Substrate and Product Inhibition of Nitrification
J. BUDAY, M. DRTIL, M. HUTŇAN, and J. DERCO
Department of Environmental Science, Faculty of Chemical Technology,
Slovak University of Technology, SK-812 37 Bratislava
Received 14 June 1999
Great effort is paid to the development of new technologies, that enable the short-cut of the
nitrification-denitrification cycle avoiding the oxidation of NO J to N0^" (nitrite route). The nitrifi­
cation of NH4" only to NO2 and its reduction to gaseous N2 offers several advantages: lower oxygen
demand for nitrification, lower demand of organic matter for the denitrification of N0^", higher
denitrification rates of N 0 ^
The aim of this work was to obtain more detailed information about the inhibitive forms (disso­
ciated or nondissociated) of the substrate/product as well as about the concentrations that cause
inhibition. The substrate—product inhibition was tested in batch tests. Each experiment at a cer­
tain pH was carried out using a set of six reactors. One of them served as a reference, while the
five others contained different concentrations of the tested compounds. Five sludges from different
municipal wastewater treatment plants were used in the experiments. The results obtained from
batch inhibition tests carried out on different sludges are briefly summarized in this work. The very
similar behaviour of different sludges is worth to note.
One of the most common technologies to remove
nitrogen from wastewaters is biological nitrificationdenitrification. Nitrification is a process, in which
NH4" is gradually oxidized to N0^" (nitritation) and
subsequently to N0^~ (nitratation) by nitrifying mi­
croorganisms. The produced N0^" are then, under
anoxic conditions, reduced to gaseous N 2 by het­
erotrophic microorganisms, utilizing the organic mat­
ter content of the wastewater. Regardless of some in­
hibitory influences, that can make difficult the use of
these biological processes, even the lack of organic
matter for denitrification can often have a negative
impact on the overall nitrogen removal efficiency. In
most of the cases, additional dosage of biodegradable
organic matter (methanol, acetic acid, organic wastes,
etc.) is used to resolve these problems. However, this
additional dosage can increase the costs of the treat­
ment process. Thus great effort is paid to the develop­
ment of new technologies, that enable the short-cut of
the nitrification-denitrification cycle avoiding the ox­
idation of N0^~ to N 0 ^ (the so-called nitrite route).
The nitrification of N H j only to N0^" and its reduc­
tion to gaseous N 2 offers several advantages
- lower oxygen demand for nitrification (only 75 %
of the amount necessary for complete nitrification to
NO3-),
- lower demand of organic matter for the denitri­
fication of N 0 ^ (only 60 % of the demand necessary
to the denitrification of N 0 ^ ) ,
- 40 % higher denitrification rates of N0^~ [1],
Chem. Papers 53 (6) 379—383 (1999)
- lower biomass yield during anoxic growth [2],
- possible adaptation of heterotrophic microorgan­
isms to high concentrations of N0^" (up to p(N0^"—
N) = 2000 mg d m " 3 at pH 8—8.5) [1].
An interesting possibility to achieve only partial
nitrification of N H j to N0^", is the SHARON pro­
cess [3]. It is based on the kinetic selection of am­
monium oxidizers in a system without sludge reten­
tion. Another possibility to perform the nitrite route
is based on the different sensibility of nitrifiers to the
substrate—product inhibition [4], where nitrite oxi­
dizers should be selectively more inhibited at lower
NH3 concentrations than ammonium oxidizers. Also,
temperatures higher than about 25 °C are reported to
favour nitrite accumulation [5].
The aim of this work was to obtain more de­
tailed information about the inhibitive forms (dis­
sociated or nondissociated) of the substrate/product
as well as about the concentrations that cause in­
hibition. The obtained results should be utilized
during the following research directed towards the
short-cut of nitrification-denitrification. Despite of
possible troubles caused by adaptation of the ni­
trite oxidizers to the substrate—product inhibition
[2, 6], there could be cases, where specific conditions
(high NH4" and N 0 ^ content of the wastewater con­
nected with high or low pH, high salinity, specific in­
hibitors strengthening the substrate—product inhibi­
tion) can enable the long-term performance of the ni­
trite route.
