Introduction to Nitrification

Biological nitrification is the conversion or oxidation of ammonium ions to nitrite ions and then to nitrate ions. During the oxidation of ammonium ions and nitrite ions, oxygen is added to the ions by a unique group of organisms, the nitrifying bacteria (Figure 6.1). Nitrification occurs in nature and in activated sludge processes. Nitrification in soil is especially important in nature, because nitrogen in absorbed by plants as a nutrient in the form of nitrate ions. Nitrification in water is of concern in wastewater treatment, because nitrification may be required for regulatory purposes or may contribute to operational problems.

Although ammonium ions and ammonia are reduced forms of nitrogen, that is, are not bonded to oxygen, it is the ammonium ion, not ammonia, that is oxidized during nitrification. The quantities of ammonium ions and ammonia in an aeration tank are dependent on the pH and temperature of the activated sludge (Figure 6.2). In the temperature range of 10° to 20 °C and pH range of 7 to 8.5, which are typical of most activated sludge processes, about 95% of the reduced form of nitrogen is present as ammonium ions.

The oxidation of ammonium ions and nitrite ions is achieved through the addition of dissolved oxygen within bacterial cells. Because nitrification or the biochemical reactions of oxygen addition occur inside biological cells, nitrification occurs through biochemical reactions.

The ammonium ions are produced in the wastewater from the

Figure 6.1 Biological nitrification. Biological nitrification in the activated sludge process consists of the removal of oxygen from an aeration tank and its addition to ammonium ions or nitrite ions. Oxygen is added to ammonium ions by the nitrifying bacterium Nitrosomonas, while oxygen is added to nitrite ions by the nitrifying bacterium Nitrobacter.

Figure 6.1 Biological nitrification. Biological nitrification in the activated sludge process consists of the removal of oxygen from an aeration tank and its addition to ammonium ions or nitrite ions. Oxygen is added to ammonium ions by the nitrifying bacterium Nitrosomonas, while oxygen is added to nitrite ions by the nitrifying bacterium Nitrobacter.

Percent Ammonia Percent Ammonium Ion

Percent Ammonia Percent Ammonium Ion

7 8 9 10 11

Figure 6.2 pH and the conversion of ammonia and ammonium ions. The relative quantities of ammonia and ammonium ions in wastewater or the activated sludge process are determined by the pH of the wastewater or aeration tank. As the pH of the wastewater decreases, ammonium ions are favored. As the pH of the wastewater increases, ammonia is favored. At a pH value of 9.4 or higher, ammonia is strongly favored.

TABLE 6.1 Organisms in the Aeration Tank That Are Capable of Nitrification

Organism

Genus

Actinomycetes

Myocbacterim

Nocardia

Streptomyces

Algae

Chlorella

Bacteria

Arthrobacter

Bacillus

Nitrobacter

Nitrosomonas

Proteus

Pseudomonas

Vibrio

Fungi

Aspergillus

Protozoa

Epistylis

Vorticella

Nitrosomonas And Nitrobacter

Figure 6.3 The nitrifying bacteria Nitrosomonas and Nitrobacter. The nitrifying bacteria of importance in the activated sludge process consist of the coccus-shaped Nitrosomonas (a) and the bacillus-shaped Nitrobacter (b). Nitrosomonas is 0.5 to 1.5 mm in size or diameter, while Nitrobacter is approximately 0.5 x 1.0 mm in size.

Figure 6.3 The nitrifying bacteria Nitrosomonas and Nitrobacter. The nitrifying bacteria of importance in the activated sludge process consist of the coccus-shaped Nitrosomonas (a) and the bacillus-shaped Nitrobacter (b). Nitrosomonas is 0.5 to 1.5 mm in size or diameter, while Nitrobacter is approximately 0.5 x 1.0 mm in size.

Figure 6.4 The protozoa Epistylis and Vorticella. Epistylis and Vorticella are stalk ciliated protozoa. Each organism possesses an enlarged anterior portion or ''head'' and a slender posterior portion or "tail."" The anterior portion has a mouth opening that is surrounded by a band of hairlike structures or cilia. The cilia beat to produce a water current to draw bacteria into the mouth opening. The tail of the organism usually is attached to a floc particle. If the tail has a contractile filament as is found in Vorticella, the protozoa is capable of springing. By springing, a stronger water current is produced for drawing bacteria into the mouth opening. Epistylis is colonial, while Vorticella is solitary (single). Species of Epistylis vary in size from 50 to 250 mm, while species of Vorticella vary in size from 30 to 150 mm.

