Water Research 38 (2004) 3179–3188

ARTICLE IN PRESS

*Correspond

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0043-1354/$ - se

doi:10.1016/j.w

Nitrification modelling in biofilms under inhibitory conditions

Shabbir H. Gheewala, Rupa K. Pole, Ajit P. Annachhatre*

Environmental Engineering and Management, Asian Institute of Technology, PO Box 4, Klongluang, Pathumthani 12120, Thailand

Received 22 May 2003; received in revised form 25 March 2004; accepted 21 April 2004

Abstract

A biofilm model has been developed for simulating nitrification in biofilms under inhibitory conditions. Nitrification

inhibition has been modelled using uncompetitive inhibition kinetics. Inhibition kinetic experiments were performed by

varying the bulk concentrations of inhibitory compound, aniline. Two sets of results were obtained with a nitrifying

biofilm that was unacclimated to aniline and another which was acclimated to aniline. Fitting of the nitrification

inhibition biofilm model to the experimental results yielded the nitrification inhibition constant, Ki; for aniline. Both the

experiments yielded a value of about 3mg/L for Ki; which was similar to that obtained during nitrification inhibition

experiments with suspended growth process carried out in an earlier study. The nitrification inhibition biofilm model is

general and can be applied to nitrification inhibition with other toxic compounds.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Aniline; Biofilm; Inhibition; Model; Nitrification

1. Introduction

The application of biofilm reactors in wastewater

treatment systems is popular in view of their high

volumetric productivity. These reactors are especially

useful for slow-growing organisms, which would other-

wise be washed out of the system, nitrifying biofilms

being a case in point. The mathematical modelling of

biofilm systems is considerably more complex than

suspended growth systems due to the interaction of

diffusion limitation and substrate uptake within the

biofilm. This results in reduced concentrations of

substrates within the biofilm. On the one hand, these

results in reduced reaction rates within the biofilm,

while, on the other hand, it can be advantageous in case

of inhibitory substances being present in the wastewater.

The concentrations and hence the inhibitory effects of

such substances would be reduced in the biofilm, thus

protecting the micro-organisms within the biofilm [1–4].

ing author. Tel/fax: +66-2-524-5644.

ess: ajit@ait.ac.th (A.P. Annachhatre).

e front matter r 2004 Elsevier Ltd. All rights reserve

atres.2004.04.018

In the present work, nitrification kinetics are studied

in a biofilm under inhibitory conditions due to aniline.

Aniline is one of the most important industrially

produced amines widely used for the manufacture of

polyurethanes, rubber and pesticides [5]. It is a useful

intermediate in the production of many other products

such as dyestuffs, drugs, photographic chemicals resins,

and varnishes. Thus, a variety of industrial effluents

have aniline as a major wastewater constituent. Aniline

inhibits the autotrophic nitrifying bacteria causing 50%

inhibition of ammonia oxidation at concentrations as

low as 1mg/L [6].

A mathematical model has been developed to simulate

nitrification kinetics in biofilms under inhibitory condi-

tions. This general model was then adapted to simulate

nitrification inhibition by aniline. A packed bed biofilm

reactor was operated using synthetic wastewater con-

taining NH4N, NaHCO3 and aniline, and its perfor-

mance vis-a-vis nitrification under inhibitory conditions

due to aniline was evaluated. Fitting the mathematical

model to the experimental data yielded the nitrification

inhibition constant for aniline. It must be emphasised

d.

ARTICLE IN PRESS

Nomenclature

Abulk bulk aniline concentration=aniline concen-

tration in biofilm (M/L3)

Avf concentration of aniline in biofilm (M/L3)

D diffusion coefficient of ammonia in biofilm

(L2/T)

Dan diffusion coefficient of aniline in biofilm (L2/

T)

DOvf concentration of dissolved oxygen (DO) in

biofilm (M/L3)

k0vf ;an zero-order reaction rate for aniline degrada-

tion in biofilm (M/L3T)

Ki nitrification inhibition constant for aniline

(M/L3)

Ks half saturation constant for ammonia oxida-

tion (M/L3)

Ks;o half saturation constant for DO (M/L3)

kvf zero-order reaction rate for nitrification in

biofilm (M/L3T)

L biofilm thickness (L)

