Lesson 8: Nitrification and Denitrification

31 Aug.,2022


nitrosomonas converts


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

Read the online lecture.





Bacteria remove nitrogen from wastewater by a two tep biological process: nitrificaiton followed by denitrificaiton. (Technically, it is a three step process: ammonification precedes nitrification and denitrification). While traveling through sewer pipes, the majority of the nitrogen contained in raw sewage (urea and fecal material) is converted from organic-nitrogen to ammonia through a process called hydrolysis.

Biological nitrification is the microbe-mediated process of oxidizing ammonia to remove nitrogenous compounds from wastewaters. Domestic sewage typically contains 20 to 40 mg/L of ammonia nitrogen (NH4-N). Organic matter containing nitrogen, e.g., protein and nucleic acid, also biodegrades to release ammonia. Releasing this ammonia into receiving streams has a direct toxic effect on fish and other animals and, in addition, causes significant oxygen depletion.

Nitrate is regarded as an undesirable substance in public water. Although it occurs naturally in water, elevated levels of nitrate in groundwater usually result from human activities, such as over use of chemical fertilizers in agriculture and improper disposal of human and animal wastes. High nitrate concentration in drinking water may cause serious problems in humand and animals. In order to protect against this effort, the US EPA established the maximum contamination level of nitrate in drinking water at 10 mg NO3-.




The net effect is that it takes 4.5 mg of oxygen to fully oxidize one mg of ammonia-N. therefore even small concentrations of ammonia can cause significant deterioration to the flora and fauna in the body of water receiving it. Thus, many domestic and industrial wastewater treatment plants are required to remove the ammonia before discharge of the treated water.



Sources of Ammonia, Nitrate, and Nitrite

Excess nitrogen in the form of ammonia in finished water can be the principal cause of nitrification since ammonia serves as the primary substrate in the nitrificaiton process. Ammonia, nitrate and nitrite can typically be found in surface water supplies as a result of natural processes. These natural sources of nitrogen generally have minimal impacts on water supply distribution systems because the concentration of nitrite nitrogen in surface and ground water is normally far below 0.1 mg/L. Other sources of nitrogen can include agricultural runoff from fertilization or livestock wastes or contamination from sewage. Ammonia also occurs naturally in some groundwater supplies, and groundwater can become contaminated with nitrogen as agriculture runoff percolates into aquifers. To protect public health, any system with source water that exceeds the inorganic contaminant MCLs for nitrate and nitrite of 10 mg/L and 1 mg/L (as Nitrogen) must treat the water to below those levels.



pH and Alkalinity

Bulk water pH value is an important factor in nitrification activity for two reasons. First, a reduction of total alkalinity may accompany nitrification because a significant amount of bicarbonate is consumed in the conversion of ammonia to nitrite. A model that was developed in 1974 indicates that 8.64 mg/L of bicarbonate (HCO3) will be utilized for each mg/L of ammonia-nitrogen oxidized. While reduction in alkalinty does not impose a direct public health impact, reductions in alkalinity can cause reductions in buffering capacity, which can impact pH stability and corrosivity of the water toward lead and copper. Secondly, nitrifying bacteria are very sensitive to pH. Nitrosomonas has an optimal pH between approximately 7.0 and 8.0, and the optimum pH range for Nitrobacter is approximately 7.5 to 8.0. Some utilities have reported that an increase in pH (to greater than 9) can be used to reduce the occurrence of nitrification.



Disinfection Practices

Chemical control or treatment of nitrifying bacteria typically involves either the maintenance of high distribution system disinfectant residuals (greater than 2 mg/L) or periodic breakpoint chlorination. Analytical survey results in the U.S. showed that greater than 90% of distribution system samples with increased nitrite and nitrate levels, indicative of nitrification, occurred in water with disinfectant residuals less than 2 mg/L. Many utilities have found that increasing disinfectant residuals by increasing chemical doses or managing water age has helped to control nitrification.

Free chlorine is more effective at inactivating ammonia-oxidizing bacteria colonies than chloramines. As a result, breakpoint chlorination is also used by utilities to treat nitrifying bacteria. Some systems using breakpoint chlorination have reported an initial increase in HPC bacteria and total coliform levels immediately following treatment that is probably attribute to biofilm sloughing.