Figure 6.4 The protozoa Epistylis and Vorticella. Epistylis and Vorticella are stalk ciliated protozoa. Each organism possesses an enlarged anterior portion or ''head'' and a slender posterior portion or "tail."" The anterior portion has a mouth opening that is surrounded by a band of hairlike structures or cilia. The cilia beat to produce a water current to draw bacteria into the mouth opening. The tail of the organism usually is attached to a floc particle. If the tail has a contractile filament as is found in Vorticella, the protozoa is capable of springing. By springing, a stronger water current is produced for drawing bacteria into the mouth opening. Epistylis is colonial, while Vorticella is solitary (single). Species of Epistylis vary in size from 50 to 250 mm, while species of Vorticella vary in size from 30 to 150 mm.

hydrolysis of urea and degradation of organic-nitrogen compounds. Hydrolysis and degradation of organic-nitrogen compounds results in the release of amino groups and the production of ammonium ions.

Although there are many organisms that are capable of oxidizing ammonium ions and nitrite ions (Table 6.1), the principle organisms responsible for most, if not all, nitrification in the activated sludge process are two genera of nitrifying bacteria, Nitrosomonas and Nitro-bacter (Figure 6.3). These genera of bacteria possess special enzymes and cellular structures that permit them to achieve significant nitrification.

The genera of nitrifying bacteria that oxidize ammonium ions to nitrite ions are prefixed Nitroso- (such as Nitrosomonas), and the genera of nitrifying bacteria that oxidize nitrite ions to nitrate ions are prefixed Nitro (such as Nitrobacter). Nitrification by organisms other than nitrifying bacteria occurs at relatively low rates, and it is not associated with cellular growth or reproduction.

The rate of nitrification achieved by nitrifying bacteria is often 1000 to 10,000 times greater than the rate of nitrification achieved by other organisms. Besides the nitrifying bacteria, there are two protozoa that are present in relatively large numbers during rapid nitrification. These protozoa are Epistylis and Vorticella (Figure 6.4). However, it is not known if these protozoa are capable of a rapid rate of nitrification, or if they simple grow in large numbers under operational conditions that are optimal for nitrification to occur through nitrifying bacteria.

Although activated sludge processes are used for nitrification, these processes are not ideal for nitrification. Due to the large population size and rapid growth of organotrophs in the aeration tank as compared to the small population size and slow growth of nitrifying bacteria, the population size of nitrifying bacteria is gradually diluted, making it difficult to achieve and maintain desired nitrification. Approximately 90% to 97% of the bacteria in the activated sludge process consists of organotrophs, while the remaining 3% to 10% of the bacteria are nitrifiers.

Nitrifying bacteria live in large variety of habitats including freshwater, potable water, wastewater, marine water, brackish water, and soil. Nitrifying bacteria are known by many names that are derived from the carbon and energy substrates (Table 7.1).

Although some genera of nitrifying bacteria are capable of using some organic compounds to obtain carbon, the principal genera of nitrifying bacteria in the activated sludge process, Nitrosomonas and Nitrobacter, use carbon dioxide or inorganic carbon as their carbon source for the synthesis of cellular material. For each molecule of carbon dioxide assimilated into cellular material by nitrifying bacteria, approximately 30 molecules of ammonium ions or 100 molecules of nitrite ions must be oxidized.

Due to the relatively large quantity of ammonium ions and nitrite ions needed to assimilate carbon dioxide, nitrifying bacteria have a very low reproductive rate. Even under the best conditions the reproductive rate of nitrifying bacteria is minimal. In the activated sludge process, nitrifying bacteria are able to increase in number only if their reproductive rate is greater than their removal rate through sludge wasting and discharge in the final e¿uent. Therefore a high MCRT is required to increase the number of nitrifying bacteria in the activated sludge process.

For the growth of one pound of dry cells, Nitrosomonas must oxidize 30 pounds of ammonium ions, while Nitrobacter must oxidize 100 pounds of nitrite ions. In contrast, for the growth of one pound

TABLE 7.1 Common Names of Nitrifying Bacteria

Name

Derivation

Ammonia-oxidizing bacteria

Oxidize ammonium ions

Ammonia-removing bacteria

Reduce the concentration of ammonium ions

Autotrophs

Obtain carbon from CO2

Chemolithoautotrophs

Obtain carbon from CO2 and energy from chemical minerals

nBOD-oxidizing bacteria

Oxidize ammonium ions Oxidize nitrite ions

nBOD-removing bacteria

Reduce the concentration of ammonium ions Reduce the concentration of nitrite ions

Nitrifiers

Oxidize ammonium ions Oxidize nitrite ions

Nitrifying bacteria

Oxidize ammonium ions Oxidize nitrite ions

Nitrite-oxidizing bacteria

Oxidize nitrite ions

Nitrite-removing bacteria

Reduce the concentration of nitrite ions

NOD-oxidizing bacteria

Oxidize ammonium ions Oxidize nitrite ions

NOD-removing bacteria

Reduce the concentration of ammonium ions Reduce the concentration of nitrite ions

of dry cells, the organotrophic bacterium Escherichia coli must oxidize only two pounds of glucose.

