Inhibition of Nitrification by Ammonia and Nitrous Acid

Author(s): A. C. Anthonisen, R. C. Loehr, T. B. S. Prakasam and E. G. Srinath

Source: Journal (Water Pollution Control Federation), Vol. 48, No. 5 (May, 1976), pp. 835-
852
Published by: Water Environment Federation
Stable URL: http://www.jstor.org/stable/25038971
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 Inhibition of nitrification by ammonia
and nitrous acid
A. C. Anthonisen, R. C. Loehr, T. B. S. Prakasam, and E. G. Srinath

pH, temperature, microbial numbers, ac

THE IMPORTANCE OFmore
been recognized for NITRIFICATION
than a cen has
tury. Engineers and scientists continue to
investigate the process to obtain a better
understanding of the fundamentals and to
use the process for the benefit of man.
Controlled nitrification may occur during
waste treatment, and engineers have begun
to incorporate the process as part of nitri
fication-denitrification systems for nitrogen
control preceding discharge of the treated
wastes.
The effectiveness of biological nitrogen
control processes depends on the ability of
nitrifying organisms to oxidize ammonia to
nitrate. The interaction of factors such as
climation, and inhibiting compounds influ
ences the rate at which nitrification takes
place. This research focuses on ammonia
and nitrite, two intermediate products
formed during the conversion of organic
nitrogen to nitrate nitrogen, and examines
their inhibitory effects on the nitrification
process.
Although seven genera of autotrophic
nitrifying organisms have been identified,1
species belonging to the genera Nitro
somonas and Nitrobacter are responsible
for most of the naturally occurring nitrifica
tion. The process may be described as

Organic
nitrogen--^ NH4+- N02- -?:-^ N03"
compounds beterotrophs nitrosomonads nitrobacter

Ammonification Nitrification

With adequate dissolved oxygen (do) of

Inhibition and
nitrification generally is not
no inhibition, nitrosomonads oxidize
a problem in amthe treatment of domestic
wastewater.
monia to nitrite and nitrobacters oxidize The thereduced forms of nitro
nitrite to nitrate. Complete nitrification
gen are generally low in concentration and
are oxidized
occurs in many situations. Results typicalcompletely with perhaps only
of batch nitrification without inhibition are
a transient accumulation of nitrite being
noted in Figure 1. observed. When there is a high concen
In most environments, any accumulation
tration of reduced nitrogen in the untreated
of nitrite has been considered wastes,
transient.
however, complete nitrification may
not occur. Inhibition
Incomplete nitrification with nitrite accu has been noted in the
mulation has been observed in activated
many sludge situprocess,2 laboratory ex
ations, however. Typical patterns
perimentsfromon the nitrification process,3'4
batch experiments in which nitrite
basic and oxida
applied soil studies,5-7 application
tion was inhibited are shown in Figure
of dairy cattle 2.
manure to soil,8 aeration of
Under some conditions both ammonia oxiand aeration of weak am
poultry waste,9
dation and nitrite oxidationmoniahave been
liquor.10
inhibited. Although many compounds may affect

-Vol. 48, No. 5, May 1976 835

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Anthonisen et al.

from observations of nitrification inhibition

in this laboratory and those reported in the
literature, (6) undertake batch and con
tinuous flow experiments to substantiate
the hypothesis, and (c) corroborate the
hypothesis and experiments with results
from independent studies. Although spe
cific wastewaters were used in this study,
the results are sufficiently general and may
be applied more broadly.
Hypothesis. Preliminary investigations
indicated that nitrites may accumulate in
nitrifying systems. Depending on operat
TIME ing conditions and initial loading rates,
varying nitrite concentrations will persist
FIGURE 1.?Schematic of nitrogen trans
without subsequent oxidation to nitrate,
formations during noninhibited nitrifica
especially in solutions or wastewater hav
tion, batch experiment.
ing high organic or ammonia nitrogen con
centrations. It was postulated that the in
hibition was related to concentrations of
the rate of nitrification, it seems that the un-ionized ammonia [free ammonia, NH3
two most important are ammonia and ni (fa)] and un-ionized nitrous acid [free
trite. When nitrobacters are the more nitrous acid, HN02(fna)].
severely inhibited, nitrite will accumulate. The reactions in the nitrification process
Ammonia also accumulates when no nitro (Figure 3) illustrate the factors involved.
somonads are present. A portion of the organic nitrogen entering
Meiklejohn 3 reviewed the inhibitory ef an aerobic waste treatment system will be
fects of many compounds and noted that converted to ammonia through biological
deamination. The ammonia will be in
The electrolytes to which the nitrifiers solution as the ammonium ion ( NH4+ ) and
are sensitive include their own sub un-ionized ammonia ( NH3 ). These will be
strates, and in each case, the substrate in an equilibrium that is affected by the
of the other species was found to be pH of the solution. When the pH in
creases, the concentration of the un-ionized
much more toxic than the organisms'
own substrate. Nitrite depressed both
respiration and growth of Nitro z
somonas. . . Nitrobacter was sensitive o
to the ammonium ion, but even more <
rr
so to free ammonia . . . Nitrate . . .
Z
was only slightly toxic to both species. L?
O
Z
Data from the above and other studies 11_14 8"
indicate that ammonia and nitrite concen Z E
UJ
trations are related to the inhibition of
o
nitrification. The pH of the solution or oc
soil also was related to the degree of in
hibition. E / XNOi
_L._
0
TIME -
Research
FIGURE 2.?Schematic of nit
General. The research approach was to with inhibition of nitrite oxida
(a) develop a guiding hypothesis or model experiment.