N ammonia flux into the biofilm (M/L2T)

pH pH in the biofilm (dimensionless)

rA nitrification rate (M/L2T)

rA;max maximum nitrification rate in the absence of

aniline (M/L2T)

rvf volumetric reaction rate in biofilm (M/L3T)

Sbulk ammonia concentration in bulk liquid (M/L3)

Svf ammonia concentration in biofilm (M/L3)

x distance into the biofilm from the surface (L)

x0 location in the biofilm at which Avf ¼ 0 (L)

xc the critical depth at which Svf ¼ 0 (L)

S.H. Gheewala et al. / Water Research 38 (2004) 3179–31883180

that although aniline has been selected as an example,

the mathematical model is general and applies to any

inhibitory compound.

2. Biofilm model

To reduce the complexity in computation, the biofilm

is assumed to be planar and homogeneous [7]. The

biofilm model has been derived in one spatial direction

only because changes in substrate concentrations per

unit length are generally orders of magnitude smaller in

other directions [8]. The biofilm is assumed to be in

steady state. Influence of liquid film diffusion on

reaction rate is neglected [9,10].

Mass balance of substrate in the slice ‘dx’ yields (Fig. 1)

dN

dx¼ �rvf : ð1Þ

Fick’s law

N ¼ �DdSvf

dx: ð2Þ

Combining Eqs. (1) and (2) we get

d2Svf

dx2¼

rvf

D: ð3Þ

The two boundary conditions for Eq. (3) are:

(1)

Since hydraulic film at the biofilm–water interface

is neglected

Svf ¼ Sbulk at x ¼ 0: ð3aÞ

(2)

Fig. 1. Substrate profile in biofilm.

If ammonia is fully penetrated and no-flow condi-

tion is assumed at support

dSvf=dx ¼ 0 at x ¼ L: ð3bÞ

On the other hand, if ammonia is partly penetrated, then

dSvf=dx ¼ 0 at x ¼ xc; ð3cÞ

where xc is the critical depth at which Svf ¼ 0: In the

above equations ‘S’ refers to ammonia.

The ammonia removal rate depends on several

parameters such as bulk NH4N and DO concentration,

pH and presence of inhibitory compounds in the

wastewater [11]. In the absence of inhibition, the

nitrification rate is as follows:

rvf ¼ kvfSvf

Ks þ Svf

DOvf

Ks;o þDOvf1� 0:0833 7:2� pHj j½ �:

ð4Þ

Eqs. (3) and (4) can be solved simultaneously to

obtain the ammonia and oxygen profiles within the

ARTICLE IN PRESSS.H. Gheewala et al. / Water Research 38 (2004) 3179–3188 3181

biofilm. The second term on the right-hand side of

Eq. (4) can be suitably modified for including inhibition

of nitrification.

Nitrification inhibition by aniline has been modelled

using uncompetitive inhibition equation [12]. Thus,

when there is inhibition due to aniline

rvf ¼ kvfSvf

Ks þ Svf þ ðSvf Avf=KiÞDOvf

Ks;o þDOvf1� 0:0833 7:2� pHj j½ �: ð5Þ

The value of Ki is equal to the concentration of the

inhibitor, which causes a decrease in reaction rate to half

of the maximum value when there is no substrate

limitation.

Degradation of aniline in the biofilm by heterotrophic

bacteria produces NH4N. However, the concentration

of NH4N produced due to aniline degradation (o1mg

N/L) is much smaller than that provided in the influent

(B35mg N/L) and is thus neglected in the model. In the

experiments, bulk NH4N and DO were maintained at

very high concentrations to avoid limitation due to

these. Also, the wastewater was adequately buffered so

that the bulk pH was in the range of 7.3–7.5.

For the condition of adequate buffer and excess

ammonia and DO, Eq. (5) reduces to

rvf ¼ kvfKi

Ki þ Avf: ð6Þ

So, from Eqs. (3) and (6) we get

d2Svf

dx2¼

kvf

D

Ki

Ki þ Avf: ð7Þ

Introducing the following non-dimensional terms:

svf ¼Svf

Sbulk; avf ¼

Avf

Abulk; z ¼

x

L;

g ¼Abulk

Ki; b2 ¼

2DSbulk

kvfL2:

We get from Eq. (7)

d2svf

dz2¼

2

b2 1þ g avfð Þ: ð8Þ

Eq. (8) depends on the concentration profile of

aniline, avf :Using Eq. (3), the profile for aniline concentration in

the biofilm is

d2Avf

dx2¼

k0vf ;an

Dan: ð9Þ

The kinetics of aniline degradation were experimen-

tally observed to be zero order [13]. Since the hydraulic

film at the biofilm–water interface is neglected, one

boundary condition for Eq. (9) is

Avf ¼ Abulk at x ¼ 0: ð9aÞ

The second boundary condition depends on whether

aniline is fully or partly penetrated into the biofilm.