379
J. BUDAY, M. DRTIL, M. HUTNAN, J. DERCO
EXPERIMENTAL
The substrate—product inhibition (in sequel as inhibition) was tested in batch tests lasting for 1—1.5 h.
Each experiment at a certain pH was carried out using
a set of six reactors (V— 200 cm 3 ). One of them served
as a reference, while the five others contained different concentrations of the tested compounds. The required concentrations of PO^", N H j , NO J , and N 0 ^
were achieved by addition of K 2 H P 0 4 , (NH 4 ) 2 S04,
NaNCb, and NaNOß solutions. The pH was continuously controlled and maintained by addition of KOH
and H2SO4 solutions. Five different sludges were used
in the experiments:
- sludge A: a sludge coming from a nitrifying municipal wastewater treatment plant (MWWTP) with
pure oxygen aeration (ca. 500 000 inhabitants),
- sludge B: a sludge coming from a MWWTP with
nitrification, denitrification, and phosphorus removal
in side stream (ca. 200 000 inhabitants),
- sludge C: a sludge coming from a nitrifyingdenitrifying lab-scale sequencing batch reactor,
- sludge D: a sludge coming from a nitrifying
MWWTP (ca. 150 000 inhabitants),
- in Fig. 2 even some results from [7] are plotted
for comparison. These results were obtained with a
sludge (sludge E) coming from a nitrifying MWWTP
100
ГГ
1
(ca. 350 000 inhabitants).
The concentrations of PO^"—P and dissolved oxygen in the reactors were 3—5 mg dm" 3 and 4—7 mg
d m - 3 , respectively. The concentrations of the activated sludge were in the range of 1—5 g dm" 3 .
All analyses were carried out according to the Standard Methods [8], except the determination of NO^~,
which was carried out using the Zambelli method [9]
(a colorimetric method with sulfanilic acid and phenol).
RESULTS A N D DISCUSSION
The concentrations of nondissociated NH 3 , HNO2,
and HNO3 were calculated on the basis of acid-base
balances [4, 10]. The inhibition was evaluated using
the equations of noncompetitive inhibition
1 =
/ =
(1)
P + Ki
P-Po
P-P0 + Щ
(2)
where: J - inhibition, Ki, K\ - inhibition constant
(concentration causing an inhibition of 50 % ) , po concentration over which inhibition starts to demon­
strate itself, p - concentration.
1—' i i i i • |
coef.ofcor=0.4
I 80
p
В 60 Б
£ 40
о
с
о
П
'£3
ÍS
D
в
с
о
с
о
•-3
Ъ
I 20
-20
:
% * •••
Li
1
1 __I 1 1 1 1 1 1
1
10
100
^(NH4+-N)/(mgdm-3)
100
coef. of cor =0.45
^ 80
о
S 60
S
£ 40
о
о 20
uJ
0.1
I I
1000
1
•í
10
100
p(NH3)/(mgdm-3)
T
1—ii
I
coef. of cor =0.91
о
S
с
о
jŕ 4^ô*
1 °
-20
-
1 1 I I I Mil
1—I I I I l l l l
1
10
100
p(NH4+-N)/(mgdm-3)
1 I
Mill
1000
0.01
0.1
1
10
píNHaVímgdm"3)
100
Fig. 1. Inhibition of nitrification by NHj—N/NH3 (inhibition of nitritation measured using sludge D, • pH = 8.9, • pH = 8.2,
A pH = 7.5, • pH = 6.8; inhibition of nitratation measured using sludges • A, A B, I C;
/ = p/(p + K{
I = (p-po)/(p-po + KD).
380
Chem. Papers 53(6)379—383 (1999)
SUBSTRATE AND PRODUCT INHIBITION OF NITRIFICATION
100
1 — Г - Г Т-Т-ГТТТ
i
1—1 1 1 M I H
r
í 80
с
p
•
Jä 60i
•s
c
o
40
' ' i i' 111A
•
'—-"Л
coef. of cor =0.41
coef.of cor =0.77
"
*
A
*
!
