ARTICLE IN PRESS

Water Research 37 (2003) 4211–4221

Biological nitrogen removal of high-strength ammonium

industrial wastewater with two-sludge system
J. Carrera*, J.A. Baeza, T. Vicent, J. Lafuente
"
Departament d’Enginyeria Qu!ımica, ETSE, Universitat Autonoma de Barcelona, Bellaterra 08193, Spain
Received 21 June 2002; received in revised form 28 April 2003; accepted 12 June 2003

Abstract

The biological nitrogen removal (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. The capacity

1
to biologically remove the nitrogen content of a real industrial wastewater with a concentration of 5000 mg N-NH+ 4 L
is demonstrated in this work. The experimental system used is based on a two-sludge system, with a nitrifying activated
sludge and a denitrifying activated sludge. This system treated real industrial wastewater for 450 days, and during this
period, it showed the capacity for oxidizing all the ammonium at average nitrification rates between 0.11 and 0.18 g N-

1
1
NH+ 4 g VSS d . Two key process parameters were evaluated: the maximum nitrification rate (MNR) and the
maximum denitrification rate (MDR). MNR was determined in continuous operation at three different temperatures:

1
1
15 C, 20 C and 25 C, obtaining values of 0.10, 0.21 and 0.37 g N-NH+ 4 g VSS d , respectively. Complete
denitrification was achieved using two different industrial carbon sources, one containing mainly ethanol and the other
one methanol. The MDR reached with ethanol (0.64 g N-NO
x g VSS

1
1
d ) was about 6 times higher than the MDR
reached with methanol (0.11 g N-NO
x g VSS
1
1
d ).
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Biological nitrogen removal (BNR); Nitrification; Denitrification; Two-sludge system; Industrial high-strength ammonium
wastewater; External carbon source; Ethanol; Methanol

1. Introduction strength ammonium wastewater, where physical–chemi-

There are many different kinds of human activity that
generate wastewater with large quantities of ammonium:
petrochemical, pharmaceutical, fertilizer and food in-
dustries, leachates produced by urban solid waste
disposal sites or waste from pig farms. Disposal of this
type of waste is a serious environmental problem
because free ammonia, diluted in water, is one of the
worst contaminators of aquatic life [1].
The BNR process is the most common method for
removing low quantities of ammonium from waste-
water, but this is not the usual treatment for high-
*Corresponding author. Tel.: +34-935812141; fax: +34-
935812013.
E-mail address: julian.carrera@uab.es (J. Carrera).
cal systems such as stripping are more frequently used.
The main problem of the biological treatment of high-
strength industrial wastewater is that high concentra-
tions of ammonium or nitrite inhibit the nitrification [2].
However, the BNR process could be an interesting way
for treating high-strength ammonium wastewater from
an environmental and economical point of view [3].
The nitrification and denitrification rates are the key
parameters in the design of a biological wastewater
treatment plant with nitrogen removal. For this reason,
it is essential to experimentally determine the maximum
nitrification rate (MNR) and maximum denitrification
rate (MDR) in similar conditions, to those of the
treatment plant on an industrial scale [4].
This study aims to define a biological treatment
system for a real industrial wastewater with an

0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0043-1354(03)00338-5
 ARTICLE IN PRESS
4212 J. Carrera et al. / Water Research 37 (2003) 4211–4221

1
ammonium concentration of 5000 mg N-NH+ 4 L . This Table 1
concentration is higher than those generally found in the Industrial high-strength ammonium wastewaters
bibliography (Table 1).
Wastewater Concentration Reference
Different configurations for BNR can be used but, in (mg N-NH+
1
4 L )
order to minimize the total volume of the system, two-
sludge systems are preferable. This paper presents one of Tannery 200–500 [5]
these systems with two separate stages; the first one is a Sludge dewatering 600–700 [6]
nitrifying activated sludge stage and the second one is a Leachate 1200 [7]
denitrifying process. Each stage consists of a reactor and Landfill leachate 2200 [8]
a settler, which produce the growth of two different
microbial populations: a nitrifying biomass and a
denitrifying biomass. Table 2
In order to treat industrial wastewater with a low Basic composition of both real industrial wastewaters
COD/N ratio, it is necessary to add an external source of
Component N-wastewater COD-wastewater
organic carbon. Different criteria have been used to (mg L
1) (mg L
1)
choose a specific external carbon source in the deni-
trification process. First, it is necessary to consider COD 0 1300–1500
which carbon compound generates the highest MDR. N-NH+4 4000–6000 0
Published references give contrasting results. Some F
30–50 0
authors suggest that acetic acid achieves higher rates Cl
500–600 700–1000
SO2