When aniline is fully penetrated: Since ammonia is

fully penetrated and no-flow condition is assumed at

support, the second boundary condition for Eq. (9) is

dAvf=dx ¼ 0 at x ¼ L: ð9bÞ

Non-dimensionalising Eq. (9) and integrating it using

the boundary conditions (Eq. (9a) and (9b)), we get

avf ¼z2

b2an�

2z

b2anþ 1; ð10Þ

where

b2an ¼2Dan Abulk

k0vf ;anL2:

The ammonia removal rate, rA; is calculated as

follows:

rA ¼ �DdSvf

dx

����x¼0

¼ �DSbulk

L

dsvf

dz

����z¼0

: ð11Þ

Hence, using Eqs. (7), (9) and (10), ammonia removal

rate, rA; is given by

rA ¼2KiDan

L k0vf ;ankvf

1ffiffiffiffiffiffiffiffiffiffiffic � 1

p tan�1 1ffiffiffiffiffiffiffiffiffiffiffic � 1

p !

;

c ¼ b2an1

gþ 1

� �: ð12Þ

When aniline is partially penetrated: Since ammonia is

partly penetrated, the second boundary condition for

Eq. (9) is

dAvf=dx ¼ 0 at x ¼ x0; ð9cÞ

where x0 is the location in the biofilm at which aniline

concentration reduces to 0.

Non-dimensionalising Eq. (9) and integrating it using

the boundary conditions (Eqs. (9a) and (9c)), we get

avf ¼z2

b2an�2zz0

b2anþ 1; z0 ¼ ban; ð13Þ

where z0 ¼ ban ¼ x0=L (dimensionless penetration depth

of aniline).

Hence, using Eqs. (8), (11) and (13), ammonia

removal rate, rA; is given by

rA ¼ kvfLbanffiffiffig

p tan�1ffiffiffig

pþ 1� ban

" #: ð14Þ

3. Materials and methods

3.1. Experimental set-up

The experimental set-up essentially consisted of the

substrate bottles, a downflow packed bed biofilm reactor

and feed as well as recirculation pumps (Fig. 2).

ARTICLE IN PRESS

BiofilmReactor

FeedPump

RecirculationPump

SubstrateBottle

SubstrateBottle

Manual 2-wayvalve

Outlet

Oxygenationbottle

DOProbe

Fig. 2. Experimental set-up of biofilm reactor.

Table 1

Composition of nutrients, ammonia and bicarbonate [15]

Stock solution Concentration in stock solution Concentration in feed

1 20 g/L KH2PO4 10mg/L KH2PO4

0.1 g/L Na2MoO4 0.05mg/L Na2MoO4

2 80 g/L MgSO4, 7H2O 40mg/L MgSO4, 7H2O

5.4 g/L FeSO4 2.7mg/L FeSO4

3 40 g/L CaCl2, 2H2O 20mg/L CaCl2, 2H2O

4 Micro-nutrients 50mL/L HCl 2.5 mL/L HCl (25%)

350mg/L ZnCl 17.5mg/L ZnCl

500mg/L MnCl2, 4H2O 25 mg/L MnCl2, 4H2O

30mg/L H3BO3 1.5 mg/L H3BO3

30mg/L CoCl2, 6H2O 1.5 mg/L CoCl2, 6H2O

120mg/L NiCl2, 2H2O 6 mg/L NiCl2, 2H2O

10mg/L CuCl2, 2H2O 0.5 mg/L CuCl2, 2H2O

180mg/L Na2MoO4, 2H2O 9 mg/L Na2MoO4, 2H2O

5 Ammonia 5.35 g/L NH4Cl (1400ppm N/L) Dilution prepared according to requirement

6 Bicarbonate 13.77 g/L NaHCO3 275.4mg/L

S.H. Gheewala et al. / Water Research 38 (2004) 3179–31883182

Synthetic wastewater was used in the experiments.

