Significance of nitrogen removal

In its various forms, nitrogen can deplete dissolved oxygen in receiving waters, stimulate aquatic plant
growth (figure __), exhibit toxicity toward aquatic life, present a public health hazard, and affect the
suitability of wastewater for reuse purposes. Wastewater effluents containing nutrients such as
nitrogen and phosphorus can cause eutrophication, the excessive growth of aquatic plants and/or
algae in lakes, streams, rivers, wetlands or any surface water subject to runoff.

Figure ___: Algae growing in a small creek

Forms of nitrogen found in wastewater:

• Nitrogenous gasses (N2O, N2)
• Organic nitrogen (urea, amino acids, fecal material)
• Ammonia (NH3 as a dissolved gas)
• Ammonium (NH4+ ions in solution)
• Nitrate (NO3 - and nitrite (NO2 - ions in solution
• Total Kjeldahl Nitrogen (TKN) is the sum of the ammonia and organic nitrogen
Total Nitrogen (TN) is the sum of TKN and nitrate and nitrite. Nitrate (NO3- is a primary
contaminant in drinking water and can cause a human health condition called
methemoglobinemia (blue baby syndrome). This is due to the conversion of nitrate to nitrite
(NO2-) by nitrate reducing bacteria in the human gastrointestinal tract.
NITRIFICATION
Nitrification is an oxidation process (loss of electrons or gain of the oxidation
state by an atom or compound takes place). This process starts with the
ammonium which gets oxidized into nitrite (NO2-), this action is performed by
the bacteria Nitrosomonas sp. Later on, this nitrite (NO2-) gets oxidized into
nitrate (NO3-), and this action is performed by the Nitrobacter sp.

The bacteria are autotrophic, and the reaction is performed under aerobic
condition. The importance of this step in the nitrogen cycle is the conversion
of ammonia into nitrate, as nitrate is the primary nitrogen source present in
the soil, for the plant. Though nitrate is toxic to the plants.

The activity of nitrifying bacteria gets slower in acidic solution, and are best at
pH between 6.5 to 8.5 and temperature vary from 16 to 35°C.

Nitrification is the biological conversion of the ammonium to nitrate nitrogen, and is a two-
step process. In the first step, aerobic bacteria known as Nitrosomonas convert ammonium to
nitrite. Another group of aerobic bacteria called Nitrobacter finish the conversion of nitrite to
nitrate. The reactions are generally coupled and proceed rapidly to the nitrate form; therefore
nitrite levels at any given time are usually low. These bacteria known as ‘nitrifiers’ are strict
aerobes, which means they must have free dissolved oxygen (O2) to perform their work, and
are active only under aerobic conditions. Complete nitrification requires approximately 4.6
pounds of oxygen for every pound of ammonium converted to nitrate. In comparison, CBOD
can be consumed with only about 1.5 pounds of oxygen. The growth rate of nitrifiers is affected
by the concentration of dissolved oxygen (DO), and at DO less than 0.5 mg/L the growth rate
is minimal. Typical operational guidelines call for a minimum DO concentration of 1.0 mg/L
at peak flow and an average daily DO concentration of 2.0 mg/L. For nitrification to proceed
the oxygen should be well distributed throughout the aeration tank and its level should not be
below 1.0 mg/L. Similar to humans, activated sludge organisms need nutrients to survive and
reproduce. Nitrifying bacteria are no different, and need calcium in their diet. Luckily, there is
usually enough calcium in the raw wastewater in the form of calcium carbonate (CaCO3) to
allow nitrifiers to survive nicely. Later in this manual, we will discuss the consequences of
insufficient calcium (alkalinity) in the wastewater. As the nitrifiers use the ammonium as an
energy source, they consume the calcium carbonate as a carbonsource.