Nitrifying bacteria obtain their energy by oxidizing inorganic substrates, namely ammonium ions and nitrite ions (Table 7.2). Nitrite ions that are the product of the oxidation of ammonium ions by Ni-trosomonas serve as the substrate for Nitrobacter (Figure 7.1). Unless nitrite ions are discharged to the activated sludge process, nitrite ions must be produced in the aeration tank in order for Nitrobacter to have an energy substrate.

TABLE 7.2 Oxidation Reactions of Nitrifying Bacteria

Genus Responsible for the

Oxidation Reaction

Oxidation Reaction

NH+ + 1.5O2 ! NO2 + H2O + 2H+

Nitrosomonas

NO2 + 0.5O2 ! NO3

Nitrobacter

Figure 7.1 Significant waste product of Nitrosomonas. Nitrite ions are the significant waste product of Nitrosomonas. These ions are important for two reasons. First, unless nitrite ions are discharged to the activated sludge process by an industry, Nitrobacter must wait for Nitrosomonas to oxidized ammonium ions and then release nitrite ions to the bulk solution in order to have an energy substrate. Second, unless Nitrobacter oxidizes nitrite ions, these ions would accumulate in the activated sludge process and would interfere with the ability of chlorine to destroy coliform organisms and filamentous organisms.

Figure 7.1 Significant waste product of Nitrosomonas. Nitrite ions are the significant waste product of Nitrosomonas. These ions are important for two reasons. First, unless nitrite ions are discharged to the activated sludge process by an industry, Nitrobacter must wait for Nitrosomonas to oxidized ammonium ions and then release nitrite ions to the bulk solution in order to have an energy substrate. Second, unless Nitrobacter oxidizes nitrite ions, these ions would accumulate in the activated sludge process and would interfere with the ability of chlorine to destroy coliform organisms and filamentous organisms.

Nitrifying bacteria belong in the family Nitrobacteracae. With some exceptions, bacteria in this family obtain carbon by assimilating carbon dioxide to a 5-carbon sugar, ribulose diphosphate, to produce a 6-carbon sugar, glucose. The assimilation of carbon dioxide results in the production of cellular material. When carbon dioxide is assimilated, the carbon dioxide is reduced by the addition of hydrogen.

There are two energy-yielding, biochemical reactions that occur during nitrification (Equations 7.1 and 7.2). More energy is derived from the first biochemical reaction, that is, the oxidation of ammo nium ions, than the second biochemical reaction, that is, the oxidation of nitrite ions.

The energy-yielding reactions occur inside the bacterial cells, and both reactions involve the use of free molecular oxygen. Since an accumulation of nitrite ions usually does not occur, the overall reaction for nitrification is controlled by the oxidation of ammonium ions to nitrite ions. The overall reaction for nitrification is a combination of the two energy-yielding reactions (Equation 7.3).

NH+ + 2O2 — Nitrifying bacteria ! NO^ + 2H+ + H2 O (7.3)

There are several intermediate compounds such as hydroxylamine (NH2OH) that are produced during nitrification. However, these compounds are short-lived and therefore are not presented in equations that describe the energy-yielding reactions of nitrification.

Although ammonium ions are used as an energy source by nitrifying bacteria, not all of the ammonium ions taken inside the bacterial cells are nitrified. Some of the ammonium ions are used as a nutrient source for nitrogen and are assimilated into new cellular material (C5H7O2N). The growth of new cells in the activated sludge process is referred to as an increase in the mixed liquor volatile suspended solids (MLVSS) (Equation 7.4).

4CO2 + HCO^ + NH+ + 4H2O ! C5H7O2N + 5O2 + 3H2O (7.4)

Carbon dioxide serves as the carbon source for the synthesis of cellular material and is made available to nitrifying bacteria as bicarbonate alkalinity. This alkalinity is produced when carbon dioxide dissolves in wastewater.

There are several genera of nitrifying bacteria. The genera can be grouped according to those that oxidize ammonium ions and those that oxidize nitrite ions (Table 7.3).

Nitrifying bacteria are not pathogenic (disease-causing) and are not common or indigenous to the intestinal tract of humans. Therefore nitrifying bacteria do not enter sewer systems and activated sludge

TABLE 7.3 Genera of Nitrifying Bacteria

Energy Substrate

Oxidized Product

Genera of Nitrifying Bacteria

Nitrosococcus

Nitrosocystis

Nitrosolobus

Nitrosomonas

Nitrosospira

Nitrobacter Nitrococcus Nitrospira processes in large numbers through domestic wastewater. Nitrifying bacteria are indigenous to soil and water. Therefore large numbers of nitrifying bacteria enter sewer systems and activated sludge processes through I/I.

The nitrifying bacteria Nitrosomonas and Nitrobacter are largely, if not entirely, responsible for nitrification in soil. Because nitrifying bacteria are destroyed by ultraviolet light, they are not found in large numbers on the surface of the soil. However, they are found in large numbers immediately beneath the surface of the soil where ultraviolet light cannot penetrate.