The concentrations of the nutrients and buffer in the

synthetic wastewater are presented in Table 1. The

wastewater flow from the substrate bottles was con-

trolled manually by a two-way valve and was fed into

the bioreactor using a peristaltic feed pump. The biofilm

reactor consisted of a cylindrical tube made of acrylic.

The filter layer containing the support media for the

biofilm (shaded region) was supported by perforated

steel plates. The dimensions of the reactor and filter

layer are given in Table 2. The effluent from the bottom

of the reactor was partly recirculated into the reactor.

ARTICLE IN PRESS

Table 2

Reactor and filter layer dimensions

Reactor with

Unacclimated biofilm Acclimated biofilm

Empty bed volume, reactor 464mL 430mL

Empty bed volume, filter layer 357mL 357mL

Media Biocarbones stones Polypropylene pellets

Bulk volume, reactora 236mL 280mL

Bulk volume, filter layera 118mL 207mL

Cross-sectional area 0.001521m2 0.001521m2

Height, filter layer 0.235m 0.235m

aWith media.

S.H. Gheewala et al. / Water Research 38 (2004) 3179–3188 3183

Sufficient recirculation ratio was maintained in order to

achieve completely mixed conditions in the reactor.

Aeration was provided to the reactor through the

oxygenation bottle in the recirculation line. The

remaining effluent was discharged after passing through

a closed chamber containing a DO probe for monitoring

the DO in the effluent. As the system was completely

mixed, the effluent DO was the same as that in the

biofilm reactor.

Initially after setting up of the reactor, it was run with

bare media with no attached biofilm to ascertain the

flow pattern of the reactor. The recirculation rate was

maintained at about nine times the influent flow rate to

achieve complete-mix conditions. Tracer experiments

were carried out to confirm the complete-mix regime and

to determine the residence time of the system.

3.2. Nitrifying biofilm

For experiments with biofilm unacclimated to aniline,

nitrifying biofilm was obtained from a domestic waste-

water treatment plant. The biofilm support media used

was Biocarbones stones (size 2.5–3.5mm, porosity

0.33).

For experiments with acclimated biofilm, seed sludge

for the nitrifying biofilm was obtained for an SBR

treating synthetic wastewater containing 200mg/L of

aniline [14]. The seed biomass had a mixed population of

heterotrophs and nitrifiers as organic carbon removal,

nitrification and denitrification occurred in the SBR [14].

The seed biomass was very well acclimated to aniline

since the SBR system was in operation for over 2 years.

The media used was cylindrical polypropylene pellets

(i.d. 3mm, o.d. 4mm, length 5mm, specific density

1.001 g/cm3, porosity 0.68). The seed sludge was

introduced in the oxygenation bottle (Fig. 2) and the

recirculation pump was operated overnight to allow the

biomass to attach to the media in the reactor. No feed

was introduced during this period. The reactor was then

fed continuously with excess of NH4Cl and NaHCO3 till

nitrification activity was established. The support media

was then washed to remove the unattached biomass so

that only the biofilm attached to the media would

remain in the system. The reactor was once again fed

with excess of NH4Cl and NaHCO3 till nitrification

activity reached the levels recorded before the removal

of unattached biomass.

3.3. Experimental procedure

The objective of the experiments was to determine

nitrification inhibition kinetics of biofilm under varying

bulk aniline concentrations and excess but constant

NH4N and DO concentrations. Eight different bulk

aniline concentrations would be used to ascertain the

nitrification inhibition kinetics.

The experiments consisted of varying the bulk aniline

concentrations by changing the influent aniline concen-

trations. At each influent aniline concentration, the

effluent aniline and NO3N values were monitored till the

steady values were reached. The influent aniline con-

centration was then altered. Thus, the effect of bulk

aniline concentration on nitrification was then ascer-

tained. The NH4Cl, NaHCO3 and DO were maintained

in excess to avoid any limitation to nitrification due to

these. A high bulk DO concentration of more than

20mg/L was maintained using pure oxygen during the

kinetic experiments.

