Denitrification of wastewater containing high nitrate and calcium concentrations.

Abstract

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO3- -N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO3- -N/gVSSh and very low residual COD in the effluent.

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Denitrification of wastewater containing high nitrate and calcium concentrations
Y. Fernández-Nava, E. Marañón*, J. Soons, L. Castrillón
Department of Chemical Engineering and Environmental Technology, University Institute of Industrial Technology of Asturias, Higher Polytechnic School of
Engineering, University of Oviedo, 33203 Gijón, Spain
a r t i c l ei n f o
Article history:
Received 22 January 2008
Received in revised form 17 March 2008
Accepted 22 March 2008
Available online 6 May 2008
Keywords:
Denitrification
SBR
High nitrate wastewater
Calcium
a b s t r a c t
The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by
denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater
treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentra-
tions led to complete denitrification in 6 h (denitrification rate: 22.8 mg NO?
as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150 mg/L. Higher
calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The
optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7 h at a maximum rate of
30.4 mg NO?
3-N=g VSS h), using methanol
3-N=g VSS h and very low residual COD in the effluent.
? 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Nitrogen compounds discharged into the environment can
cause serious problems such as the eutrophication of rivers and
deterioration of water sources, as well as hazards to human health.
Furthermore, nitrates can also form nitrosamines and nitrosa-
mides, potentially carcinogenic compounds (Hu et al., 1999; Ono
et al., 2000; Forman et al., 1985).
The World Health Organisation (WHO) establishes the limit for
nitrates in drinking water at 50 mg NO3/L (or 11.3 mg NO3-N/L).
However, the US Environmental Protection Agency (EPA) has set
this limit at 10 mg NO3/L (2.3 mg NO3-N/L). The European Commu-
nity, on the other hand, whose guidelines are followed by the
Spanish Ministry of Health, has established the maximum admissi-
ble levels of nitrates plus nitrites in drinking water at 50 mg NO3/L
(Directive 98/83/EC). As regards discharges, the admissible concen-
tration depends on the receiving environment, usually ranging be-
tween 10 and 30 mg NO3-N/L for discharges into fresh water and
50 mg NO3-N/L for discharges into seawater. These limits are lower
if the discharges occur in ‘‘sensitive areas”, ranging between 10 and
15 mg Total-N/L (Directive 91/271/EEC).
Nitrate pollution of soil and waters (surface waters and ground-
water) are mainly associated with agricultural and livestock farm-
ing activities and, in certain areas, with specific industrial
activities. Nitrate wastes containing over 1000 NO3-N mg/L are
generated during the production of cellophane, explosives, fertiliz-
ers and pectin and in the metal finishing industries (Bilanovic et al.,
1999; Zala et al., 1999; Watanabe et al., 2001).
One of the surface treatment industries that has taken on in-
creased importance in recent times is that of stainless steel manu-
facturing due to the greater consumption of products of this type in
the chemical, petrochemical, building and food industries. Waste
waters containing high concentrations of metals, nitrates and fluo-
rides are generated in the stainless steel manufacturing process.
These waste waters are treated at the plant itself, undergoing a
precipitation process, generally with Ca(OH)2, to remove the fluo-
rides and metals in the form of sludge, thus obtaining treated
wastewater which still contains high nitrate concentrations (be-
tween 500 and 1000 NO3-N mg/L) as well as dissolved calcium as
a consequence of the aforementioned treatment.
The processes used for treating nitrate-rich wastewater include
reverse osmosis, ion exchange, catalysis and biological nitrate
reduction (denitrification). In contrast to those processes which
only concentrate the nitrate, such as reverse osmosis and ion ex-
change, denitrification converts nitrate to free nitrogen. Besides,
both the aforementioned physical–chemical processes are non-
selective. Biological denitrification, on the other hand, affords a
cost-effective process for the removal of nitrates (Dahab, 1987;
Shrimali and Singh, 2001; Leakovic et al., 2000).
Heterotrophic denitrification requires anoxic conditions in or-
der to reduce nitrate to nitrite and subsequently to nitrogen gas,
according to the following sequence (Koren et al., 2000; Tchoba-
noglous et al., 2003):
NO?
3! NO?
When the wastewater does not contain biodegradable organic
carbon compounds, these have to be added. Methanol is the most
commonly employed external carbon source because of being easy
2! NOðgÞ ! N2OðgÞ ! N2ðgÞ
0960-8524/$ - see front matter ? 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.03.048
* Corresponding author. Tel.: +34 985 182027; fax: +34 985 182337.