The process of nitrification produces acids. This acid formation, along with the calcium
carbonate reduction, can lower the pH of the MLSS and cause a decline in the growth rate of
nitrifying bacteria. The optimum pH for Nitrosomonas and Nitrobacter organisms is between
7.5 and 8.5 and nitrification stops at pH levels at or below 6.0. Approximately 7.14 pounds of
alkalinity (as CaCO3) are consumed per pound of ammonia oxidized to nitrate.
Water temperature also affects the rate of nitrification. Nitrification reaches a maximum rate at
liquid temperatures between 30 and 35 degrees C (86oF and 95oF). At temperatures of 40oC
(104oF) and higher, nitrification rates fall to near zero. Conversely, cold water temperatures
can also affect the rate of nitrification. As water temperature decreases, the nitrifiers slow
down. Many wastewater treatment plants increase the MLVSS amounts during the winter
months to increase the amount of aerobic autotrophic bacteria available to perform nitrification
efficiently.
Nitrifying bacteria are sensitive organisms, and react quickly to environmental changes. Rapid
changes in liquid temperature can shock nitrifiers and other organisms we rely upon to treat
our wastewater. As seasons change, cold fronts can bring swift changes in air temperatures.
Water temperatures change fast as well, and treatment plants with mechanical aeration
equipment where the MLSS is thrown into the air (figure 4) can see temperature decreases
much faster than facilities with submerged, compressed air diffusers. We can see the effects of
a MLSS temperature drop of one degree or more per day as cloudiness in secondary clarifiers,
and an increase in effluent ammonium levels.

Sludge Age and Mixed Liquor amounts are also integral components in the nitrification
process. When performing sludge age calculations (MCRT or SRT) to determine the detention
time required for nitrification, the capacity of the oxic (aerated) portion of the plant should be
used. Since anoxic or fermentation basins are not aerated and nitrifying organisms are strict
aerobes, the capacity of these basins should not be included in calculations for oxic SRT.
Extended aeration (package type) wastewater plants are more capable of nitrification than
contact-stabilization and some other activated sludge modifications due to the high sludge age
and long periods of aeration. Toxicity and sources of inhibition to microorganisms present
problems to operators and nitrifying organisms. Some of the most toxic compounds to nitrifiers
include cyanide, thiourea, phenol and heavy metals such as silver, mercury, nickel, chromium,
copper and zinc. Some of these compounds can enter a wastewater treatment plant from landfill
leachate and septage. Nitrifying bacteria can also be inhibited by high concentrations of free
forms of their own substrate. Nitrite oxidizing bacteria are sensitive to free nitrous acid, and
ammonium oxidizing bacteria are sensitive to free ammonia (NH3). Increased levels of free
ammonia can decrease nitrifier growth rates. Some treatment plants that may have increased
influent organic nitrogen and ammonia levels include plants that serve highway rest areas and
wastewater plants serving schools.

DENITRIFICATION
Denitrification is the reduction process, where the nitrate is removed in the
form of nitrogen and is converted to nitrogen gas. The action is performed by
bacteria like Bacillus, Aerobacter, Lactobacillus, Spirillum, Pseudomonas.

The bacteria are heterotrophs, and the action is completed under anaerobic
condition. Even the small amount of oxygen may hamper the process, but
there is a need of organic carbon. Denitrification is useful for wastewater
treatment, aquatic habitats.
The denitrification is performed best at pH between 7.0 to 8.5 and at the
temperature between 26 to 38°C.

