Benefits of Denitrification

The benefits of denitrification are significant if denitrification is used properly. The benefits include protecting the quality of the receiving water, permit compliance, strengthening of the floc particles, control of undesired filamentous growth, return of alkalinity to the treatment process, and cost-savings for the treatment or degradation of cBOD.

By denitrifying, the quantity of nitrite ions and nitrate ions discharged to the receiving water is greatly reduced. The ions are used as a nitrogen nutrient by many aquatic plants. The reduction in the quantity of these ions discharged to the receiving water helps to prevent the overabundant growth of aquatic plants and its resulting eu-trophication.

Nitrite ions are highly toxic to aquatic life. Denitrification reduces the amount of nitrite ions discharged to the receiving water. The reduction of nitrite ions discharged to the receiving water reduces tox-icity concerns related to aquatic life.

Activated sludge processes that have a total nitrogen discharge limit must denitrify. Successful denitrification ensures permit compliance.

Denitrification promotes the formation of firm and dense floc particles. Firm and dense floc particles are resistant to shearing action and have desired settling characteristics. An anoxic environment produced through denitrification favors the growth of facultative anaerobic, floc-forming bacteria and discourages the growth of strict aerobic, filamentous organisms and weak facultative anaerobic, fila mentous organisms. Strict aerobic, filamentous organisms can only use free molecular oxygen. Therefore their growth is stopped, or the organisms die under an anoxic environment.

Although weak facultative, anaerobic filamentous organisms can use free molecular oxygen and nitrite ions or nitrate ions, these filamentous organisms cannot compete successfully for nitrite ions or nitrate ions with facultative anaerobic, floc-forming bacteria. Therefore the growth of these filamentous organisms is stopped, or the organisms die under an anoxic environment.

Nearly all activated sludge processes that denitrify must nitrify. Nitrification results in a loss of alkalinity. Denitrification returns alkalinity to the activated sludge process. By denitrifying, much of the alkalinity lost during nitrification can be returned to the activated sludge process through denitrification.

By using nitrite ions and nitrate ions produced during nitrification to degrade cBOD, an activated sludge process need not supply dissolved oxygen to the aeration tank. The use of nitrite ions and nitrate ions instead of dissolved oxygen results in cost-savings, that is, decreased electrical cost for operation of the aeration equipment.

The Gram stain uses a simplistic staining technique to differentiate bacteria into two large groups, Gram-negative bacteria and Grampositive bacteria. The basis for this differentiation is due to the physical and chemical composition of the cell walls of bacteria to retain and release stains.

Christain Gram, a Danish physician, who was working on a method to differentiate pathogenic bacteria from mammalian tissue, developed the Gram stain in 1883. All bacteria, except one genus, respond to the Gram stain technique. Following staining, Gramnegative bacteria are pink-red when examined under the microscope, while Gram-positive bacteria are blue-purple when examined under the microscope.

There are several Gram staining techniques. The technique most commonly used in wastewater laboratories for the identification of different bacteria is the Hiicker method. This method employs the use of four reagents. These reagents consist of crystal violet, Gram's iodine, a decolorizing agent, and safranin. These reagents can be purchased from most chemical suppliers to water and wastewater laboratories. The stains are applied to a smear of bacteria on a microscope slide in the following order: crystal violet, Gram's iodine, decolorizing agent, and safranin. The color of Gram-negative and Gram-positive bacteria after the application of each reagent is presented in Table I.1. The Gram stain results of the bacteria are observed after the safranin addition.

Appendix I


TABLE I.1 Bacterial Response to Each Reagent of the Gram Stain Technique


Gram-Negative Bacteria

Gram-Positive Bacteria

Crystal violet



Gram's iodine



Decolorizing agent






Appendix II


The food/microorganism ratio, or F/M, is a measurement of the food entering the activated sludge process and the microorganisms in the aeration tank(s). Each activated sludge process has an F/M at which it operates best. This F/M may fluctuate throughout the year according to changes in operational conditions, such as industrial discharges, permit requirements, and temperature.