E-mail address: emara@uniovi.es (E. Marañón).
Bioresource Technology 99 (2008) 7976–7981
Contents lists available at ScienceDirect
Bioresource Technology
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Author's personal copy
assimilated by denitrifying bacteria and its low cost (Tam et al.,
1992; Akunna et al., 1993; Clifford and Liu, 1993; Christensson
et al., 1994; Mailloux et al., 2002).
Only a small number of research studies have been published to
date on the denitrification of wastewater containing nitrate at con-
centrations higher than 600 mg NO3-N/L (Constantin and Fick,
1997; Glass and Silverstein, 1998, 1999; Austerman-Haun et al.,
1999; Dhamole et al., 2007; Cyplik et al., 2007; Nair et al., 2007).
As regards the wastewater generated in the stainless steel pickling
process, research has mainly focussed on the application of mem-
brane processes (Kim et al., 2007).
Biological denitrification of high nitrate waste is a slow process.
To increase the rate of denitrification, parameters such as pH, tem-
perature, COD=NO?
must be optimized. Glass and Silverstein (1998) reported that at
near-neutral and alkaline pH values from 6.5 to 9, denitrification
of high nitrate wastewater using sodium acetate as carbon source
was affected by pH. At pH 6 7.0, denitrification of water containing
an initial nitrate concentration of 1350 mg NO3-N/L was com-
pletely inhibited. As the pH increased from 7.5 to 8.5 and 9.0, the
accumulation of nitrite increased significantly, although the overall
time required to completely remove nitrate and nitrite was more
or less constant at 9 h for MLSS = 6–8 g/L.
Gradual acclimation of sludge by means of stepwise increases in
wastewater nitrate concentration is one of the methods used to de-
velop the suitable consortium to treat high strength nitrate waste.
Glass and Silverstein (1999) reported that, after gradual acclima-
tion of the activated sludge, complete denitrification of high nitrate
(8200 mg NO3-N/L) high-salinity (ionic strength = 3.0) wastewater
took 22 h at pH 9 with a MLSS concentration of 37 g/L using so-
dium acetate as carbon source in a bench-scale SBR. Dhamole
et al. (2007) reported an acclimation strategy by means of a step-
wise increase in the nitrate concentration for the treatment of high
nitrate waste (?9000 mg NO3-N/L) using sodium acetate as carbon
source in a sequencing batch reactor (SBR). Complete denitrifica-
tion using acclimated sludge (MLSS = 44 g/L) was achieved in only
6 h. During the acclimation period, an increase in the nitrite peak
value from zero to 5907 mg NO?
The formation of alkalinity during the denitrification process, in
conjunction with the high concentrations of calcium present in the
wastewater generated in the stainless steel industry, gives rise to
the formation of CaCO3precipitates and other calcium precipitates
owing to the presence of fluorides that it was not possible to com-
pletely remove in the prior treatment process and the presence of
phosphates,addedasasourceofphosphorousforthedenitrification
process.Theseprecipitatesmayformaroundthe biomassflocs,thus
reducing their activity. This reduction in biomass activity was ob-
servedby Yuetal. (2000)inanaerobicreactors;theseauthorsfound
that the biomass growth rate, and hence its activity, presented an
optimum value in relation to the concentration of calcium present.
The present paper studies the removal by biological denitrifica-
tion of the nitrates present in wastewater containing high concen-
trations of calcium, similar to the wastewater generated in the
rinsing process of pickled products from the stainless steel indus-
try. The process was carried out in SBR reactors. This type of reac-
tor was chosen because it occupies less space, as the biological
degradation and the sedimentation of the biomass takes place in
the same tank, in contrast with what occurs in conventional stirred
tank reactors (CSTRs). Furthermore, the former are more efficient
in recovering biomass, they facilitate the change in scale and have
been shown to be effective in high nitrate wastewater denitrifica-
tion processes (Veydovec et al., 1994). The use of different inocula
was studied, as well as the effects of the presence of calcium on the
denitrification rate and biomass growth and the effect of the
COD=NO?
as the external carbon source.
3-N and biomass concentration of the process
2-N=L was observed.
3-N ratio employed in the process when using methanol
2. Methods
2.1. Experimental system
The 3 L volume glass closed reactors used for the laboratory
experiments were provided with mechanical stirrers IKA/WERKE
(Eurostar digital model) in order to improve contact between the
microorganisms and the synthetic wastewater. At the beginning
of each operation cycle, the reactor containing the inoculum
(0.5 L) was loaded with 2 L of synthetic wastewater by means of
a peristaltic pump (Watson-Marlow SCIQ 323). At the end of the
denitrification reaction period, the stirrer was turned off and set-
tling of the biomass commenced. When both phases, biomass
and supernatant, were completely separate, the supernatant was
unloaded by pumping. The SBR system was operated in the follow-
ing sequential phases: loading period (40 min), anoxic reaction
period (6–22 h, depending on the operational conditions being
tested), settling period (30 min), and unloading period (40 min).