Each experimental run with one particular concentra-

tion of aniline was carried out for at least six hydraulic

retention times (HRTs) as it was observed that this was

the minimum time required to wash out all the NO3N

from the previous run [13]. The run was not carried

beyond nine HRTs as it was observed that the

nitrification rate started to decrease gradually after

stabilising at 6–7 HRTs, probably due to long-term

toxicity effect of aniline. Also, for higher concentrations

of aniline, each run was separated by feeding NH4N and

NaHCO3 only (with nutrients) during subsequent days.

This was done to ensure that the inhibition effect of

aniline from the previous run did not affect the

subsequent one and the nitrification activity of the

ARTICLE IN PRESSS.H. Gheewala et al. / Water Research 38 (2004) 3179–31883184

biofilm was maximum at the beginning of each

inhibition run. This also prevented the excessive growth

of aniline degraders, which was observed when experi-

ments with aniline were carried out continuously for 3–4

days.

4. Results and discussion

4.1. Theoretical results

The ammonia removal rates, rA; are given by Eqs. (12)

and (14). The maximum ammonia removal rate will

occur when there is no aniline inhibition and is given by

rA;max ¼ kvfL: The theoretical dimensionless ammonia

removal rate, rA=rA;max; is plotted as a function of the

dimensionless bulk aniline concentration, g; in Fig. 3.

This is the expected outcome of the nitrification

inhibition biofilm kinetic experiments with aniline. The

first part of the curve in Fig. 3, governed by Eq. (14), is

for the case when aniline in partly penetrated into the

biofilm because the bulk aniline concentrations are low.

As bulk aniline concentration increases, the degree of

penetration of aniline into the biofilm increases till at a

critical bulk aniline concentration, obtained from ban ¼1; aniline is fully penetrated. The curve beyond that

critical bulk aniline concentration is governed by

Eq. (12).

The dimensionless ammonia removal rate, rA=rA;max;is also plotted for various dimensionless penetration

ratios of aniline, ban (Fig. 4). ban ¼ 1 represents the

condition when aniline is just fully penetrated into the

biofilm. In this case, ammonia removal in the whole

biofilm is affected by inhibition due to aniline. ban > 1

also represents the condition where aniline is fully

penetrated into the biofilm and hence, ammonia removal

Ammonia Removal Rate as

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 0.5 1 1.5

Dimensionless Bulk A

Dim

ensi

on

less

Am

mo

nia

Rem

ova

l Rat

e

Transition of anilinepenetration

from partial to full

Concen

Fig. 3. Ammonia removal rate as a func

in the whole biofilm is inhibited by aniline. However, in

this case, aniline concentrations inside the biofilm are

higher than in the case of ban ¼ 1: Hence, rate of

reduction of ammonia removal rate, as represented by

slope of the curves in Fig. 4, is higher than that for

ban ¼ 1: The curves for ban > 1 thus lie below the curve

for ban ¼ 1: bano1 represents the condition when aniline

is partly penetrated into the biofilm. In such a case, that

part of the biofilm which does not have any aniline,

performs ammonia removal at its maximum rate (since

there is no limitation of ammonia or DO) whereas the

remaining part, which is influenced by aniline inhibition,

performs ammonia removal at a reduced rate. Hence,

rate of reduction of ammonia removal rate, as repre-

sented by slope of the curves in Fig. 4, is lower than that

for ban ¼ 1: Thus, the curves for bano1 lie above the

curve for ban ¼ 1:

4.2. Experimental results and model verification

4.2.1. Unacclimated biofilm

Inhibition kinetic experiments were carried out with

aniline as the inhibitory compound. The biofilm was not

acclimated to aniline. Bulk DO was maintained at about

30mg/L. Thus, any changes in effluent NO3N were

attributed to inhibition of nitrifying bacteria in the

biofilm due to aniline. The concentration of aniline in

the influent was varied (0–47mg/L) and samples taken

when steady state was achieved for each particular

aniline concentration. The samples were then analysed

for NH4N, NO3N and aniline. The analysis revealed

that aniline was virtually not degraded at all. Thus,

effluent NO3N concentration depended solely on influ-

ent aniline concentration. Fig. 5 shows the expected

profiles of NH4N and aniline in the biofilm.

a Function of Bulk Aniline

2 2.5 3 3.5

niline Concentration

tration

tion of bulk aniline concentration.

