Lesson 21:




In this lesson we will answer the following questions:

  • How nitrogen is broken down.
  • What is nitrates and nitrites?
  • How does the treatment plant deal with nitrates?



Reading Assignment

Along with the online lecture, read chapters 18 and 19 in Simplified Procedures for Water Examination.




Nutrients, such as nitrogen and phosphorus, are essential for plant and animal growth and nourishment, but the overabundance of certain nutrients in water can cause several adverse health and ecological effects. Nitrogen exists in the atmosphere at nitrogen gas (N2). In nitrogen fixation, bacteria convert N2 into ammonia, a form of nitrogen usable by plants. When animals eat the plants, they acquire usable nitrogen compounds.

Nitrogen, in the forms of nitrate, nitrite, or ammonium, is a nutrient needed for plant growth. About 78% of the air that we breathe is composed of nitrogen gas, and in some areas of the United States, certain forms of nitrogen are commonly deposited back to earth as acid rain.

Nitrogen is used in agriculture to grow crops, and on many farms the landscape has been modified to maximize output. Fields have been leveled and modified to efficiently drain off excess water that may fall as precipitation or from irrigation practices. This makes the nitrogen fertilizers flow into the nearby waterways, polluting the streams. When these fertilizers are carried as runnoff to streams, they can result in algal blooms, called eutrophication.

Although nitrogen is abundant naturally in the environment, it is also introduced through sewage and fertilizers. Chemical fertilizers or animal manure is commonly applied to crops to add nutrients. Nitrate can get into water directly as the result of runoff of fertilizers containing nitrate. Some nitrate enters water from the atmosphere, which carries nitrogen-containing compounds derived from cars and other sources. More than 3 million tons of nitrogen are deposited in the U.S. each year from the atmosphere, derived either naturally from chemical reactions or from the combustion of fossil fuels, such as coal and gasoline. Nitrate can also be formed in water bodies through the oxidation of other forms of nitrogen, including nitrite, ammonia, and organic nitrogen compounds such as amino acids. Ammonia and organic nitrogen can enter water through sewage effluent and runoff from land where manure has been applied or stored.



Nitrogen Cycle

Nitrogen enters the living world by way of bacteria and other single-celled prokaryotes (do not have a nucleus), which convert atmospheric nitrogen (N2) into biologically usable forms in a process called nitrogen fixation. Some species of nitrogen-fixing bacteria are free-living in soil or water, while others are beneficial symbionts that live inside of plants.

Nitrogen-fixing microorganisms capture atmospheric nitrogen by converting it to ammonia (NH3), which can be take up by plants and used to make organic molecules. The nitrogen-containing molecules are passed to animals when the plants are eaten. They may be incorporated into the animal's body or broken down and excreted as waste, such as the urea found in urine.


Nitrogen doesn't remain in the bodies of living organisms forever. Instead, it's converted from organic nitrogen back into N2 gas by bacteria. This process often involves several steps in terrestrial (land) ecosystems. Nitrogenous compounds from dead organisms or wastes are converted into ammonia by bacteria. The ammonia is then converted into nitrites and nitrates. In the end, the nitrates are made into N2 gas by denitrifying prokaryotes.

Nitrogen-containing compounds act as nutrients in streams in rivers. Nitrate reactions (NO3-) in fresh water can cause oxygen depletion. Thus, aquatic organisms depending on the supply of oxygen in the stream will die. The major routes of entry of nitrogen into bodies of water are municipal and industrial wastewater, septic tanks, and animal wastes. Bacteria in water quickly convert nitrites (NO2-) to nitrates (NO3-).



Nitrogen as a Limiting Nutrient

In natural ecosystems, many processes, such as primary production and decomposition, are limited by the available supply of nitrogen. In other words, nitrogen is often the limiting nutrient, the nutrient that's in shortest supply and thus limits the growth of organisms or populations. A nutrient is limiting if, by adding more of it, growth increases. For example, it will cause plants to grow taller than if nothing were added. If a non-limiting nutrient was added instead, it won't have an effect on the plan'ts growth.

Let's look at an example: If we added nitrogen to half the bean plants in a garden and found that they grew taller than untreated palnts, that would suggest nitrogen was limiting. If, instead, we didn't see a difference in growth in our experiment, that would suggest that some other nutrient besides nitrogen must be limiting.

Nitrogen and phosphorus are the two most common limiting nutrients in both natural ecosystems and agriculture. That's why if you look at a bag of fertilizer, you will see it contains a lot of nitrogen and phosphorus.