836 Journal WPCF

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ammonia will increase. The pH increase
may occur naturally or by the addition of
a base.
It was postulated that, at certain concen
trations, un-ionized ammonia (fa) is com
pletely inhibitory to both nitrosomonads
and nitrobacters and that at lower concen
trations fa will be less inhibitory to nitro
somonads than to nitrobacters. The latter
situation will cause nitrite to accumulate
and persist.
As nitrite oxidation occurs, there is a re
lease of hydrogen ions that decreases the
pH to an extent related to the buffering
capacity of the system. The nitrite formed
will exist in equilibrium with un-ionized
nitrous acid (fna). As the pH decreases,
the concentration of fna will increase.
Two processes work to reduce fa in
hibition. As the pH decreases, the am
monia equilibrium will adjust and the con
centration of fa will decrease. In addition,
the total ammonia concentration will de
crease as it is oxidized to nitrite. These
reductions tend to relieve inhibition of the
nitrobacters caused by fa, promoting oxi
dation of nitrite to nitrate.
There are, however, situations in which
nitrite is not oxidized to nitrate under con
ditions generally favoring complete nitrifi
cation, that is, in the absence of fa and
with adequate aeration. The continued
presence of nitrite in the absence of fa
was postulated as being a result of fna
inhibition because as the pH decreases
occur as a result of ammonia oxidation the
fna concentration increases. It was hy
pothesized that certain concentrations of
fna would be inhibitory to both the nitro
somonads and nitrobacters.
These conditions may be portrayed
graphically (Figure 4) to indicate the fac
tors that are involved and to identify situ
ations in which nitrification inhibition will
or will not occur. Figure 4 does not pre
sent fa and fna concentrations directly,
but as a relationship between pH and the
concentrations of total ammonia and nitrite
nitrogen, respectively. Total ammonia and
nitrite nitrogen may be measured directly
by available analytical methods. The fa
and fna concentrations may be determined
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Nitrification
ORGANIC
AMMONIA EQUILIBRIUM
NITROGEN NH++OH';=?
_ NH3+ H20
NITROUS ACID EQUILIBRIUM
NH4+ 1.5 o2?*-h2o+h+ + |h++ N0;?=?H
N0? + 0.5 02-*-NOJ
FIGURE 3.?Nitrification reactions
equilibria.
by knowing the total ammonia and nitrite
nitrogen concentrations and by using equa
tions or figures in the Appendix.
The total ammonia nitrogen and the ni
trite nitrogen concentrations are plotted on
a logarithmic scale, while pH is repre
sented on a linear scale. Figure 4 permits
fa and FNA relationships to approximate
straight lines at different but interrelated
values of pH and total ammonia nitrogen,
and of pH and nitrite nitrogen.
Presenting the relationships in this man
ner provides an operational chart that may
be used to evaluate a nitrifying system.
Ammonia nitrogen, nitrite nitrogen, and
pH data may be plotted to determine
whether inhibition to nitrification does or
is likely to exist. Conditions to relieve any
inhibition also may be identified.
Based on the concepts presented earlier,
there should be at least four zones on the
chart. Zone 1 represents the condition
when the fa concentration is high enough
to inhibit both nitrosomonads and nitro
bacters. No nitrification will occur and
ammonia will accumulate in the system.
At lower concentrations of fa, only nitro
bacters may be inhibited and nitrite ac
cumulation will occur. This condition is
represented by Zone 2.
At still lower fa concentrations, neither
nitrobacters nor nitrosomonads will be in
hibited and complete nitrification will occur
(Zone 3). In the absence of any fna
inhibition, complete nitrification should
represent conditions to the left of line [B].
Inhibition of nitrification by fna may occur,
and Zone 4 represents the latter condition.
The boundaries of the zones are noted as
[A], [B], and [C] in Figure 4 as sharp
separations. Because of factors such as
-Vol. 48, No. 5, May 1976 837
Anthonisen et al.

o
o
z rr
?J
o z
o
rr <
i z
o
?J
<
rr

o
O
o

INCREASING pH -*
FIGURE 4.?Postulated relationships o
nitrous acid inhibition to nitrifying

acclimation, numbers In of the predictive

active phas
organisms,
and the effect of temperature on to
chart was used reaction
assess
rates, however, it is likely that
changes boundary
necessary to c
conditions will consist of ranges
hibitory rather than
conditions. The
sharp separations. were particularly val
Research approach. thatTotheidentify
inhibitorythe
cond
numerical values ofcould
the boundaries noted
be controlled.
in Figure 4, values ofusing
fa andthe
fnaoperational
that were
hibitory
inhibitory to nitrification wereconditions
determined.we
lieved.
The data were collected Theseseparate
in three studies
phases: exploratory, predictive,
organisms and
were notcor
per
and would resume
roborative. The exploratory nitr
and predic
inhibition over
tive studies were conducted was removed
a 4-yr
The third
period under laboratory, pilotphase,
plant, corr
and
field conditions. included data from exp
The exploratory phase included batch
by independent resear
and continuous flowcultural wastes,
laboratory and
and pilot
plant aeration units in which
These nitrite
data were not ac
spe
cumulated. Data from
ward the exploratory
the phenomenon
phase permitted thetion; however, of
formulation thea dat
de
for similar
tailed operational chart the purpose of corr
to Figure 4.