The food value or food supply entering the activated sludge process consists of the BOD loading or pounds discharged to the aeration tank(s). The BOD loading is calculated by multiplying the concentration (mg/l) of BOD entering the aeration tank by the influent aeration tank flow in millions of gallons per day (MGD) by the weight constant of 8.34 pounds per gallon of wastewater (Equation II.1).

BOD mg/l x Flow (MGD)x 8.34 pounds/gal wastewater

The microorganism value or amount of microorganisms in the activated sludge process consists of the pounds of mixed liquor volatile suspended solids (MLVSS) in the on-line aeration tank(s). The pounds of MLVSS is calculated by multiplying the concentration (mg/l) of MLVSS by the aeration tank(s) volume in million gallons

(MG) by the weight constant of 8.34 pounds per gallon of wastewater (Equation II.2).

MLVSS (mg/l)x Aeration tank volume (MG)

x 8.34 pounds/gal wastewater = pounds MLVSS (II.2)

The F/M of an activated sludge process can be calculated by dividing the pounds of food as BOD applied to the microorganisms or MLVSS present in on-line aeration tanks (Equation II.3)

F/M = Pounds BOD to aeration tank/Pounds MLVSS in aeration tank (II.3)


The hydraulic retention time or HRT is the amount of time in hours for wastewater to pass through a tank, such as an aeration tank. Changes in the HRT of an activated sludge process can affect biological activity. For example, decreasing HRT adversely affects nitrification, while increasing HRT favors nitrification and the solubliza-tion of colloidal BOD and particulate BOD.

The HRT of an aeration tank is determined by dividing the volume of the aeration tank in million gallons by the flow rate through the aeration tank (Equation II.4). The flow rate through the aeration tank must be expressed as gallons per hour (gph).

HRT (hours) = (Volume of aeration tank, gal) / (Flow rate, gph)


The mean cell residence time or MCRT is the amount of time, in days, that solids or bacteria are maintained in the activated sludge system. The MCRT is known also as the solids retention time (SRT). To calculate the MCRT of an activated sludge process, it is necessary to know the amount or pounds of solids or suspended solids in the activated sludge system and the amount or pounds of suspended solids leaving the activated sludge system.

To determine the pounds of suspended solids in the activated sludge system, the pounds of mixed liquor suspended solids (MLSS) must be calculated. The MLSS consists of all solids in the aeration tank(s) and secondary clarifier(s). Therefore the pounds of MLSS in an activated sludge systems consists of the concentration (mg/l) of MLSS times the volume (MG) of the aeration tank(s) and clarifier(s) times the weight constant of 8.34 pounds per gallon of wastewater (Equation II.5).

Pounds of MLSS

= MLSS mg/l x(Volume of aeration tanks + Clarifiers, MG) x 8.34 pounds/gal wastewater (II.5)

To determine the pounds of suspended solids leaving the activated sludge process, the amount or pounds of suspended solids loss through wasting and discharge in the secondary e¿uent must be calculated. Therefore the pounds of suspended solids leaving the activated sludge process consists of pounds of activated sludge wasted per day and the pounds of activated sludge or secondary e¿uent suspended solids discharged per day (Equation II.6).

Pounds of suspended solids leaving activated sludge process = Wasted sludge (mg/l)x Wasted sludge flow (MGD) x 8.34 pounds/gal wastewater + Secondary e¿uent suspended solids (mg/l) x E¿uent flow (MGD)x 8.34 pounds/gal wastewater (II.6)

The mean cell residence time of an activated sludge process can be calculated by dividing the pounds of suspended solids or MLSS in the activated sludge system by the pounds per day of suspended solids leaving the activated sludge system (Equation II.7).

Suspended solids in system, pounds

Suspended solids leaving system per day

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