Absence of Sunlight

Although monochloramine will degrade when exposed to the atmosphere at varying rates depending on the amount of sunlight, wind, and temperature, nitrifiers are very sensitive to near UV, visual, and fluorescent light; consequently, nitrification episodes occur in the dark (in covered reservoirs, pipelines, taps, etc.).



Operations Activities

Nitrifying bacteria are slow growing organisms, and nitrification problems usually occur in large reservoirs or low-flow sections in the system. Operational practices that ensure short residence time and circulation within the system can minimize nitrification problems. Low circulation areas of the system , such as dead-ends and reservoirs are prime areas for nitrification occurrence since detention time and sediment buildup can be much greater than in other parts of the system.

Water temperature has a strong effect on the growth rate of nitrifying bacteria. Numerous instances have been documented that nitrification episodes are more common during the warmer months. Most strains of nitrifiers grow optimally at temperatures between 25 and 30°C, but nitrification has occurred over a wide range of temperatures. Operations activities that lead to decreased water age may also result in decreased bulk water temperatures.




The Microbial Populations

As shown in the nitrification process, ammonia is first oxidized to nitrite ions, then the nitrite ions are oxidized to nitrate ions. Each oxidation is carried out by a different group of bacteria, the ammonia oxidizing bacteria (AOB) and the nitrite oxidizing bacteria (NOB). Each group of bacteria has multiple species and a wastewater treatment process may contain several species of each group. Most textbook descriptions of the nitrification process refer to Nitrosomonas species as the AOB and Nitrobacter species as the NOB and this simplification can serve the process operator and troubleshooter well as the two groups have well characterized growth conditions. Nitrifying bacteria are autotrophs, they use inorganic sources of carbon (such as carbon dioxide and carbonate ion) to produce biomass in contrast to the great majority of the other microbes in the system (heterotrophs) which typically use a variety of organic substances both as an energy and carbon source. The autotrophs grow and reproduce much more slowly than the heterotrophs, e.g. Nitrosomonas may reproduce (divide) once in eight hours compared to a fast-growing heterotroph that may divide every 20 minutes. In addition, the autotrophs are more sensitive to the growth conditions such as pH, temperature and the presence of toxic compounds.

To maintain nitrifying microbes in a process, the sludge age must be kept high enough to retain a sufficient population of these organisms. Under toxic and/or cold weather conditions, the growth rate of natural nitrifying populations tends to slow appreciably, causing nitrifiers to wash out of the system. Thus, it can be a problem to maintain ammonia removal if such conditions persist.

Proper nutrition for nitrifying bacteria also requires other elements such as calcium, copper and iron to name a few. Maintaining optimal conditions is not always practical since it can be cost prohibitive. Instead several measures can be used to help maintain the nitrifying population in the face of deteriorating conditions. These include:

  • Maintaining a higher than normally desired biomass concentration in the biotreater aeration zone, and then building up an even higher sludge concentration in the biotreater to help hold the slow growing autotrophs in the system under aticipated adverse conditions (like colder weather).
  • Using bioaugmentation with separately grown, concentrated microbial inocula to augment the natural seeding and growth of the autotrophs in the system.
  • Keeping tight control over the pH of the system. Lower pHs (acidic conditions) are particularly adverse.
  • Maintaining excess dissolved oxygen in the aeration zone at all times. The autotrophs compete with heterotrophs for dissolved oxygen and the heterotrophs are more efficient at scavenging oxygen at low concentrations. A dissolved oxygen concentration of 2 mg/L should be maintained.
  • Keeping high concentrations of substances known to be toxic to the autotrophs such as excessively high ammonia concentrations or toxic heavy metal ions such as copper and chromium out of the wastewater entering the system. This can be helpful in industrial settings such as petroleum refineries where relatively high concentrations of ammonia are present in the untreated wastewater and where separation of side streams is more feasible.
  • Enforcing industrial pretreatment standards for domestic sewer discharges of substances known to negatively affect the slower growing nitrifiers.


Bacteria remove nitrogen from wastewater by a two step biological processes: nitrification followed by denitrification. Technically, it is a three step process: ammonification precedes nitrification and denitrification. While traveling through sewer pipes, the majority of the nitrogen contained in raw sewage is converted from organic-nitrogen to ammonia through a process called hydrolysis. In the majority of situations, more ammonium than ammonia is created during ammonification. The actual ratio is influenced by pH amd temperature.





