Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists

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plant, forming nodules (knoblike growths). Root nodules help the host plant by providing

ﬁxed nitrogen (often, the limiting nutrient for plants), while providing organic substrates

(often, the limiting nutrient for heterotrophs) produced by the plant for the bacteria. The

actinomycete Frankia also produces root nodules, most commonly in nonleguminous

woody plants such as the alder and bayberry.

Other nitrogen-ﬁxing bacteria form associations with plants without nodule formation.

Clostridium and Desulfovibrio, for example, grow in the root zone of eelgrass, a shallow

saltwater plant, and Azotobacter and Azospirillum grow in the rhizosphere of some

grasses, including corn. Nitrogen-ﬁxing cyanobacteria may also form symbiotic relation-

ships with fungi, as in some lichens, as well as with plants such as the water fern Azolla

(used agriculturally to ﬁx nitrogen in rice paddies). This great diversity of organisms and

interactions further demonstrates the great adaptability of life!

Nitrogen ﬁxation requires an uncommonly high metabolic investment of energy and

reducing power to break molecular nitrogen’s strong trivalent bond (almost twice the

energy of oxygen’s double bond). The conversion of a single molecule of N

into 2

mol of reduced nitrogen requires eight protons, eight electrons, and between 16 and 24

ATP (Figure 13.19).

The highly specialized nitrogenase enzyme complex, which includes dinitrogenase,

employs cofactors containing iron and usually molybdenum or, occasionally, vanadium.

Other iron-containing enzymes and ATP are needed to transfer the required electrons to

the nitrogenase system. The reducing power (electrons) must come, in turn, from an

electron-donating substrate (usually organic, although some nitrogen ﬁxers can use sul-

ﬁde, hydrogen, or carbon monoxide). Although the conversion of two nitrogen atoms

from an oxidation state of 0 to 3 requires only six electrons, the process actually requires

eight, producing a molecule of hydrogen gas (H

There is some variation in the enzymes among different species, but even the nitro-

genases of aerobic bacteria are typicall y highly sensitive to irreversible inactivation by

oxygen. How, then, can these aerobes ﬁx nitrogen? Obviously, they must have means

of preventing the contact of oxygen with the enzymes. One approach, taken by Klebsiella,

which is a facultative anaerobe, is to ﬁx nitrogen only under anaerobic conditions. Other

Dinitrogen tetrahydride

Dinitrogen dihydride

Nitrogen

Ammonia

Dinitrogen

Oxidation

State

ecies

Nitrogen

Dinitrogenase

-3

-2

-1

Nitrogen Fixation

Dinitrogenase

Figure 13.19 Nitrogen ﬁxation pathway.

418

MICROBIAL TRANSFORMATIONS

strategies include producing a physical barrier, forming a protective complex with another

protein, and/or maintaining high rates of respiration to deplete oxygen near and within the

cell. Thus, another advantage of being within a root nodule is the more restricted ﬂux of

oxygen. Klebsiella and some other free-living bacteria can also produce a thick slime

layer. Being in the rhizosphere, where root exudates produce a higher concentration of

respirable organic substrates, helps limit oxygen concentration.

Several of the ﬁlamentous cyanobacteria ﬁx nitrogen only in a few differentiated cells

(unusual in prokaryotes) with thick walls, called heterocysts (see Section 10.5.4 and

Figure 10.20). The nitrogen is then distributed to the other cells in the ﬁlament, which

in return provide organic substrates to the nonphotosynthetic heterocyst. Many cyanobac-

teria may also prefer reduced levels of oxygen (perhaps 10% of saturation).

Most nitrogenases will also cometabolize other triple bonds, such as those of acetylene

(HC



CH) and hydrogen cyanide (HC



N). In fact, acetylene reduction to ethylene



) is often used as an indire ct means of measuring nit rogen-ﬁxing activity.

Dissimilatory Nitrate Reduction, Including Denitriﬁcation A wide variety of aerobic

prokaryotes are also able to utilize nitrate (NO



) as a terminal electron acceptor in the

absence of oxygen. In many case s, including with strains of Escherichia coli, Bacillus,

Staphylococcus, Spirillum , some actinomycetes, Aquifex, and some archaea, the product

of this dissimilatory nitrate reduction is nitrite ðNO



Þ. A few of these organisms are able

to further reduce the nitrite to ammonia, but this type of ammoniﬁcation (as opposed to

the mineralization of organic nitrogen) appears to be of only minor importance in most

environments.