Activated sludge processes are seeded with Nitrosomonas and Ni-trobacter through I/I, and these genera of nitrifying bacteria also are largely, if not entirely, responsible for nitrification in the activated sludge process. The principle species of nitrifying bacteria responsible for the oxidation of ammonium ions and nitrite ions are Nitrosomonas europeae and Nitrobacter agilis, respectively. Other genera of nitrifying bacteria are of secondary importance in the activated sludge process.

Nitrosomonas and Nitrobacter are Gram-negative bacteria and are strict aerobes that require free molecular oxygen or dissolved oxygen in order to oxidize substrate. Gram-negative bacteria stain red when dried smears of the bacteria are exposed to a series of dyes. Oxygen is used to carry and remove electrons from the bacterial cell as they are released during the oxidation of ammonium ions and nitrite ions.

Although nitrifying bacteria can grow and reproduce in the presence of most organic compounds, some simplistic forms of organic compounds can inhibit their activity, that is, inhibit nitrification. These inhibitory compounds include alcohols and acids. Some organic com-

TABLE 7.4 Basic Physiological and Structural Features of Nitrosomonas and Nitrobacter

Feature

Nitrosomonas

Nitrobacter

Carbon source

Inorganic (CO2)

Inorganic (CO2)

Cell shape

Coccus (spherical)

Bacillus (rod-shaped)

Cell size, mm

0.5 to 1.5

0.5 x 1.0

Habitat

Soil and water

Soil and water

Motility

Yes

No

Oxygen requirement

Strict aerobe

Strict aerobe

pH growth range

5.8 to 8.5

6.5 to 8.5

Reproduction mode

Binary fission

Budding

Generation time

8 to 36 hours

12 to 60 hours

Temperature growth

5° to 30 °C

5° to 40 °C

range

Sludge yield

0.04 to 0.13 pound of cells

0.02 to 0.07 pound of cells

per a of NHJ oxidized

per a of NOg oxidized

Cytomembranes

Present

Present

pounds that have amino groups, such as methylamine (CH2NH2), also inhibit the activity of nitrifying bacteria.

With some exceptions, nitrifying bacteria are obligate (strict) auto-trophs. Because they are obligate autotrophs, some simplistic forms of organic compounds that remain in the aeration tank inhibit nitrifying bacteria, that is, inhibit nitrification. Therefore a large and diverse population of organotrophs must be present in the aeration tank in order to oxidize these simplistic forms of organic compounds.

Basic physiological and structural characteristics of Nitrosomonas and Nitrobacter are provided in Table 7.4. Important structural features listed in Table 7.4 are the cytomembrane.

The cytomembranes of nitrifying bacteria intrude from the cell membrane into the peripheral or inner region of the cell (Figure 7.2). The cytomembranes are the active site for the oxidation of ammonium ions and nitrite ions. Here the oxidation of the ions occurs at relatively high rates.

In an activated sludge process the oxidation of ammonium ions and nitrite ions consists of their adsorption to the surface of the cytomembranes and their oxidation by enzymes on the surface of the cytomembranes. After these ions have been oxidized, their respective waste or product (nitrite ions and nitrate ions) are released to the bulk solution (Figure 7.3). As the ions are oxidized, energy is obtained and stored in the form of high-energy, phosphate bonds.

Figure 7.2 Cytomembranes of nitrifying bacteria. The cytomembranes of the nitrifying bacteria Nitrosomonas (a) and Nitrobacter (b) are the active sites for the nitrification of ammonium ions and nitrite ions. The cytomembranes consist of extensions or an accordionlike folding of the cell membrane away from the cell wall and toward the center of the cytoplasm.

Figure 7.2 Cytomembranes of nitrifying bacteria. The cytomembranes of the nitrifying bacteria Nitrosomonas (a) and Nitrobacter (b) are the active sites for the nitrification of ammonium ions and nitrite ions. The cytomembranes consist of extensions or an accordionlike folding of the cell membrane away from the cell wall and toward the center of the cytoplasm.

Nitrobacter

Figure 7.3 Oxidation of ammonium ions and nitrite ions on the cytomembranes. It is on the cytomembranes of Nitrosomonas and Nitrobacter where ammonium ions and nitrite ions, respectively, come in contact with the enzymes that add oxygen to each ion. Ammonium ions removed from the bulk solution are oxidized on the cytomembranes of Nitrosomonas, while nitrite ions removed from the bulk solution are oxidized on the cytomembranes of Nitrobacter.

Nitrobacter

Figure 7.3 Oxidation of ammonium ions and nitrite ions on the cytomembranes. It is on the cytomembranes of Nitrosomonas and Nitrobacter where ammonium ions and nitrite ions, respectively, come in contact with the enzymes that add oxygen to each ion. Ammonium ions removed from the bulk solution are oxidized on the cytomembranes of Nitrosomonas, while nitrite ions removed from the bulk solution are oxidized on the cytomembranes of Nitrobacter.