838 Journal WPCF

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 Nitrification

The following are the experiments con other wastes used in the exploratory and
ducted in each of the phases: predictive phases were solutions of the
actual wastes noted. The weak ammonia
I. Exploratory studies
liquor was obtained from a coking plant
A. Batch systems
in Pennsylvania.
1. Laboratory scale: The data encompass a broad range of
a. Synthetic wastes
microbial environments, that is, agricul
b. Dilute poultry manure tural, industrial, and municipal waste
c. Stored oxidation ditch mixed
waters and the soil. This variety helped
liquor to identify the common basis of nitrification
2. Pilot plant scale:
inhibition in biological systems.
Oxidation ditch-poultry manure
Analyses. All experiments in the ex
B. Continuous flow systems
ploratory and predictive phases took place
Laboratory scale: Dilute poultry
manure at ambient temperatures. The analyses
II. Predictive studies performed for each experiment depended
on the nature of the experiment. Analyses
A. Batch systems included total solids, volatile solids, chem
1. Laboratory scale: ical oxygen demand (cod), total Kjeldahl
Dilute mink manure
nitrogen (tkn), NH4+-N, N02-N, NO3-N,
2. Pilot plant scale: pH, do, oxygen uptake rates, and micro
Oxidation ditch-mink production
wastes scopic observations.
3. Full scale: The analytical procedures used were
those described by "Standard Methods" 15
Oxidation ditch-poultry manure and Prakasam et al.16 The do concentra
B. Continuous flow systems
tions and oxygen uptake rates were deter
1. Laboratory scale:
mined with a do probe, cod was analyzed
Weak ammonia liquor by the rapid method.17
2. Pilot plant scale: It is convenient to refer to the organisms
a. Oxidation ditch-poultry ma responsible for converting ammonium to
nure
nitrite as nitrosomonads and those that
b. Diffused aeration-poultry manitrite to nitrate as nitrobacters.
convert
nure
3. Full scale: When these terms are used, it is not in
tended to imply that there may not have
a. Oxidation ditch-poultry ma been other genera of nitrifying organisms
nure
that were involved. Specific bacteria were
b. Oxidation ditch-dairy not
cattle
isolated or identified. Nitrification in
manure
III. Corroborative studies the poultry manure systems was shown to
be autotrophic 22 and it was assumed to be
A. Batch systems
autotrophic in the other systems as well.
1. Laboratory scale:
Nitrification studies Results and Discussion
2. Pilot plant scale: General. Data from the combined ex
Flushing system-poultry manure
B. Other systems periments were voluminous and would
1. Soil nitrification studies result in excessive length and detail if
presented in this paper; however, a detailed
2. Agricultural waste treatment
studies description of each experiment is avail
3. Municipal waste treatment able.18 This section will present the over
studies all results and indicate how the data were
obtained and used.
The synthetic waste was a mixture of The data identifying potentially inhibi
various concentrations of soluble organics, tory fa or fna concentrations were obtained
ammonium sulfate, and the mixed liquor in the following manner. The boundaries
from a nitrifying oxidation ditch. The of the zones in Figure 4 may be viewed in

-Vol. 48, No. 5, May 1976 839

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Anthonisen et al.

two ways: concentrations at which inhibi cumulated and the pH decreased,

tion begins or concentrations at which in concentration increased to a value
hibition is relieved. The view depends on hibited the nitrobacters. At this poi
the condition of the system being evaluated. ammonia concentration no longer de
For example, if a system is in Zone 1, it and nitrite concentration remain
may be expected that, when conditions stant. The pH and nitrite concentr
change to decrease the fa concentration this point were used to help define
such that it is located to the left of bound ary [A].
ary [C], inhibition to nitrosomonads should Many factors were considered before
be relieved. Similarly, if the system is in specific experimental results were used to
Zone 3 and the fa concentration increases define boundaries [A] and [C]. All units
so that it is located to the right of boundary were seeded with organisms from an active,
[B], inhibition of nitrobacters may com completely-nitrifying unit, and it was as
mence. sumed that any lag before ammonia oxida
Both batch and continuous flow units
tion was caused by inhibition rather than
acclimation. If there was no initial delay
were used in the exploratory and predictive
phases. Typical results (Figure 5) in the ammonia
from a oxidation, it was assumed
batch unit illustrate how boundary thatcondi
the initial fa concentration was below
tions [A] and [C] were defined. Each level. If the ammonia con
the inhibitory
batch unit was seeded with activelycentration
nitrifyreached zero when the nitrite
concentration reached a constant level, it
ing sludge from a well-defined, completely
nitrifying control unit. The initial was assumed
am that lack of substrate rather
monia concentrations varied in the than
units.
an inhibitory fna concentration caused
the nitrite
Initially ammonia was lost from the sys to remain constant. Because
tem by volatilization and reducedeach unitfa
the was seeded with mixed liquor
concentration to a level that permitted
from a completely nitrifying unit, it was
assumed to
nitrosomonads to oxidize the ammonia that both nitrosomonads and ni
nitrite. The pH value and total ammoniawere present and could accom
trobacters
concentration at the beginning ofplish nitrification unless they were in
nitrite
accumulation were used as valueshibited.
to help Data from the predictive phase
define boundary [C]. As the nitrite ac latter assumption.
verified the
Similarly, data from such studies also
could be used to identify when inhibitory
^-VALUES NOTING conditions were relieved. Judgment was
/ BOUNDARY [C]
an important factor in determining when
^VALUES NOTING data were applicable to define the three
/ BOUNDARY [A] boundary conditions.
Typical results from continuous flow sys
tj -PH tems (Figure 6) illustrate how data were
1^-NO?-N obtained to define the boundary conditions.
Periodic increases in nitrite concentration
occurred as the fa concentration fluctuated.
In Figure 6, the fa concentration at which
the nitrite accumulation began was as
-?NHj-N sumed as that inhibitory to nitrosomonads
and as a value defining boundary [B]. In
- _N0-3-N a comparable manner, fa or fna values
TIME -? defining boundaries [C] and [A] could be
obtained from analyzing deviations from
FIGURE 5.?Patterns of nitrification in a
equilibrium conditions.
batch system with data identifying bound phase, continuous flow
In the predictive
aries [A] and [C.] systems were allowed to establish equi

