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Environmental Technology
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Improving the Biological Nitrogen Removal Process

in Pharmaceutical Wastewater Treatment Plants: A
Case Study
M. Torrijos , J. Carrera & J. Lafuente
Published online: 11 May 2010.

To cite this article: M. Torrijos , J. Carrera & J. Lafuente (2004) Improving the Biological Nitrogen Removal Process
in Pharmaceutical Wastewater Treatment Plants: A Case Study, Environmental Technology, 25:4, 423-431, DOI:
10.1080/09593332508618462

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 Environmental Technology, Vol. 25. pp 423-431
© Selper Ltd, 2004

IMPROVING THE BIOLOGICAL NITROGEN REMOVAL

PROCESS IN PHARMACEUTICAL WASTEWATER
TREATMENT PLANTS: A CASE STUDY

M. TORRIJOS, J. CARRERA* AND J. LAFUENTE

Department of Chemical Engineering, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain

(Received 28 January 2004; Accepted 16 February 2004)

ABSTRACT

The Biological Nitrogen Removal (BNR) process of some pharmaceutical wastewater treatment plants has important
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operational problems. This study shows that, in order to solve these problems, the design of industrial BNR processes
should start by analysing three key parameters: the characteristics of the wastewater load, the determination of the
maximum TKN removal rate and the detection of toxic or inhibitory compounds in the wastewater. A case study of this
analysis in pharmaceutical wastewater is presented here. In this case, the conventional TKN analytical method does not
make an accurate characterisation of the wastewater load because it measures a concentration of 100 mg TKN l-1 whereas the
real concentration, determined with a modified TKN analytical method, is 150-500 mg TKN l-1. Also, the TKN removal of the
treatment system is insufficient in some periods because it falls below legal requirements. This problem might be a
consequence of the wrong characterisation of wastewater during the design process. The maximum TKN removal at 27 ºC
(24 mg N g VSS-1 d-1 or 197 mg N l-1 d-1) was evaluated in a pilot-scale plant. This value is six times greater than the average
NLR applied in the full-scale plant. Finally, some of the components of the wastewater, such as p-phenylenediamine, might
have inhibitory or toxic effects on the biological process. P-phenylenediamine causes a large decrease in the nitrification rate.
This effect was determined by respirometry. This methodology shows that the effect is mainly inhibitory with a contact time
of 30 min and if the contact time is longer, 14 hours, a toxic effect is observed.

Keywords: Biological nitrogen removal, pharmaceutical wastewater, nitrification rate, respirometry, inhibition or toxicity.

INTRODUCTION treatment of some industrial wastewater, such as

There are many different kinds of human activity that
generate wastewater with large quantities of nitrogenous
compounds: petrochemical, pharmaceutical, fertiliser and
food industries, leachates produced by urban solid waste
landfills or waste from pig farms. Disposal of these
compounds is a serious environmental problem because free
ammonia diluted in water is one of the worst pollutants of
aquatic life [1]. For this reason, the European Directive for the
treatment of urban wastewater 91/271/EEC explicitly
demands the removal of ammonium from wastewater.
The BNR process is the most common method for
removing low quantities of ammonium from wastewater, but
this is not the usual treatment for high-strength ammonium
wastewater, where physical-chemical systems such as
stripping are more frequently used. Nevertheless, the BNR
process could be an interesting method of treating high-
strength ammonium wastewater from an environmental and
economical point of view [2, 3]. However, the biological
pharmaceutical water, has important operational problems.
First of all, the previous characterisation of wastewater for the
treatment design is sometimes inaccurate. This error is a
result of the heterogeneous composition of these effluents and
the presence of certain complexly structured nitrogenous
compounds. Secondly, the TKN removal capacity of the BNR
systems may be insufficient in some periods of the year. This
problem is usually a consequence of the wrong
characterisation of wastewater in the design process. Finally,
the nitrification process is susceptible to inhibition caused by
inhibitory or toxic compounds in the influent. Nitrification
inhibition by industrial compounds is a serious problem for
some small and large wastewater treatment plants [4, 5, 6, 7].
The continuous presence of an inhibitor or toxic component
could discard the biological treatment. If the presence is
occasional, it is essential to quantify its effect on the biological
process in order to choose the best alternative.
A case study is presented here of the analysis of the key
parameters in the BNR process of a pharmaceutical industry.