Figure 7.4 Asexual reproduction. Asexual reproduction or binary fission is the principle means by which bacteria reproduce. Binary fission or the simple splitting of a bacterium into two bacteria results in very rapid growth of the bacterial population. This growth is often referred to as a periodic doubling of the population size (i.e., 1, 2, 4, 8, 16, 32, 64, 132,...).

Nitrifying bacteria may grow and reproduce as individual cells or small aggregates embedded in slime. In the activated sludge process, nitrifying bacteria are found adsorbed on the surface of the floc particles, suspended in the bulk solution, and on the biological growth on the sides of the aeration tank and clarifiers. Only the nitrifying bacteria that are exposed to free molecular oxygen nitrify.

Nitrifying bacteria reproduce asexually (Figure 7.4). Nitrosomonas reproduces by binary fission or simply splitting in half (Figure 7.5), while Nitrobacter reproduces by budding (Figure 7.6).

Although nitrifying bacteria are poor floc-forming bacteria, they are incorporated in the floc particles through two means. First, nitrifying bacteria that possess a compatible charge are quickly removed

Nitrosomonas Floc
Figure 7.5 Binary fission in Nitrosomonas. When Nitrosomonas reproduces, the cell simply divides into two cells. This division occurs as the cell is ''pinched'' in at the center into two new cells.
Figure 7.6 Budding in Nitrobacter. Like yeast cells, Nitrobacter reproduces by budding. When budding occurs, new cellular material from the original or parent cells ''pushes'' free to form a new or daughter cell.
Nitrification Mechanism

Figure 7.7 Adsorption of nitrifying bacteria to floc particles. Nitrifying bacteria are poor floc-forming bacteria and are adsorbed to the surface of floc particles through two mechanisms. First, if the surface charge of the nitrifying bacteria is compatible for adsorption, the nitrifying bacteria are added directly to the growing floc particle. Second, if the surface charge of the nitrifying bacteria is not compatible for adsorption, the surface charge of the nitrifying bacteria is made compatible for adsorption through the coating action of secretions released by ciliated protozoa. The secretions from rotifers, free-living nematodes, and other multicellular organisms also help to remove nitrifying bacteria from the bulk solution to the surface of floc particles.

Figure 7.7 Adsorption of nitrifying bacteria to floc particles. Nitrifying bacteria are poor floc-forming bacteria and are adsorbed to the surface of floc particles through two mechanisms. First, if the surface charge of the nitrifying bacteria is compatible for adsorption, the nitrifying bacteria are added directly to the growing floc particle. Second, if the surface charge of the nitrifying bacteria is not compatible for adsorption, the surface charge of the nitrifying bacteria is made compatible for adsorption through the coating action of secretions released by ciliated protozoa. The secretions from rotifers, free-living nematodes, and other multicellular organisms also help to remove nitrifying bacteria from the bulk solution to the surface of floc particles.

from the bulk solution and adsorbed to the surface of the floc particle. Second, nitrifying bacteria that do not possess a compatible charge are adsorbed to the floc particle when their surface charge is made compatible. The secretions of sticky sugars and starches by ciliated protozoa, rotifers, and free-living nematodes that coat the surface of nitrifying bacteria alters their surface charge and permits their adsorption to the floc particles (Figure 7.7).

Because nitrifying bacteria obtain a relatively small amount of energy from the oxidation of ammonium ions and nitrite ions, reproduction or generation time is slow, and a small population of organisms or MLVSS is produced per pound of ammonium ions and nitrite ions oxidized. The population size of nitrifying bacteria within the acti vated sludge is very small in comparison to the population size of organotrophs.

The difference in population size between nitrifying bacteria and organotrophs is due to two reasons. First, in most municipal and industrial activated sludge processes, the concentration of carbonaceous wastes greatly exceeds the concentration of nitrogenous wastes. Therefore more substrate is available to grow more organotrophs. Second, organotrophs obtain more energy for reproduction than do nitrifying bacteria when they oxidize their respective substrates. Therefore organotrophs can reproduce more quickly than nitrifying bacteria can reproduce.

Compared to organotrophs the generation time of nitrifying bacteria is much longer. The generation time of most organotrophs within the activated sludge process is 15 to 30 minutes. Under favorable conditions the generation time of nitrifying bacteria within the activated sludge process is 48 to 72 hours.

Within the nitrifying bacterial population there also is a difference in population sizes between Nitrosomonas and Nitrobacter. The population size of Nitrosomonas is larger than Nitrobacter. Because Nitrosomonas obtains more energy from the oxidation of ammonium ions than Nitrobacter obtains from the oxidation of nitrite ions, Nitrosomonas has a shorter generation time and is able to increase quickly in numbers as compared to Nitrobacter (Table 7.5). A larger population size of Nitrosomonas than Nitrobacter in the activated sludge process provides for more ammonium ion oxidizing ability than nitrite ion oxidizing ability.