840 Journal WPCF

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 Nitrification

z
o

<
UJ
UJ
rr

300 h

E
Z 200
O
<
rr

uj 100 /BEGINNING OF
o
z
o N02-N
N02-N ACCUMULATION
o

97 98 99 100 101 102 103 104 105 106 107 108 109 110 III
DAY OF STUDY
FIGURE 6.?Transient accumulation of nitrite in continuous-flow study, Unit

librium conditions in one of the fourcontents

zones were diluted such that the fn
and then conditions, generally pH,concentration
were was less than 0.1 mg/1. Both
adjusted purposefully to have conditions
nitrifying organisms responded. Increase
ammonia
representative of another designated zone oxidation occurred with a simu
taneous decrease in pH. This change in
occur. Such adjustments yielded additional
data on the boundary conditions and veri that the nitrifying organisms were
dicated
fied that the inhibition could be controlled
not permanently inhibited by the inhibitor
fnaand
by varying the process parameters concentrations.
operating conditions. In the continuous flow laboratory studie
Exploratory phase. The studies identi
(IB), units maintained with an fa concen
fied previously as the exploratory tration
phase greater than 0.3 mg/1 continuall
were used to identify the fa and fna accumulated
con nitrites, while units in whic
centrations describing boundaries [A],the
[B],fa concentrations were less than 0.
and [C]. Boundary concentrations mg/1 obconsistently had oxidation of the i
tained from these studies are summarized
fluent ammonia to nitrate and showed only
in Table I. transient accumulations of nitrite. Thes
During most of the pilot plant-scale studies
oxi (IB) demonstrated that nitrifyin
dation ditch study (IA2), the fa or fna
systems placed in a mode of nitrite a
concentrations were greater than 1.0 cumulation
mg/1 (Zone 2) could be returned t
and apparently in a range that resulted in
a nitrate-accumulating mode (Zone 3) by
inhibition of nitrobacters. Nitrites accumu
relieving the fa inhibition.
lated, and at no time did nitrates increase.
The data in Table I were portrayed in
diagram
In the latter portion of the study, the ditch similar to the operational char

-Vol. 48, No. 5, May 1976 841

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Anthonisen et al.

TABLE I.?FA and FNA Concentrations Obtained from Exploratory Studies that
Defined Inhibitory Boundary Conditions

Boundary
Unit Study* Unit
[A] [C]
FNA (mg/1) FA (mg/1)

1 0.6 IB
2 1.4 7
3 0.5 140
1 0.4
2 1.6
3 1.0 31
4 0.5 57
5 0.8 90
6 1.5
7 1.5 11
8 1.6 27
9 2.4 44
10 64
1 145
2 13
3 0.2 16
4 23
7 0.3 28
10 0.2 150
11 0.6 142

* Notation refers to that used in the identification of research components, that is, I Ala is the batch labor
tory study using synthetic wastes in the exploratory phase.

(Figure 7). Each boundary conditionofisammonia concentration evaluated and

shown as a band rather than a sharp by sep testing the hypotheses with additional
aration. The width of the band is influ types of wastes. The data resulted from
enced by the many factors other thanlaboratory-,
fa pilot plant-, and full-scale units.
or fna inhibition that affected the bio Figures 8 and 9 illustrate typical results.
logical systems. The ambient tempera Study IIA1 was a laboratory batch study.
tures of the systems ranged from 10?Ato unit with an initial total ammonia nitro
23?C. The active mass of the nitrifying gen concentration of 4,100 mg/1 was seeded
organisms in the systems may have varied.with the mixed liquor from a completely
The widths of the boundary conditionsnitrifying
be unit and the nitrogen transforma
tween the zones are [A], 0.2 to 2.6 fna tions monitored. When the unit demon
mg/1; [B], 0.1 to 1.0 fa mg/1; and [C],strated
10 fna inhibition, the mixed liquor
to 150 fa mg/1. was separated into units A and B. Unit A
Predictive phase. These studies were was maintained as a control and the pH of
made to verify the boundaries identifiedunit
in B adjusted to lower the f,na concen
the exploratory phase. In each study, contration. When the fna inhibition was re
ditions were specifically adjusted to bring
lieved ( unit B ), the nitrite was oxidized to
nitrate (Figure 8). The control unit con
about different nitrification patterns. The
boundaries in Figure 7 were used to tinuedan to have the fna concentration in the
ticipate conditions of inhibition and then
inhibitory range, and no nitrite oxidation
to relieve the inhibition by adjusting occurred.
the
total ammonia or nitrite nitrogen concen Figur? 9 represents data from a continu
tration and/or the pH. These studies ous ex study in which the pH and total am
panded the results obtained in themonia
ex concentration were adjusted to
achieve an inhibitory fa concentration and
ploratory studies by increasing the range