Human Impacts on Nitrogen Cycling

We humans may not be able to fix nitrogen biologically, but we certainly can industrially. About 450 million metric tons of fixed nitrogen are made each year, most of which goes to making fertilizers we use on our lawns, gardens, and agricultural fields. Human activity releases nitrogen into the environment by two means: combustion of fossil fuels and use of nitrogen-containing fertilizers in agriculture. Both processes increase levels of nitrogen-containing compounds in the atmosphere. High levels of atmospheric nitrogen, other than N2, are associated with harmful effects, like the production of acid rain (as nitric acid (HNO3)) and contributions to the greenhouse effect (as nitrous oxide (N2O)).

Also, when artificial fertilizers containing nitrogen and phosphorus are used in agriculture, the excess fertilizer may be washed into lakes, streams, and rivers by surface runoff. A major effect from fertilizer runoff is eutrophication. In this process, nutrient runoff causes overgrowth, or a "bloom" of algae or other microorganisms. Without the nutrient runof, they were limited in their growth by availability of nitrogen or phosphorus.

Eutrophication at a wastewater outlet


Eutrophication can reduce oxygen availability in the water during the nighttime because the algae and microbes that feed on them use up large quantities of oxygen in cellular respiration. This can cause the death of other organisms living in the affected ecosystems, such as fish and shrimp, and result in low-oxygen, species-depleted areas called dead zones.





Nitrates and Nitrites

Nitrates (NO3) in the water are from fertilizer runoff, leaky cesspools, sewage treatment plants, manure runoff, and car exhausts. In nature, they are generally formed by the action of bacteria on ammonia and on compounds which contain nitrogen.

Nitrites (NO2) are a relatively short-lived form of nitrogen that quickly becomes converted to nitrates by bacteria. Nitrites produce a serious illness in fish, known as "brown blood disease", even though they don't exist for very long in the environment.



Nitrites also react directly with hemoglobin in human blood and other warm-blooded animals to produce methemoglobin. Methemoglobin destroys the ability of red blood cells to transport oxygen. This condition is especially serious in babies under three months of age. It causes a condition known as methemoglobinemia or "blue baby syndrome". Water with nitrite levels exceeding 1.0 mg/L should not be used for feeding babies.


Nitrates have the same effect on aquatic plant growth as phosphates and thus the same negative effect on water quality. The plants and algae are stimulated, which provide food for fish. This may cause an increase in the fish population. But, if algae grow too wildly, oxgyen levels in the water will be reduced and fish will die.

Because nitrates can exist for short times in the altered form of nitrites, and because nitrites can cause serious illness to both wildlife and humans, acceptable nitrate levels for drinking water have been established as 10 mg/L (ppm). Unpolluted water generally has a nitrate reading of less than 0.1 mg/L.

Water naturally contains less than 1 milligram of nitrate-nitrogen per liter and is not a major source of exposure. Higher levels indicate that the water has been contaminated. Common sources of nitrate contamination include fertilizers, animals wastes, septic tanks, municipal sewage treatment systems, and decaying plant debris.

The ability of nitrate to enter well water depends on the type of soil and bedrock present, and on the depth and construction of the well. State and federal laws set the maximum allowable level of nitrate-nitrogen in public drinking water at 10 mg/L (10 parts per million).



Nitrification vs Denitrification

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 gets oxidized into nitrate (NO3). This action is performed by the Nitrobacter sp.

The bacteria are autotrophic (can produce its own food), and the reaction is performed under aerobic condition (requires oxygen). 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 and 8.5 and a temperature variance from 16 to 35°C.

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, and Pseudomonas.


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


Comparison Chart

Basis for Comparison Nitrification Denitrification
Meaning The part of nitrogen cycle where ammonium is converted into nitrate. The level where reduction of nitrate is made into nitrogen gas.
The process involves Nitrifying bacteria like Nitrobacter, Nitrosomonas

Grows slowly

Requires aerobic conditions
Denitrifying bacteria like Spirillum, Lactobacillus, Pseudomonas, Thiobacillus

Grows rapidly

Requires anaerobic conditions
The microbes Autotrophic Heterotrophic
Precursor Ammonium Nitrate
End product Nitrate Nitrogen
pH and Temperature pH 6.5 - 8.5

Temperature: 16 - 35°C
pH 7.0 - 8.5

Temperature: 26 - 38°C
Importance Provides nitrate to the plant, which acts as teh important nitrogen source Is used in wastewater treatment and is beneficial for aquatic habitats.