The difference in generation times between Nitrosomonas and Ni-trobacter directly affects nitrification. The difference is in part responsible for the buildup of nitrite ions during unfavorable operational conditions including cold temperature, hydraulic washout, low dissolved oxygen level, start-up, toxicity, and slug discharge of soluble cBOD.

In the aeration tank of municipal and industrial activated sludge processes a relatively high MCRT (> 8 days) is required in order to provide an opportunity for the slow growing and poorly flocculating population of nitrifying bacteria to increase in size. Even with a relatively high MCRT, the population size of the nitrifying bacteria is normally less than 10% of the total bacterial population in the aeration tank.

Although the ultimate population size of nitrifying bacteria is dependent on the amount of substrate (ammonium ions and nitrite ions)

TABLE 7.5 Increase in Population Sizes of Nitrosomonas and Nitrobacter over 72 Hours

Nitrosomonas Nitrobacter

TABLE 7.5 Increase in Population Sizes of Nitrosomonas and Nitrobacter over 72 Hours

Nitrosomonas Nitrobacter

Hours

Divisions

Bacteria

Divisions

Bacteria

0

0

1

0

1

8

1

2

0

1

16

2

4

1

2

24

3

8

2

4

32

4

16

2

4

40

5

32

3

8

48

6

64

4

16

56

7

128

4

16

64

8

256

5

32

72

9

512

6

64

Note: The table assumes that the Nitrosomonas population and the Nitrobacter population begin with one bacterium each, no bacterium dies, and Nitrosomonas and Nitrobacter reproduce at optimum generation times in the laboratory, 8 hours for Nitrosomonas and 12 hours for Nitrobacter. After 72 hours (three days) the population size of Nitrosomonas is eight times larger than the population size of Nitrobacter. The difference in population sizes over time and the difference in energy obtained from the oxidation of substrates permits ammonium ions to be more easily oxidized than nitrite ions in the activated sludge process.

Note: The table assumes that the Nitrosomonas population and the Nitrobacter population begin with one bacterium each, no bacterium dies, and Nitrosomonas and Nitrobacter reproduce at optimum generation times in the laboratory, 8 hours for Nitrosomonas and 12 hours for Nitrobacter. After 72 hours (three days) the population size of Nitrosomonas is eight times larger than the population size of Nitrobacter. The difference in population sizes over time and the difference in energy obtained from the oxidation of substrates permits ammonium ions to be more easily oxidized than nitrite ions in the activated sludge process.

available, the growth and reproduction of the population is strongly influenced by several operational factors including dissolved oxygen, alkalinity and pH, temperature, inhibition, toxicity, and mode of operation.

For example, the growth rate of nitrifying bacteria is directly influenced by temperature. With increasing temperature, the growth of the nitrifying bacteria accelerates, and nitrification is achieved with little difficulty. With decreasing temperature, the growth of the nitrifying bacteria slows, and nitrification is achieved with much difficulty. Therefore during cold temperatures a buildup of nitrite ions may occur as Nitrobacter oxidizes nitrite ions more slowly than Nitrosomonas oxidizes ammonium ions.

The presence of nitrifying bacteria in an activated sludge process can be identified by the production of nitrite ions or nitrate ions across the aeration tank. Nitrifying bacteria can be cultivated on selective agar. However, due to the slow generation time of nitrifying bacteria and their poor isolation and colony development on agar media, it is often difficult to identify nitrifying bacteria on selective agar.

In the activated sludge process, numerous organisms, particularly bacteria, work together to oxidize and remove wastes. The successful operation of the activated sludge process involves the management of abundant and active populations of organotrophs and nitrifying bacteria. Proper interactions between organotrophs and nitrifying bacteria are required to remove carbonaceous BOD (cBOD) and nitrogenous BOD (nBOD).

Organotrophs remove soluble cBOD, particulate BOD (pBOD), and colloidal BOD (coBOD). Particulate BOD and colloidal BOD are forms of cBOD. The organotrophs are known by several names (Table 8.1). The names also are derived from their carbon and energy substrates.

Organotrophs often are referred to as heterotrophs because they obtain the carbon they need for cellular growth from organic wastes, and not from carbon dioxide. Because they derive energy from the oxidation of organic wastes, they are called chemotrophs. Therefore organotrophs are chemoorganotrophs; namely they derive carbon and energy from the oxidation of organic compounds. A comparison of carbon and energy substrates of organotrophs and nitrifying bacteria is provided in Table 8.2.