2.2. Stainless-steel wastewater
Wastewater from the stainless steel industry was characterised
over a period of one month, during which two samples were col-
lected each week and analyzed in triplicate. The composition of
this wastewater is shown in Table 1. Considerable variation in its
characteristics was observed, possibly due to variation in the
industrial process as well as in the pre-treatment of the wastewa-
ter with lime. To forestall this variation in the composition of the
wastewater influencing the experimental results of the denitrifica-
tion process under study, synthetic wastewater was used (pH: 8.5,
fluoride: 5 mg/L, nitrate-N: 700 mg/L, sulphate: 200 mg/L, calcium:
150 mg/L, chloride: 177 mg/L). These concentrations were em-
ployed due to being the most common in the samples analyzed
during characterisation of the industrial wastewater. No metal
ion was introduced, since the presence of metals is practically
inappreciable after pre-treatment with lime.
2.3. Inocula
Two different inocula were studied: sludge from the biological
treatment of leachate (LTP) generated at a Municipal Solid Waste
Landfill; and sludge from a Sewage Treatment Plant (SWP). The
leachate treatment consists of a pressurized nitrification–denitrifi-
cation process followed by ultrafiltration to separate the sludge
Table 1
Composition of stainless steel pickling rinse wastewater after lime treatment
ParameterAverage MaximumMinimumSD
pH
Suspended solids
COD
Fluoride
Nitrate-N
Nitrite-N
Ammonium-N
Sulphate
Chloride
Calcium
Magnesium
Total iron
Nickel
Manganese
Total chromium
Copper
Cadmium
Zinc
7.1
15.6
65.1
13.3
455.5
69.0
7.1
155.4
180.3
375.3
5.3
0.05
0.14
0.07
0.04
0.04
<0.01
0.28
9.6
39.0
335.4
100.0
1179.5
170.0
14.6
200.0
222.4
1041.8
6.8
0.07
0.26
0.32
0.10
0.20
<0.01
0.55
5.9
4.0
22.5
4.8
87.5
4.0
0.2
128.2
139.5
143.5
3.9
0.01
0.07
0.02
0.02
0.01
<0.01
0.03
1.0
12.0
82.4
25.0
263.4
45.8
3.7
39.0
41.5
222.9
0.8
0.02
0.06
0.08
0.03
0.06
<0.01
0.23
Units: mg/L, except pH.
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(Biomembrat process). The biological treatment in the Sewage
Treatment Plant consists of an activated sludge process with re-
moval of nitrogen and phosphorus (UCT configuration).
2.4. Start-up period
Prior to the commencement of experiments, the sludge
underwent a two-week acclimation period (10 operating cycles
with an anoxic reaction period of 24 h) by introducing 750 mL
of inoculum (sludge) and 2000 mL of synthetic wastewater into
the reactor. During start-up, the reactor was fed with synthetic
wastewater diluted 50% with drinking water with the aim of
progressively acclimating the biomass to the high NO?
centration of the wastewater to be treated (Glass and Silverstein,
1999; Dhamole et al., 2007). All the processes were performed at
room temperature (20 ± 1 ?C) in an anoxic environment. Experi-
ments were carried out without pH adjustment, since pH values
of 9.5 were reached, which allow high denitrification yields
when treating high nitrate wastewater (Glass and Silverstein,
1999).
The COD/NO3-N ratio initially employed was 4, using methanol
as carbon source (Clifford and Liu, 1993; Christensson et al., 1994);
phosphorous was also added as a nutrient in the form of Na2HPO4
at a N/P ratio of 10.
3-N con-
2.5. Study of the influence of calcium concentration on the
denitrification process
Calcium was added to the synthetic wastewater to study the ef-
fect of calcium concentration on reactor performance resulting in
total concentrations of around 150, 350, 450 and 550 mg Ca/L in
the feed solution. Methanol was used as a source of carbon
(COD/NO3-N ratio of 4); phosphorous was also added as a nutrient
in the form of Na2HPO4, (N/P ratio of 10).
The bioreactor was operated during 40 cycles for each of the
calcium concentrations under study.