However, under similar conditions, anaerobic respiration by a fairly broad array of

otherwise aerobic proka ryotes, such as some Pseudomonas, Thiobacillus denitriﬁcans,

Paracoccus, and a few archaea, results in a sequential conversion (Figure 13.20) of nitrate

Nitric oxide

Nitrogen

Nitrous oxide

Dinitrogen

Oxidation

State

Species

Nitrogen

Nitrate reductase

Nitrate

Nitrite reductase

Nitric oxide

reductase

Nitrite

Nitrous oxide

reductase

Denitrification

Figure 13.20 Denitriﬁcation pathway.

NITROGEN 419

to nitrite to elemental nitrogen gas (N

). The term denitriﬁcation is applied to this pro-

cess because N

is unusable by most organisms, so that these reactions represent a loss of

available (ﬁxed) nitrogen to an aquatic or soil environment. (Similarly, the release of

small amo unts of the gaseous intermediates NO and N

O represent losses of nitrogen

from the system.)

A general reaction for oxidation of a carbohydrate through denitriﬁcation can be writ-

ten as

O þ NO



O þ OH



ð13:10Þ

If the organic materia l oxidized is methan ol, the reaction can be written instead as

OH þ NO



O þ OH



ð13:11Þ

Example 13.5 An anaerobic groundwater contains 100 mg/L of carbohydrate with an

empirical formula of CH

O. How much nitrate would have to be added to meet the stoi-

chiometric requirement for complete oxidation of this contaminant?

Answer From equation (13.10), 1.0 mol of nitrate would be needed per 1.25 mol of

O, which has an apparent molecular weight of 1ð12 Þþ2ð1Þþ1ð16Þ¼30:

required NO



-N ¼ 100 mg CH

O=L

1 mol CH

30 g CH



1 mol NO



1:25 mol CH



14 g NO



1 mol NO





¼ 37:3mg=LNO



-N

Under anaerobic conditions we often expect unpleasant odors to develop. When nitrate

is present, however, this typically does not happen. Further, the active organisms are

mainly aerobes that have the additional ability to respire using nitrate as an alternative

terminal electron acceptor. Thus, sanitary engineers, and subsequently, environmental

engineers and scientists, have traditi onally referred to conditions in which oxygen is

absent but nitrate is present as anoxic rather than anaer obic. However, please be aware

that most biologists and others outside our ﬁeld do not make this distinction, using anae-

robic and anoxic as synonyms to refer to any system in which oxygen is absent.

Although most denitriﬁers are organotrophs, some are lithotrophs utilizing hydrogen or

sulﬁde. Recently, autotrophic nitriﬁcation (Section 13.2.2) also has been linked to deni-

triﬁcation, in a process dubbed anammox (anoxic ammonia oxidation). In oxidizing

ammonia as an energy source, the organisms involved can reduce nitrate to nitrite and

then reduce the nitrite to nitrogen gas:

þ NO



! N

þ 2H

O ð13:12Þ

This process does not seem to play a major environmental role in nitrogen cycling. How-

ever, it may be important to the organisms involved in some circumstances, particularly

when aerobic conditions (under which the nitrate is produced) are followed by anoxic

ones. This occurs in soils that are periodically ﬂooded, as well as in some wastewater

treatment plants.

In general, denitriﬁcation does not occur in the presence of oxygen. Since approxi-

mately 20% less energy is available to an organism when it is utilizing nitrate, there

420 MICROBIAL TRANSFORMATIONS

has been strong selective pressure for microbes to develop control mechanisms to sup-

press nitrate reduction in the presence of O

. However, denitriﬁcation can occur in anoxic

microenvironments (microscopically localized environments having different physical

and chemical condi tions) within heterogeneous aerobic systems, such as moist soils

and microbial colonies.