The biological conversion of ammonium to nitrate nitrogen is called nitrification. Nitrification is a two-step process. Bacteria known as Nitrosomonas convert ammonia and ammonium to nitrite. Next, bacteria called Nitrobacter finish the conversion of nitrite to nitrate. Biological nitrification is the process in which Nitrosomonas bacteria oxidize ammonia to nitrite and Nitrobacter bacteria oxidize nitrite to nitrate. This process results in the overall conversion of ammonia to nitrate. These microorganisms are autotrophic, which means they derive their carbon source from inorganic carbon, such as carbon dioxide and bicarbonate. Most other types of organisms in activated sludge are heterotrophic, which means they derive their carbon source from the organic matter in the wastewater. Environmental conditions of pH, alkalinity, temperature, dissolved oxygen concentration and organic loading affect the nitrification process in activated sludge plants.

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", meaning they must have free dissolved oxygen to perform their work. Nitrification occurs only under aerobic conditions at dissolved oxygen levels of 1.0 mg/L or more. At dissolved oxygen (DO) concentrations less than 0.5 mg/L, the growth rate is minimal. Nitrification requires a long retention time, a low food to microorganism ratio (F/M), a high mean cell residence time (measured as MCRT or Sludge Age), and adequate buffering (alkalinity). A plug-flow, extended aeration tank is ideal. Temperature is also important.

Nitrification is temperature sensitive. The optimum temperature for nitrification is generally considered to be 30°C.

Temperature Effect upon Nitrification >45°C Nitrification ceases 28-32°C Optimal temperature range 16°C Approximately 50% of nitrification rate at 30°C 10-30°C Significant reduction in nitrification rate - 20% of rate <5°C Nitrification ceases


What is the key factor for achieving nitrification? Mean Cell Residence Time (MCRT). As temperature increases, nitrifier growth rate increases. As nitrifier growth rate increases, required MCRT decreases. For every 10°C increase in temperature, nitrifier growth rate doubles, required MCRT is cut in half and required MLSS concentration is also reduced. As dissolved oxygen increases, nitrifier growth rate increases up to DO levels of about 5 mg/L. Maintain dissolved oxygen concentration at 2.0 mg/L or higher for optimum nitrification. Nitrification consumes alkalinity and lowers pH in the activated sludge mixed liquor. pH below 6.5 or above 8.0 can significantly inhibit nitrification.


The nitrification process produces acid. This acid formation lowers the pH of the biological population in the aeration tank and can cause a reduction of the growth rate of nitrifying bacteria. The optimum pH for Nitrosomonas and Nitrobacter 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. The nitrificaiton reaction (that is, the conversion of ammonia to nitrate) consumes 7.1 mg/L of alkalinity as CaCO3 for each mg/L of ammonia nitrogen oxidized. An alkalinity of no less than 50-100 mg/L is required to insure adequate buffering.

Water temperature also affects the rate of nitrification. Nitrification reaches a maximum rate at temperaures between 30 and 35°C (86 and 95°F). At temperatures of 40°C (104°F) and higher, nitrification rates fall to near zero. At temperatures below 20°C, nitrification proceeds at a slower rate, but will continue at temperatures of 10°C and less. However, if nitrification is lost, it will not resume until the temperature increases to well over 10°C.

Some of the most toxic compounds to nitrifiers include cyanide, thiourea, phenol and heavy metals such as silver, mercury, nickel, chromium, copper and zinc. Nitrifying bacteria can also be inhibited by nitrous acid and free ammonia.

To review, the nitrogen cycle starts with nitrogen (N2) in the air. Algae use this nitrogen to make organic nitrogen. The organic nitrogen in the algae moves through the food chain and ends up in our plant influent. Now that it's in the plant, what do we do with it? The first step at the treatment plant is to get the ammonium ion away from the organic. This is done in two different ways. The first way is simply time. With a little time, sewage becomes stagnant and the ammonium group just falls off. The second way is that the microbes that like to eat carbon chains will do so and leave the ammonium group behind. Now that the ammonium is detached from the carbon chain, it is called ammonia.