2.6. Study of the influence of the COD/N ratio on the denitrification
process
In order to adjust the amount of carbon source needed to
achieve high denitrification efficiency without increasing the
COD of the effluent, lower COD/N ratios were tested than the one
used in the study of the influence of calcium concentration. The
composition of the water was the same as in the previous study.
Phosphorous was added as a nutrient in the form of Na2HPO4at
a N/P ratio of 10. The biomass concentration inside the reactor
was kept between 3.12 and 3.40 g VSS/L.
The bioreactor was operated during 20 cycles for each one of
the ratios under study.
2.7. Analytical methods
Nitrate concentration was monitored spectrophotometrically at
420 nm using the sodium salicylate method (Rodier, 1978). Nitrite
detection was determined at 585 nm using the ferrous sulphate
method (HACH manual, adapted from McAlpine and Soule,
1933). COD (colorimetric method with closed reflux), fluoride
(potentiometry), total (TSS) and volatile (VSS) suspended solids
were measured by gravimetry according to Standard Methods
(APHA et al., 2001). The spectrophotometric readings were ob-
tained on a HACH DR 2010 spectrophotometer. The concentration
of fluoride was determined using an ORION 96-09 fluoride-selec-
tive electrode. The concentration of dissolved oxygen (DO) and
pH were measured with a YSI 55/25 FT oximeter and a CRISON
pH25 pH-meter.
3. Results and discussion
3.1. Results with the different inocula
Complete nitrate removal was achieved in 6 h when using
sludge from the LTP as inoculum (3.12 g VSS/L). The experimental
results provided a linear fit, which indicates a zero-order kinetics
with respect to nitrate concentration, obtaining a nitrate-removal
rate of 22.8 mg NO3-N/g VSS h. When the bioreactor was running,
nitrite-nitrogen levels in the effluent were found to be high only
during the first operating cycles, up to levels of 35 mg/L. Values
of 2 mg NO?
When denitrification was performed using biomass from the
SWP (7.8 g VSS/L), complete denitrification was achieved in 10 h.
The kinetic study showed a zero-order kinetics with a nitrate-re-
moval rate of 7.3 mg NO3-N/g VSS h, much lower than that ob-
tainedwhen usinginoculum
concentrations were not observed either in the cycles carried out.
As mentioned previously, prior acclimation of the sludge to high
nitrate concentrations increases the denitrification rate. The in-
creased efficiency of the sludge from the leachate treatment plant
may be due to the prior acclimation that this sludge has undergone
in the Biomembrat process, during which it has been in contact
with high nitrate concentrations of around 1500 mg NO3-N/L (Mar-
añón et al., 2006). This does not occur in the case of sludge from
municipal sewage treatment, in which the concentrations of ni-
trate do not usually exceed 50 mg NO3-N /L.
2-N=L and below were obtained in all the other cycles.
fromtheLTP.Highnitrite
3.2. Influence of calcium concentration on the denitrification process
After start-up, the reactor showed good denitrification charac-
teristics: complete denitrification was achieved at all calcium con-
centrations studied. Denitrification rates at 50, 150, 450 and
550 mg Ca/L were determined measuring NO3-N concentrations
over time (Fig. 1). Each time point is the average of triplicate mea-
surements and three profiles were made on separate days for each
experimental Ca variation. The highest specific denitrification rate
was found for a calcium concentration of 150 mg/L, being 22.8 mg
NO3-N/g VSS h. The rate decreased for lower and higher calcium
concentrations, being 21.1, 15.5 and 11.0 mg NO3-N/g VSS h for
50, 450 and 550 mg Ca/L, respectively.
As regards the biomass growth rate, this also showed an opti-
mum for 150 mg Ca/L, as can be seen in Fig. 2. Increasing the cal-
cium concentration from 50 to 150 mg Ca2+/L produced a 1.4-fold
increase in the biomass growth rate, whereas further increases in
calcium up to 550 mg/L led to a decrease in the growth rate of
around 8-fold with respect to 150 mg/L. However, this decrease
0
200
400
600
800
024
time (hours)
68 10
NO3
--N (mg/l)
50 mgCa/l; 2.1 g VSS/l
150 mgCa/l; 3.1 g VSS/l
450 mgCa/l; 8.4 g VSS/l
550 mgCa/l; 10.2 g VSS/l
15.5 mg NO3-N/g VSS·h
11.0 mg NO3-N/g VSS·h
22.8 mg NO3-N/g VSS·h
21.1 mg NO3-N/g VSS·h
Fig. 1. Denitrification kinetics in batch tests for different calcium concentrations in
wastewater (Y error bar is less than 5%).