842 Journal WPCF

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 Nitrification

10 10"
Ti?rn?rrn?i?n?i?i?r
\ \ \ / * '
\ \ \ / \ / \ /
\ \ \ i V
\ \ y A
\x \\ A
' \ /' Vfa
' \ I50mg/I
_ 10" \ \ i \ I \ 10"
E
O?
E
x X \ \ O
o

\/\?\\'
Z er
UJ
o
o
CE

z
A ^ 10 mg/1
\ \A\
o
UJ
FNA ? r.\. i \ ^ H
2
<
2.8 mg/1
? io2 H10" \ *
\ ? ^ 1.0 mg/1 <
-J

\ h

/ - V-- x \
/ 1 ^?
/ \_
\
\
/
FNA
I 02 w^ ^A^p^ [BJ \ \
10 1_I 1/ I_I_l_Vl_k\j_I_ 10
PH
FIGURE 7.?Boundary conditions identified with data from
exploratory phase of research.

thus modify the conditions from Zone 3 to
Zone 2 (Figure 4). The dashed line repre
sents the expected boundary condition be
tween the two zones. After the adjustment,
nitrite was not oxidized completely, indi
cating that the nitrobacters were inhibited
as a result of the change. In this case, only
partial inhibition occurred because some
nitrate nitrogen was produced.
The study with the weak ammonia liquor
(IIB1) was done to see if the hypothesis
was applicable to a waste primarily of an
inorganic rather than organic nature and
because difficulties in obtaining complete
nitrification of weak ammonia liquor have
been reported.10 By operating the unit to
avoid nitrification inhibition conditions and
maintaining a 20-hr hydraulic detention
time and 100 percent sludge recycle to
retain adequate amounts of nitrifying or
ganisms, the system accomplished complete
nitrification under equilibrium conditions.
Table II represents the average of analyses
obtained during the 2-wk equilibrium pe
riod. The rate of ammonium oxidation
during this period was 52 mg/l/hr. A sig
nificant aspect was the development of a
population of nitrifying organisms that was
capable of completely oxidizing an indus
trial waste with 1,200 mg/1 total ammonia
nitrogen to nitrate nitrogen.
The data in Table III summarize the fa
and fna concentrations obtained in the
predictive phase that described the bound

-Vol. 48, No. 5, May 1976 843

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Anthonisen et al.
300
UNIT A UNIT B
200h V^NH4-N NO ADJUSTMENT pH ADJUSTMENT
TO REDUCE FNA

200

-N0,-N

E 100 ^NH4"N
z
o
K
< BOUNDARY CONDITIONS <

i rV'r_j
QC
AS IN FIGURE 7-\
?J
?
O
Jj 10 15 20 25 30 35 40
o O
o
1.0

o.ifc-*?-^

.01 J_L
10 15 20 25 30 35 40 10 15 20 25 30 35 40
DAY OF STUDY

FIGURE 8.?Data from Study IIA1, predictive

BEFORE pH ADJUSTMENT AFTER pH ADJUSTMENT

10 E

EXPECTED BOUNDARY [b]-"^

_?L_J_I_l_I_I_I_L
Z 400
O
<
?z 300 UJ
S
I
Id
O NOj-N
z
Q
g 200 <

100

85 87 89 91 -u? J N02-N
I 1.
93 109 125 127
I I 1129 131 133
DAY OF STUDY
FIGURE 9.?Effect of un-ionized ammonia on nitrite accumulation, continuous
flow predictive study.

844 Tournai WPCF

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 Nitrification

TABLE IL?Equilibrium Data of Continuous with those from the above phases. The
Flow Predictive Study IIB1?Weak studies documented nitrogen transforma
Ammonia Liquor
tions in soil, municipal waste treatment,
Concentration (mg/1) and agricultural waste treatment. Al
Parameter though these studies were not directed
Influent
Unit Mixed Clarifier specifically at nitrification inhibition, they
Liquor Effluent
contained data that could be evaluated in
NH4-N 1,170 0 0 terms of fa and fna and their effect on
Organic 30 94 Trace nitrification.
TKN 1,200 94 0
N02-N 2.1 1J 1.4 The studies of Duisberg and Buehrer19
NO4-N 0 1,200 1,200 determined the comparative influence of
Suspended injection and surface application of fer
Solids 113 2,050* 80 tilizer ammonia on nitrification in a sandy
loam soil. The tests were conducted in the
* 76.5 percent volatile. laboratory and greenhouse at incubation
temperatures of 30?C. The fa concentra
tions from this study were calculated by
ary conditions. These data were incor using the equations in the Appendix and
porated (Figure 10) in the diagram de temperature of 30?C. The effect of tem
veloped from data of the exploratory phase. perature on the fa concentration is sig
The boundary lines noted in Figure 10 are nificant. The fa concentration at 30 ?C is
the same as those in Figure 7. The data about twice that at 20 ?C for the same pH
points from the predictive phase (Table and total ammonia nitrogen concentration.
III) fall within the predicted boundary of No nitrification occurred until the fa
inhibition, thus reinforcing the basic hy levels decreased to the levels noted as
pothesis. boundary [C] in Table IV, at which time
Corroborative phase. This corroboration nitrite began to accumulate. At fa con
was done after completion of the above centrations to the left of boundary [B],
phases and was done to determine the nitrite was oxidized to nitrate. At no time
degree to which the previous results could did fna concentrations increase to the in
be extended. Studies in the literature that hibitory level (Zone 4). The fa concen
contained data on nitrification inhibition trations that defined boundaries [C] and
were examined and the results compared [B] are presented in Table IV.