4 15000–20000 300–800
than glucose, methanol or ethanol [9]. However, other
authors achieved similar results with acetic acid to those
achieved with methanol [10,11]. Some references in-
dicate that ethanol reaches higher rates than methanol formed. Therefore, an external carbon source was also
[12,13], although another study indicates the opposite used.
[14]. The basic composition of the two real industrial
It is also necessary to consider the costs and wastewaters are summarized in Table 2. As can be seen,
availability of the external carbon source. If the source the concentration of ammonium in the N-wastewater

1
is a chemical compound (ethanol, methanol, and acetic ranged from 4000 to 6000 mg N-NH+ 4 L , while the

acid), it will be available at a market price. If there are
plans to build an industrial scale treatment plant, the
external carbon source should be cheap and be produced
in sufficient quantities to guarantee the continuous
operation of the wastewater treatment plant. These
requirements can be achieved using carbonaceous by-
products, which are currently generated by an industry,
but that are not considered waste products.
This paper focuses on the study of the nitrification
and denitrification stages of a two-sludge system, with
the specific aim of determining the MNR and the MDR.
In addition, the influence of temperature on the
nitrification process and different external carbon
sources on the denitrification process were also studied.
2. Materials and methods
2.1. Description of wastewaters
concentration of COD was 1300–1500 mg COD L
1 in
the COD-wastewater. Most of this organic matter was
ethanol, and thus easily biodegradable. The N-waste-
water also contained high concentrations of chloride
and sulphate anions.
2.2. Description of the external carbon sources
Two external carbon sources were used, both
by-products of two industrial processes. The first
by-product was a mixture of waste from alcoholic
drinks production, and the main carbon source was
ethanol. The second by-product was a waste from a
chemical industry, made up of a mixture of methanol,
isopropylic alcohol and acetone. Its main component
was methanol. Table 3 shows the composition of the two
by-products. Throughout this study, they have been
called the ‘ethanol mixture’ (that formed by waste from
alcoholic drinks) and the ‘methanol mixture’ (that
formed from chemical waste).

In addition to the high-strength ammonium waste-
water (N-wastewater), the process included the treat-
ment of a second industrial wastewater that contained
mainly organic matter (COD-wastewater) and useful for
the denitrification. However, this organic matter was
insufficient to denitrify all the nitrate that had been
2.3. Monitoring and control system
Every reactor of the treatment plant had in-line
sensors (dissolved oxygen (DO), pH, ORP, temperature)
connected to probe controllers. All these controllers and
the mechanical elements of the pilot plant were
 ARTICLE IN PRESS
J. Carrera et al. / Water Research 37 (2003) 4211–4221 4213

connected to a PC, through different data acquisition itoring, data backup, and control of key process
cards (Advantech PCL726, PCL813 and PCLD885). parameters (flows, pH, DO and temperature). The pH
Specific software was developed in C language in order control was based on an ON/OFF-algorithm acting over
to automate all the system. It was based on previous a solid dispenser that adds sodium carbonate. The DO
software developments [15] and included graphic mon- control was based on a digital PID algorithm pro-
grammed in the computer, which modifies the airflow
using a mass flowmeter (Bronkhorst Hi-Tec, 0–20
Table 3 Ln min
1). The treatment plant is located indoors,
Percentage composition of the by-products used as external permitting temperature regulation through a heating,
carbon sources ventilation and air-conditioning system.
By-product Component Percentage
(in weight)
2.4. Experimental settings
Waste from alcoholic drinks Ethanol 8.7
(‘ethanol mixture’) The experimental work was carried out in a two-
Glucose 0.8 sludge, treatment plant with two separate stages:
Glycerol 0.5
nitrification and denitrification. Fig. 1 shows a diagram
Water 90.0
of the treatment plant. The N-wastewater was fed into
Chemical waste (‘methanol Methanol 60.0 the nitrifying activated sludge system, made up of a 27 L
mixture’) aerobic reactor and a settler. The aerobic reactor used
Acetone 10.0 the automatic pH control with a setpoint of 7.5. The DO
Isopropylic 10.0 was maintained at 3 mg O2 L
1 through the PID
alcohol controller. The temperature was maintained at 20 C
Water 20.0 for 300 days, then it was changed at 15 C for 30 days

Alkalinity recirculation

Carbonate ORP DO ORP DO

dosage pH Temp pH Temp
Effluent

Settler Settler

N-wastewater
Aerobic Anoxic Nitrogen gas
stripping

Sludge recirculation
Sludge recirculation
Stirrers

COD-wastewater Mass
flowmeter

External carbon source Frequency

converters

Air Probes
Pumps
PC pH control
Water and sludge line
Air line
Signal line

Fig. 1. Layout of the treatment plant.