A A
A
•
•
•
A
-
c
o
J5
20
1c
c
.c
c
i и nl
i i i i mil—Aj._T.t_i i iul
1
0.1
10
i i i i nu
100
1000
0.000001
0.01
0.0001
3
p(HN02)/(mgdm- )
3
p(N0 2 *-N)/(mgdm' )
100
coef. ofcor.=0.55
100
"
•
•
•
A
10
100
/ H N O ^ - N ^ m g dm' 3 )
1000
• i-i-tiiu—i
1 =
Tret -
r
100%
Tref
(3)
where: J - inhibition, r - nitritation/nitratation rate,
rref - nitritation/nitratation rate in the reference re­
actor.
Correlation coefficients were calculated according
to [11] as
х
coef. of cor. = •
х
пТ, У~Т, Т,У
y/[nZ*2 - ( £ * ) 2 ] > Ľ 2 / 2 - Q » 2 ]
(4)
where: n - number of measurements, x - concentration
of the inhibitive substance, and у - inhibition.
Inhibition of Nitrification b y N H + / N H 3
The tested ranges of pH and NH4"—N concen­
trations were 6.8—9 and 0—250 mg d m - 3 , respec­
tively. The reference reactors contained at the begin­
ning of the experiments píNH^"—N) = 1 mg dm" 3
Chem. Papers 53 (6) 379—383 (1999)
60
A
A
c
o
s
2
c
40
A
A
• A
20
•cs
g
3
c
0
1 MIH
0.001
0.01
0.1
p(HN0 2 )/(mgdm" 3 )
F i g . 2. Inhibition of nitrification by N O ^ — N / H N 0 2 (A sludge A, • sludge В, О sludge E;
(p-po)/(p-po + K<)).
Eqn (2) was used in cases, where there was clearly
found that up to a certain concentration no inhibition
occurred (inhibition occurred only at concentrations
exceeding p 0 - see Fig. 1). The inhibition was calcu­
lated using the following equation
A
•
A
A
0.0001
80
•
4
. T^I i ^ . i i c
•
1
J = p/(p + Ki),
I =
and p(NO^~—N) = 15 mg d m - 3 (reference reactor for
the nitratation), and p(NH|—N) = 13 mg d m - 3 (reference reactor for the nitritation). The average concentration of the examined substance in the reference
reactor is signed with a dashed line (in all of the figures). It is obvious from Fig. 1 (the horizontal axes
of all of the figures are in logarithmic scale) that the
inhibition of nitritation and nitratation was caused by
the nondissociated form, the NH 3 .
This becomes evident, as soon as the NH4"—N concentrations are converted to the NH3 ones (see the coefficient of correlation). As the inhibition started only
over a certain concentration (So), both equations of
noncompetitive inhibition were utilized to describe it
(both equations were utilized even for the HNO2 and
HNO3 inhibition). The very similar behaviour of three
different sludges is worth to note.
Inhibition of Nitrification by N O 2 / H N O 2
The tested ranges of pH and NO-7—N concentrations were 6.8—8.9 and 0—415 mg dm" 3 , respectively. The reference reactors contained at the beginning of the experiments ^(NHj—N) = 15 mg dm" 3
and p(NO^~—N) = 15 mg dm" 3 (reference reactor for
the nitratation), and ^(NHj—N) = 15 mg dm" 3 (ref-
381
J. BUDAY, M. DRTIL, M. HUTNAN, J. DERCO
60
- coef. ofcor.=0.98
50
A
-
40
30
20
У
0
• * *
1
10
100
p(N03--N)/(mgdm-3)
lltllllL
J__l 1 1 M I H
1 1 1 1Hill
43
B
'Б
c
o
c
o
43
ja
л
c
-10
1-10^
1
7
1-10"
1-10"*
3
p(HN03)/(mgdnT )
1-10"*
1000
- 10
e:
c
o
1-10'
80
80
coef. ofcor.=0.40
•
60
-
á,
•
60
Á
•
-
£ 40
o
c
o
"
B
- 40
Á
•
a 20
c
o
'43
o
c
o
Á
20
•
43
11 111..
