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 nitrifi­cation 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 N0^

The aim of this work was to obtain more detailed information about the inhibitive forms (disso­ciated 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 cer­tain 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 nitrification-denitrification. Nitrification is a process, in which NH4" is gradually oxidized to N0^" (nitritation) and subsequently to N0^~ (nitratation) by nitrifying mi­croorganisms. The produced N0^" are then, under anoxic conditions, reduced to gaseous N2 by het­erotrophic microorganisms, utilizing the organic mat­ter content of the wastewater. Regardless of some in­hibitory 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 treat­ment process. Thus great effort is paid to the develop­ment of new technologies, that enable the short-cut of the nitrification-denitrification cycle avoiding the ox­idation of N0^~ to N 0 ^ (the so-called nitrite route). The nitrification of N H j only to N0^" and its reduc­tion 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 denitri­fication of N 0 ^ (only 60 % of the demand necessary to the denitrification of N 0 ^ ) ,

- 40 % higher denitrification rates of N0^~ [1],

- 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 pro­cess [3]. It is based on the kinetic selection of am­monium oxidizers in a system without sludge reten­tion. Another possibility to perform the nitrite route is based on the different sensibility of nitrifiers to the substrate—product inhibition [4], where nitrite oxi­dizers 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 de­tailed information about the inhibitive forms (dis­sociated or nondissociated) of the substrate/product as well as about the concentrations that cause in­hibition. 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 ni­trite oxidizers to the substrate—product inhibition [2, 6], there could be cases, where specific conditions (high NH4" and N 0 ^ content of the wastewater con­nected with high or low pH, high salinity, specific in­hibitors strengthening the substrate—product inhibi­tion) can enable the long-term performance of the ni­trite route.

Chem. Papers 53 (6) 379—383 (1999) 379

J. BUDAY, M. DRTIL, M. HUTNAN, J. DERCO

E X P E R I M E N T A L

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 cm3). One of them served as a reference, while the five others contained differ­ent concentrations of the tested compounds. The re­quired concentrations of PO^", NHj , NO J , and N 0 ^ were achieved by addition of K 2 HP0 4 , (NH4)2S04, 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 nitrifying-denitrifying 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

(ca. 350 000 inhabitants). The concentrations of PO^"—P and dissolved oxy­

gen in the reactors were 3—5 mg dm"3 and 4—7 mg dm - 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 NH3, 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 =

/ =

P + Ki

P-Po P-P0 + Щ

(1)

(2)

where: J - inhibition, Ki, K\ - inhibition constant (concentration causing an inhibition of 50 %), po -concentration over which inhibition starts to demon­strate itself, p - concentration.

100

I 80 p В 60 Б £ 40 о

I 20

-20

100

^ 80 о S 60 S £ 40 о о 20

1 ° -20

-

:

ГГ 1 1—' i i i i • |

coef.ofcor=0.4 П

D

% * • • • Li 1 1 __I 1 1 1 1 1 1 1 I I

-

10 100 ^(NH4

+-N)/(mgdm-3) 1000

coef. of cor =0.45

•í jŕ 4^ô*

uJ 1 1 I I I M i l 1—I I I I l l l l 1 I M i l l

0.1 1 10 100 p(NH4

+-N)/(mgdm-3) 1000

1 10 100 p(NH3)/(mgdm-3)

T 1—i i I

coef. of cor =0.91

0.01 0.1 1 10 píNHaVímgdm"3)

100

с о '£3

ÍS в с о с о •-3 Ъ

о

S

с о

Fig. 1. Inhibition of nitrification by NHj—N/NH3 (inhibition of nitritation measured using sludge D, • pH = 8.9, • pH 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).

= 8.2,

380 Chem. Papers 53(6)379—383 (1999)

SUBSTRATE AND PRODUCT INHIBITION OF NITRIFICATION

100

í 80 с p Jä 60 i •s 4 0

c o

.c c

20

0.1

i 1 — Г - Г Т

-

-

-Т-ГТТТ 1—1 1 1 M I H r

coef. of cor =0.41

•

' ' i i ' 111A • '—-"Л

" * * ! • • A •

A A A A

i и nl i i i i mil—Aj._T.t_i i iul i i i i n u

1 10 100

p(N02*-N)/(mgdm'3)

coef.of cor =0.77

1000 0.000001 0.0001 0.01 p(HN02)/(mgdm-3)

c o

J5 1c c

100

10 100 /HNO^-N^mg dm'3)

coef. ofcor.=0.55

•

"

4 A

A A

. T^I i ^ . i i c • i - i - t i i u — i

A •

A

•

•

A •

A

1 M I H

•

•

A

A

1000 0.0001 0.001 0.01 0.1

p(HN0 2 )/(mgdm" 3 )

100

80

60

40

20

0

1

c o

s 2 c •s c g 3 c

Fig . 2. Inhibition of nitrification by N O ^ — N / H N 0 2 (A sludge A, • sludge В, О sludge E; J = p/(p + Ki), (p-po)/(p-po + K<)).