 ARTICLE IN PRESS
4214 J. Carrera et al. / Water Research 37 (2003) 4211–4221
and finally changed to 25 C. The sludge retention time
(SRT) was kept at around 25 days.
The effluent of the nitrifying activated sludge system
was one of the three influents to the denitrifying
activated sludge system (Fig. 1). The other two were
the COD-wastewater and the external carbon source.
The denitrification stage was made up of a 27 L reactor
without aeration; a 15 L aerated tank and a settler. The
nitrogen gas formed in the anoxic reactor was stripped
in the aerated tank, thus favouring the sedimentation
stage that follows. The temperature was the same as for
the aerobic reactor. The pH was not controlled and its
value was about 8.0–9.0. The SRT was kept at around
15 days.
The nitrogen-loading rate (NLR), nitrification and
denitrification rates were defined as
 
N
NHþ4 in
NLR ¼ ; ð1Þ
HRTreactor ½VSSreactor
    
N
NHþ4 in
N
NH4 out
þ
rnitrification ¼ ; ð2Þ
HRTreactor ½VSSreactor
    
N
NO
x in
N
NOx out

The solid dispenser was calibrated to give an exact
rdenitrification ¼ ; ð3Þ
HRTreactor ½VSSreactor
where HRT is the hydraulic retention time, [VSS]reactor
the biomass concentration, [N-NH+ 4 ]in the influent
ammonium concentration, [N-NH+ 4 ]out the effluent
ammonium concentration, [N-NO
x ]in the influent oxi-
dized nitrogen concentration and [N-NO
x ]out the
effluent oxidized nitrogen concentration.
2.5. Analytical methods
The analysis of total suspended solids (TSS), volatile
suspended solids (VSS), sludge volumetric index (SVI),
alkalinity and ammonium were carried out using the
methodology described in APHA’s Standard Methods
[16]. The analyses of chloride (Cl
), sulphate (SO2
4 )
nitrite (NO



2 ), nitrate (NO3 ) and fluoride (F ) were
carried out 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 C, 15 kV from a
negative source, indirect UV detection at 254 nm and
5 min of analysis.
3. Results and discussion
The nitrifying and denitrifying reactors of the present
study were inoculated with biomass emanating from a
preliminary study using a modified Ludzack-Ettinger
configuration that treated these industrial wastewaters
for a year [17]. Both systems started operating with the
same microbial population and then developed towards
specialized biomass (owing to the specific experimental
conditions). The two-sludge plant was operated con-
tinuously for 450 days, using real, high-strength
industrial wastewater.
3.1. Nitrification
3.1.1. pH control
The N-wastewater contained less alkalinity than
stoichiometrically required for nitrification. In order to
solve this problem, an automatic system for pH control
was installed for adding solid sodium carbonate to the
nitrifying reactor. This control supplies the necessary
alkalinity so that the pH conditions favour the process.
The optimal pH for the nitrification process is in the
range of 7.5–8.5 [18]. It was chosen a value of 7.5, since
at higher pH, the equilibrium ammonium–ammonia
would be displaced to ammonia, which can inhibit the
process. The addition of sodium carbonate also pro-
vided suitable conditions for the nitrifying microorgan-
isms, by avoiding potential restrictions in the use of one
of their substrates: inorganic carbon.
amount of sodium carbonate (1.3 g Na2CO3 per dosage).
As the control system kept a register of the total dosages
per day, it was possible to quantify the total amount of
carbonate used per day. During the experiments, it was
checked to see if the carbonate consumption agreed with
the theoretical values of alkalinity destruction, accep-
table for design of nitrification systems (7.1 g CaCO3 per
g N-NH+ 2
+
4 consumed, or 4.26 g CO3 /g N-NH4 ) [18].
The experimental value obtained was 6.170.6 g Na2CO3
per g N-NH+ 4 consumed, which corresponds to
4.470.4 g CO2
+
3 /g N-NH4 (a value which is very close
to the theoretical).
From day 140 of operation, part of the water coming
out of the denitrifying system was recirculated back to
the nitrifying system. Part of the alkalinity generated by
the denitrifying system is recovered with this recircula-
tion, thus decreasing the amount of added carbonate.
3.1.2. Sludge evolution
The initial biomass concentration of the nitrifying
reactor was about 4000 mg VSS L
1. Fig. 2 shows the
VSS and TSS concentrations of this reactor and its SVI.
The VSS concentration was kept around 35007700 mg
VSS L
1 throughout the study. The VSS/TSS ratio was
around 4878%. Nevertheless, this ratio decreased
below the average value between 200 and 250 days; this
decrease was due to the change in the external alkalinity
source. The original sodium carbonate was changed to
another cheaper source, calcium hydroxide; the addition
of calcium ion in the nitrifying reactor caused the
precipitation of sulphate and chloride salts. These salts
increased the inorganic composition of the sludge and
 ARTICLE IN PRESS
J. Carrera et al. / Water Research 37 (2003) 4211–4221 4215