Ammonia is an interesting thing. It actually has two different forms. Most people think of ammonia in the form NH3. This form is actually ammonia gas. The form of ammonia that separated from the organic is NH4, the ammonium ion. These two can change back and forth depending on the pH in the water. If the pH is high, more NH3 is present. If the pH is neutral or low, most of the ammonia is in the form of ammonium ion. Now that we have ammonia we move on to the next step. In the aeration tank and the trickling filter there are all kinds of different little microbes to eat the carbon-containing foods. But there is only one particular little microbe that eats the ammonia. It's name is Nitrosomonas. It eats ammonium ions for its energy food source. In the natural biological process, Nitrosomas eats ammonium ions and produces nitrite or NO2 as a product. Once they Nitrosomas produces nitrite the Nitrobacter comes along and quickly makes it into nitrate or NO3. The Nitrobacter is much faster at doing its work., changing nitrite to nitrate as fast as it gets it. This two step process in which ammonia is changed to nitrite and then nitrate is called nitrification.

Why do we need to do nitrification in the wastewater treatment plant? For one thing, ammonia left in the treated effluent can be toxic to fish. Also, the nitrifiers in the receiving waters will be working to convert that ammonia to nitrate. That conversion will use up oxygen. By nitrifying the plant effluent, the oxygen demand on the receiving waters will be reduced.

Suspended growth reactors are wastewater treatment processes in which the microorganisms and bacteria treating the wastes are suspended in the wastewater being treated, such as the various modes of the activated sludge treatment process. Suspended growth reactors are commonly used for the biological conversion of ammonia to nitrate (i.e. biological nitrification). The biological nitrification process can be classified as either single-stage or separate-stage. In the single-stage process, nitrification and carbonaceous oxidation (BOD removal) occur within the same basin, and the BOD5/TKN (Total Kjeldahl Nitrogen) ratio of the primary effluent is typically greater than 5.

TKN is the sum of organic nitrogen and ammonia nitrogen. The concentration of TKN in typical domestic wastewater ranges from 20 to 85 mg/L as Nitrogen. In the separate-stage process, carbonaceous oxidation and nitrification occur in separate tanks, and the BOD5/TKN ratio is typically between 1 and 3.

Single-stage and Separate-stage Biochemical Nitrification Processes



Suspended Growth Reactors

Wastewater treatment processes in which the microorganisms and bacteria treating the wastes are suspended in the wastewater being treated. The wastes flow around and through the suspended growths. The various modes of the activated sludge process make use of suspended growth reactors. These reactors can be used for BOD (biochemical oxygen demand) removal, nitrification, and denitrification. There are a variety of operational configuration for suspended growth reactors, but not all of them are suitable for biological nitrification.


Extended Aeration

Extended aeration plants will perform the best in regards to achieving nitrification because of their long aeration and mean cell residence time (MCRTs). The extended aeration process is one modification of the activated sludge process which provides biological treatment for the removal of biodegradable organic wastes under aerobic conditions. Air may be supplied by mechanical or diffused aeration to provide the oxygen required to sustain the aerobic biological process. Mixing must be provided by aeration or mechanical means to maintain the microbial organisms in contact with the dissolved organics. In addition, the pH must be controlled to optimize the biological process and essential nutrients must be present to facilitate biological growth and the continuation of biological degradation. The image below depicts the process flow for a typical extended aeration plant.


Extended aeration systems are typically manufactured to treat wastewater flow rates between 0.002 to 0.1 MGD. Extended aeration plants are usually started up using "seed sludge" from another sewage plant. It may take as many as two to four weeks form the time it is seeded for the plant to stabilize. Extended aeration package plants are typically used in small municipalities, suburban subdivisions, apartment complexes, highway rest areas, trailer parks, small institutions, and other sites where flow rates are below 0.1 MGD. These systems are also useful for areas requiring nitrification.



Sequencing Batch Reactors (SBR)

A sequencing batch reactor (SBR) is a variation of the activated sludge process. As a fill and draw or batch process, all biological treatment phases occur in a single tank. This differs from the conventional flow through activated sludge process in that SBRs do not require separate tanks for aeration and sedimentation. SBR systems contain either two or more reactor tanks that are operated in parallel, or one equalization tank and one reactor tank. Package SBRs are typically manufactured to treat wastewater flow rates between 0.01 and 0.2 MGD. There are normally five phases in the SBR treatment cycle: fill, react, settle, decant, and idle. The length of time that each phase occurs is controlled by a programmable logic controller (PLC), which allows the system to be controlled from remote locations.

