Nitrification occurs by various species of autotrophic microorganisms. While energy is derived from the oxidation reaction of ammonia nitrogen (NH4-N), the carbons required to build the cell mass are derived from dissolved CO2. Since the relatively small energy stored in N-H bondings is used to produce the C-C bondings that bear higher energy, multiple N-H bondings are required to produce one C-C bonding. As a result, the autotrophic biosolids yield (YA) is much lower than that of heterotrophic counterparts (YH). In original ASM#1 (Henze, 1987), YA was assumed at 0.24 COD/g N while YH was assumed at 0.67 g COD/g COD.

In biological wastewater treatment, autotrophic biosolids yields are not only low , but also nitrogen contents in the wastewater are typically much lower than carbon contents, e.g. TKN/COD ratio is around 0.1 in municipal wastewater. As a consequence, autotroph biomass is only 2-5% of the total biosolids in municipal wastewater treatment. Therefore autotrophic biosolids yields are often neglected when estimating excess biosolids production.

Nitrification is a two-step process as follow, where ammonium nitrogen (NH4+) is oxidized to nitrite (NO2) and nitrite is further oxidized to nitrate (NO3). Since nitrate formation from nitrite is far faster than nitrite formation from ammonia, only a negligible amount of nitrite exists at steady state in a typical reaction condition.


Nitritation by Nitrosomonas (Slow) : NH4+ + 1.5 O2  →  NO2 + 2H+ + H2O
Nitratation by Nitrobacter (Fast) : NO2 + 0.5O2   →  NO3
Overall reaction : NH4+  +  2 O2   →  NO3 + 2H+ + H2O
                  Molecular weight(Dalton)        14 (N only)   2×32
                  Alkalinity (eqiv. CaCO3)     0            0             0        -2          0

According to above equations, two moles of acidity are produced, which also means two moles of alkalinity are consumed, when one mole of NH4+ is oxidized. Here, NH4+ is considered neutral since it consists of one alkalinity (NH3) and one acidity (H+).  The specific oxygen requirement for ammonia nitrogen oxidation can be calculated based on above equations. Assuming no new autotrophic microorganisms are produced during the reaction, 4.57 mg O2 (=2×32/14) and 7.14 mg alkalinity as CaCO3 (= 2×50/14) are consumed to oxidize 1 mg NH4-N.

In practical situations, however, some nitrogen atoms are used to produce new microorganisms. The following equations represent nitritation and nitratation reactions in typical nitrification condition (Tchobanoglous, 2002). Due to the partial loss of nitrogen to the new cells, the O2 and alkalinity consumptions based on the treated NH4-N are slightly lower than those values above. Although the exact values vary system by system due to the varying autotrophic sludge yields, equivalent oxygen and alkalinity consumptions are often assumed at 4.3 mg O2/mg N and 6.8 mg CaCO3/mg N.

   Nitritation :

55NH4+ + 76 O2 + 109 HCO3  à  C5H7O2N (cell) + 54NO2 + 57H2O + 104H2CO3

   Nitratation :

400NO2 + NH4+ + 4H2CO3 + HCO3 +1950O2 à C5H7O2N (cell) +3H2O +400NO3

The autotrophic bacteria responsible for nitrification not only grow slower than heterotrophic bacteria, but also are more susceptible to the environmental changes such as temperature, pH, and the toxic chemicals in wastewater. High ammonia concentration can inhibit nitrification, but the threshold concentration varies depending on pH. In general high level ammonia is more toxic at low pH because unionized dissolved NH3 can penetrate microbial cells easier to cause physiological problems. High nitrite (NO2) level in mixed liquor indicates a problem in nitratation, which is normally a fast reaction, but it is not clear whether the nitrite itself is toxic to the nitrobacter that is responsible for nitratation.

pH and alkalinity effect

The optimum pH for nitrification is known to be around 7.5, which is the middle ground of the two optimum pH forNitrosomonas and Nitrobacter that grow fastest at pH 7.8-8.0 and at pH 7.3-7.5, respectively. It is well know that nitrification rate slows down at a pH below 7.0 and it ceases at around pH 6.0. However, it must be noted that the diminished nitrification with decreasing pH is only true when pH drops or fluctuates rapidly. If mixed liquor is acclimated at a low pH for extended period of time, nitrification occurs even at a pH below 6.0 (Tarre, 2004). Though full scale studies are rare, many lab-scale studies have shown the possibilities that some of the minor species in Nitrosomonas and Nitrobacter genera grow fast at low pH and replaces major species. It is also possible that the same species playing a role at high pH adapts to low pH.

Excessive pH fluctuation can occur at low alkalinity, which in turn decreases the activity of nitrifiers. Therefore, high alkalinity is crucial to prevent pH from excessive fluctuations especially when TKN loading fluctuates. Although the concentration of dissolved CO2, which is used as building blocks of autotrophs (or nitrifiers), decreases as alkalinity decreases, there is no enough evidence to prove low CO2 concentration in mixed liquor directly hinders the growth of autotrophs in field condition.

Temperature effect

It is well known that the growth of nitrifiers, which is already slow, slows down further at low temperature, which possibly makes nitrifiers washed out. As shown in Fig. 1, the maximum growth rate of nitrifiers decreases less than a half when temperature decreases from 14 oC to 6 oC in long-term tests. Maximum nitrification rate per cell mass also decreases with temperature at the same temperature range as can be seen in Fig. 2, where maximum nitrification rate is somewhat higher when the temperature variation starts from high to low in a short term experiment.

However, the slower growth rate at lower temperature in Fig. 1 does not directly mean nitrification rate decreases proportionally in continuously running biological systems due to the following reasons.

  • First, there is a great deal of redundancy in nitrifier population due to the long SRT in biological nutrient removal (BNR) processes especially in MBR. In MBR, the high solids retention time (SRT) of 12-30 days enables the enrichment of autotrophs. Therefore, poor nitrification is not commonly observed in MBR treating municipal wastewater as long as there are no drastic changes in water pH, temperature, TKN loading, etc.
  • Second, if there is any residual ammonia left over as a result of a slower nitrification, nitrifier can grow faster due to the available food, NH4+-N. This self-correcting mechanism can keep nitrification efficiency high as long as there is no drastic changes in reaction condition.


Fig. 1. Long term effect of temperature on ammonium oxidizing organisms. Growth rates arebased on 12 weeks of operation of 3 pilot plants close to washout of the organisms at 6, 10 and 14 8 oC. In the equation, T represents liquid temperature in Celsius. (Reproduced from Gujer, 2010)

image0042l3k4jFig. 2. Short term effects of temperature on ammonium oxidizing organisms based on batch results with activated sludge grown at 6 and 14 oC. In the equation, T represents liquid temperature in Celsius. (Reproduced from Gujer 2010)

Dissolved oxygen effect

Nitrification rate is also affected by dissolved oxygen (DO) level. DO of 1 mg/L is considered as a minimum requirement to prevent any inhibition caused by the insufficient oxygen level (Tchobanoglous, 2002). At lower DO than 1 mg/L, nitrification slows down in general. At below 0.5 mg/L DO, nitrification nearly stops and denitrification starts to occur.

However, if low DO condition sustains stably, biological systems can eventually adapt to the low DO and nitrification occurs at below 0.5 mg/L. In one full scale dairy wastewater treatment plant, author observed nitrification was accomplished nearly 100% at a DO below detection limit while oxidation reduction potential (ORP) was at persistently around 0 mV. Therefore, it appears that the most crucial factor affecting nitrification efficiency in biological wastewater treatment is the stability of operational condition.


© Seong Hoon Yoon