TABLE III.?FA and FNA Concentrations Obtained from Predictive Studies that
Defined Inhibitory Boundary Conditions

Boundary
Unit Study* Unit
[A] [C]
FNA (mg/1) FA (mg/1)

2.1 56 IIB1
2.0 76
0.6 64
2.3 60
1.2 110
1.3 80 IIB3b
1.8 50
0.4
1.0
0.3
0.8

* Notation refers to that used in the identification of research components, that is, IIA1 is the batch
laboratory study using dilute mink wastes in the predictive phase.

-Vol. 48, No. 5, May 1976 845

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Anthonisen et al.

ti?rn?rrn i \?i n i?"?4

\\ \
\\ i \\i/
\/
\ \ \/ X
\ \ '\ / \ O?
E
.O3?JZ
O
O
?C

V: V \ x o
2

A*/
/ V "\Nx
<

10* <

' / \ x I
O

/ M / \ ? \ \
' / N ?N \
/ / \ x x
i '_U_l_1_I
6 7
M ! M_1 10

pH
FIGURE 10.?Boundary conditions identified with data from
predictive phase of research.

The studies of Broadbent et al.20 evalu
ated the transformation and movement of
urea in soils. Urea nitrogen was added to
14 samples of California soils, and the
samples were incubated at 24 ?C for 8 wk.
Twelve samples demonstrated some inhibi
tion as persistence of either ammonia or
nitrite. The fa and fna concentrations in
these samples generally were in the in
hibitory range.
Few municipal waste treatment studies
contain data illustrating nitrite accumula
tion. The research of Taylor et al.,21 how
ever, does include data from laboratory
experiments to determine the effect of in
creasing ammonia concentrations on the
activated sludge process. Results of their
Experiments 2 and 3 are presented in Fig
ure 11. Both experiments demonstrated
nitrite accumulation when the fa concen
trations were in the inhibitory range. As
the nitrites accumulated, the fna concen
tration increased and reached concentra
tions that were within boundary [A]. In
hibition of nitrosomonads by fna may have
caused the total ammonia accumulation in
these units.
Three studies dealing with the aeration
of poultry wastes18' 22>23 had fa or fna
concentrations that apparently reached in
hibitory levels, resulting in either the ac
cumulation of nitrite or ammonia or the
cessation of ammonium oxidation. Data
from these studies that helped identify the

846 Journal WPCF

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 NITRIFICATION

boundaries between the four zones are in TABLE IV.-FA and FNA Concentrations
Table IV. Obtained from Corrobative Studies
that Defined Inhibitory
The data from Table IV were placed Boundary Conditions
(Figure 12) on the operational diagram.
The boundary lines on Figure 12 are the Boundary
same as those on the previous diagrams Study
(Reference No.) [A] [B] [C]
(Figures 7 and 10). The data from the FNA (mg/I) FA (mg/i) FA
corroborative phase fit well with the results
from the previous studies. 19 2.7 24
3.5 32
SUMMARY 2.5 21
4.6 24
The accumulation of nitrite during the 20 131
biological oxidation of ammonia is not a 21 0.8 _
recently discovered phenomenon. An ob 0.3
18 0.3 _ 50
servation of such accumulation was noted 22 0.5 _ 20
almost a century ago (1879), before the 23 2.8 100
isolation of the nitrifying organisms.24 Ni 1.7 28
trite accumulation has since been observed
to occur in a variety of microbial habitats
such as the soil, natural waters, and sys inhibition of a
tems treating municipal, industrial, and tion by un-io
agricultural wastes. Such accumulations acid. The rese
generally have been explained as the result preparation of
of a difference in the reaction rates of the described inhib
ammonia- and nitrite-oxidizing organisms. concentrations
The described research indicated that nitrogen, nitr
accumulation of ammonium and nitrite ions separate zones
in biological systems could be the result of of nitrification