7978
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in growth rate at higher calcium levels may have also been caused
by a shortage of electron acceptors (nitrate) at these higher bio-
mass levels, thus hindering further growth. Nonetheless, growth
continues to be linear, whereas an exponential decrease would
be more logical when there is a shortage of nitrate. Complete deni-
trification was reached after 8 h for a VSS concentration of 3.12 g/L
and after 5 h for 8.44 g/L.
These results are partially in agreement with those of Yu et al.
(2000), who reported that calcium lowers the activity of anaerobic
sludge at any concentration, but that the biomass growth rate (and
therefore also the net activity of the sludge) shows an optimum
with respect to calcium.
During the experiments, nitrite-nitrogen levels in the effluent
were found to be high only during the first experiments, up to lev-
els of 35 mg/L. All the other trials showed levels of below 5 mg/L. In
general, the later experiments presented values of 2 mg/L and be-
low, likewise indicating the good denitrifying performance of the
reactor regardless of the calcium concentration.
As is well known, denitrification produces alkalinity. Fig. 3
shows the alkalinity of the effluent for the different calcium con-
centrations in the wastewater. The alkalinity of the effluent de-
creases as the calcium concentration increases. This effect, as
well as the decrease in pH, is due to the precipitation of CaCO3,
which removes carbonate from the (active) system, thus lowering
its buffering capacity.
This effect can also be observed by determining the fixed sus-
pended solids (FSS) in the biomass, which increase with the cal-
cium content in the influent, thus diminishing the VSS/FSS ratio
(Fig. 4). At calcium concentrations of 550 mg/L, the VSS/TSS ratio
decreased to about 25%.
The results obtained did not show any inhibition due to the
presence of fluoride ions. In this respect, ecotoxicity tests carried
out in industrial wastewater showed this effluent not to be ecotox-
ic (EC50 > 100%), even with fluoride ion concentrations of up to
10 mg/L (European Commission, 2006). The fluoride concentra-
tions in the effluent proved to be highly dependent on the calcium
concentration in the wastewater.
Higher calcium concentrations will lead to more CaF2precipita-
tion. The following concentrations of fluoride in the effluent were
found for the reactor conditions: 4.7 mg F?/L for calcium concen-
trations of 50 mg/L, 3.5 mg F?/L for 150 mg/L and 0.3 mg/L for cal-
cium concentrations greater than 350 mg/L.
From the species present in the reactor, the following calcium
precipitates can be formed, in the following order of solubility:
Ca3ðPO4Þ2< CaF2< CaCO3< CaHPO4< CaSO4
Initially, between 50% and 75% of the calcium precipitated
immediately in the reactor due to the pH value (around 8) and to
the presence of phosphate and fluoride. By the end of the process,
the higher pH obtained and the presence of carbonates produced
during denitrification led to a further decrease in calcium concen-
tration. Therefore, between 90% and 96% of the calcium ions were
removed as a result of precipitation.
The results obtained indicate that the presence of calcium in this
typeofwastewaterdecreasesthedenitrificationratewhenthelevels
of dissolved calcium exceeded 150 mg/L. This fact highlights the
need to suitably adjust the dose of Ca(OH)2employed in the pre-
treatment stage for this water, such that the amount of lime added
permits the precipitation of metals and the majority of fluoride ions
without much residual calcium remaining in solution.
3.3. Influence of COD/N ratio on the denitrification process
As previously mentioned, methanol was used as external car-
bon source. However, the amount to be added must be carefully
determined, since excessive added methanol increases the COD
of the effluent, besides constituting an extra cost. According to dif-
ferent studies (Clifford and Liu, 1993; Christensson et al., 1994; Fo-
glar and Briški, 2003; Foglar et al., 2005; Cyplik et al., 2007), the
COD/NO3-N ratio employed in denitrification processes when
using methanol as carbon source ranges between 3.7 and 5.2.
Fig. 5 and Table 2 show the results obtained in the denitrifica-
tion of high nitrate wastewater for the different COD/N ratios stud-
ied. Each time point is the average of triplicate measurements and
three profiles were made on separate days for each experimental
COD/N ratio. For the lower ratios employed (COD/N equal to 3.4
and 3.6), deceleration of the denitrification rate was observed as
the nitrate concentration and COD decreased below a certain level.
For a COD=NO?
30.4 mg NO?
cess, which subsequently decreased to 17.8 mg NO?
the following 4 h. In both intervals, the denitrification rate fitted
a straight line, thus indicating zero-order kinetics with respect to
nitrate concentration. Employing this ratio, the treated effluent
presented concentrations of 13 mg NO?
after 7 h of treatment.