Nitrate Assimilation A wide variety of bacteria, archaea, fungi, algae, and most plants

can utilize nitrate as their source of nitrogen. However, to do this, they must reduce it to an

oxidation state of 3 (the level of ammonia and organic nitrogen). This is referred to as

assimilatory nitrate reduction. While dissimilatory reduction can be linked to energy-

yielding respiration, assimilatory reduction requires energy. As a result, the biomass yield

of organisms growing on nitrat e as their nitrogen source will be slightly lower than when

ammonia is used. Hence, an organism that is able to use both ammonia and nitrate will

utilize ammonia preferentially when it is available. On the other hand, the presence of

oxygen does not inhibit assimilatory reduction.

13.2.2 Nitrogen Oxidation

The oxidation of ammonia to nitrite, and nitrite to nitrate, is known as nitriﬁcation. There

is no known biochemical oxidation of nitrogen gas, although there are abiotic mechanisms

(electrochemical, photochemical, thermal). Similarly, there is no known pathway for

direct conversion of ammonia to nitrogen gas (th e reverse of nitrogen ﬁxation).

Autotrophic Nitriﬁcation Most nitriﬁcation results from the activity of aerobic, auto-

trophic chemolithotrophs, referred to as nitrifying bacteria (Figure 13.21). They utilize

reduced nitrogen (ammonia or nitrite) as their energy source, carbon dioxide as their car-

bon source, and oxygen as their electron acceptor.

The overall oxidation really consists of two separate steps, carried out by different bac-

teria (Figure 13.22). Ammonia oxidation involves the transformation of ammoni a to

nitrite, a six-electron transfer (3toþ3), and is sometimes called nitrite formatio n or

Figure 13.21 Clustered nitrifying cocci within activated sludge ﬂoc.

NITROGEN 421

nitritiﬁcation. In the second step, nitrite oxidation or nitratiﬁcation, nitrite is converted

to nitrate, a two-electron change (þ3toþ5). Similarly, the bacteria can be referred to as

ammonia oxidizers (or nitritiﬁers) and nitrite oxidizers (nitratiﬁers). No known nitri-

ﬁer can oxidize ammonia all the way to nitrate.

Balanced equations for the individual reac tions and their sum can be written as

! NO



þ H

O þ 2H

ð13:13Þ



! NO



ð13:14Þ

þ 2O

! NO



þ H

O þ 2H

ð13:15Þ

Note that the equations are written with ammonium and nitrite as the reactants, since it

is these ionic forms that are expected to be predominant at neutral pH values. However,

there is some evidence that the forms actually used by the bacteria are nonionized

ammonia (NH

) and nitrous acid (HNO

). Also note that 2 mol of oxygen is used and

that 2 mol of strong acid is produced from 1 mol of weak acid.

Known organisms carrying out the ﬁrst (nitritiﬁcation) step are the proteobacteria

Nitrosomonas (b), Nitrosospira (b), and Nitrosococcus (g). The second (nitratiﬁcation)

step is performed by the proteoba cteria Nitrobacter (a), Nitrococcus (g), and Nitrospina

(d), and by Nitrospira, a member of the Xenobacteria. Thus, although all of the nitriﬁers

were once included in the same family because of their activities, it is now reco gnized that

they are phylogenetically diverse. Also, at one time most nitriﬁers were assumed to be

either Nitrosomonas or Nitrobacter. However, it is now recognized, based on genetic tech-

niques, that although these are the most often cultured species, they are not necessarily the

most common or most active in the environment. Hence, nitrifying activity should not be

assigned to these genera unless they are actually identiﬁed.

As shown in Figure 13.22, ammonia monooxygenase (AMO) is the enzyme responsi-

ble for catalyzing the ﬁrst reaction of nitriﬁcation, in which ammonia is oxidized to hydro-

xylamine. Hydroxylamine oxidoreductase then produces a transi ent intermed iate

Nitrogen

State

Species

Hydroxylamine

Oxidation

Nitroxyl

Nitrogen

Nitrite

Ammonia

-1

-3

NOH

Monooxygenase

+3+3

+1+1

-3

−3

−1

OHNH

NOHNOH

(AMO)

Ammonia

oxidoreductase

Hydroxylamine

(HAO)

Nitrogen

State

Oxidation

Species

Nitrogen

Nitrate

Nitrite

−

Nitrite

Oxidase

−

(a)(b)

Figure 13.22 Steps in autotrophic nitriﬁcation: ðaÞ ammonia oxidation; ðbÞ nitrite oxidation.