14000 100 200

TSS
VSS 90
12000 % VSS/ TSS
average % VSS/ TSS 80

TSS and VSS (mg·L )

VSS/ TSS percentage

-1
SVI 150
10000 70

SVI (mL·g-1)
60
8000
50 100
6000
40

4000 30
50
20
2000
10
0 0 0
0 50 100 150 200 250 300 350 400 450
Time (days)

Fig. 2. TSS, VSS, % VSS/TSS and SVI of the nitrifying sludge system throughout the study.

7000
without recirculation with recirculation
100
6000
90

Percentage of ammonium removal

5000 [N-NH4+ ] influent 80
[N-NH4+ ] effluent 70
[N] (mg·L-1)

[N-NOx- ] effluent
4000 60
% ammonium removal
50
3000
40
2000 30
20
1000
10
0 0
0 50 100 150 200 250 300 350 400 450
Time (days)

Fig. 3. Nitrogen concentration in the influent and the effluent of the nitrifying sludge system and the percentage of ammonium
removal throughout the study.

the VSS/TSS ratio decreased by 30–35%. In order to centration of the nitrifying system, but not a decrease in
correct this problem, the alkalinity source was changed the NLR. Table 4 shows the operational parameters of
again to sodium carbonate and the VSS/TSS ratio the two runs. The average nitrification rate was very
reached the average value of the study. The nitrifying high in both runs. The ammonium removal percentage
sludge showed good settleability throughout the study, was also very high throughout the study, ranging from
with SVI values below 50 mL g
1. 90% to 100%.
The comparison of ammonium concentration in the
3.1.3. Nitrogen removal influent with oxidized nitrogen concentration (nitrate
Fig. 3 shows the concentrations of all nitrogen plus nitrite) in the effluent, confirmed that the ammo-
compounds in the influent and the effluent of the nium removal is due to the nitrification process,
nitrifying system and the ammonium removal percen- since both concentrations are about equal. In
tage during the 450 days of operation. There are two addition, the carbonate consumption obtained (Section
periods, the first one without alkalinity recirculation and 3.1.1) agreed with the nitrification stoichiometry. These
the second one with alkalinity recirculation (the effluent results reject the concept of ammonia removal by
of the denitrifying system, see Fig. 1). This recycle stripping or other biological ways like ANAMMOX
involved the dilution of the influent ammonium con- process [19].
 ARTICLE IN PRESS
4216 J. Carrera et al. / Water Research 37 (2003) 4211–4221

Table 4
Operational parameters for the nitrifying system throughout the study

Run Period [N-NH+4 ]influent [N-NH+4 ]effluent Influent flow HRT VSS Average nitrification rate
(days) (mg N L
1) (mg N L
1) (L d
1) (days) (mg L
1) (g N-NH+4 g VSS

1
1
d )

1 140 48007450 80750 2.7–3.0 9–10 35007500 0.13–0.15

2 310 19007400 40720 5.4–9.0 3–5 35007800 0.11–0.18

3.1.4. Maximum nitrification rate 0.15 2000

The experiments to determine the MNR were done
under similar conditions to those of an industrial scale

NLR and nitrification rate

(g N-NH4+·g VSS ·d )
-1 -1
1500

[N-NH4 ] (mg·L )
treatment plant, i.e. continuous. The problem with

-1
0.10
doing these experiments was the high nitrogen concen-
tration in the N-wastewater, because if the NLR is 1000

+
Nitrification rate
higher than the MNR, a large accumulation of 0.05 NLR
[N-NH4+ ] influent
ammonium can take place. This accumulation can 500
[N-NH4+ ] effluent
inhibit the process [2] and the observed nitrification rate
would not be the maximum. Therefore, the experiments 0.00 0
were carried out with a gradual and controlled increase 0 5 10 15 20
(a) Time (days)
in the NLR, so that the nitrification rate was the as close
as possible to the NLR. Only when the NLR was slightly
0.4 2500
above the MNR, was there some accumulation of
ammonium in the system; however, the observed
NLR and nitrification rate

2000
(g N-NH4+·g VSS ·d )
-1 -1

0.3
nitrification rate was still at maximum.