In the fill phase, raw wastewater enters the basin, where it is mixed with settled biomass from the previous cycle. Some aeration may occur during this phase. Then, in the react phase, the basin is aerated, allowing oxidation and nitrification to occur. During the settling phase, aeration and mixing are suspended and the solids are allowed to settle. The treated wastewater is then discharged from the basin in the decant phase. In the final phase, the basin is idle as it waits for the start of the next cycle. During this time, part of the solids are removed from the basin and disposed of as waste sludge. An SBR system does not require an RAS system, as both aeration and settling occur in the same tank. This prevents any sludge from being lost during the react step and eliminates the need to return sludge from the clarifier to the aeration chamber. Below is a diagram showing the process flow for a sequencing batc reactor:


Package plant SBRs are suitable for areas with little land, stringent treatment requirement, and small wastewater flows. More specifically, SBRs are appropriate for RV parks or mobile homes, campgrounds, construction sites, rural schools, hotels and other small applications. SBRs are also suited for sites that need minimal operator attendance and that have a wide range of inflow and/or organic loadings. Industries with high BOD loadings will find SBRs useful for treating wastewater. These systems are also suitable for facilities requiring nitrification, denitrification, and phosphorous removal.



Oxidation Ditches

An oxidation ditch, a modified form of the activated sludge process, is an aerated, long term, complete mix process. Many systems are designed to operate as extended aeration systems. Typical oxidation ditch treatment systems consist of a single or multi-channel configuration within a ring, oval, or horseshoe-shaped basin. Horizontally or vertically mounted aerators provide aeration, circulation, and oxygen transfer in the ditch. Package oxidation ditches are typically manufactured in sizes that treat wastewater flow rates between 0.01 and 0.5 MGD. Below is a flow diagram for an oxidation ditch.


Depending on the system size and manufacturer type, a grit chamber may be required. Once inside the ditch, the wastewater is aerated with mechanical surface or submersible aerators that propel the mixed liquor around the channel at velocities high enough to prevent solids deposition. The aerator ensures that there is sufficient oxygen in the fluid for the microbes and adequate mixing to ensure constant contact between the organisms and the food supply. Oxidation ditches tend to operate in an extended aeration mode consisting of long hydraulic and solids retention times which allow more organic matter to break down.

Oxidation ditches are suitable for facilities that require nutrient removal, have limitations due to the nature of the site, or want a biological system that saves energy with limited use of chemicals unless required for further treatment. Oxidation ditch technology can be used to treat any type of wastewater that is responsive to aerobic degradation. This technology is particulary useful for schools, small industries, housing developments, and small communities. Ultimately, this technology is most applicable for places that have a large amount of land available.



Total Kjeldahl Nitrogen (TKN)

The nitrogen cycle is the means by which atmospheric nitrogen is made available in various forms to living organisms. From the basic molecules of ammonia, nitrate, and nitrite to the more complex amino acids and proteins, nitrogen is essential for living organisms to function. It is also an important part in the smooth operation of many wastewater treatment plants. In order for the cycle to operate smoothly it is vital to know the amount of nitrogen contained in the various phases of the cycle. Total Kjeldahl Nitrogen (TKN) analysis provides the opportunity to quantify the amount of nitrogen contained in organic form. Because there are a large variety of organic compounds which contain nitrogen, a single test cannot be formulated that will cause each one to respond in an equal manner. The digestion serves to overcome that issue by converting all target nitrogen forms to a single compound, namely ammonia. This allows for all the nitrogen to be analyzed as one species. Unfortunately, qualitative analysis is not possible. The results must be expressed simply as TKN. TKN digestion will only give you results of the total organic nitrogen plus ammonia. TKN is usually requested to gain knowledge as to the total nitrogen content of the sample. Thus the other nitrogen anayses are often run as well. The relationships are given below:

Total Nitrogen = TKN + (Nitrate + Nitrite)

Total Organic Nitrogen = TKN - Total Ammonia)