423
 The full-scale plant is an activated sludge process for nitrogen
and COD removal, with a modified Ludzack-Ettinger
configuration. The volume of the anoxic reactor is 131 m3 and
that of the aerobic reactor is 393 m3. The influent flow is 100
m 3 d- 1 and the HRT is 5.2 days. The full-scale plant was
designed to treat an industrial wastewater of 100 mg TKN l-1
and 6000 mg COD l-1. Nevertheless, there are ammonium
accumulations over 250 mg N-NH4+ l-1 during certain periods
of the year. The reason why the effluent ammonium
concentration is higher than the influent may be the use of a
wrong methodology to determine the average influent TKN
concentration. In addition, the ammonium accumulations
could be the result of the deficient TKN removal capacity
design of the system or the presence of a toxic/inhibitory
compound in the wastewater.
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Figure 1. Plan of the treatment pilot plant.
424
MATERIAL AND METHODS
Pilot-scale Plant
The pilot-scale plant consists of an anoxic reactor (18
litres), two aerobic reactors (27 litres each) and a settling tank
(Figure 1). The volume ratio between the anoxic and aerobic
reactors is the same as that of the full-scale plant. Each
mechanical unit of the pilot-plant (pumps, level detectors,
etc.) is controlled by a PLC, which enables automation of all
these elements. Each pilot-plant reactor has in-line sensors
(DO, pH, ORP, temperature) connected to probe controllers.
A control and monitoring computer supervises the PLC (via
RS-232). This computer also acquires data from the probe
controllers (via RS-485) and it controls the DO through the
 manipulation of the pneumatic control valves of each aerobic
reactor (via 4-20 mA loop). This control loop is based on a
digital PID algorithm programmed in the computer. The pH
control of the first aerobic reactor is based on an ON/OFF-
algorithm acting over a solid dispenser that adds sodium
carbonate [8].
The pilot-plant was inoculated with biomass from the
full-scale plant. The NLR and the rTKN of the pilot-plat were
defined as:
[ TKN] in
NLR = (i)
HRT ⋅ [VSS]
[ TKN] in − [ TKN] out
rTKN = (ii)
HRT ⋅ [VSS]
Respirometry
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This study used an LSS respirometer. In this
respirometer, the gas and liquid phases are static and the DO
is measured in the liquid phase [9]. The volume of the
respiration vessel is 100 ml and it is magnetically stirred. In
the respiration vessel, pH, DO and temperature are measured
in the liquid phase through the DO probe (WTW-Cellox 325)
Figure 2. Diagram of the respirometric procedure.
425
and the pH probe (WTW-Sentix 81), which are connected via
RS-232 to the PC. The respiration vessel is submerged in an
isothermal bath. The chosen temperature set point was 25 ºC
and the pH was maintained at between 8.0-8.2.
Before any OUR determination, the biomass is aerated
until it reaches a level of 7-8 mg DO l-1. The procedure starts
with the evaluation of the endogenous OUR (OURend) without
substrate. After that, a pulse of substrate (5 mg N-NH4+ l-1) is
added in order to evaluate the first exogenous OUR (OURN).
The OURN is considered the sum of the OURend and the OUR
resulting from the nitrification (OURnit). The r N is calculated
using the OURnit and the stoichiometry of nitrification
reaction. When all the ammonium is oxidized and the
biomass returns to an endogenous state, a pulse of
toxic/inhibitor (variable concentration) is added in order to
calculate the second exogenous OUR (OURH). The OURH is
considered the sum of the OURend and the OUR resulting
from the toxic/inhibitor (OURTI). After a certain contact time
between the toxic/inhibitor and the biomass, another pulse of
substrate (5 mg N-NH4+ l-1) is added and the measurement is
the third exogenous OUR (OURN + H ). The OUR N + H is
considered the sum of the OURend, the OURTI (both previously
evaluated) and the OUR resulting from the nitrification
affected by the toxic/inhibitor (OURnit-TI). The nitrification
rate affected by the toxic/inhibitor (rN-TI) is calculated
using the OURnit-TI and the nitrification stoichiometry. A
diagram of the respirometric procedure is shown in figure 2.
 The percentage of TI is defined as the decrease in the rN at a
toxic/inhibitor concentration:
r −r
% TI = N N− TI ⋅ 100 (iii)
rN
Analytical Methods
The analysis of total suspended solids (TSS), volatile
suspended solids (VSS), sludge volume index (SVI), alkalinity,
chemical oxygen demand (COD) and ammonium were made
using the methodology described in Standards Methods [10].
Nitrates were measured by capillary electrophoresis using a
WATERS Quanta 4000E CE. The electrolyte used was a
WATERS commercial solution. The conditions of the analysis
were: temperature of 20 ºC, 15 kV from a negative source,
indirect UV detection at 254 nm and 5 minutes of analysis.
The biomass composition was determined by the UAB
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increase the ammonium concentration in the system. The
main nitrogenous components of this pharmaceutical
wastewater are: carnitine hydrochloride (β-hydroxy-γ-
(trimethylammonio)butyrate) (C7H16ClNO3), chloramphenicol
α-succinate (C16H16Cl2N9O8) and trimethylamine (C3H9N). The
conventional TKN method was modified to solve this
analytical problem. The modified method is a double
digestion. The conditions of the first digestion are: 10 ml
H 2SO4, 1 g catalyst, 2 hours and T = 350 ºC. The conditions of
the second digestion are stronger than the first digestion:
20 ml H2SO 4, 2 g catalyst, 6 hours and T = 350 ºC. The TKN
concentrations obtained using this modified method for the
three pure components are presented in Table 1.
The percentage of hydrolysed carnitine using the first
digestion is only 6 %. This percentage increase to 55 % for the
second digestion. The hydrolysed succinate is approximately
the same for both digestions, around 70 %. Finally, the
trimethylamine is totally hydrolysed in the first digestion. So,
the only increase in the TKN concentration of a wastewater
sample between both digestions is a result of the carnitine.