For a COD=NO?
(29.8 mg NO?
cess (Table 2), subsequently decreasing to 16.6 mg NO?
Likewise in this case, the experimental data fit a straight line in
both intervals. Using this ratio, complete denitrification was
achieved in 7 h, although the treated effluent still contained a dis-
solved COD concentration of 290 mg/L.
3-N ratio of 3.4, a maximum denitrification rate of
3-N=g VSS h was obtained in the first 3 h of the pro-
3-N=g VSS h in
3-N=L and 72 mg COD/L
3-N ratio of 3.6, the maximum denitrification rate
3-N=g VSS h) was obtained in the first 4 h of the pro-
3-N=g VSS h.
0
0.5
1
1.5
2
2.5
3
0102030 40
assay number
VSS/VSSo
Ca= 50 mg/L
Ca= 150 mg/L
Ca= 350 mg/L
Ca= 450 mg/L
Ca= 550 mg/L
Fig. 2. Evolution of VSS concentrations in batch tests for different calcium
concentrations.
0
500
1000
1500
2000
2500
3000
3500
01020 3040
assay number
mg CaCO3/L
Ca= 50 Ca= 150Ca= 350 Ca= 450Ca= 550
Fig. 3. Alkalinity of the treated wastewater for different calcium concentrations.
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Both COD/N ratios are lower than the ones employed by other
researchers in denitrification processes when using methanol as
carbon source (Table 3). For both ratios, it was observed that the
decrease in denitrification rate was linked to an increase in COD
consumption, as can be seen in the values of the relation CODcon-
sumed/Nremoved(Table 2). For example, for a COD=NO?
to 3.6, 2.4 mg of COD were consumed during the first 4 h for every
mg of removed N, whereas the removed COD increased to 4.2 mg
for every mg of removed N when the denitrification rate decreased.
It would thus appear that for each low COD=NO?
ists a threshold value below which competition arises between the
3-N ratio equal
3-N ratio, there ex-
denitrifying bacteria and other heterotrophic bacteria that may be
present in the reactor.
No deceleration at all was observed for a COD=NO?
equal to4, thedenitrification
NO?
achieved in just over 7 h, although the dissolved COD concentra-
tion was very high (490 mg/L).
3-N ratio
22.8 mg obtained being
3-N=g VSS h. Using this ratio, complete denitrification was
4. Conclusions
Rinse wastewater generated in the stainless steel manufactur-
ing process contains high concentrations of nitrates (between
600 and 1000 mg NO?
logical treatment of this wastewater by denitrification using bio-
mass from a landfill leachate treatment plant enables the
concentration of nitrates to be reduced below established dis-
charge limits in surface waters of non-sensitive areas.
3-N=L) and of calcium (up to 1000 mg/L). Bio-
0
10
20
30
40
50
60
12
25
40
52
64
78
86
98
110
122
134
143
159
170
183
196
assay number
TSS, VSS (g/L)
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
VSS/TSS (%)
TSSVSS VSS/TSS
50 g/L
150 g/L
350 g/L450 g/L
550 g/L
Fig. 4. Evolution of TSS and VSS in batch tests for different calcium concentrations.
0
100
200
300
400
500
600
700
012345678
time (hours)
NO3
- -N (mg/L)
COD/N=4
COD/N=3.6
COD/N=3.4
Fig. 5. Denitrification kinetics for different COD/N ratios using methanol as carbon
source (Y error bar is less than 5%).
Table 3
Values found in the literature for specific denitrification rates obtained during the
study of the denitrification process at different COD/N ratios using methanol as
carbon source
COD/
N
ratio
NO3-N
influent
(mg/L)
Specific
denitrification rate
(mg NO3-N/g VSS h)
Reactor typeReference
5.220021.0Continuous flow
reactor
Continuous
anoxic
chemostat
SBR
Foglar et al.
(2005)
Christensson
et al. (1994)
4.2150 91.0
4.0700 21.9a
Clifford and
Liu (1993)
Christensson
et al. (1994)
4.0150 32.0Continuous
anoxic
chemostat
Continuous flow
reactor
SBR
3.7 20017.0a
Foglar et al.
(2005)
The present
study
The present
study
3.6 70029.8b
3.4700 30.4b
SBR
aCalculated values.
bMaximum denitrification rate.
Table 2
Denitrification rates, COD consumption and composition of the effluent at different
COD/N ratios after 6 h reaction time
COD/
N
CODconsumed/NO3-
Nremoved(mg/mg)
rNO3-N max
(mg N/g VSS h)
NO3-N
effluenta(mg/
L)
COD
effluenta
(mg/L)
3.4
3.6
4.0
2.5
2.4
2.9
30.8
29.8
22.8
1372
290
470
0
0
a7 h reaction time.