Total Inorganic Nitrogen = (Nitrate + Nitrite) + Total Ammonia

TKN = Total Organic Nitrogen + Total Ammonia




Aerobic Versus Anoxic

When you think of a tank that has aerobic conditions, you know that there is plenty of oxygen in the tank. Likewise, an anaerobic tank is one without oxygen. There is one other thing to consider. If dissolved oxygen in the tank is gone, the tank is not necessarily anaerobic. Both nitrite (NO2) and nitrate (NO3) have "chemically combined oxygen". If aerobic microbes run out of dissolved oxygen they get a little creative and break down these nitrogen chemicals to get the oxygen they need. If a tank has no dissolved oxygen but it DOES have nitrate in it, the tank condition is called anoxic. The aerobic microbes can still function when the tank conditions are anoxic. This is sometimes what happens in the final settling tank of an activated sludge process. In the clarifier, the aerobic microbes use up the dissolved oxygen left from aeration and then start to use the oxygen from the nitrate. As the nitrate gets broken down, the microbes use the oxygen and leave the nitrogen. The nitrogen is in the form of nitrogen gas. At first glance this doesn't seem to be a problem. Nitrogen gas is non-toxic and should just float to the surface of the clarifier and go into the air. The problem is that the microbes inside the floc particles are the ones to run out of DO first, then start using nitrate and making nitrogen gas. When this happens, the nitrogen gas bubbles cause the floc particle to rise to the surface with nitrogen bubbles inside. The process of microbes converting nitrate to nitrogen gas is called denitrification. Denitrification is a good process to have but not in the clarifier.

The process of denitrification is not done by the nitrogen microbes. Instead, it is certain carbon eaters that change the nitrate to nitrogen gas because they need the oxygen. The product of denitrification, the nitrogen gas, is also the end of the nitrogen cycle.



Now that we know how the nitrifiers work by themselves, we need to look at how they work with the rest of the microbe population. As we already know there are small microbes, known as bacteria, and big microbes, known as protozoans. Nitrifiers are part of the small microbe population. These small microbes are separated into two categories. The microbes that eat the organic materials with carbon in them are known as carbonaceous. The second group eat nitrogen cmpounds and are therefore called nitrifiers. It is much easier to get carbon eating microbes to eat than it is to get nitrogen eating microbes to eat.

Carbonaceous microbes are the young adults of the population. They eat a lot and likewise reproduce. The nitrifiers are the "senior citizens" of the small microbe population. They are older, pickier about eating conditions and reproduce much less than the carbonaceous microbes do. In terms of activated sludge, the nitrifiers prefer longer sludge retention times. If the average age of the microbes is kept too short, the carbonaceous microbes will do great, but the nitrifiers won't start to eat. If the average age of the microbes is too old, the nitrifiers will do great, but the carbonaceous microbes start to die. From this we can more clearly see why a two-stage secondary process works best.

Activated sludge and trickling filters both work well as a first stage process to lower CBOD. The extent of nitrification in trickling filters depends on a variety of factors including temperature, DO, pH, presence of inhibitors, filter depth and media type, loading rate, and wastewater BOD. Low-rate trickling filters allowed the development of a high-nitrifying population. If two filters were used, heterotrophic growth occurs in the first filter and nitrification in the second filter.

RBCs are typically staged anyway. In order to get a consistently nitrified effluent, five RBC stages are recommended. RBC biofilm has an initial adsorption of microbes to the disk surface to form 1-4 mm thick biofilm that is responsible for BOD removal in rotating biological contactors. The rotating disks provides a large surface area for the attached biomass. The first stages of an RBC mostly removes organic materials, whereas subsequent stages remove the NH3 form of nitrogen as a result of nitrification, when the BOD is low enough. Ammonia oxidizers can not effectively compete with the faster-growing heterotrophs that oxidize organic matter. Nitrification occurs only when the BOD is reduced, and increases with rotation speed. RBC performance is negatively affected by low dissolved oxygen in the first stages and by low pH in the later stages where nitrification occurs.

Another big difference between the carbonaceous and nitrogenous microbes is where they come from. The carbonaceous microbes come in with the domestic sewage. Nitrifiers are only available from the soil. During the winter months the carbonaceous microbes continue to restock in the plant. If the nitrifiers in the plant get washed out or killed, there is no way to replace them until the ground thaws. This can make winter nitrification difficult. Nitrifiers only grow in the top inch of exposed soil, so a large pile would not be helpful. For this reason, and the fact that the nitrifiers work a lot slower in the colder winter temperatures, effluent ammonia limits are often less stringent in the winter than in the summer.





Now that your plant is nitrifying, what do you need t odo to make it denitrify? Answer: Establish anoxic conditions somewhere in the activated sludge process. Pre-denitrification uses an anoxic zone at the beginning of the activated sludge tanks.