In the Treatment Plant

So, why should we remove nitrogen from wastewater in the treatment plant? In its various forms, nitrogen can deplete dissolved oxygen in receiving waters, stimulate aquatic plant growth, like algae, 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. As part of the Safe Drinking Water Act (SDWA), EPA has set the maximum contaminant level (MCL) for nitrate in drinking water at 10 mg/L. Typical effluent permit limits for nitrogen compounds in wastewater effluent vary, but most all are based on the location of final effluent discharge.

Depending on environmental variables such as temperature and pH, nitrogen enters a treatment plant in various forms. Starting at the toilets in our homes to the main sewer line out in the street, nitrogen is mostly in the form of organic nitrogen (urea, amino acids, fecal material). Through a process called hydrolysis, the particulate organic nitrogen begins to convert to ammonia or ammonium. Hydrolysis is the converion of particulate organic material into forms that are small enough to be taken up and consumed by bacteria. The amount of ammonia or ammonium formed depends on the liquid pH and temperature. Ammonification is also accomplished by bacterial decomposition.

Typically, by the time the sewage enters the wastewater treatment plant, much of the organic nitrogen has been converted to ammonium, roughly a 60/40 split. As an operator, you would do very little to control this, since ammonification is a natural process that occurs through the collection system all the way to the head works of our treatment plants.

Ammonia stripping towers are used by some industries and systems to remove nitrogen from the waste stream by intentionally raising the pH to 11, or higher, with calcium oxide (lime), pumping the wastewater/lime solution into a tower packed with plastic or wood media and allowing the ammonia gas to be released from the solution as the treated wastewater splashes over the media and flows downward through the tower. This type of physical/chemical method of nitrogen removal is not common in domestic wastewater treatment plants, but may be found at industrial waste treatment plants.




The biological conversion of ammonium to nitrate nitrogen is called nitrification (aerobic digestion), which is a two-step process. Bacteria, known as Nitrosomonas convert ammonia to ammonium (NH4) to nitrite (NO2). Next, bacteria called Nitrobacter finish the conversion of nitrite (NO2) to nitrate (NO3).

These bacteria are known as "nitrifiers" and are strict "aerobes", meaning they must have free dissolved oxygen to perform their work. Nitrification occurs only under aerobic conditions at dissolved oxygen (DO) levels of 1.0 mg/L or more. At DO concentrations less than 0.5 mg/L, the growth rate is minimal. The optimal pH for these bacteria is between 7.5 and 8.5; most treatment plants are able to effectively nitrify with a pH of 6.5 to 7.0. Nitrification stops at a pH below 6.0. It has been determined that the nitrification reaction (the conversion of ammonia to nitrate) consumes 7.1 mg/L of alkalinity as calcium carbonate (CaCO3) for each mg/L of ammonia nitrogen oxidized. An alkalinty of no less than 50-100 mg/L is required to ensure adequtae buffering.

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, which will affect how the treatment plant operates, depending on the unit used.

For nitrification to proceed, the oxygen should be well distributed throughout the aeration tank and its DO level should not be below 1.0 mg/L. Similar to us, 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.

Sludge Age and Mixed Liquor amounts are also integral components in the nitrification process. When performing sludge age calculations to determine the detention time required for nitrification, the capacity of the aerated portion (oxic) of the plant should be used. There should be enough time in the aeration tanks to complete nitrification. Typically a retention time higher than 10 days will allow for nitrification. There also needs to be enough alkalinity to allow nitrifiers to complete their work. Influent alkalinity should be 200-250 mg/L, and aeration tanke effluent should have at least 50 mg/L as CaCO3 remaining. Ammonia present as a dissolved gas can inhibit nitrifying bacteria if present in high levels. Ammonia concentratiosn of 60 mg/L and higher can harm these sensitive bacteria.





The biological reduction of nitrate (NO3) to nitrogen gas (N2) by facultative heterotrophic bacteria, is called denitrification (anaerobic digestion). Heterotrophic bacteria need a carbon source as food to live and obtain their carbon and energy from biodegrading influent wastewater containing carbon. We measure the influent carbon by sampling for and running a carbonaceous biochemical oxygen demand (CBOD) test.Facultative bacteria can get their oxygen by taking dissolved oxygen out of the water or by taking it off of nitrate molecules.