[N-NH4+ ] (mg·L-1)
Three experiments were carried out, at 15 C, 20 C 1500
and 25 C. Fig. 4(a) shows the results of the first 0.2
experiment in which the temperature was 15 C. The Nitrification rate 1000
experiment began with a NLR of 0.06 g N-NH+
NLR
4 g 0.1 [N-NH4+ ] influent
VSS
1 d
1. Since no accumulation of ammonium [N-NH4+ ] effluent 500

occurred, the NLR was increased to 0.13 g N-NH+ 4 g

0.0 0
VSS
1 d
1. This NLR was clearly higher than the MNR 0 5 10 15 20

1
at 15 C, since accumulation of 150 mg N-NH+ 4 L
(b) Time (days)

was produced. The MNR was calculated as an average 0.6 2000

of the rates measured between days 5 and 16, i.e. a
period of three HRTs, and was found to be 0.1070.01 g 0.5
NLR and nitrification rate
(g N-NH4+·g VSS ·d )
-1 -1

1
1
N-NH+ 4 g VSS d . The results of the second and the 1500

[N-NH4+ ] (mg·L-1)
0.4
third experiments are shown in Figs. 4(b) and (c),
respectively. The MNR in both experiments was 0.3 1000
Nitrification rate
evaluated using the same procedure of the first experi- NLR
0.2 [N-NH4+ ] influent
ment. The MNR in the second experiment (T ¼ 20 C) [N-NH4+ ] effluent 500
was calculated using the rates measured between 13 and 0.1
20 days, i.e. a period of two HRTs, and was found to be

1
1 0.0 0
0.2170.01 g N-NH+ 4 g VSS d . The MNR in the 0 5 10 15 20

third experiment (T ¼ 25 C) was calculated using the (c) Time (days)

rates obtained between 12 and 20 days, i.e. a period of
two HRTs, and was found to be 0.3770.03 g N-NH+
VSS
1 d
1. These values show the influence of tempera-
ture on nitrification rates. The temperature coefficient
obtained, adjusting these rates to an Arrenhius-type
equation (rnitT1 ¼ rnitT2 yðT1
T2 Þ ), is y ¼ 1:1470:03:
Table 5 compares the MNR obtained at 25 C in this
study, with different published dates of systems for
treating high-strength, ammonium wastewaters. The
MNR at 25 C was chosen since most published data
Fig. 4. Experimental determination of the maximum nitrifica-
4 g tion rate at: (a) 15 C, (b) 20 C and (c) 25 C.
was obtained at that temperature. The table shows that
this study’s MNR is clearly higher than those for BNR
single-sludge systems. This is a logical result, since in the
single-sludge systems there is a negative influence of the
influent COD/N ratio on the achievable MNR [17,25].
 ARTICLE IN PRESS
J. Carrera et al. / Water Research 37 (2003) 4211–4221 4217

Table 5
Nitrification rates in systems for treating high-strength ammonium wastewater

Technology T ( C) Nitrification rate Reference

1
1
3
1
(g N-NH+
4 g VSS d ) (g N-NH+
4 m d )

Nitrifying biofilm 30 0.35 — [20]

Nitrifying biofilm 30 — 5.0 [21]
Nitrifying biofilm 28 — 0.55 [22]
Nitrifying biofilm 28–32 0.18–0.21 0.8–1.0 [6]
Nitrifying sludge 28–32 0.14–0.18 0.6–0.8 [6]
Nitrifying sludge 25 0.37 1.3 This study
Single-sludge 27 0.16 — [23]
Single-sludge 25 0.05 — [24]
Single-sludge 25 0.03 — [17]

More specifically, the MNR obtained with the

1
1
nitrifying system, 0.37 g N-NH+4 g VSS d , is 12 times
higher than the MNR obtained with the same waste-
water in a single-sludge system, with nitrification–

