# Nitrification

Nitrification is the process by which ammonium (NH4+) or ammonia (NH3) is oxidized into nitrite (NO2-) by ammonia-oxidizing bacteria or AOB, often Nitrosomonas spp, and the NO2- further oxidized into nitrate (NO3-) by nitrite-oxidizing bacteria or NOB, often Nitrobacter spp.

The two processes of nitrification are called nitritation and nitratation. The bacterial groups are both chemo-litho-autotrophic - that is their only energy source is chemical energy, their electron-doner is an inorganic compound and their carbon source is plain carbon dioxide (CO2), or in practice bicarbonate (HCO3-).

## Nitritation

This is the first step of the nitrification, and the process by which NH4+ is oxidized into NO2-. This process can be divided into three sub-processes:

Step 1: NH3 is oxidized into hydroxyl-amine (NH2OH) with the help of the enzyme mono-oxygenase.
Step 2: NH2OH is oxidized into NO2-
Step 3: Electrons and oxygen and free hydrogen ions are converted into water.

Step 1 can be written as: NH3 + O2 + 2 H+ + 2 e- => NH2OH + H2O
Step 2 can be written as: NH2OH + H2O => NO2- + 5H+ + 4e-
Step 3 can be written as: ½ O2 + 2 H+ + 2 e- => H2O

The total reaction is Σ: NH3 + 1.5 O2 => NO2- + H+ + H2O

This reaction effects an acidification of the environment. Examples on how to calculate the degree of acidification using the charge balance equation is explained further down.

## Nitratation

The nitratation is the second part of the nitrification. The NOB uses the enzyme nitrite oxidoreductase (NOR) to conduct the process. The reaction is happening in two steps (here step 4 and step 5).

Step 4: NH3 NO2- is oxidized into NO3- with the help of the NOR enzyme
Step 5: Remaining oxygen, electrons and protons assembles into water.

Written using chemical equations the reactions are:

Step 4 can be written as: NO2- + H2O => NO3- + 2 H+ + 2 e-
Step 5 can be written as: ½ O2 + 2 H+ + 2 e- => H2O
The total reaction is Σ: NO2- + ½ O2 => NO3-

Now that we know that nitrification equals nitritation plus nitratation, we can add Σ1,2,3 to Σ4,5. This provides us with the total nitrification reaction.

It is Σ1-5: NH3 + 2 O2 => NO3- + H+ + H2O.

Above it is assumed that it is NH3 that is the substrate for nitrification. In the literature it is generally accepted that although there is much more NH4+ than NH3 present in places where nitrification takes place (optimum pH is less than 8), it is NH3 that is the substrate for the bacteria (Anthonisen et al. 1976 & Suzuki et al. 1974), not NH4+.

The two processes, nitritation and nitratation can of course run at different velocities. And if the nitritation is faster than the nitratation, there will be a build-up of NO2- and its corresponding acid HNO2, and if nitrataion is fastest only small amounts of NO2- will be detectable. In fact it is problematic if NO2- is allowed to build up as HNO2 is poisonous to both AOB and NOB.

## Energetics of nitrification and minimum generation time of AOB and NOB

Experience has shown that when biofilters are set-up to remove NH3 from ventilation air there is often a lag in NOB activity - which is seen by a build-up of NO2-.

The reason might be that there's less energy in Σ1-3 than in Σ4-5.

The energy from Σ1-3: NH4+ + 1.5 O2 => NO2- + 2H+ + H2O is 274.91 kJ/mol while the energetics of
Σ4-5: NH3 + 2 O2 => NO3- + H+ + H2O is 74.16 kJ/mol
In the first of these two equation more energy is available (please note that I added a H+ on both sides, don't worry, it doesn't matter).

When there's so much more energy in Σ1-3 than in Σ4-5 it is probable that AOB grows faster than NOB, hence there is a build-up of NO2- until the NOB's catches up.

Growth experiments, providing optimal conditions for both AOB and NOB also shows that AOB can grow faster than NOB (this is more a real proof than the argumentation above). The minimum doubling time of AOB is 7-8 hours while the minimum doubling time of NOB is 10-13 hours (Philips et al. 2002).

## Inhibition by NH3 and HNO2

The two main inhibitors of the nitrification process is nitrous acid and ammonia, the former being the most important. It is rather difficult to access the inhibition of any compound on any process as there may be many factors that cannot be controlled. Such factors include bacterial adaption to conditions, specific AOB and NOB strain in question, other inhibitors present, pH in the medium and the ionic strength of the medium.

Despite all of these precautions, it is actually possible to say something qualified about HNO2 inhibition and NH3 inhibition. An important work in that respect was done by Anthonisen (1976). He made a graph illustrating inhibition of AOB and NOB by HNO2 and NH3. After the graph I will elaborate a bit about the problems with such a graph and the terms necessary to use when describing such a graph. It is by no means a critique of Anthonisen.

Fig 1. From a master thesis project - based on Anthonisen et al. 1976

Four different zones can be found in the picture: Zone 1, Zone 2, Zone 3 and Zone 4. There are also boundaries between the zones. They are called [A], [B] and [C]. These boundaries are intervals so that the e.g. [A] is a range of HNO2 concentrations from 0.2 mg/L; 2.8 mg/L, (dashed lines).