Analytical Service using elemental organic microanalysis This increase represents 50 % of the TKNcarnitine, so the
(NCHS) by means of Carlo Erva EA1108. TKNcarnitine of the sample can be calculated as:

RESULTS AND DISCUSSION

[ TKN] 6h − [ TKN] 2h
Characterisation of Wastewater Load
[ TKN] carnitine = 0.5
(iv)

The average TKN of the industrial wastewater The hydrolysed carnitine for the second digestion is 55 %, so
measured using the conventional method described in the total TKN of the sample can be calculated as:
Standards Methods [10] is 100 mg TKN l-1 but it is clearly
incorrect because there are accumulations of over 250 mg N-
NH4+ l-1 during some periods. The conventional method does
[ TKN] total = [ TKN] 6h + (1 − 0.55) ⋅ [ TKN] carnitine (v)
not make a complete chemical hydrolysis of some complex
nitrogenous organic molecules, although the heterotrophic
microorganisms of the biological process could Finally, the combination of the equations (iv) and (v) is the
easily hydrolyse these compounds to generate energy and total TKN of the sample:

Table 1. Analytical conditions employed and TKN concentrations obtained for carnitine, succinate and trimethylamine.

Blank Sample H2SO4 Catalyst Digestion TKN

Sample T (ºC)
(mg N l-1) volume (ml) volume (ml) (g) time (h) (mg N l-1)
Carnitine 100 6
50 10 1 2 350
100 7
100 54
50 20 2 6 350
100 57
Succinate 100 71
50 10 1 2 350
100 72
100 66
50 20 2 6 350
100 66
Trimethylamine 100 100
50 10 1 2 350
100 107

426
 [ TKN] 6h − [ TKN] 2h
[ TKN] total = [ TKN] 6h + 0.45 ⋅
0.5
Applying the modified method, the real TKN
concentration of the wastewater was found to be in the range
of 150-500 mg TKN l-1. This value explains the high
concentration of N-NH4+ in the effluent during some periods.
This method cannot measure 30 % of the succinate of the
sample but this is the minor component and this
approximation does not imply a significant error in the real
TKN. This new methodology can be applied to any
pharmaceutical wastewater if the main nitrogenous
components are identified.
Determination of the Maximum TKN Removal Rate
An experiment at 27 ºC was carried out in the pilot-
plant in order to determinate the r TKNmax of the system. The
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experiment began with the average NLR applied in the full-
(vi)
scale plant (4 mg N g VSS- 1 d- 1). This NLR was gradually
increased until an ammonium accumulation was detected. In
these conditions, the observed nitrification rate is r TKNmax.
Table 2 shows the experimental conditions.
Figure 3 shows the NLR of each run and the
ammonium concentration in the influent and the effluent of
the pilot-plant. The rTKNmax (24 mg N g VSS-1 d-1 or 197 mg N l-1
d-1) was six times higher than the average NLR applied in the
full-scale plant. Another way to assess the nitrification
capacity of the pilot plant is to calculate the actual maximum