1
1
denitrification (0.03 g N–NH+ 4 g VSS d ) with an
influent COD/N ratio of 3.4 [17]. In order to compare
the MNR of this study with the nitrifying biofilm rates, a
nitrification rate is needed by unit of volume and not by
mass unit. In the experiment at 25 C, the MNR by unit
of volume was 1.3 g N m
3 d
1. Comparison of data
from this work to that of immobilized nitrifying biomass
systems, provides an even greater disparity in the results.
The reason for such different results might be the
heterogeneity of the industrial wastewater. Although
they are all classified as high-strength ammonium
wastewaters, each one has its own peculiarities that
influence a biological treatment process. The industrial
wastewater studied here contains a high concentration
of ammonium, as well as high concentrations of
sulphate, chlorine and a certain amount of fluoride
(Table 2). These components can influence biological
treatment; for example, high chlorine [5] or fluoride [26]
concentrations can inhibit the nitrification process.
3.1.5. Inhibition of the nitrification by substrate
The results obtained during the first run confirmed the
difficulty of working with an influent concentration of

1
5000 mg N-NH+ 4 L , since a small drop in the removal
percentage resulted in an accumulation of up to 500 mg

1
N-NH+ 4 L in the nitrifying reactor. The reason for the
decrease in the removal percentage was a direct result of
the NLR being increased above the nitrification rate of
the system. The free ammonia concentration, at 500 mg

1
N-NH+ 
4 L , 20 C and pH=7.5, was about 7.7 mg
NH3 L
1. This free ammonia concentration can cause
the inhibition of the ammonium oxidizing and the nitrite
oxidizing bacteria [8]. This inhibition also resulted in the
accumulation of 1500 mg N-NO
2L

1
and the decrease
in the nitrification rate, from 0.15 to 0.10 g N-

1
1
NH+ 4 g VSS d . In order to mitigate this inhibition,
the NLR was decreased when the ammonium concen-
tration in the nitrifying reactor reached values ap-

1
proaching 300 mg N-NH+ 4 L .
3.2. Denitrification
The effluent of the nitrification system was treated in
the denitrifying system, using two different external
carbon sources, during different periods. In order to
study the effects of both external carbon sources on the
denitrification rate, the MDR of the system, in steady
state, was evaluated.
3.2.1. Denitrification with ‘ethanol mixture’ as external
carbon source
The first external carbon source to be tested was the
‘ethanol mixture’. This external carbon source contained
mainly the same organic matter as the COD-wastewater
that the industry generates. Fig. 5(a) shows the influent
and effluent nitrogen concentrations (nitrate plus nitrite,
although nitrite contribution was almost negligible).
The temperature was maintained at 20 C and the
external carbon source flow was adjusted to produce an
influent COD/N ratio of 5 g COD g N
1, thus ensuring
that the system was not limited by the organic matter.
The 200 days of the external carbon source study were
divided into six runs. Table 6 summarizes the values of
the operational parameters and gives the average value
of the nitrogen and biomass concentrations for each run,
along with the standard deviation. Errors in the
denitrification rate were calculated on the assumption
that the error associated with each concentration was
the standard deviation.
During runs 1 and 2, the system was limited by the
amount of nitrogen supplied by the nitrification system.
In order to operate at faster rates, solid sodium nitrate
was added quantitatively to the denitrifying reactor in
runs 3–6.
In runs 3 and 4, the system was still limited by the
substrate since the effluent nitrogen concentration
 ARTICLE IN PRESS
4218 J. Carrera et al. / Water Research 37 (2003) 4211–4221

fluctuated between 0 and 30 mg N-NO

1

x L . During run these conditions was the MDR: 0.6470.10 g N-NO
x g
4, the biomass concentration in the reactor increased to VSS
1 d
1. This rate was maintained for 21 days, so it
an average value of 9700 mg VSS L
1. This high biomass can be assumed that the steady state was reached.
concentration produced a large amount of nitrogen gas, Operating at such a high rate led to operational
which provoked rising problems in the settler. In run 5, problems due to the large quantities of nitrogen gas
this problem was overcome by a reduction in the produced. For this reason, the influent concentration
biomass concentration in the system, down to an was reduced. In these new conditions the denitrification
average value of 3600 mg VSS L
1. In this run, the rate dropped to 0.2270.07 g N-NO
x g VSS

1
1
d and
denitrification was not limited by substrate, since the stayed at that value for 55 days with a high performance
effluent nitrogen concentration always exceeded 60 mg level, without causing any major operational difficulties.
N-NO