ARTICLE IN PRESS

Ammonia Removal Rate as a Function of Bulk Aniline Concentration

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 3 6 9 12 15

γ=Abulk /Ki

r A/r

A,m

axβan

=0.25

an=0.1

β an =1.5

βan =3

β an=2

β an =1

βan=0.5

βan=0.8

β

Fig. 4. Effect of aniline penetration ratio on ammonia removal rate.

Bulk Liquid Support

Aniline bulk

Biofilm

NH4Nbulk NH4Nbiofilm

Aniline biofilm

Fig. 5. Profiles of aniline and NH4N.

S.H. Gheewala et al. / Water Research 38 (2004) 3179–3188 3185

Since the aniline concentration in the biofilm is the

same as that in the bulk water, the nitrification rate from

Eq. (6) reduces to

rA ¼ rA;maxKi

Ki þ Abulk

� �: ð15Þ

Based on this assumption, the nitrification rates were

plotted for different bulk aniline concentrations (Fig. 6).

Fig. 6 clearly indicates that the mathematical model fits

the experimental data well (R2 ¼ 0:975). The value of

50% inhibition, Ki; is 3.2mg/L, which is in accordance

with the data from previous experiments with suspended

growth process [12]. This value is, however, very low

compared to other compounds such as methanol

(116.0mg/L), acetone (804.2mg/L) and formalin

(61.5mg/L) [16]. This shows the high toxicity of aniline

to the nitrifying population.

4.2.2. Acclimated biofilm

The results of the inhibition kinetic experiments are

shown in Fig. 7. The trend of the results is similar to the

theoretical development presented in Fig. 3. Each

experimental point in Fig. 7 corresponds to experimental

run with a particular aniline concentration. When

aniline was fed to the biofilm reactor along with

NH4N, NaHCO3 and nutrients, its bulk concentration

started to increase and bulk NO3N concentration started

to decrease due to inhibition. Typical profiles of aniline

build-up and consequent reduction in bulk NO3N

concentration are presented in Fig. 8. After the bulk

aniline concentration reached its steady maximum value

in the reactor, the NO3N concentration reached a steady

minimum value. This steady NO3N concentration

yielded the nitrification rate at the particular bulk

aniline concentration. Seven such experimental runs

were conducted to yield nitrification rates at various

bulk aniline concentrations (0–17mg/L) (Fig. 7). Fitting

Eqs. (12) and (14) to the experimental data yielded Ki

value of 3.0mg/L for aniline. This value is similar to that

obtained during experiments with unacclimated biofilm.

This value also compares favourably with earlier

experiments with aniline inhibition of nitrification in

activated sludge process where it was observed that

nitrification activity nearly stopped when aniline con-

centration in the bulk increased beyond 4mg/L [17,18].

The transition of aniline penetration in the biofilm from

partial to full takes place at a bulk aniline concentration

ARTICLE IN PRESS

0.00

0.05

0.10

0.15

0.20

0.0 10.0 20.0 30.0 40.0 50.0

Bulk Aniline Conc. (mg/L)

Nit

rifi

cati

on

Rat

e (g

N/m

2 .d)

Outlier

rA = rA,max[Ki /(Ki+Abulk)] K i = 3.2 mg/L

R2 = 0.975

1

2

34

5

67

8

Nitrification Inhibition Kinetics

Fig. 6. Nitrification inhibition curve for unacclimated biofilm.

Nitrification Inhibition Kinetics

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 2 4 6 8 10 12 14 16 18

Bulk Aniline Conc. (mg/L)

Nit

rifi

cati

on

Rat

e (g

N/m

2 .d)

1 2

3

4

5

6

7

K i = 3.0 mg/L

R2 = 0.97

Fig. 7. Nitrification inhibition curve for acclimated biofilm.

S.H. Gheewala et al. / Water Research 38 (2004) 3179–31883186

of 3.4mg/L. The decrease of nitrification rate with

increasing bulk aniline concentrations is sharp at low

bulk aniline concentrations as indicated by the high

slope in the initial part of the curve in Fig. 7. At high

bulk aniline concentrations, the slope decreases showing

that the nitrification rate is not significantly affected by

increase in bulk aniline concentration. This is because

once the bulk aniline concentration reaches a high value,

the aniline is fully penetrated into the biofilm. Since the

whole of the biofilm is affected by aniline inhibition,

further increase in bulk aniline concentration does not

change the nitrification rate considerably. The metho-

dology for model verification is presented in Fig. 9. The

various parameters such as kvf ; D; Dan and L were

predefined—kvf and L obtained from kinetic experi-

ments, and D and Dan from the literature [19]. The

parameters k0vf ;an and Ki were given an initial input

value which was later on varied during model fitting.