autotrophic rate (µA,T) as proposed by Nowak et al. [11]. The
µ A,T augmented from 0.3 d-1 at run I to 1.5 d- 1 at run V.
However, some of the ammonium accumulations in the full-
scale plant occur in winter, when the minimum temperature
in the reactor is 12 ºC. The temperature effect is commonly
described using an Arrhenius-type equation [12]:

Table 2. Experimental conditions of the pilot-plant in determination of the maximum TKN removal rate.

NLR
HRT [TKN]in [VSS] Fin Influent
Run mg N Mg N FIR/Fin FER/Fin T (ºC)
(days) (mg N l-1) (mg l-1) (l d-1) COD/N
g VSS-1 d-1 l-1 d-1
I 4.0 ± 0.2 17 ± 1 10 165 4200 7.2 31:1
II 10 ± 2 51 ± 10 5 250 5100 14.4 23:1
III 14 ± 1 80 ± 6 3.4 270 5700 20.9 4 2 23:1 25-30
IV 20 ± 6 130 ± 39 3.8 500 6500 19.1 11:1
V 36 ± 5 295 ± 41 1.6 470 8200 46.1 11:1

Figure 3. NLR, COD and nitrogen concentration in the pilot-scale plant through the determination of the maximum TKN
removal rate.

427
 rTKN max,T1 = rTKN max,T 2 ⋅ θ
( T1− T 2) % Nitrification =
N removed − N assimilated
⋅ 100 (x)
(vii)
N removed

The temperature coefficient employed, (θ = 1.127), was

% Assimilation = 100 − % Nitrification (xi)
chosen from a similar process to that presented in this study
[13]. The rTKNmax at 12 ºC is 4.0 mg N g VSS-1 d-1 (or 33 mg N l-1
This nitrogen mass balance was performed in two
d-1) the value of the average NLR applied in the full-scale
different periods (run I and run V). Table 3 shows the result of
plant. Therefore, despite the analytical difference, the full-
these mass balances. The most important way of TKN
scale plant should have a good enough capacity for TKN
removal is nitrification, a way that increases when the system
removal. The nitrate and COD concentrations in the effluent
is operated for a long time in favourable conditions for
were always below 50 mg N-NO3- l-1 and 500 mg O2 l-1,
nitrification. The percentage nitrification in run V is 77 %, so
respectively (figure 3). This suggests that the range of the
the rNmax is 18.5 mg N g VSS-1 d-1 (or 152 mg N l-1 d-1) at 27 ºC.
nitrate and COD removal efficiencies was 85-100 %.
In a BNR process with a high influent COD/N ratio, a
Detection of Toxic or Inhibitory Compounds by Respirometry
significant part of the TKN removal is a result of the nitrogen
assimilation in the growth of the heterotrophic biomass.
The final key parameter in the BNR process of
Consequently, the nitrification rate can only be calculated if a
pharmaceutical wastewater is the detection of compounds
nitrogen mass balance is performed.
with inhibitory or toxic effects during the biological process.
One of the most employed techniques for determining and
The nitrogen removed per day is:
quantifying the toxicity or inhibition of a compound is
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respirometry [14, 15, 16, 17]. In this case, respirometry was