Denitrification is an anaerobic respiration process in which nitrate serves as the electron

acceptor. In simpler terms, denitrification occurs when free dissolved oxygen levels are
depleted and nitrate becomes the primary bound oxygen source for facultative heterotrophic
microorganisms. Heterotrophic organisms can obtain their carbon and energy from the same
source – by biodegrading influent wastewater containing carbon. We measure the influent
carbon by sampling for and running a carbonaceous biochemical oxygen demand (CBOD) test.
When bacteria break apart nitrate (NO3-) to gain the oxygen (O2), the nitrate is reduced to
nitrite (NO2-) then quickly to nitrous oxide (N2O), and finally nitrogen gas (N2) as the
oxygen is stripped away by the microorganisms. Since nitrogen gas has low water solubility, it
tends to escape as gas bubbles once the surrounding liquid is saturated with nitrogen. These
gas bubbles can become bound in the settled sludge floc in clarifiers and cause the sludge to
rise to the surface.
Conditions Required for Effective Denitrification
Conditions that affect the efficiency of denitrification include nitrate concentration, anoxic
conditions, presence of organic matter, pH, temperature, alkalinity and the effects of trace
metals.
Since denitrifying bacteria are facultative organisms, they can use either dissolved oxygen or
nitrate or sulfate as an oxygen source for metabolism and oxidation of organic matter. If
dissolved oxygen and nitrate are present, facultative bacteria prefer to use the dissolved oxygen.
This will occur since dissolved oxygen is readily available and yields more energy to the
organisms. Therefore it is imperative to keep dissolved oxygen levels as close to zero as
possible in anoxic basins or timed anoxic cycles. Excessive dissolved oxygen in basins
designed as anoxic is possible through aerated return sludge (air lift type RAS), excessive
splashing of liquid streams into anoxic basins, and air diffusion being used for tank mixing
instead of using mixing pumps or mixing devices.
Another important aspect of denitrification is the presence of organic matter to drive the
denitrification reaction. Organic matter may be in the form of raw wastewater, foodprocessing
wastes, or chemical sources such as methanol, ethanol, acetic or citric acid.
When these sources are not present, bacteria may depend on internal (endogenous) carbon
reserves as the source of organic matter. This material is released during the death phase of
organisms and may not be a consistent enough source of carbon to drive denitrification to
completion. Whatever organic source is used to drive the denitrification reaction, it should be
fed consistently and at a rate to keep denitrification levels maximized. Conversely, it is
important to avoid raising effluent CBOD values and avoid spending excessive money on
organic sources such as methanol.
An advantage of denitrification is the production of alkalinity and an increase of pH.
Approximately 3.0 to 3.6 mg of alkalinity (as CaCO3) is produced per milligram of nitrate
reduced to nitrogen gas. Optimum pH values for denitrification are 7.0 to 8.5. Temperature
affects the growth rate of denitrifying organisms, with increased growth rate at higher
temperatures. Denitrification can occur between 5 to 30oC (41oF to 86oF), and these rates
increase with temperature and type of organic source present. The highest growth rate can be
found when using methanol or acetic acid. A slightly lower rate using raw wastewater will
occur, and the lowest growth rates are found when relying on endogenous carbon sources at
low water temperatures. Denitrifying organisms are generally less sensitive to toxic chemicals
than nitrifiers and recover from toxic shock loads faster than nitrifiers.
Nitrification, an oxidation process
Once the ammonium reaches the aeration tank, the aerobic autotrophic bacteria (nitrifiers) get
to work. As long as there is enough DO left over after CBOD removal by aerobic and
facultative bacteria, the nitrifiers will consume calcium carbonate while oxidizing the
ammonium as an energy source. Nitrosomonas bacteria take the lead by converting the
ammonium (NH4+) to nitrite (NO2-), then the Nitrobacter step in to finish off the conversion
of nitrite to a final fully oxidized form called nitrate (NO3-). While the calcium carbonate is
being consumed, nitrous acid is being formed and the total alkalinity of the liquid begins to