For NH3 (lines that are not dashed), there are the boundaries [B] and [C]. For [B] it is that left of [B] there is complete nitratation. Inhibition by NH3 only begins somewhere in [B], and right of [B] nitratation rates gradually declines due to NH3 inhibtion. Nitratation is conducted by NOB's or Nitrobacter spp.. For nitritation it is boundary [C] that is the most important. Left of [C] nitritation is not affected by NH3 at all, but somewhere in the interval inhibition begins and when NH3 exceeds approx. 150 mg/L inhibition by NH3 is definitely taking place. Nitritation, the first step of nitrification, is conducted by AOB's are Nitrosomonas spp.

For HNO2 there is no inhibition whatsoever, by HNO2, right of [A], where as left of [A], when the concentration of HNO2 exceeds 2.8 mg/L, inhibition is definitely something that should be accounted for.

There are three zones, their span is shown with green lines. In Zone 3 there is neither any HNO2 nor NH3 inhibition and the nitrification processes can run at full speed. In zone 2, only AOB (nitrosomonas spp) is inhibited by NH3 and there is no HNO2 inhibition. In zone 1, only NOB (nitrobacter) is also inhibited by NH3, but not by HNO2. In Zone 4, both AOB and NOB are inhibited by HNO2 and only the AOB may be inhibited by NH3.

There are different interpretations of Anthonisen, yet they are probably due to confusion about what is meant by nhibition is beginning and inhibition is commencing. Better, but impossible on a general level, would be to state how tough the inhibition really is by showing some inhibition equations.

There is evidence that inhibition by both HNO2 and NH3 is not so severe as Anthonisen suggest is his well-cited paper from 1976. For instance, Buday (1999) investigated at which NH3 levels the inhibition of NH3 was at 50%. His conclusion was that NH3 inhibition could be described with two types of inhibition equations, for one of his two equations inhibition was at 50% at 16 mg/L and using the other equations the inhibition was mg/L at 20 mg/L. Both values are well passed interval [B] from Anthonisen.

## pH effects of nitrification

From the overall equation Σ1-5: NH3 + 2 O2 => NO3- + H+ + H2O. it can be seen that some H+ is created in the process. This indicated that nitrification will result in a lowered pH which is also the case.

The way pH is calculated in aqueous solutions is by using charge balance equations. The charge balance states that the total charge in a system must always be zero. The charge balance equation can be solved for H+ so that the charge is always zero. For more information about the charge balance equation please visit pH scale, a website about pH, the pH scale and how to calculate pH.

## Nitrogen cycle

In this section about the Nitrogen Cycle I will focus on the Nitrogen cycle in relation to nitrification.

## Modeling nitrification

Nitrification can be modeled or simulated, of course. The process is in itself very simple, the difficult part being which parameters to put into a model.

Below is a code showing how nitrification can be modeled in Java.

import java.math.BigDecimal;

//Ammoniaoxidation, calculates the rate of ammonia oxidation.

public class AmmoniaOxidation

{

public static BigDecimal p1(BigDecimal μmax_aob,BigDecimal KS_nh3,BigDecimal NH3,BigDecimal HNO2,BigDecimal KI_hno2,BigDecimal KS_O2,BigDecimal O2,BigDecimal Xaob,BigDecimal VOL)

{

}

}

The thing going is that the class ammoniaoxidation is called using several arguments that helps the method named p1 to return a value which is the rate of ammonia oxidation. The arguments are μmax_aob, which is the specific and maximum attainable oxidation rate possible per unit of ammoniaoxidizers (AOB), denoted XAOB. The choice of units is not simple - I recommend using chemical oxygen demand, like in the ADM1, or moles carbon.

Other arguments are KS_nh3 and NH3 which are the Monod half-saturation constant and the ammonia concentration. To calculate the inhibition imposed on the process, HNO2 and KI_hno2 are called also. The first is just the nitrous acid concentration and the latter is an inhibition constant. To regulate the ammonia oxidation so that it is low at low oxygen partial pressures, KS_O2 and O2 are called also; the name of these variables are self-explanatory. It runs like a part of the equation correcting for O2 pressure, and KS_O2 basically explain the O2 affinity for AOB. Finally VOL is called, its the volume of liquid in which the process is taking place.

I did not include ammonia inhibition in the above equation. The equation was used in a situation where NH3 inhibition on AOB's was not an issue.

In the end something is returned - it is a value that is returned into a variable used for further calculations.

The equation is better seen using an equation. Here it is:

For the nitratation its exactly the same, but the values used are not the same. Please e-mail me if you would like to see a source code file. The equation is given here:

In this the latter equation I put in a NOB in many cases just to say, that the values here are not the same as in the case of nitritation.

## References

ANTHONISEN AC, LOEHR RC, PRAKASAM TBS, et al. INHIBITION OF NITRIFICATION BY AMMONIA AND NITROUS-ACID JOURNAL 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)