10
100
1000
MO
1-1 o 4
1-10'7
p(HN0 3 )/(mgdm- 3 )
-1
3
p(N03--N)/(mgdm- )
1-10" £
F i g . 3 . Inhibition of nitrification by N 0 3 —N/HNO3 (inhibition measured using sludge D, • pH = 8.5, • pH = 7.7, • pH
/ = p/(p + Äi),
/ = (p - Po)/(p - Po + K<)).
6.8;
T a b l e 1. Forms and Concentrations of N H J / N H 3 , N O ^ " / H N 0 2 , and N O ^ / H N 0 3 Causing Inhibition of Nitrification
Form causing inhibition
NH+
no
no
Nitritation
Nitratation
NH 3
yes
yes
HN03
yes
no
NO"
no
yes
HNO2
yes
no
NO"
no
yes
Concentration causing inhibition
NH3
Nitritation
beginning
inhibition
inhibition
Nitratation beginning
inhibition
inhibition
of
of
of
of
of
of
the inhibition (po/(mg d m - 3 ) )
50 % (Ki - eqn ( I ) ) / ( m g d m " 3 )
50 % {K[ - eqn (2))/(mg d m " 3 )
the inhibition (po/{mg d m - 3 ) )
50 % {Ki - eqn (I))/(mg d m " 3 )
50 % (К*. - eqn (2))/(mg d m " 3 )
1
46
30
1
20
16
NO"—N
NO~—N
HNO2
8 x 10"8
7 x 10"6
6 x IQ"6
1 x Ю-4
0.0109
0.0105
198
HN03
205
Note: In cases of inhibition by dissociated forms it was not possible to determine po from the obtained results.
erence reactor for the nitritation). As it is obvious
from Fig. 2, the inhibition of nitritation was caused by
the nondissociated HNO2. The behaviour of different
sludges, like previously in the case of NH 3 inhibition,
was again quite uniform. Contrary to the commonly
382
accepted theory of HNO2 inhibition of nitratation, the
dissociated NO^" was found to cause the inhibition of
nitrite oxidizers. The inhibitive effect of a certain con­
centration of NO^~ was not significantly strengthened
by lowering the pH.
Chem. Papers 53 (6) 379—383 (1999)
SUBSTRATE AND PRODUCT INHIBITION OF NITRIFICATION
I n h i b i t i o n of N i t r i f i c a t i o n b y N O 3 / H N O 3
T h e tested ranges of pH and N 0 ^ ~ — N concen­
- 3
trations were 6.8—8.5 and 0—400 mg d m , respec­
tively. T h e reference reactors contained at t h e begin­
- 3
ning of t h e experiments p ( N H j — N ) = 1 mg d m
- 3
and p(NO^~—N) = 15 mg d m
(reference reactor for
- 3
t h e n i t r a t a t i o n ) , a n d ^ ( N H j — N ) = 15 mg d m
(ref­
erence reactor for t h e n i t r i t a t i o n ) . T h e nondissociated
HNO3 was found t o cause t h e inhibition of nitritation
(see Fig. 3), while t h e n i t r a t a t i o n was inhibited by t h e
dissociated NO^~
CONCLUSION
T h e nondissociated N H 3 , H N 0 2 , and H N 0 3 were
found as inhibitive forms for t h e nitritation. T h e
nondissociated HNO2 a n d HNO3 were expected t o in­
hibit t h e n i t r a t a t i o n [4], b u t on t h e basis of our results
t h e nondissociated NH3 a n d t h e dissociated NO^~ and
N0^" were found t o inhibit t h e n i t r a t a t i o n . Inhibitive
concentrations of s u b s t r a t e / p r o d u c t , as well as t h e in­
hibition constants are summarized in Table 1. Com­
paring t h e inhibition constants, n i t r a t a t i o n seems t o
be more sensitive t o NH3 inhibition t h a n nitritation.
All these experiments were carried out as batch tests,
so t h e behaviour of t h e nitrifying microorganisms in
long-term experiments can differ due to acclimatiza­
tion. T h e s u b s t r a t e — p r o d u c t inhibition will be stud­
ied further in long-term experiments.
Chem. Papers 53 (6) 379—383 (1999)
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383