1.0 LIMITS OF A
BOUNDARIES FA F N
[A]
- -- -- V~~~~~~

o .01 L in ,B]FNA
0
.t 400
I-~N03-
Z ~ NON 2
lWi 300\
0

o 200

100 NO3-N
- ~~~~N2-N N4- N
0 100 200 300 400 500 0 100 200 300 400 500
CONCENTRATION OF AMMONIA IN FEED, mg/l
FIGURE 11.-Data of Taylor et al.21

Vol. 48, No. 5, May 1976 84

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Anthonisen et al.

10

\/\/\A\--
v \/ v x 5
1 x / \" \ Ce] 1 ^

/ / \ N
I I \ \ \
/ M / \ [b] x
<

\
I I \ \ \
__?_i_a_i_i_lA
5 6 7 8 9
PH
FIGURE 12.?Data from corroborative phase.

The data from all experiments reported in
this paper are presented in Figure 13.
The fa inhibited nitrobacters at concen
trations substantially lower than those that
inhibited nitrosomonads. It was this dif
ferential that caused nitrites to accumulate
without subsequent oxidation to nitrate.
The inhibition by fa or fna was not per
manent and could be relieved by adjusting
the operational conditions such that the fa
or fna concentrations were less than in
hibitory concentrations. Dilution, pH ad
justment, and denitrification, specifically for
fna, were successful in reducing the in
hibitory concentrations.
Many environmental and operational
factors, some of which were identified
earlier in this paper, will affect the fa and
fna concentrations that are inhibitory. The
inhibitory concentrations noted in Figure
13 may vary in a specific situation. How
ever, the fact that the inhibitory concen
trations and width of the described bands
were verified with different wastes, over
significant periods of time, and with un
controlled as well as controlled conditions
indicates that the widths of the bands and
the range of inhibiting concentrations are
reasonable and probably widespread.
The most important point, however, does
not lie in the exact fa and fna values iden
tified as inhibitory concentrations. Rather,
the important aspects of this study are that
it (a) identifies ranges of fa and fna in
which nitrification inhibition is likely, (b)
identifies causes of incomplete nitrification,
(c) indicates ways to reverse the inhibition,
and (d) provides the designer and treat
ment plant operator with better informa
tion to control the nitrification process.
When nitrification seems to be incomplete
or will not begin, the possibility of inhibi
tion should be investigated by determining
the fa and fna concentrations and com
paring them with the ranges noted in
Figure 13. If inhibition is a possibility,
steps should be taken to reduce the fa and
fna concentrations to noninhibitory levels.
Conclusions
1. The research results clearly indicated
the inhibitory effects of fa and fna on the
nitrification process.
2. The fa and fna concentrations, rather

848 Journal WPCF

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 Nitrification

KT

7T^7
10
T1-IY~1 I l
\ \
/
\
\\ // wV
\ * A\
\ ' \ I \ 150 mg/1
\ I \ I \ 103 E
Z
o? UJ
E o
o
z
UJ \H ce
o
o
ir N\ // \
\ /
# \v AA
\
/ N FA?*\a A\A <
z
A X I0mg/l \ *M z

^/ *\
!- A.V
o
UJ 2
FNA? 2
2.8 mg/1
[c] <

5 io2 ? " \l0f

l.0mg/l \ * _ 2 -1
10* <

' A-- \ \\
O

//FNA^.
w //\.CA?.\
.\ \
/ 0.2mg/^ 0 ^\ [B] \
10 AL I ^1 m\m\ I_L 10

PH
FIGURE 13.?Summary of data from all experiments showing
boundary conditions of zones of nitrification inhibition.

than total ammonia or nitrite ion concen
trations, inhibit nitrification.
3. The concentrations of fa that inhibit
nitrosomonads are greater than those that
inhibit nitrobacters. The ranges of fa con
centrations that begin to inhibit the nitrify
ing organisms are: fa inhibition to nitro
somonads, 10 to 150 mg/1 and fa inhibition
to nitrobacters, 0.1 to 1.0 mg/1.
4. The inhibition of nitrifying organisms
was initiated at concentrations of fna be
tween 0. 22 and 2.8 mg/1.
5. The following may affect the inhibi
tory fa and fna concentrations in a nitrifi
cation system: acclimation of the nitrifiers
to fa and fna, temperature, and the num
ber of active nitrifying organisms.
6. An operational chart may be used to
assess the performance of nitrifying
systems.
7. Nitrification patterns may be modified
by using operating procedures that increase
or reduce the inhibitory effects of fa and
fna as appropriate.
8. fa and fna inhibition has been shown
to occur with municipal, industrial, and
agricultural wastes and with fertilizers in
the soil.
Acknowledgments
Credits. This paper was presented at the
47th Annual Conference of the Water Pol
lution Control Federation, Denver, Colo.,
Oct. 6-11, 1974.

-Vol. 48, No. 5, May 1976 849

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Anthonisen et al.

Authors. A. C. Anthonisen, R. C. Loehr, trophic Oxidation of Ammonium and Hy

T. B. S. Prakasam, and E. G. Srinath were, droxylamine." Soil Sei. Soc. Amer. Proc,
24, 448 (1960).
respectively, research associate; director,
12. Aleem, M. I. H., "The Physiology and Chemo
Environmental Studies Program; senior re autotrophic Metabolism of Nitrobacter
search associate; and research associate, agilis." Ph.D. thesis, Cornell Univ., Ithaca,
Department of Agricultural Engineering, N. Y. (1959).
Cornell University, Ithaca, N. Y., at the 13. Boon, B., and Laudelot, H., "Kinetics of Ni
time of this study. A. C. Anthonisen is trite Oxidation by Nitrobacter winograd
now with MONTECO Environmental Man skyi." Biochem. Jour., 85, 440 (1962).
agement Associates, Montgomery, N. Y.; 14. Pokallus, R. S., "Toxicity of Nitrite to Nitro