N removed = Fin ⋅ [ TKN] in − Fout ⋅ [ TKN] out
The nitrogen assimilated per day by the heterotrophic
biomass can be calculated as (it is assumed that the biomass is
mainly heterotrophic and its concentration is in steady-state in
every run):
N assimilated = gVSS purge ⋅ %N biomass + gVSS out ⋅ %N biomass
The percentage of nitrification and nitrogen assimilation
can be calculated using equations (x) and (xi).
(viii)
used to evaluate the percentage of TI (equation iii) of the
p-phenylenediamine (C6H 8N 2). This compound is sometimes
present in the wastewater and may partly be responsible for
some ammonium accumulations in the full-scale plant.
The percentage of TI was evaluated at five different
concentrations of p-phenylenediamine (1, 3, 5, 7 and 10 mg l-1),
with a contact time between the p-phenylenediamine and the
(ix)
biomass of 30 min. The effect of p-phenylenediamine in the
nitrification process can be described by the inhibition kinetic
models in Table 4 [18].

Table 3. Biomass composition and nitrogen mass balance in runs I and V.

Biomass composition (%)

Run Nremoved (g d-1) Nassimilated (g d-1) % Nitrification % Assimilation
C N H
I 43.8 8.8 6.5 5.0 1.7 66 34
V 45.2 9.6 6.6 16.4 3.8 77 23

Table 4. Inhibition kinetic models [18]. Figure 4 shows that experimental data is in accordance with
the behaviour predicted by the Aiba model and the
Kinetic model Equation Levenspiel model of product inhibition with a correlation
  [ TI] β coefficient (r2) of 0.99 in both models. The experimental data is
Levenspiel % TI = 1 − 1 −  
  K Le  not in accordance with the behaviour predicted by the other
models. The kinetic constants of the inhibition models are
Aiba % TI = 1 − exp
(− KA ⋅[TI]) shown in Table 5.
  γ

% TI = 1 − 1 − 
[ TI]  
 
These results describe the effect of p-phenylenediamine
Luong
K  in the nitrification process with a contact time of 30 min.
  Lu  
However, these results cannot be used to assess whether the
 K  decrease in the nitrification rate is a result of toxicity or
Non-competitive % TI = 1 −  Nc 

 K Nc + [ TI] 
inhibition. The procedure was modified to determine only the
toxic effect. This modification involved removing the
p-phenylenediamine after the contact time with the biomass.

428
Downloaded by [UPM] at 06:58 30 December 2014

Figure 4. % TI on nitrification for different p-phenylenediamine concentrations and predicted behaviour by Aiba and Levenspiel
kinetic models. The experimental conditions are: T = 25 ºC, pH = 8.0-8.2 and 30 min of contact time. Errors bars
indicate ± standard deviation.

Table 5. Kinetic constants of the adjusted models for the Table 6. % T/I with and without washing of the biomass
inhibition of nitrification by p-phenylenediamine (T after contact time with the p-phenylenediamine.
= 25 ºC).
[p-phenylenediamine] Contact Washing after %
Kinetic model Coefficients (mg l-1) time contact time T/I
KLe = 24 ± 17 (mg l-1) 7 30 minutes Non 90 ± 1
Levenspiel
β=7±5 7 30 minutes Yes 20 ± 5
Aiba KA = 0.31 ± 0.01 (l mg ) -1
7 14 hours Yes 92 ± 1