The experiments yielded Abulk during the various runs

and the corresponding nitrification rates, Expt: rA: Foreach value of Abulk, the penetration of aniline (partial or

full) was ascertained and the corresponding theoretical

nitrification rate, Model rA; predicted using Eqs. (12)

and (14). The residual of the experimental and

theoretical nitrification rate for each Abulk was calcu-

lated. The sum of residuals was minimised by varying

k0vf ;an and Ki using the SOLVER function in MS Excel.

5. Conclusions

A general biofilm model was developed for simulating

nitrification in biofilms under inhibitory conditions.

For the model, mass transfer and substrate uptake

ARTICLE IN PRESS

Profiles of Aniline and Nitrate

0

5

10

15

20

25

30

0 1 2 3 4 5 6

Time (HRT)

Co

nce

ntr

atio

ns

(mg

/L)

Aniline Nitrate-N

Influent Aniline Concentration = 20.2 mg/L

Fig. 8. Typical profiles of aniline and NO3N in an inhibition kinetic experiment.

Aniline PenetrationFullPartial

(Model rA - Expt. rA)2

Stop

Calculate Model rA

using Equation 12Calculate Model rA

using Equation 14

Min

imis

e re

sidu

als

usin

g SO

LV

ER

fun

ctio

n in

MS

Exc

el

by v

aryi

ng K

ian

d k 0

vf,a

nva

lues

1. Parameter values:Constant: kvf, D, Dan, LVariable: k0vf,an, Ki

2. Measured values:Abulk, Expt. rA

Minima of residuals

Aniline PenetrationFullPartial

(Model rA - Expt. rA)2

Stop

Calculate Model rA

using Equation 12Calculate Model rA

using Equation 14

Min

imis

e re

sidu

als

usin

g SO

LV

ER

fun

ctio

n in

MS

Exc

el

by v

aryi

ng K

ian

d k 0

vf,a

nva

lues

1. Parameter values:Constant: kvf, D, Dan, LVariable: k0vf,an, Ki

2. Measured values:Abulk, Expt. rA

Minima of residuals

Aniline PenetrationAniline PenetrationFullPartial

Σ(Model rA - Expt. rA)2

StopStop

Calculate Model rA

using Equation 12Calculate Model rA

using Equation 12Calculate Model rA

using Equation 14Calculate Model rA

using Equation 14

Min

imis

e re

sidu

als

usin

g SO

LV

ER

fun

ctio

n in

MS

Exc

el

by v

aryi

ng K

ian

d k 0

vf,a

nva

lues

1. Parameter values:Constant: kvf, D, Dan, LVariable: k0vf,an, Ki

2. Measured values:Abulk, Expt. rA

1. Parameter values:Constant: kvf, D, Dan, LVariable: k0vf,an, Ki

2. Measured values:Abulk, Expt. rA

Minima of residuals

Fig. 9. Algorithm for verification of the nitrification inhibition

biofilm model.

S.H. Gheewala et al. / Water Research 38 (2004) 3179–3188 3187

interactions were considered in a planar biofilm with

uniform properties (density, diffusivity, biomass con-

centration, etc.). Nitrification inhibition was modelled

using uncompetitive inhibition kinetics. A completely

mixed, downflow, packed bed biofilm reactor was

operated in the continuous-flow mode for nitrification.

Its performance vis-a-vis nitrification under inhibitory

conditions due to aniline was evaluated. Two sets of

experiments were conducted using nitrifying biofilm (i)

unacclimated and (ii) acclimated to aniline. The overall

objective of the experimentation in each case was to

evaluate the performance of nitrifying biofilm under

inhibitory conditions through nitrification kinetic ex-

periments. To obtain meaningful results, a proper

methodology of experimentation was developed by

repeated trials and improvisation of the experimental

procedure. Maximum nitrification rates of about 3.5 gN/

m2 d were obtained under non-inhibitory conditions.