When Nitrosomanas and Nitrobacter convert ammonia to nitrate, there is a chemical reaction that can be used to describe the overall change:

NH4 + 2O2 → NO3 + 2H + H2O


Biological denitrification is the process in which microorganisms reduce nitrate to nitrite and nitrite to nitrogen gas. Heterotrophic bacteria normally present in activated sludge perform this conversion when there is no molecular oxygen or dissolved oxygen, and there is sufficient organic matter. The bacteria derive their oxygen from the oxygen contained in the nitrate. The nitrogen gas produced is in the form of nitric oxide (NO), nitrous oxide (N2O) or nitrogen gas (N2). The net removal of nitrogen is accomplished by stripping the nitrogen gas formed during denitrification out of the wastewater in a subsequent aeration process.

Dissolved oxygen inhibits denitrification. As DO increases, denitrification rate decreases. Maintain DO below 0.3 mg/L in the anoxic zone to achieve denitrification.

From this we see that the process requires oxygen and produces hydrogen ions (H). Adding hydrogen ions to water is the same as adding acid to water. It can make the pH go down. Nitrifiers stop working if the pH gets much below 6.8. The pH of the aeration influent will stop nitrifiers unless some chemical is already in the water to soak up those hydrogen ions. That "chemical" is called alkalinity. Alkalinity actually refers to a number of chemicals that collectively do the job of absorbing acid. One of those chemicals is calcium carbonate (CaCO3). The measurement of alkalinity in mg/L is actually given as the concentration of calcium carbonate that would absorb just as much acid as all those in the water sample collectively do. Alkalinity is critical to absorb or "buffer" the pH during nitrification. For every pound of ammonia converted, there will be well over seven pounds of alkalinity used up. If there isn't more than enough alkalinity in the aeration influent, the pH will drop like a rock and so will the rate of nitrification. If there isn't enough alkalinity in the aeration influent, calcium carbonate or another acid absorbing chemical will need to be added.

Optimum pH values for denitrification are between 7.0 and 8.5. Denitrification is an alkalinity producing process. The biological reduction of nitrate (NO3) to nitrogen gas (N2) by facultative heterotrophic bacteria is called denitrification. Heterotrophic bacteria need a carbon source as food to live. 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 microbes. The process is performed under anoxic conditions, when the dissolved oxygen concentration is less than 0.5 mg/L, ideally leass 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 lower water solubility, it escapes into the atmosphere as gas bubbles. A carbon source is required for denitrification to occur.

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, bacteria will use the dissolved oxygen first. That is, the bacteria will not lower the nitrate concentration. Denitrification occurs only under anaerobic or anoxic conditions. Another important aspect of denitrification is the requirement for carbon; that is, the presence of sufficient organic matter to drive the denitrification reaction. Organic matter may be in the form of raw wastewater, or supplemental carbon. 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. Denitrifying organisms are generally less sensitive to toxic chemicals than nitrifiers, and recover from toxic shock loads quicker than nitrifiers.

Temperature affects the growth rate of denitrifying organisms, with greater rate at higher temperatures. Denitrification can occur between 5 and 30°C (41 to 86°F), 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. Wastewater cannot be denitrified unless it is first nitrified.

How will you now when all the nitrate is used up? The next place the microbes will go for their oxygen source is the sulfate. As the sulfates are used up, sulfides will combine with hydrogen to form hydrogen sulfide and this stinks like rotten eggs. Why would you want to perform denitrification at your facility? The obvious reason would be total nitrogen limits in your discharge permit, others include alkalinity and oxygen recovery, the desire to produce a high stabilized effluent, and a reduction of problems with rising sludge in your clarifier.

Post-denitrication uses an anoxic zone at the end of the activated sludge tanks. Denitrification is slower in a post-denitrification zone than in a pre-denitrification zone.


What could be done to increase the denitrificaiton rate in a post-denitrification zone? Step-feed denitrification uses alternating periods of aerobic and anoxic conditions. Primary effluent is fed at multiple points along the tank to provide a carbon source for denitrification.