422

MICROBIAL TRANSFORMATIONS

(nitroxyl) while forming nitrite . Luckily, hydroxylamine itself rarely accumulates, as it is

a potential mutagen. Nitrite oxidation is catalyzed by nitrite oxidase.

AMO shows some similarity to methane monooxygenase (MMO), the enzyme used by

methanotrophs (Section 10.5.6) to oxidize methane. In fact, many ammonium oxidizers

and methanotrophs can aerobically cometabolize each other’s substrate as well as a num-

ber of other compounds, including trichloroethylene.

Compared to oxidation of organic compounds, relatively little energy is available to

nitriﬁers. It takes about 35 mol of NH

for ammonia oxidizers to ﬁx 1 mol of CO

Nitrite oxidizers require even more substrate, about 100 mol of NO



per mole of CO

ﬁxed. Since the nitrite typically comes from the ammonia (100 mol of ammoni a produces

100 mol of nitrite), this means that ammonia oxidizers are usually more abundant than

nitrite oxidizers. Also, cell yields based on their energy source are much lower for nitri-

ﬁers than for most heterotrophs, often in the range 5 to 20% rather than 50 to 60%.

The fact that nitriﬁers appear to belo ng to only a few genera suggests that there may be

more limitations on their activity than would be true if they were more diverse. In fact,

compared to many heterotrophs, nitriﬁers are slow growing. Under ideal conditions, mini-

mum doubling times are around 8 hours. Also, there are no known thermophilic nitriﬁers,

so that autotrophic nitriﬁcation does not occur in systems with temperatures above

42



C. Furthermor e, since there are no known sporeformers, elevated temperatures actu-

ally kill the nitriﬁers; this means that activity is slow to return to a system (being depen-

dent on reinvasion or reinoculation) even once elevated temperatures decrease. Optimum

temperature is usually around 28 to 30



C, and activity is usually minimal at temperatures

below 10



Similarly, pH can be limiting. Optimum pH values are around 7.5 to 8, with almost no

activity below pH 6. This may in part be because of the unavailability of nonionized

ammonia at low pH values. Also, nitrite is more toxic at low pH, where it is present as

nonionized nitrous acid. At high pH, toxicity from ammonia becomes a problem. On the

other hand, although they are aerobic [with the exception of the anammox process, equa-

tion (13.12)], nitriﬁers can survive for prolonged periods under anaerobic conditions and

are effective at utilizing low concentrations of oxygen. In other words, they have a low K

(half-saturation coefﬁcient, Section 11.7.2) value for dissolved oxygen, typically below

0.5 mg/L. Similarly, they requi re only small amounts of their energy sources to approach

maximum activity rates (K

values for ammonia- or nitrite-N of 1 mg/L or less).

A close relationship between ammonia and nitrite oxidizers can be expected, since the

product of the ﬁrst group is the substrate for the second. Thus, the two groups are typically

located in close physical association. Ammonia oxidizers are usually more abundant,

since about three times as much energy is available from ammonia oxidation as nitrite

oxidation. Typically, only traces of nitrite are seen in the environment. Thus, nitriﬁcation

is often treated as though it was a single step, involving one group of bacteria. However,

accumulations of nitrite can occur under transient conditions, particularly since the nitrite

oxidizers appear to be a little more sensitive to low pH and high concentrations of ammo-

nia and nitrite.

Heterotrophic Nitriﬁcation Some heterotrophic bacteria and fungi are able to oxidize

nitrite to nitrate, and/or occasionally, ammonia to nitrite. This does not appear to

provide any beneﬁt to the organism and hence is considered a type of cometabolism.

Perhaps in some cases this represents as similatory nit rate reduct ion enzymes working

in reverse.

NITROGEN 423

It is generally believed that heterotrophic nitriﬁcation plays only a small role in nitro-

gen cycling. However, in some environments, such as acid soils, in which autotrophic

nitriﬁcation is severely inhibited, it may have a local effect.