Bulk aniline concentrations for the unacclimated biofilm

experiments were in the range of 0–47mg/L while those

for acclimated biofilm were in the range of 0–17mg/L.

Fitting the experimental results to the model yielded

inhibition coefficient, Ki; for aniline. In the case of

unacclimated biofilm, Ki of 3.2mg/L was observed while

for acclimated biofilm, the Ki was 3.0mg/L. The

transition of aniline penetration in the acclimated

biofilm from partial to full took place at a bulk aniline

concentration of 3.4mg/L. The biofilm nitrification

inhibition model is general and can be applied to other

inhibitory compounds. Also, the methodology devel-

oped in this study could be used for model validation.

Acknowledgements

This research was carried out as part of the AIT-DTU

Twinning Project funded by Danish International

Development Assistance (Danida). The authors are

thankful to Danida for funding of this project.

References

[1] Stevens DK. Interaction of mass transfer and inhibition in

biofilms. J Environ Eng 1988;114(6):1352–8.

[2] Gaudy Jr AF, Lowe W, Rozich A, Colvin R. Practical

methodology for predicting critical operating range of

biological systems treating inhibitory substances. J Water

Pollut Control Fed 1988;60:77.

ARTICLE IN PRESSS.H. Gheewala et al. / Water Research 38 (2004) 3179–31883188

[3] Gheewala SH, Annachhatre AP. Efficiency of nitrifying

biofilms under shock load conditions. Presented at the

Third IWA World Water Congress, Melbourne, Australia,

17–21 April 2002.

[4] Gheewala SH, Annachhatre AP. Efficacy of biofilms under

toxic conditions. J Environ Eng 2003;129(6):576–9.

[5] Manahan SE. Toxicology chemistry. Chelsea, MI: Lewis

Publishers Inc; 1989.

[6] Hockenbury MR, Grady CPL. Inhibition of nitrification—

effects of selected organic compounds. J Water Pollut

Control Fed 1977;49:768–77.

[7] Characklis WG. Process analysis. In: Characklis WG,

Marshall KC, editors. Biofilms. New York: Wiley; 1990.

[8] Wanner O, Gujer W. A multispecies biofilm model.

Biotechnol Bioeng 1986;28(3):314–28.

[9] Christiansen P, Hollesen L, Harremoes P. Liquid film

diffusion on reaction rate in submerged biofilters. Water

Res 1995;29(3):947–52.

[10] Harremoes P, Henze M. Biofilters. In: Henze M, Harre-

moes P, Jansen JLC, Arvin E, editors. Wastewater

treatment: biological and chemical processes. 2nd ed.

Berlin: Springer; 1997.

[11] Jansen JLC, Harremoes P, Henze M. Treatment plants for

nitrification. In: Henze M, Harremoes P, Jansen J, Arvin

E, editors. Wastewater treatment: biological and chemical

processes. 2nd ed. Berlin: Springer; 1997.

[12] Gheewala SH. Biodegradation of aniline. M. Eng. thesis,

Asian Institute of Technology, Thailand, 1995.

[13] Gheewala SH. Modelling of nitrification in biofilms under

inhibitory conditions due to aniline. Doctoral dissertation,

Asian Institute of Technology, Thailand, 2001.

[14] Karn SK. Biological nitrogen removal under inhibitory

conditions in a sequencing batch reactor. M. Eng. thesis,

Asian Institute of Technology, Thailand, 1998.

[15] Janning KF. Hydrolysis and oxidation of particulate

organic matter in biofilters. Ph.D. thesis, Department of

Environmental Science and Engineering, Technical Uni-

versity of Denmark, 1998.

[16] Oslislo A, Lewandowski Z. Inhibition of nitrification in the

packed bed reactors by selected organic compounds.

Water Res 1985;19(4):423–6.

[17] Than K, Gheewala SH, Annachhatre AP. Modelling of

nitrification inhibition due to aniline in suspended growth

processes. Water Environ Res 2002;74(6):531–40.

[18] Gheewala SH, Annachhatre AP. Biodegradation of Ani-

line. Water Sci Technol 1997;36(10):53–63.

[19] Lide DR. CRC handbook of chemistry and physics, 73rd

ed. Boca Raton, FL: CRC Press; 1992.