Addition of bioaugmentation products

Increase aeration tank dissolved oxygen concentration Increase hydraulic retention time (HRT) Increase primary clarifier efficiency

Install biological holdfast (ringlace) system

Bacterial cultures rapidly remove cBOD and improve or initiate nitrification

Promotes rapid cBOD removal and improved nitrification Provides more time for nitrification Removes more colloidal and particulate cBOD and lowers dissolved oxygen demand in the aeration tank

Increases bacterial populations for removing cBOD quickly and nitrifying without overloading the secondary clarifier liquor effluent contains <1 mg/liter ionized ammonia (NH4+) and <1 mg/liter nitrite (NO2-).

There are two biochemical reactions that are responsible for the loss of alkalinity during nitrification. The minor reaction is the used of bicarbonate alkalinity as the carbon substrate for the synthesis of cellular materials and reproduction. The major reaction is the production of free nitrous acid (HNO2) that destroys alkalinity.

When ionized ammonia is oxidized to nitrite during nitrification, hydrogen protons (H+) are produced (Equation 10.5). When hydrogen protons combine with nitrite, free nitrous acid is produced (Equation 10.6). As nitrous acid is produced, alkalinity is destroyed, and the pH of the mixed liquor drops.

Although alkalinity is lost during nitrification, some alkalinity is returned naturally to the activated sludge process through deamination of organic nitrogen compounds and denitrification (Equation 10.7). Deamination of organic nitrogen compounds results in the production of ionized ammonia. Ionized ammonia represents an increase in alkalinity. Through denitrification (i.e., the use of nitrate to degrade soluble cBOD), alkalinity is produced in two forms. First, the production of hydroxyl ions (OH-) returns alkalinity directly to the process. Second, the release of carbon dioxide that dissolves in the wastewater returns alkalinity indirectly through the formation of the bicarbonate ion (HCO3-).

Nitrifying bacteria are active over a wide range of pH values, 5 to 8.5 (Table 10.7). Although the optimum pH range for nitrification is 7.3 to 8.5, most activated sludge processes nitrify at a near neutral pH value, 6.8 to 7.2. At pH values greater than 7.3, undesired operational conditions may occur in the activated sludge process. These undesired operational conditions include the following:

84 NITRIFYING BACTERIA TABLE 10.7 pH and Nitrification pH Effect on Nitrification

4.0-4.9 Nitrifying bacteria present but inactive; limited nitrification occurs through the activity of organotrophic bacteria

5.0-6.7 Nitrifying bacteria are active but activity is sluggish

6.8-7.2 Desired pH range for nitrification in the activated sludge process 7.3-8.0 Rate of nitrification assumed be constant

8.1-8.5 Optimum pH range for nitrification (e.g., in laboratory work with nitrifying bacteria only)

*Pound of ionized ammonia oxidized per pound ML VSS per day FIGURE 10.5 Effect of dissolved oxygen concentration upon the rate of nitrification.

*Pound of ionized ammonia oxidized per pound ML VSS per day FIGURE 10.5 Effect of dissolved oxygen concentration upon the rate of nitrification.

• A decrease in enzymatic activity in organotrophic bacteria resulting in decreased cBOD removal efficiency

• The development of a phosphorus deficiency through the precipitation of orthophosphate with calcium ions

• The development of weak and buoyant floc particles

Because nitrifying bacteria are strict aerobes, nitrification can occur only in the presence of free molecular oxygen (O2). Approximately 4.6mg of oxygen are consumed per milligram of ionized ammonia oxidized to nitrate.

Insignificant nitrification occurs at dissolved oxygen concentrations lower than 0.5mg/liter (Figure 10.5), while significant nitrification occurs at dissolved oxygen concentrations between 2 and 3mg/liter (Table 10.8). Improved nitrification may occur at dissolved oxygen concentrations higher than 3 mg/liter, if organ-otrophic bacteria remove cBOD more rapidly and provide more time for nitrification.

TABLE 10.8 Dissolved Oxygen Concentration and Nitrification

Dissolved Oxygen (mg/liter)

Effect on Nitrification


Nitrification initiated but insignificant


Rate of nitrification begins to accelerate


Rate of nitrification is significant


Sustained nitrification


Maximum rate of nitrification


Nitrification may improve, if organotrophic bacteria remove cBOD

more rapidly


Forms of Toxicity to Nitrifying Bacteria


Description or Example

Free chlorine residual

Hypochlorous acid (HOCl) or hypochlorous ion (OCl-)


Heavy metals


Phenols and recognizable, soluble cBOD




Free ammonia or free nitrous acid


Ultraviolet radiation



TABLE 10.10

Examples of Recognizable, Soluble cBOD

Organic Compound Formula Number of Carbon Units




CH3NH2 1






(CH3)2CHOH 3




(CH3)3COH 4

Ethyl acetate

CH3CO2C2H5 4



There are several forms of toxicity to nitrifying bacteria (Table 10.9).These forms of toxicity include two unique forms for nitrifying bacteria. The unique forms of toxicity are (1) recognizable, soluble cBOD and (2) substrate.

Nitrifying bacteria are obligate autotrophs. As obligate autotrophs, they are dependent upon inorganic carbon for their carbon substrate. As obligate autotrophs, their enzymatic ability to oxidize ionized ammonia and nitrite is inhibited in the presence of several specific, short-chain (1-4 carbon units) alcohols and amines (Table 10.10). These inhibitory organic compounds are referred to as recognizable, soluble cBOD. These compounds may be discharged to the sewer system or may be produced in the sewer system or various treatment tanks under anaerobic (septic) conditions. Only when these organic compounds are either degraded to a low concentration or degraded completely does nitrification occur in the activated sludge process.

Although ionized ammonia and nitrite are the energy substrates for nitrifying bacteria, their accumulation in the activated sludge process can cause substrate tox-icity to nitrifying bacteria. Substrate toxicity occurs when the ionized ammonia concentration in the aeration tank is >480mg/liter.

With increasing pH, ionized ammonia is converted to free ammonia (Equation 10.8). The accumulation of ammonia is a function of the ionized ammonia concentration and aeration tank pH.

With decreasing pH, nitrite is converted to free nitrous acid (Equation 10.9). The accumulation of nitrous acid is a function of nitrite concentration and aeration tank pH. Substrate toxicity due to the accumulation of nitrous acid occurs because ionized ammonia is oxidized to nitrite more quickly than nitrite is oxidized to nitrate:

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