13.2.3 Nitrogen in Environmental Engineering and Science

If nitrogen is second only to carbon in terms of the complexity of its cycling, it probably

also is second only to carbon in terms of its importance in environmental engineering and

science. The range of concerns includes wastewater and potable water treatment, surface

and groundwater contamination, agriculture, sludge and solid waste management, human

and environmental toxicity, and bioremediation. What is more, these problems may be

interdependent. For example, in agriculture, nitroge n is often the limiting nutrient for

many important crops (e.g., wheat, corn, cotton), so that loss of ﬁxed nitrogen is a

major concern. However, overapplication of chemical fertilizers, manures, or wastewater

treatment sludges, which typically are high in nitrogen, can lead to groundwater (from

leaching) and/or surface water (from runoff) contamination. In fact, nitrate is the leading

groundwater contaminant in the United States, mostly as a result of agricultural

practices.

Oxygen Demand As noted in equation (13.15), complete stoichiometric oxidation of

ammonium requires 2 mol of O

per mole of ammonium-N. This translates into

64=14 ¼ 4:57 mg O

=mg NH

-N oxidized to nitrat e. Because the organisms also reduce

and assimilate some N for cell constituents, the actual ratio is a little lower, typicall y

4:33 mgO

=mg NH

-N (3.22 for ammonium oxidation to nitrite and 1.11 for converting

nitrite to nitrate). This represents the N-BOD (Section 13.1.3).

Consider typical sewage (Table 13.4), with a BOD of 200 mg/L and a reduced nitrogen

content of 30 mg/L (half organic-N and half ammonium-N). During secondary treatment

(biological treatme nt required under federal law; see Chapter 16), the oxygen required to

reduce the BOD to 20 mg/L (90% removal) is 180 mg/L. For the nitrogen, typically the

organic-N is converted to ammonium, and about 5 mg/L is assimilated, leaving 25 mg/L.

If this remaining nitrogen is oxidized, it would require 25 4:33 ¼ 108 mg=L of oxygen,

an increase of 60% of the original carbonaceous demand. Some industrial wastewaters

may have much higher ammonium concentrations (even 1000s of mg/L) and thus repre-

sent even greater N-BODs.

TABLE 13.4 Typical Carbonaceous and Nitrogenous Biochemical Oxygen Demand

of Treated

and Untreated Sewage

C-BOD TKN N-BOD

Total BOD N-BOD N-BOD

(mg/L) (mg/L) (mg/L) (mg/L) (% of C-BOD) (% of total)

Sewage 200 30 130 330 65 39

Efﬂuent nitriﬁed 20 1 4.3 24 22 18

Efﬂuent unnitriﬁed 20 25 108 128 540 84

Assuming secondary treatment with 90% C-BOD removal and assimilation of 5 mg=LNH

-N. The remaining

TKN is either oxidized to nitrate (nitriﬁed) or remains unoxidized (unnitriﬁed). Total Kjeldahl nitrogen (TKN)

consists of organic-N (usually quickly ammoniﬁed) þ ammonium-N.

Based on 4.33 mg O

demand/mg NH

-N oxidized.

424 MICROBIAL TRANSFORMATIONS

Originally, biological sewage treatment plants produced nitriﬁed efﬂuents, since it was

recognized that the presence of nitrate indicated thorough stabilization of the wastewater.

However, once the BOD test became available, plants realized that they could reduce the

energy costs associated with supplying oxygen (a major portion of the overall cost of

treatment) by providing only carbonaceous removal. Thus, most U.S., plants for many

years were designed and operated so as not to nitrify.

However, this meant that ammonium was being discharged to the receiving water,

where it might also undergo nitriﬁcation and exert an N-BOD. Two classic papers in

the 1960s, one examining the Thames estuary in London and the other the Grand River

in Michigan, showed that this did in fact occur in some waters, serious ly depleting DO.

However, in some other receiving waters there was little evidence of nitriﬁcation. It now

appears that nitriﬁcation can be expected in smaller rivers and streams, where the nitriﬁers

grow mainly attached to streambed surfaces (including vegetation), and in estuaries,

where plankton ic nitriﬁers have time to grow. In larger rivers, where streambed surfaces

are relatively small compared to the water volume, nitriﬁers appear to settle out and to

grow too slowly to oxidize most of the ammonia.

Ammonium may also enter streams from agricultural runoff, stormwater from subur-

ban and urban areas (including erosion from fertilized lawns), and from natural organic

inputs, such as falling leaves and bird droppi ngs. Of course, this nitrogen also can repre-

sent an oxygen demand.