Ammonia Stripping

Ammonia stripping is the removal of nitrogen from wastewater when the nitrogen is in gaseous ammonia form. Ammonia is a volatile substance, which means that is has a tendency to leave the wastewater and enter the atmosphere. Ammonia (NH3) and ammonium (NH4) exist in equilibrium with each other based on the pH. Most of the ammonia-nitrogen in municipal wastewater is in the ammonium form because of its neutral pH range (between 6 and 8). Therefore, chemicals such as lime or sodium hydroxide must be added to raise the pH to the 10.5 to 11.5 range. This will effectively "convert" the ammonium in the wastewater to ammonia. The stripping effect is achieved by introducing the high pH wastewater into th etop of a tower packed with fixed media (or "packing"). Air is blown into the bottom of the tower and flows in a countercurrent fashion with the incoming wastewater. The intimate contact between wastewater droplets and fresh air encourages the ammonia to volatilize from the wastewater to the exiting air stream.

Breakpoint chlorination is the chemical oxidation ammonia to nitrogen gas (N2) by the addition of chlorine. The "breakpoint" is the chlorine dosage, which results in an increase in the free chlorine residual with increasing chlorine dosages. Breakpoint chlorination requires relatively high chlorine dosages per unit of ammonia present in the wastewater. In general, about 10 pounds of chlorine are required to oxidize one pound of ammonia-nitrogen. Because of the high chlorine demand, breakpoint chlorination is not used as the primary ammonia (or nitrogen) removal process. In activated sludge, biological methods, such as nitrification/denitrification, are used to remove the bulk of the nitrogen and breakpoint chlorination is used as a final polishing step to remove the residual nitrogen.




In a two-stage secondary process, the first stage is used to reduce the BOD and the second stage is used to reduce the concentration of ammonia. The nitrificaiton process requires a slow-growing nitrifying bacteria with sludge that has been aged for a long time and high dissolved oxygen concentration. In addition, they were susceptible to inhibition by a wide range of compounds at concentrations so low as not to affect the heterotrophic bacteria. For these reasons, it would seem sensible to separate the processes of carbonaceous removal and nitrogen removal into separate reactors. If the second stage unit is activated sludge, for example, it would seem from this that the operator should try to keep as many nitrifiers in the second stage mixed liquor as possible. Nitrifiers are unfortunately poor settling microbes. They don't stick together too well in a floc and, therefore, don't settle well in a clarifier. For this reason we need to have some carbonaceous microbes around to form good floc particles for settling. If there are too many carbon eaters, there might not be enough nitrifiers to reduce the ammonia concentration. As you can see, the balance between the carbonaceous microbes and the nitrifiers is very important.

The best way to balance the microbes is to balance their food. The carbon eater's food can be measured as BOD. The nitrifier's food is measured as TKN. If, for example, there is twice as much BOD in the water as there is TKN, there are going to be about twice as many carbonaceous microbes as there are nitrifiers. If there are too many nitrifiers, the ammonia concentration will be reduced, but the effluent will be cloudy from non-settling microbes. If there are too many carbonaceous microbes, the effluent will be clear but nitrification will slow down or even stop. If the BOD concentration is divided by the TKN concentration, the number should be larger than 2. If the value is less than 2, the effluent will be cloudy. Operating with a value between 2 and 6 in a second-stage nitrification unit should produce a clear effluent with well reduced ammonia concentrations. If the BOD:TKN ratio is below 2, it can be raised by allowing some of the primary effluent to be bypassed around the first stage to raise the second stage influent BOD concentration.

One problem that operators of a separate stage nitrification unit face is that the amount of chlorine needed to disinfect the effluent will go up after nitrification has started. Denitrification will change effluent nitrate into nitrogen gas. We just don't want this going on in the clarifiers.





Nitrification is a microbial process by which reduced nitrogen compounds (primarily ammonia) are sequentially oxidized to nitrite and nitrate. Ammonia is present through either naturally-occurring processes or through ammonia addition during secondary disinfection to form chloramines.

Nitrate (NO3-) and nitrite (NO2-) 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 (NH3+) to nitrate (NO3-). Denitrification is the conversion of nitrate (NO3-) to nitrogen gas (N2). 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.





Discuss, in detail, the three types of suspended growth reactors used for nitrification/denitrification. You must be logged into Canvas to submit this assignment. Make sure you choose the appropriate semester.





Answer the questions in the lesson quiz. You must be logged into Canvas to take this quiz. You may take the quiz up to three times; an average will be taken for final grade calculation. Make sure you choose the appropriate semester.