I =

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 calcu­lated using the following equation

Tret - r

1 = Tref

100% (3)

where: J - inhibition, r - nitritation/nitratation rate, rref - nitritation/nitratation rate in the reference re­actor.

Correlation coefficients were calculated according to [11] as

coef. of cor. = • пТ,хУ~Т,хТ,У

y/[nZ*2 - (£*) 2]>Ľ2/ 2 - Q » 2 ] (4)

where: n - number of measurements, x - concentration of the inhibitive substance, and у - inhibition.

Inhibit ion of Nitrification by N H + / N H 3

The tested ranges of pH and NH4"—N concen­trations were 6.8—9 and 0—250 mg d m - 3 , respec­tively. The reference reactors contained at the begin­ning of the experiments píNH^"—N) = 1 mg dm"3

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 NH3.

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.

Inhibi t ion 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-

Chem. Papers 53 (6) 379—383 (1999) 381

J. BUDAY, M. DRTIL, M. HUTNAN, J. DERCO

10 100

p(N03--N)/(mgdm-

3)

-

-

coef. ofcor.=0.98

A

У

• * * 1 l l t l l l l L J__l 1 1 MIH 1 1 1 1 H i l l 1

-

1000 1-10"* 1-10'7

1-10"* 1-10"

p(HN0

3)/(mgdnT

3)

60

50

40

30

20

10

0

-10

e: c o

4 3

B 'Б c

o c o

43 ja

л c

1-10̂

80

60

£ 40 o c o a 20

10 100

p(N03--N)/(mgdm-3)

1000 MO-1 1-10'7 1-1 o 4

p(HN03)/(mgdm-3)

-

-

• " •

•

•

11 111. .

• coef. ofcor.=0.40

á,

Á

Á

Á

80

60

- 40

20

1-10"£

c o '43

B

o c o 43

Fig . 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;

Tab le 1. Forms and Concentrations of NHJ /NH3 , NO^"/HN0 2 , and N O ^ / H N 0 3 Causing Inhibition of Nitrification

Form

NH+ NH3

Nitritation no yes Nitratation no yes

causing inhibition

NO" no yes

Concentration

Nitritation beginning of the inhibition (po/(mg d m - 3 ) ) inhibition of 50 % (Ki - eqn ( I ) ) / (mg d m " 3 ) inhibition of 50 % {K[ - eqn (2))/(mg d m " 3 )

Nitratation beginning of the inhibition (po/{mg d m - 3 ) ) inhibition of 50 % {Ki - eqn (I)) / (mg d m " 3 ) inhibition of 50 % (К*. - eqn (2))/(mg d m " 3 )

causing

N H 3

1 46 30

1 20 16

HNO2 yes no

inhibition

N O " — N

198

N O " no yes

HNO2

1 x Ю - 4

0.0109 0.0105

N O ~ — N

205

H N 0 3

yes no

H N 0 3

8 x 1 0 " 8

7 x 1 0 " 6

6 x I Q " 6

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 NH3 inhibition, was again quite uniform. Contrary to the commonly

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 con­centration of NO^~ was not significantly strengthened by lowering the pH.

382 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 R E F E R E N C E S

The tested ranges of pH and N0^~—N concen­trations were 6.8—8.5 and 0—400 mg d m - 3 , respec­tively. The reference reactors contained at the begin­ning of the experiments p ( N H j — N ) = 1 mg d m - 3

and p(NO^~—N) = 15 mg d m - 3 (reference reactor for the ni t ratat ion), and ^ ( N H j — N ) = 15 mg d m - 3 (ref­erence reactor for the nitr i tat ion). The nondissociated HNO3 was found to cause the inhibition of nitritation (see Fig. 3), while the ni tratat ion was inhibited by the dissociated NO^~

C O N C L U S I O N

The nondissociated N H 3 , H N 0 2 , and H N 0 3 were found as inhibitive forms for the nitritation. The nondissociated HNO2 and HNO3 were expected to in­hibit the ni t ratat ion [4], but on the basis of our results the nondissociated NH3 and the dissociated NO^~ and N0^" were found t o inhibit the nitratat ion. Inhibitive concentrations of substrate/product, as well as the in­hibition constants are summarized in Table 1. Com­paring the inhibition constants, nitratat ion seems to be more sensitive to NH3 inhibition than nitritation. All these experiments were carried out as batch tests, so the behaviour of the nitrifying microorganisms in long-term experiments can differ due to acclimatiza­tion. The subst ra te—product inhibition will be stud­ied further in long-term experiments.

1. Abeling, U. and Seyfried, C. F., Water Sei. TechnoL 26, 1007 (1992).

2. Turk, O. and Mavinic, D. S., J.W.P.C.F. 61, 1440 (1989).

3. Jetten, M. S. M., Horn, S. J., and van Loosdrecht, M. C. M., Water Sei. TechnoL 35, 171 (1992).

4. Anthonisen, A. C , Loehr, R. C , Prakasam, T. B. S., and Srinath, E. G., J.W.P.C.F. 48, 835 (1976).

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Chem. Papers 53 (6) 379—383 (1999) 383