In denitriﬁcation, nitrate is used as an alternate electron acceptor to oxygen. Thus

in systems in which carbon oxidation and nitriﬁcation take place, denitriﬁcation can

result in ‘‘recovery’’ of some of the oxygen utilized for nitriﬁcation. For example, in

equation (13.10), 1 mol of nitrate is ‘‘used’’ to oxidize 1.25 mol of carbohydrate. This

nitrate required 2 mol of oxygen when it was produced from ammonia [equation

(13.15)]. If the same amount of carbohydrate were oxidized by oxygen, 1.25 mol of O

would be required. Thus, using the nitrate saves using 1.25 mol of O

, essentially ‘‘reco-

vering’’ 62.5% (100  1.25/2) of the oxygen that was used to form the nitrate. Some

advanced wastewater treatment plants that nitrify take advantage of this to reduce aeration

costs while removing nitrogen as N

Toxicity of Nitrogen Compounds If neither the treatment plant nor the receiving water

nitriﬁes, the ammonia from discharges and runoff wi ll be present in the stream. This can

be a serious problem in that nonionized ammonia can be extremely toxic to aquatic organ-

isms. It is recommended that nonionized ammonia concentrations in freshwater remain

below 0.02 mg/L (20 ppb) to protect the most sensitive water uses (such as trout produc-

tion), or below 0.04 mg/L in most other cases. (These values are an exception to the gen-

eral reporting convention for nitrogen and are based on NH

rather than NH

-N.) The

amount of nonionized ammonia is strongly dependent on pH (Section 13.2), but at the

slightly alkaline pH values of some streams, these levels can be exceeded. Thus, whether

the stream nitriﬁes or not, there may be good arguments to require nitriﬁcation within the

treatment plant, and it is becoming more common again.

Example 13.6 In Example 13.4, a water sample had 3.0 mg/L total ammonia- þ

ammonium-N. If that sample was taken from a stream below a wastewater treatment

plant, does the stream meet an nonionized NH

standard of 0.04 mg/L?

Answer The nonionized NH

concentration calculated for that example was

0.047 mg/L NH

-N. Thus, the standard is not being met.

NITROGEN 425

Ammonia is also toxic to microorganisms, including nitriﬁers, and at high levels can

interfere with treatment. As long as the nitriﬁers are active, feed concentrations to a reac-

tor can be high, but a sudden increase in concentration or pH can lead to failure. The

increased concentration of nonionized ammonia slows down nitriﬁer act ivity, decreasing

ammonia oxidation rates and leading to a rapid buildup in concentration and further

increased toxicity. Typically, well-designed and well-operated reactors can handle up to

500 mg/L (or even higher) inﬂuent total ammonia concentrations, depending on pH,

because the concentration within the reactor will remain well below 100 mg/L.

Nitrite is also highly toxic to aquatic organisms. U.S. recommendations at one time

were to maintain concentrations of nitrite-N below 0.06 mg/L, and some European coun-

tries have suggested a 0.02 mg/L limit. Fortunately, nitrite rarely accumulates in the envir-

onment, since it is an intermediate for both nitriﬁcation and denitriﬁcation. Apparently for

this reason, there is no longer a U.S. nitrite criterion.

Both nitrite and nitrate may be toxic to humans, and drinking water standards have

been set at 1 mg/L for nitrite-N and 10 mg/L for nitrate-N. Upon ingestion, nitrate

may be reduced to nitrite in the digestive tract. Once absorbed into the bloodstream, nitrite

binds (preferentially over oxygen) with hemoglobin, leading to a mild to severe asphyxia

known as methemoglobinemia. This is of particular concern for bottle-fed infants, in

whom it is called ‘‘blue baby disease’’, as it can be fatal. Nitrite can also react with certain

amines released during degradation of amino acids, forming nitrosamines, some of which

are potent carcinogens.

Nutrient Enrichment Usually, either nitrogen or phosphorus is the nutrient that limits

the amount of photosynthetic growth that can occur in a water body. Typically, while

phosphorus is limiting in lakes and reservoirs, nitrogen is the limiting nutrient in streams

and coastal waters. Since a variety of organisms can utilize each form, addition of nitro-

gen as either ammonium or nitrate whe re N is limiting can lead to excessive growth of

plants, algae, and/ or cyanobacteria, speeding the process of eutrophication (Section

15.2.6). This is a major reason that some wastewater treatment plants are required to prac-

tice some form of advanced treatment to remove nitrogen, not merel y to nitrify it.