1
x L . The denitrification rate obtained under This last run showed that this system can operate at high
denitrification rates for an extended period. The COD/N
ratio consumed during denitrification, using the ‘ethanol
mixture’, was 4.370.4 g COD g N
1.
3000
5 Table 7 compares the MDR obtained by this study
[N-NOx- ] (mg·L-1)

2500 [N-NOx- ]influent with those of several published works. It can be seen that
[N-NOx- ] effluent
Average concentration
the MDR of this study were higher than those obtained
2000 4 6 by single-sludge systems, with nitrification–denitrifica-
tion. The reason is that in this system, no oxygen is sent
1500 3
to the anoxic reactor so there is no aerobic consumption
1000 2 of organic matter. This produces a higher percentage of
1 denitrifying bacteria in the system. However, the MDR
500 reached in batches, with pure cultures of denitrifying
bacteria, is higher than that of this study.
0
0 50 100 150 200
(a) Time (days)
3.2.2. Denitrification with ‘methanol mixture’ as external
2500 carbon source
[N-NOx- ]influent From day 200 of operation, the external carbon
[N-NOx- ] effluent
source was changed to the ‘methanol mixture’. It was
[N-NOx- ] (mg·L-1)

2000
Average concentration
diluted with tap water in order to maintain the same
3
1500 HRTs employed with the ‘ethanol mixture’. Fig. 5(b)
2 shows influent and effluent nitrogen concentrations
1000 1 (nitrate plus nitrite, although nitrite contribution was
almost negligible) of the denitrifying system with
500 ‘methanol mixture’ as the carbon source.
The temperature was kept at 25 C and the influent
0 COD/N ratio at 5 g COD g N
1, to ensure that the
0 20 40 60 80 100 120 140 system was not limited by the organic matter. The 140
(b) Time (days)
days of operation with this external carbon source were
Fig. 5. Influent and effluent nitrogen concentrations of the divided into three periods. Table 8 summarizes the
denitrifying system. (a) With ‘ethanol mixture’ as a carbon values of the operational parameters and shows the
source and (b) with ‘methanol mixture’ as carbon source. average values of the nitrogen and biomass concentrations

Table 6
Operational parameters for the denitrifying system with ‘ethanol mixture’

Run Period [N-NO
x ]influent [N-NO
x ]effluent Influent flow HRT VSS Average denitrification rate
(days) (mg N L
1) (mg N L
1) (L d
1) (days) (mg L
1) (g N-NO
x g VSS

1
1
d )

1 50 450750 46710 19.1 2.2 80007300 0.0270.02

2 41 750760 40720 24.7 1.7 84007400 0.0570.02
3 16 1240730 33720 26.3 1.6 81007500 0.0970.03
4 13 1820720 17710 35.0 1.2 97007600 0.1570.03
5 21 2630740 110760 38.2 1.1 36007200 0.6470.10
6 55 16007120 30710 28.0 1.5 47007300 0.2270.07
 ARTICLE IN PRESS
J. Carrera et al. / Water Research 37 (2003) 4211–4221 4219

Table 7
Denitrification rates with ethanol

Technology Process T ( C) Denitrification rate Reference

(g N-NO
x g VSS

1
1
d )

Single-sludge Continuous 20 0.02–0.12 [14]

Single-sludge Continuous 20 0.29a [27]
Single-sludge Continuous 20 0.39a [28]
Denitrifying sludge Continuous 20 0.64 This study
Denitrifying pure culture Batch 25 3.3 [12]
a
Original experimental data at 15 C, corrected using a temperature coefficient of 1.10.

Table 8
Operational parameters for the denitrifying system with ‘methanol mixture’

Run Period [N-NO
x ]influent [N-NO
x ]effluent Influent flow HRT VSS Average denitrification rate
(days) (mg N L
1) (mg N L
1) (L d
1) (days) (mg L
1) (g N-NO
x g VSS

1
1
d )

1 19 900730 590730 23.3 1.8 47007100 0.0470.03

2 64 10907100 5207200 21.0 2.0 35007200 0.0870.04
3 57 13507200 90760 32.2 1.3 58007300 0.1770.06

Table 9
Denitrification rates with methanol

Technology Process T ( C) Denitrification rate Reference

(g N-NO
x g VSS

1
1
d )

Single-sludge Continuous 30 0.14 [3]