The biomass is washed with deionised water and
resuspended in water with micronutrients. After that, the
nitrification rate is re-evaluated. Two contact times were
tested using the modified procedure: 30 min and 14 hours.
Both tests were carried out with 7 mg
p-phenylenediamine and compared to the procedure without
washing of the biomass (Table 6). After 30 min of contact time,
the effect of the p-phenylenediamine is mainly inhibitory
because the biomass recovers the activity when the inhibitor is
removed. On the other hand, the effect of p-phenylenediamine
is mainly toxic after 14 hours because the biomass does not
resume its activity after the removal of the inhibitor. Another
explanation for the apparent toxicity of p-phenylenediamine
could be that this substance is adsorbed by the nitrifying
biomass. This phenomenon is known for allythiourea (ATU)
l -1
of that it is an adsorbed biodegradable inhibitor of the
nitrification process [4].
The disposal of p-phenylenediamine in the wastewater
causes a critical problem in the BNR process. The apparent
toxicity reaches a total with 7 mg l-1 and a contact time of 14
hours whereas the HRT of the full-scale plant is 5.2 days.

429
 CONCLUSIONS would like to thank Jaume Cubells for his support.

This work shows that the design of BNR processes for

industrial wastewaters should begin with an analysis of these NOMENCLATURE
three key parameters: wastewater characterisation, maximum
TKN removal rate and presence of toxic or inhibitory BNR = biological nitrogen removal
compounds. In particular, this case study of a pharmaceutical COD = chemical oxygen demand
wastewater shows that: DO = dissolved oxygen
The conventional TKN analytical method does not Fin = influent flow
make an accurate wastewater characterisation because it Fout = effluent flow
measures a concentration of 100 mg TKN l-1 whereas the real FIR/Fin = internal recycle/influent flow ratio
concentration, determined using a modified TKN analytical FER/Fin = external recycle/influent flow ratio
method, is 150-500 mg TKN l-1. This new methodology can be HRT = hydraulic retention time
applied to any pharmaceutical wastewater if the main KLe = Levenspiel inhibition constant
nitrogenous components are identified. KA = Aiba inhibition constant
The rTKNmax at 27 ºC (24 mg N g VSS-1 d-1 or 197 mg N l-1 KLu = Luong inhibition constant
d-1) was evaluated in a pilot-scale plant. The temperature KNc = Non-competitive inhibition constant
effect reduces the rTKNmax to 4 mg N g VSS-1 d-1 at 12 ºC. This LSS = Liquid Static Static Respirometer
value is the average NLR applied in the full-scale plant. NLR = nitrogen loading rate
Therefore, despite the analytical difference, the full-scale plant OUR = oxygen uptake rate
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should have a good enough capacity for TKN removal. 77 % θ = Temperature coefficient
of TKN removal is via nitrification while the remainder is µA,T = actual maximum autotrophic growth rate
assimilated by the heterotrophic biomass. PID = proportional integral derivative controller
The p-phenylenediamine causes a significant decrease rTKN = TKN removal rate
in the nitrification rate. This effect was determined by rTKNmax = maximum TKN removal rate
respirometry. After short contact time (30 min), the effect is rN = nitrification rate
mainly inhibitory and can be described by the Aiba and rNmax = maximum nitrification rate
Levenspiel inhibition kinetic models. However, the effect is rN-TI = nitrification rate affected by toxic/inhibitor
apparently toxic after longer contact time (14 hours) and the SVI = sludge volumetric index
biomass does not resume its activity. The disposal of p- % TI = toxicity or inhibition percentage
phenylenediamine is the main operational problem of the full- [TI] = toxic or inhibitor concentration
scale plant. TSS = total suspended solids
TKN = total Kjeldahl nitrogen
ACKNOWLEDGMENTS [TKN]in =influent TKN concentration
[TKN]out = effluent TKN concentration
This work has been supported by CICYT (project VSS = volatile suspended solids
REN2000-0670/TECNO). The Department of Chemical [VSS]reactor = reactor biomass concentration
Engineering UAB is member of the Centre de Referència en β = Levenspiel coefficient
Biotecnologia de la Generalitat de Catalunya. The authors γ = Luong coefficient

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