Alkalinity Consumption As can be seen from equation (13.13), each mole of ammo-

nium oxidized to nitrite produces 2 mol of acidity. This uses up 2 mol of the alkalinity

(acid-neutralizing, or pH-buffering, capacity) of the water in which it occurs. If the

water does not initially have enough alkalinity, this will result in a drop in pH. This

can, in turn, inhibit nitriﬁcation as the pH drops as low as 5.5, as well as resulting

in other harmful effects in wastewater treatment or perhaps soil. (Ammonia concentra-

tions are usually diluted enough that this level of acidiﬁcation of a stream will not

occur.) To prevent this, nitrifying t reatment plants may have to a dd supplem ental

alkalinity to raise the pH to more desirable or permitted levels (usually, > 6.5).

Acidity produced by nitriﬁcation can also be one of the reasons for adding lime

(usually, it is really crushed limestone, CaCO

) to agricultural ﬁelds or fertilized

lawns and gardens.

Example 13.7 In a wastewater treatment plant, 20 mg/L of ammonium-nitrogen is

nitriﬁed to nitrate. How much alkalinity is consumed?

Answer From equation (13.13), 2 mol of alkalinity is needed for every mole of

nitrogen oxidized. By convention, alkalinity is expressed as CaCO

equivalents, with

426 MICROBIAL TRANSFORMATIONS

2 m ol of acid neutralized per mole (100 g). Thus 1 mol of CaCO

is needed for every

mole of ammonium:

Alkalinity consumed

¼ 20 mg=LNH

-N

1 mol NH

-N

14 g NH

-N



1 mol CaCO

1 mol NH

-N



100 g CaCO

1 mol CaCO



¼ 20ð7:14 mg CaCO

=mg NÞ¼143 mg=LCaCO

alkalinity

Denitriﬁcation produces 1 mol of alkalinity for each mole of nitrite or nitrate reduced

to nitrogen gas [e.g., equations (13.10) and (13.11)]. Thus half of the alkalinity lost

through nitriﬁcation can be recovered by denitriﬁcation while removing the nitrogen

(as a gas) from the syst em. This will often eliminate the need for alkalinity addition in

wastewater treatment, but would usually be undesirable in agricul ture, where the nitrogen

loss is typically a conce rn.

Disinfection Nitrite and chlorine rapidly react to form nitrate and chloride, which has no

disinfecting power. For example, in drinking water, where hypochlorous acid is the pre-

dominant active chlorine species;



þ HOCl ! NO



þ H

þ Cl



ð13:16Þ

Thus, every milligram of NO



-N uses up 71 =14 ¼ 5:1 mg of active chlorine (which is

always expressed on the basis of Cl

, with molecular weight of 71).

Ammonia also reacts with chlorine, but the products depend on the relative concentra-

tions of the two. At low Cl/N molar ratios ( 1), mainly monochloramine (NH

Cl),

forms:

þ HOCl ! NH

Cl þ H

O þ H

ð13:17Þ

As the Cl/N ratio increases, dichloramine (NHCl

) forms:

Cl þ HOCl ! NHCl

þ H

O ð13:18Þ

Mono- and dichloramine are referred to as combined chlorine. Although they still repre-

sent active chlorine, they have much less disinfecting power than HOCl (1to2%as

effective). This is one reason that disinfection of was tewater (which usually contains

ammonia) is less effective than disinfection of drinking water (which typically does not).

Finally, at still higher ratios, the chlorine oxidizes the ammonia to produce nitrogen gas

or nitrate, or occasionally, trichloramine (NCl

), depending on pH and the speciﬁc ratio.

NHCl

þ HOCl ! N

and=or NO



and=or NCl

ð13:19Þ

This is referred to as breakpoint chlorination (Figure 13.23), and these products have

no disinfecting power. Thus, depending on the Cl/N ratio, adding more chlorine can actu-

ally result in a decrease in the remaining active chlorine, or chlorine residual. Once all of

the ammonia is converted to nitrogen gas, nitrate, or trichloramine, any additional chlor-

ine addition results in an increase in the chlorine residual.

NITROGEN 427