7980
Y. Fernández-Nava et al./Bioresource Technology 99 (2008) 7976–7981

Page 7

Author's personal copy
The presence of calcium in this water has a negative effect on
the denitrification process. An increase in fixed solids in the biolog-
ical sludge may lead to a decrease in its activity, as evidenced by
the biomass growth rate, which decreases for calcium concentra-
tions in the wastewater of above 150 mg/L. Under these conditions,
the denitrification rate obtained was 22.8 mg NO?
decreasing for higher concentrations of calcium. This finding high-
lights the need to carry out strict control of the dose of calcium
hydroxide employed in pre-treating this wastewater.
Another parameter to be taken into account is the COD/N ratio
required for the denitrification process. The addition of organic
matter in the form of methanol with a COD/N ratio P 3.6 enables
complete denitrification to be achieved in just over 7 h. However,
COD concentrations in the effluent are high for direct discharge
(290–470 mg/L). When the COD/N ratio was decreased to 3.4,
residual NO?
were obtained, respectively, after 7 h of processing. These values
would allow discharge of the treated effluent even into surface
waters of some sensitive areas.
3-N=g VSS h,
3-N and COD concentrations of 13 mg/L and 72 mg/L
Acknowledgements
We gratefully acknowledge support from the European Union
via Contract ECSC-7210-PR-358, ‘‘Membrane-bioreactor system
for treatment of nitrates in pickling process waste water” and the
Consejería de Educación y Ciencia, Principado de Asturias, Spain
(Project FC-05-IB-130). We also wish to thank COGERSA and the
Consorcio de Aguas de Asturias (CADASA) for providing the sludge
samples used as inoculum, and Mr. Paul Barnes for proof reading
the English version of the manuscript.
References
Akunna, J., Bizeau, C., Moletta, R., 1993. Nitrate and nitrite reductions with
anaerobic sludge using various carbon sources: glucose, glycerol, acetic acid,
lactic acid and methanol. Water Research 27 (8), 1303–1312.
APHA, AWWA, WPCF, 2001. Standard Methods for the Examination of Water and
Wastewater, 20th ed. Public Health Association, Washington, DC.
Austerman-Haun, U., Meyer, H., Seyfried, C.F., Rosenwinkel, K.H., 1999. Full scale
experiences with anaerobic/aerobic treatment plants in the food and beverage
industry. Water Science Technology 40 (1), 305–312.
Bilanovic, D.,Battistoni,P.,Cecchi,F.,
Denitrification under high nitrate concentration and alternating anoxic
conditions. Water Research 33 (15), 3311–3320.
Christensson, M., Lie, E., Welander, T., 1994. A comparison between ethanol and
methanol as carbon sources for denitrification. Water Science and Technology
30 (6), 83–90.
Clifford, D., Liu, X., 1993. Biological denitrification of spent regenerant brine using a
sequencing batch reactor. Water Research 27 (9), 1477–1484.
Constantin, H., Fick, M., 1997. Influence of C-sources on the denitrification rate of a
high-nitrateconcentratedindustrialwastewater.WaterResearch31(3),538–589.
Council Directive 91/271/EEC of 21 May 1991 on urban waste water treatment.
Official Journal of the European Communities L/135, 30.5.1991, pp. 40–52.
Council Directive 98/83/CE of 3 November 1998 on the quality of water intended for
human consumption.
Cyplik,P.,Grajek,W.,Marecik,R.,Króliczak,P.,Dembczyn ´ski,R.,2007.Applicationofa
membranebioreactortodenitrificationofbrine.Desalination 207, 134–143.
Pavan, P.,Mata-Álvarez, J.,1999.
Dahab, M.F., 1987. Treatment alternatives for nitrate contaminated groundwater
supplies. Journal of Environmental Systems 17 (1), 65–75.
Dhamole, P.D., Nair, R.R., D’Souza, S.F., Lele, S.S., 2007. Denitrification of high
strength nitrate waste. Bioresource Technology 98, 247–252.
European Commission, 2006. Membrane-bioreactor system for treatment of
nitrates in pickling process wastewater. Technical steel research. Factory –
wide control, social and environmental issues, Report EUR 22430 EN.
Foglar, L., Briški, F., 2003. Wastewater denitrification process – the influence of
methanol and kinetic analysis. Process Biochemistry 39, 95–103.