Single-sludge Continuous 10 0.06 [10]
Single-sludge Continuous 20 0.17–0.48 [14]
Single-sludge Continuous — 0.03 [29]
Denitrifying sludge Continuous 25 0.17 This study
Denitrifying sludge Batch 22 0.29 [11]
Denitrifying pure culture Continuous 20 0.55 [30]
Batch 25 1.3
Denitrifying pure culture Batch 25 2.2 [12]

in each of the periods, along with the standard
deviation. Errors in the denitrification rate were
calculated assuming that the error associated to each
concentration is the standard deviation. With this
carbon source, there was no sign of limitation by
nitrogen, so it was not necessary to add solid sodium
nitrate to achieve the MDR.
The average denitrification rate increased from
0.0470.03 g N-NO
x g VSS

1
1
d to 0.1770.06 g N–
NO
x g VSS
1
1
d in about 80 days. This increase was
due to the acclimation time needed for the denitrifying
biomass to the ‘methanol mixture’. This period agrees
with the acclimation time to methanol, which has been
reported to be between 50 and 100 days [10,11]. The
COD/N ratio consumed, using the ‘methanol mixture’,
was 3.970.5 g COD g N
1.
Table 9 compares the MDR obtained using the
‘methanol mixture’ with different published studies that
used pure methanol. The range of denitrification rates in
single-sludge systems with nitrification–denitrification is
quite large, with values higher and lower than those of
this study. The results for denitrifying sludge systems
and pure cultures are all higher than those of the current
study. This lower rate can be due to the carbon source
used for the present study, since pure methanol was not
utilized.
3.2.3. Comparison of maximum denitrification rates
In order to compare the MDR obtained with the two
carbon sources, it was necessary to correct the MDR of
the ‘methanol mixture’, since that rate was determined at
25 C and that of the ‘ethanol mixture’ at 20 C. The
temperature coefficient, determined experimentally with
the same biomass [31], was 1.10. Using that correction,
the MDR at 20 C for the ‘methanol mixture’ would be
about 0.11 g N-NO
x g VSS

1
1
d , while for the ‘ethanol
mixture’, it was 0.64 g N-NO
x g VSS

1
1
d . Therefore,
the MDR with ‘ethanol mixture’ appears to be about 6
 ARTICLE IN PRESS
4220 J. Carrera et al. / Water Research 37 (2003) 4211–4221
times higher than the MDR with the ‘methanol mixture’.
This ratio is higher than other bibliographic values
[12,13], in which MDR with ethanol is about 1.5–3.5
times higher than MDR with methanol. This difference
can be due to the composition of the ‘methanol mixture’
used, which includes acetone (10%) and isopropylic
alcohol (10%).
4. Conclusions
The results of this study show that it is possible to
remove the ammonium of an industrial wastewater with

1
up to 5000 mg N-NH+ 4 L and high concentration of
sulphates and chlorides, using a biological, two-sludge
system with nitrification and denitrification.
The nitrification rate achieved with the activated
sludge nitrifying system was very high. The MNR
obtained at 15 C, 20 C and 25 C were 0.10, 0.21 and

1
1
0.37 g N-NH+ 4 g VSS d , respectively. The nitrifying
sludge showed good settleability throughout the study,
with SVI values below 50 mL g
1. The ammonium
removal percentage was very high throughout (90–
100%), regardless of the concentration of the influent.
The main problem for the treatment of this industrial
wastewater, with a high concentration of ammonium,
was possible inhibition by substrate of the nitrification
process.
The effluent of the nitrifying system was treated in the
activated sludge denitrifying system, using two different
external carbon sources, during different periods. The
first carbon source was mainly ethanol and the second
one was a mixture of methanol (60%), acetone (10%)
and isopropylic alcohol (10%). In order to study the
effects of both external carbon sources on the denitri-
fication rate, the MDR in steady state was evaluated.
The MDR obtained with the first carbon source was
about 0.64 g N-NO
x g VSS

1
1
d at 20 C, while the
MDR obtained with the second carbon source was
0.17 g N-NO
x g VSS

1
1
d at 25 C. If the temperature
coefficient is taken into account, the denitrification rate
with ethanol was about six times higher than that
obtained with methanol.
Acknowledgements
This work has been supported by CICYT (project
REN2000-0670/TECNO). The Generalitat de Catalunya
provided financial support for Julia! n Carrera through a
pre-doctoral fellowship. We wish to express our
gratitude to the company FREIXENET S.A. The
Departament d’Enginyeria Qu!ımica (UAB) is a member
of the CERBA.
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