decline. By the time the wastewater has reached the end of the aeration tank system,
• The CBOD has been oxidized to low levels by aerobic and facultative bacteria as
a carbon food source
• The incoming ammonia/ammonium has been oxidized to nitrite, then to nitrate
• Ammonia (NH3) and ammonium (NH4+) levels have decreased, nitrate (NO3-)
levels have increased
• Dissolved oxygen demand has decreased, residual DO has increased
• Total alkalinity (as calcium carbonate) has decreased
• pH may have decreased
• A total of 6.1 pounds of oxygen has been consumed by aerobic and facultative
bacteria to oxidize CBOD and ammonia
• Significant bacterial floc has been formed, trapping suspended and colloidal
solids, including particulate organic nitrogen
Denitrification, a reduction process
Here’s where the process gets a little tricky. After the aeration tank, the MLSS normally
enters a secondary clarifier for settling and separation of the solids from the treated water.
If there is a carbon source available for bacteria to consume, little or no dissolved oxygen
and nitrate is present, denitrification can take place.
In the settled activated sludge that becomes the sludge blanket in the clarifier, the free
available dissolved oxygen is rapidly used up by aerobic and facultative bacteria. Once
free DO is gone, nitrification basically stops. If there is sufficient carbon present in the
form of CBOD or carbon from within the bacteria itself, facultative organisms will continue
to thrive. Since there is little to no free DO present, they use a different internal enzyme to
allow the use of a combined oxygen source instead of free oxygen. The most readily available
and easily reduced oxygen is found within the nitrate compound, NO3-.
Facultative bacteria are able to consume carbon in a oxygen depleted environment, using
nitrate as an alternative oxygen supply. A bacterium reduces the nitrate molecule by taking one
oxygen atom off the nitrate (NO3-), reducing it to nitrite (NO2-). Another bacterium takes one
more oxygen atom from the nitrite, reducing it to nitrous oxide (N2O). Finally, another
bacterium takes the last oxygen reducing it to nitrogen gas (N2).
Where does the nitrogen gas go? Some of it dissolves into the liquid solution, however once
the water is saturated with nitrogen gas, no more nitrogen can be dissolved in the water.
Microscopic bubbles of nitrogen form, becoming larger bubbles as they bump into each other.
Some of the bubbles rise directly to the tank surface, some of the bubbles remain trapped in the
MLSS floc they formed inside of. Once enough of these bubbles accumulate inside the floc
particle, the bubbles make the floc particle positively buoyant causing the particle to rise to the
surface. Some of the floc may be neutrally buoyant, where it remains suspended mid-water,
neither rising nor settling. If enough of these nitrogen bubbles accumulate inside a dense floc
particle, or inside a clump of sludge, the whole clump of sludge may rise to the surface. This
is commonly referred to as sludge ‘pop-ups’ or clumping.
It has been noted that nitrification and denitrification can take place simultaneously within an
aeration tank (Hope, GRU, 1999). Since the outer layer of bacteria making up a floc particle
are exposed directly to oxygen from aeration, this layer stays mostly aerobic. Oxygen may not
penetrate deep within the floc particle, causing this area to become oxygen deficient or anoxic.

Comparison between Nitrification and Denitrification

BASIS FOR
NITRIFICATION DENITRIFICATION
COMPARISON

Meaning The part of nitrogen cycle Denitrification is the level

where ammonium where reduction of nitrate
(NH4+) is converted into (NO3-) is made into
nitrate (NO3-) is called nitrogen gas (N2).
nitrification.

The process Nitrifying bacteria like Denitrifying bacteria like

involves Nitrobacter, Spirillum, Lactobacillus,
Nitrosomonas. Pseudomonas, Thiobacillus.

Grows slowly. Grows rapidly.

Requires aerobic Requires anaerobic

condition. condition.

The microbes Autotrophic. Heterotrophic.

Precursor Ammonium . Nitrate.

End product Nitrate. Nitrogen.

pH and The process occurs at the The process occurs at the

Temperature pH between 6.5 to 8.5 pH between 7.0 to 8.5 and
and temperature between temperature between 26 to
16 to 35 degree C. 38 degree C.

Importance Provides nitrate to the Denitrification is used in

plant, which acts as the wastewater treatment and
important nitrogen is beneficial for aquatic
source. habitats.