Foglar, L., Briški, F., Sipos, L., Vukovic ´, M., 2005. High nitrate removal from synthetic
wastewater with the mixed bacterial culture. Bioresource Technology 96, 879–
888.
Forman, D., Al-Dabbagh, S., Doll, R., 1985. Nitrates, nitrites and gastric cancer in
Great Britain. Nature 313, 620–625.
Glass, C., Silverstein, J., 1998. Denitrification kinetics of high nitrate concentration
water: pH effect on inhibition and nitrite accumulation. Water Research 32 (3),
831–839.
Glass, C., Silverstein, J., 1999. Denitrification of high-nitrate, high-salinity
wastewater. Water Research 33 (1), 223–229.
Hu, H.Y., Goto, N., Fujie, K., 1999. Concepts and methodologies to minimize
pollutantdischarge forzero-emission
Technology 39 (10–11), 9–16.
Kim, Y.H., Hwang, E.D., Shin, W.S., Choi, J.H., Ha, T.W., Choi, S.J., 2007. Treatments of
stainless steel wastewater containing a high concentration of nitrate using
reverse osmosis and nanomembranes. Desalination 202, 286–292.
Koren, D.W., Gould, W.D., Bédard, P., 2000. Biological removal of ammonia and
nitrate from simulated mine and mill effluents. Hydrometallurgy 56, 127–
144.
Leakovic, S., Mijatovic, I., Cerjan-Stefanovic, S., Hodzic, E., 2000. Nitrogen
removal from fertilizer wastewater by ion exchange. Water Research 34
(1), 185–190.
Mailloux, M., Tartakovsky, B., Milette, D., Guiot, S.R., Peisajovich, A., Olivier, L.,
Bélenger, C., Lorrain, M.J., 2002. Evaluation of a carbon-source stimulated
bioremediation technology for the remediation of a nitrate-contaminated
aquifer at an airport site, ground and water: theory and practice. In:
Proceedings of the 55th Canadian Geotechnical and Third Join IAH-CNC and
CGS Groundwater Specially Conferences, Niagara Falls, Ontario, October 20–
23.
Marañón, E., Castrillón, L., Fernández, Y., Fernández, E., 2006. Anaerobic treatment
of sludge from a nitrification–denitrification landfill leachate plant. Waste
Management 26, 869–874.
McAlpine, R., Soule, B., 1933. Qualitative Chemical Analysis. D. Van Nostrand
Company, Inc., New York. pp. 476–575.
Nair, R.R., Dhamole, P.B., Lele, S.S., D’Souza, S.F., 2007. Biological denitrification of
high strength nitrate waste using preadapted denitrifying sludge. Chemosphere
67, 1612–1617.
Ono, Y., Somiya, I., Oda, Y., 2000. Identification of a carcinogenic heterocyclic amine
in river water. Water Research 34 (3), 890–894.
Rodier, J., 1978. Análisis de las aguas, Aguas naturales, aguas residuales y aguas de
mar. Omega Ed., Barcelona, pp. 180–182.
Shrimali, M., Singh, K.P., 2001. New methods of nitrate removal from water.
Environmental Pollution 112, 351–359.
Tam, N.F.Y., Wong, Y.S., Leung, G., 1992. Effect of exogenous carbon sources on
removal of inorganic nutrient by the nitrification–denitrification process. Water
Research 26 (9), 1229–1236.
Tchobanoglous, G., Burton, F.L., Stensel, H.D., 2003. Wastewater Engineering
Treatment and Reuse. McGraw Hill Inc., New York. pp. 616–623.
Veydovec, W., Silverstain, J., Cook Jr., N., Figueroa, L., Hund, R., Lehmukhl, G., 1994.
Denitrification inhibition by high nitrate waste. In: Proceedings of the ASCE
National. EE Conference, ASCE, New York, NY, pp. 415–422.
Watanabe, T., Motoyama, H., Kuroda, M., 2001. Denitrification and neutralization
treatment by direct feeding of an acidic wastewater containing copper ion and
high-strength nitrate to a bio-electrochemical reactor process. Water Research
35 (17), 4102–4110.
Yu, H.Q., Tay, J.H., Fang, H.H.P., 2000. The roles of calcium in sludge granulation
during UASB reactor start-up. Water Research 35 (4), 1052–1060.
Zala, S., Nerurkar, A., Desai, A., 1999. Biotreatment of nitrate-rich industrial effluent
by suspended bacterial growth. Biotechnology Letters 21, 481–485.
production.Water Science and
Y. Fernández-Nava et al./Bioresource Technology 99 (2008) 7976–7981
7981