Denitrification occurs when oxygen levels are depleted and nitrate becomes the primary oxygen source for microorganisms. The process is performed under anoxic conditions, when the dissolved oxygen concentration is less than 0.5 mg/L, ideally less than 0.2. When bacteria break apart nitrate (NO3) to gain the oxygen (O2), the nitrate is reduced to nitrous oxide ((N2O), and, in turn, nitrogen gas (N2). Since nitrogen gas has low water solubility, it escapes into the atmosphere as gas bubbles. Free nitrogen is the major component of air, thus its release does not cause any environmental concerns. These gas bubbles can become bound in the settled sludge floc in clarifiers and cause the sludge to rise to the surface.

Optimum pH values for denitrification are between 7.0 and 8.5. Denitrificaiton is an alkalinity producing process. Approximately 3.0 to 3.6 pounds of alkalinity (as CaCO3) is produced per pound of nitrate, thus partially mitigating the lowering of pH caused by nitrification in the mixed liquor. Since denitrifying bacteria are facultative organisms, they can use either dissolved oxygen or nitrate as an oxygen source for metabolism and oxidation of organic matter. If dissolved oxygen and nitrate are present, these bacteria perfer to use the dissolved oxygen. This will occur since DO 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.

Another important aspect of denitrification is the requirement for carbon, or the presence of sufficient organic matter to drive the reaction. Organic matter may be in the form of raw wastewater, or supplemental carbon. 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, it should be fed consistently and at a rate to keep denitrification levels maximized.

Temperature affects the growth rate of denitrifying organisms, with greater growth rate at higher temperatures. Denitrification can occur between 5 and 30°C, 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. Wastewater cannot be denitrified unless it is first nitrified.



Total Kjeldahl Nitrogen (TKN)

Total Kjeldahl Nitrogen (TKN) is the EPA approved parameter used to measure organic nitrogen and ammonia. Nitrogen follows a cycle of oxidation and reduction. Most of the nitrogen in untreated wastewater will be in the forms of organic nitrogen and ammonia nitrogen. Lab tests are used to determine both of these forms. The sume of these two forms of nitrogen is also measured and is known as TKN. Wastewater will normally contain between 20 to 85 mg/L of nitrogen. Organic nitrogen will normally be in the range of 8 to 35 mg/L, and ammonia nitrogen will be in the range of 12 to 50 mg/L. Organic nitrogen compounds in wastewater undergo microbial conversions through processes called nitrification and denitrication. Wastewater treatment plants typically measure both the TKN and the ammonia concentrations at various points in their wastewater system.


When analyzing the amount of nitrogen in a system, the following relationships are given to help calculate how well the system is working:

Total Nitrogen = TKN + (Nitrate + Nitrite)
Total Organic Nitrogen = TKN - Total Ammonia
Total Inorganic Nitrogen = (Nitrate + Nitrite) + Total Ammonia
TKN = Total Organic Nitrogen + Total Ammonia




Treatment Processes

There are several wasy to encourage bacteria to perform the work of nitrogen removal. Some processes may be desined specifically for nitrification/denitrification using separate aeration and anoxic tanks (selectors) and others may use timers controlling aeration blowers by turning them on and off.

Watch the following video about removing nitrogen from water: https://www.youtube.com/watch?v=BosHU4ARR9w





Nitrate and nitrite are naturally occurring inorganic ions that are part of the nitrogen cycle. Microbial action in soil or water decomposes wastes containing organic nitrogen into ammonia, which is then oxidized to nitrite and nitrate. Water naturally contains less than 1 milligram of nitrate-nitrogen per liter and is not a major source of exposure. Higher levels indicate that the water has been contaminated. Common sources of nitrate contamination include fertilizers, animals wastes, septic tanks, municipal sewage treatment systems, and decaying plant debris. State and federal laws set the maximum allowable level of nitrate-nitrogen in public drinking water at 10 mg/L (10 parts per million). Nitrification is the conversion of ammonia to nitrate . Denitrification is the conversion of nitrate to nitrogen gas . Total Kjeldahl Nitrogen or TKN is defined as total organic nitrogen and ammonia nitrogen. If you can't perform this test, you still need to monitor the nitrogen cycle at the plant.





Complete the questions for Assignment 21, which deals with the Nitrate/Nitrite labs in your lab book.  This assignment grade will automatically submit to the gradebook. If you are unhappy with your grade, close the quiz out and re log back in for your new grade to submit. I will take your highest grade.




Read the Nitrate/Nitrite labs in your lab book and do the assignment listed above.




Answer the questions in the Lesson 21 Quiz. Your score will automatically submit to the gradebook. If you are unhappy with your score; close the quiz completely out, re log back in and take it again. The new score will submit to the gradebook. I take your highest score when calculating final grades.