T. B. S. Prakasam is project manager, somonas Europea." Ph.D. thesis, Butgers
Univ., New Brunswick, N. J. (1963).
Metropolitan Sanitary District of Greater 15. "Standard Methods for the Examination of
Chicago, Chicago, 111.; and E. G. Srinath is Water and Wastewater." 13th Ed., Amer.
staff research chemist, Union Carbide Pub. Health Assn., Washington, D. C.
Corp., Tonawanda, N. Y. (1971).
16. Prakasam, T. B. S., et al., "Evaluation of
References Methods for the Analysis of Physical, Chem
1. Breed, R. S., et al, "Bergey's Manual of De ical, and Biochemical Properties of Poultry
terminative Bacteriology." 7th Ed., Williams Wastewaters." Paper presented at Special
and Wilkins Co., Baltimore, Md. (1957). Meeting of ASAE Committee SE-412,
2. Tomlinson, T. G., et al, "Inhibitation of Nitri Chicago, 111. (Dec. 1972).
fication in the Activated Sludge Process of 17. Jeris, J. S., "A Rapid COD Test." Water ?
Sewage Disposal." Jour. Appl Bacteriol Wastes Eng., 4, 89 (1967).
29, 266 (1966). 18. Anthonisen, A. C, "The Effects of Free Am
3. Meiklejohn, J., "Some Aspects of the Physi monia and Free Nitrous Acid on the Nitrifi
ology of the Nitrifying Bacteria." "Auto cation Process." Ph.D. thesis, Cornell Univ.,
trophic Microorganisms." University Press, Ithaca, N. Y. (1974).
Cambridge (1954). 19. Duisberg, P. C, and Buehrer, T. B., "Effect
4. Gibbs, W. M., "The Isolation and Study of of Ammonia and Its Oxidation Products on
Nitrifying Bacteria." Soil Sei., 8, 427 Rate of Nitrification and Plant Growth."
(1919). Soil Sei., 78, 37 (1954).
5. Martin, W. P., et al, "Threshold pH Value 20. Broadbent, F. E., et al, "Transformations and
for the Nitrification of Ammonia in Desert Movement of Urea in Soils." Soil Sei. Soc.
Soils." Soil Sei. Soc. Amer. Troc., 7, 223 Amer. Proc, 22, 303 (1958).
(1942). 21. Taylor, R. L., et al, "Effects of High Concen
6. Chapman, H. D., and Liebig, F., Jr., "Field trations of Nitrogen on Activated Sludge."
and Laboratory Studies of Nitrite Accumu Jour. Water Poll. Control Fed., 28, 177
lation in Soils." Soil Sei. Soc. Amer. Troc., (1956).
16, 276 (1952). 22. Loehr, R. C, et al, "Development and Demon
7. Stojanovic, B. J., and Alexander, M., "Effect stration of Nutrient Removal from Animal
of Inorganic Nitrogen on Nitrification." Wastes." EPA-R2-73-095, Environ. Protec
Soil Set., 86, 208 (1958). tion Technol. Ser. (Jan. 1973).
8. Olsen, R. J., et al, "Effect of Manure Applica 23. Dunn, G. C, and Robinson, J. B., "Nitrogen
cation Aeration, and Soil pH on Soil Ni Losses Through Denitrification and Other
trogen Transformations and on Certain Soil Changes in Continuously Aerated Poultry
Test Values." Soil Sei. Soc. Amer. Troc., Manure." Proc. Agr. Waste Management
34, 222 (1970). Conf., Cornell Univ., Ithaca, N. Y., 545
9. Prakasam, T. B. S., and Loehr, R. C, "Micro (1972).
bial Nitrification and Denitrification in 24. Warington, R., "On Nitrification-Part II."
Concentrated Wastes." Water Res., 6, 859 Jour. Chem. Soc, 35, 429 (1879).
(1972).
10. Barker, J. E., and Thompson, R. J., "Bio
logical Removal of Carbon and Nitrogen APPENDIX?Determination of FA
from Coke Plant Wastes." Office of Res. and FNA
and Monitoring, U. S. EPA, EPA-R2-73
167 (Apr. 1973). The fa concentration is a function of the
11. Engel, M. S., and Alexander, M., "Auto total ammonia concentration, pH, and tern

850 Journal WPCF

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 Nitrification

0.001 0.1 1.0 3.0 100

UNIONIZED AMMONIA, mg/1 (NH3)

FIGURE A-l.?Diagram for determination of un-ionized am

concentrations.

perature of the solution as follows: in which K?> = the ionization constant of

17 the ammonia equilibrium equation and
Kw = the ionization constant of water. Both
fa as NH3 (mg/1) = ?
constants vary with temperature. The ratio
total ammonia as N (mg/1) X 10pH of Kh:Kw may be related to temperature:
X Kh/Kw + 10PH (A1) Kb:Kw = e(6'344/273+?c> (A2)
1000

0.001 0.01 0.1 0.6 1.0 10 100

UNIONIZED NITROUS ACID , mg/1 (HN02>

FIGURE A-2.?Diagram for determination of un-ionized nitrous acid

concentrations.

-Vol. 48, No. 5, May 1976 851

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Anthonisen et al.

To avoid cumbersome calculations to related to temperature by

determine fa, a diagram based on Equation
Al may be developed for specific tempera Ka = e(-2'30?/273+oc> (A4)
tures. By taking the logarithm of each side
of Equation Al, one obtains a straight line Equation A3 also may be developed into
relationship that will yield a series of paral a diagram to facilitate more rapid deter
lel lines for each pH value when plotted mination of fna (Figure A2). Detailed
on log-log paper (Figure Al). information on the development of these
The fna concentration is related to the equations and figures is available.18
total nitrite concentration, pH, and tem The following examples illustrate the use
perature as follows: of Figures A-l and A-2:
46 1. Total ammonia nitrogen concentration
fna as HNO2 (mg/1) = ?
= 200 mg/1
NQ2--N (mg/1) pH = 7.5
X Ka X lop? (A3) Therefore, fa = 3.0 mg/1
2. Nitrite nitrogen concentration = 200
in which Ka = the ionization constant of mg/1
the nitrous acid equilibrium equation and pH = 6.4
also varies with temperature. Ka may be Therefore, fna = 0.6 mg/1

852 Journal WPCF

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