By using organic wastes as a substrate for carbon and energy, or-ganotrophs reduce the quantity of cBOD in the activated sludge process. By using carbon dioxide as a carbon substrate and ammonium ions and nitrite ions as energy substrates, nitrifying bacteria decrease

TABLE 8.1 Common Names of Organotrophs

Name Derivation cBOD-oxidizing bacteria Oxidize organic wastes cBOD-removing bacteria Reduce the concentration of organic wastes

Chemoorganotrophs Obtain carbon and energy from organic wastes

Heterotrophs Do not obtain carbon from carbon dioxide

Organotrophs Obtain carbon and energy from organic wastes alkalinity and pH in the aeration tank and reduce the quantity of nBOD in the activated sludge process.

In order for nBOD to be reduced, cBOD first must be reduced to a relatively low concentration (< 40 mg/l) to ensure that adequate degradation of those soluble and simplistic forms of cBOD that inhibit the activity of nitrifying bacteria occurs. Therefore nitrifying bacteria are dependent on organotrophs to reduce cBOD.

Organotrophs are found in freshwater, potable water, wastewater, marine water, brackish water, and soil. They also are associated with plants and the fecal waste from humans and animals. Organotrophs enter sewer systems and activated sludge processes through domestic wastewater and I/I. Some organotrophs are disease-causing organisms.

Although it is difficult to enumerate and characterize the viable numbers of organotrophs within soil and fecal wastes, population sizes of these organisms are estimated as several billion per gram of soil or fecal waste. In the activated sludge process, organotrophs proliferate to relatively large numbers, approximately 60,000,000 per milliliter of bulk solution in the aeration tank and 20,000,000,000 per gram of MLVSS.

Organotrophs oxidize organic wastes through aerobic respiration or anaerobic respiration. Bacteria that can oxidize organic wastes through aerobic respiration only are strict or obligate aerobes; that is,

TABLE 8.2 Comparison of Carbon and Energy Substrates for Organotrophs and Nitrifying Bacteria

Carbon/Energy Substrate Organotrophs Nitrifying Bacteria

Carbon source Carbon source removal Energy source Energy source removal

Organic wastes Decrease cBOD Organic wastes Decreases cBOD

CO2 as alkalinity Decreases alkalinity/pH NH+ and NO2 Decreases nBOD

the bacteria can only use free molecular oxygen. Bacteria that can oxidize organic wastes using free molecular oxygen, if it is available, or another molecule, such as nitrite ions or nitrate ions, if free molecular oxygen is not available are facultative anaerobes.

Although facultative anaerobes can use free molecular oxygen or another molecule to oxidize organic wastes, their preference always is for free molecular oxygen. There are two reasons for this preference. First, the organotrophs obtain a larger quantity of energy by oxidizing the organic wastes with free molecular oxygen rather than another molecule. Second, the organotrophs produce more offspring (greater reproduction) by oxidizing the organic wastes with free molecular oxygen rather than another molecule. Within the activated sludge process approximately 80% of the organotrophs are facultative anaerobes (Table 8.3).

The dominant genera of organotrophs in the activated sludge process are determined by wastewater composition. For example, protein-aceous wastes favor the proliferation of Bacillus, while carbohydrate wastes favor the proliferation of Pseudomonas.

TABLE 8.3 Common Genera of Organotrophs in the Activated Sludge Process

Genus

Strict Aerobes

Facultative Anaerobes

Achromobacter

x

Acinetobacter

x

Actinomyces

x

Aerobacter

x

Arthrobacter

x

Bacillus

x

Beggiatoa

x

Cornynebacterium

x

Enterobacter

x

Escherichia

x

Flavobacterium

x

Klebsiella

x

Micrococcus

x

Nocardia

x

Proteus

x

Pseudomonas

x

Sphaerotilus

x

Thiothrix

x

Zoogloea

x

Collodial BOD, Protein Containing Sulfur and Phosphorus

Solublization by Exoenzymes 1

Soluble cBOD

Solublization by Exoenzymes 1

Soluble cBOD

Figure 8.1 Oxidation of soluble cBOD. Bacterial cells rapidly absorb soluble cBOD. Colloidal BOD such as proteins must be solublized through the production and release of bacterial exoenzymes. Bacterial cells rapidly absorb, once sol-ublized, the resulting soluble cBOD. Inside the bacterial cells, endoenzymes oxidize the soluble cBOD into new cells, and several inorganic compounds including carbon dioxide (CO2), water (H2O), ammonium ions (NH%), phosphate ions (PO2and sulfate ions (SO%~). Proteins that contain phosphate groups and thiol groups (-SH), serve as the compounds that yield phosphates and sulfates when oxidized.

The dominant bacteria are selected by the compatibility of bacterial enzyme systems (ability to degrade substrates) and the substrates present in the wastewater. Enzyme systems are the "tools" or cellular machinery that bacteria use to degrade substrate. An enzyme system may degrade one substrate or a variety of substrates. However, no enzyme system can degrade all substrates, and no substrate can be degraded by all enzyme systems.

During the oxidation of cBOD, electrons are released from the substrate. The electrons are removed from the cell by free molecular

Organotrophs
Nitrifying Bacteria Ciliated Protozoa

Organotrophs Insoluble and Complex Forms of cBOD

Organotrophs Insoluble and Complex Forms of cBOD

Figure 8.2 Removal of insoluble and complex forms of cBOD. Insoluble and complex forms of cBOD, namely particulate BOD and colloidal BOD, are adsorbed directly to the surface of floc particles if these forms of cBOD have compatible surface charge. These forms of cBOD are adsorbed indirectly to the surface of floc particles if their surface charge is made compatible through the coating action of secretions from ciliated protozoa and other higher life forms.

oxygen, nitrite ions, or nitrate ions. If nitrite ions or nitrate ions are used to remove the electrons, molecular nitrogen (N2) is produced. The production of molecular nitrogen from nitrite ions or nitrate ions during respiration is denitrification. For example, when nitrate ions are used to degrade methanol (CH3OH), an organic substrate is degrade through anoxic respiration to produce carbon dioxide, water, molecular nitrogen, and new bacterial cells (Equation 8.1).

CH3OH + NO^ — Bacillus ! 3CO2 + N2 + 9H+ + 3OH" (8.1)

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Cellulomonas with the exoenzyme cellulase

C^glucose^^

C glucose J)

Figure 8.3 Solublization and oxidation of cellulose. Cellulose is a starch having many glucose units bonded by special chemical bonds. Cellulose is insoluble in wastewater. Cellulose can be solublized and oxidized under appropriate operational conditions in the activated sludge process. If cellulose is adsorbed to the surface of the floc particles, and the bacterium Cellulomonas is present in the floc particle, then Cellulomonas produces and releases the exoenzyme cellulase. When cellulase comes in contact with cellulose, the special chemical bonds are broken, and individual glucose units are released. These glucose units quickly dissolve in the wastewater and are absorbed by Cellulomonas as well as other organotrophs. Once absorbed, cellulose is oxidized. The oxidation of cellulose results in the production of new organotrophs, carbon dioxide (CO2), and water (H2O).

Figure 8.3 Solublization and oxidation of cellulose. Cellulose is a starch having many glucose units bonded by special chemical bonds. Cellulose is insoluble in wastewater. Cellulose can be solublized and oxidized under appropriate operational conditions in the activated sludge process. If cellulose is adsorbed to the surface of the floc particles, and the bacterium Cellulomonas is present in the floc particle, then Cellulomonas produces and releases the exoenzyme cellulase. When cellulase comes in contact with cellulose, the special chemical bonds are broken, and individual glucose units are released. These glucose units quickly dissolve in the wastewater and are absorbed by Cellulomonas as well as other organotrophs. Once absorbed, cellulose is oxidized. The oxidation of cellulose results in the production of new organotrophs, carbon dioxide (CO2), and water (H2O).

Molecular nitrogen is insoluble in wastewater, and when released in the secondary clarifier, it often becomes entrapped in the floc particles or solids. The entrapment of molecular nitrogen results in a loss of compaction of the solids and a loss of solids from the secondary clarifier.

When cBOD enters an activated sludge process, the organotrophs quickly absorb the soluble, simplistic forms of cBOD, such as acids and alcohols. This cBOD is then oxidized inside the bacterial cells (Figure 8.1). The insoluble and complex forms of cBOD are adsorbed to the slime on the surface of the bacterial cells (Figure 8.2). These forms of cBOD may be solublized and absorbed by the bacterial cells if sufficient retention time exists.

Exoenzymes are enzymes that are produced inside the cytoplasm of the bacterial cell and released through the cell membrane and cell wall to solublize the complex forms of cBOD (pBOD and coBOD) embedded in the slime. In order for the bacterial cells to produce and release exoenzymes, solublize complex forms of cBOD, and degrade the cBOD, approximately six hours of hydraulic retention time or HRT (Appendix II) are required in the aeration tank.

The solublization of cellulose (pBOD) is accomplished by the bacterium Cellulomonas. This bacterium produces the exoenzyme cellu-lase that is specific for the solublization of cellulose. When cellulose is solublized, soluble sugar (glucose) is produced (Figure 8.3). The sugar is easily absorbed by Cellulomonas as well as other organotrophs and is degraded.

Organotrophs also are capable of oxidizing some xenobiotics or synthetic compounds such as pesticides and chlorinated hydrocarbons. In addition to the oxidation of cBOD, some organotrophs such as Achromobacter, Aerobacter, Bacillus, Escherichia, Flavobacterium, and Pseudomonas initiate floc formation in the activated sludge process. The floc particles that develop eventually will contain large numbers of not only organotrophs but also nitrifying bacteria. Together these two groups of bacteria remove cBOD and nBOD from the activated sludge process.

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