Bioresource Technology 127 (2013) 415–421

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Bioresource Technology
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Evaluation of integrated anaerobic/aerobic fixed-bed sequencing batch biofilm

reactor for decolorization and biodegradation of azo dye Acid Red 18: Comparison
of using two types of packing media
E. Hosseini Koupaie a, M.R. Alavi Moghaddam a,⇑, S.H. Hashemi b
a
Civil and Environmental Engineering Department, Amirkabir University of Technology, Hafez Ave., Tehran 15875-4413, Iran
b
Environmental Science Research Institute, Shahid Beheshti University, Tehran, Iran

h i g h l i g h t s

" Comparison of pumice stones and polyethylene media as biofilm support was performed.
" First-order decolorization kinetics with respect to dye concentration was observed.
" Detection and quantification of the dye metabolites by HPLC analysis were conducted.
" SEM analysis was used to investigate the attached-growth biofilm morphology.
" More than 92% of COD, 96% of dye and 63% of 1-naphthylamine-4-sulfonate was removed.

a r t i c l e i n f o a b s t r a c t

Article history: Two integrated anaerobic/aerobic fixed-bed sequencing batch biofilm reactor (FB-SBBR) were operated to
Received 5 July 2012 evaluate decolorization and biodegradation of azo dye Acid Red 18 (AR18). Volcanic pumice stones and a
Received in revised form 28 September 2012 type of plastic media made of polyethylene were used as packing media in FB-SBBR1 and FB-SBBR2,
Accepted 3 October 2012
respectively. Decolorization of AR18 in both reactors followed first-order kinetic with respect to dye con-
Available online 13 October 2012
centration. More than 63.7% and 71.3% of anaerobically formed 1-naphthylamine-4-sulfonate (1N-4S), as
one of the main sulfonated aromatic constituents of AR18 was removed during the aerobic reaction phase
Keywords:
in FB-SBBR1 and FB-SBBR2, respectively. Based on statistical analysis, performance of FB-SBBR2 in terms
Acid Red 18
Biodegradation
of COD removal as well as biodegradation of 1N-4S was significantly higher than that of FB-SBBR1. Spher-
Decolorization ical and rod shaped bacteria were the dominant species of bacteria in the biofilm grown on the pumice
Fixed-bed sequencing batch biofilm reactor stones surfaces, while, the biofilm grown on surfaces of the polyethylene media had a fluffy structure.
(FB-SBBR) Ó 2012 Elsevier Ltd. All rights reserved.
Packing media

1. Introduction serious environmental problems due to their toxicity, mutagenicity

Azo dyes account for more than 50% of all colorants used world-
wide and are the most common synthetic dyes discharged into the
environment (Meng et al., 2012). The release of azo dye-containing
wastewaters not only can affect the transparency and aesthetic
appearance of natural water, but also can obstruct sunlight pene-
tration diminishing photosynthesis and oxygen solubility (Jonstrup
et al., 2011; Meng et al., 2012). Previous researchers also proved
that several azo dyes and their aromatic constituents can create
⇑ Corresponding author. Tel.: +98 912 2334600; fax: +98 21 66414213.
E-mail addresses: ehssan.hosseini.k@gmail.com (E. Hosseini Koupaie), alavi@
aut.ac.ir, alavim@yahoo.com (M.R. Alavi Moghaddam), h_hashemi@sbu.ac.ir (S.H.
Hashemi).
~ao Umbuzeiro
and carcinogenicity effects to aquatic life (De Arag
et al., 2005; Tan et al., 2005).
Several physicochemical methods such as coagulation/floccula-
tion, electrolysis, adsorption, membrane filtration, ion-exchange,
irradiation and advanced oxidation have been tested for removal
of azo dyes from wastewaters (Singh and Arora, 2011). None, how-
ever, has appeared as a panacea, because most of these methods
transfer contaminants into another phase rather than degrade
them to less toxic and harmful products (Moussavi and Heidarizad,
2010). High energy consumption, expensiveness and generation of
hazardous sludge which require safe disposal are other drawbacks
of using these treatment methods (Pandey et al., 2007). As a result,
biological processes have aroused interest due to their cost
effectiveness, ability to produce less sludge, reliability and

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.003
416 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421
environmental benignity (Baban et al., 2010; Champagne and
Ramsay, 2010; Kolekar et al., 2012).
Two stage (combined) anaerobic–aerobic process is one of the
most accepted strategies for biological treatment of azo dye-con-
taining wastewaters. During the anaerobic stage, decolorization
occurs through a microbiologically nonspecific process in which
the azo bond is reductively cleaved to aromatic amines (Singh
and Arora, 2011). These resultant aromatic amines are also ex-
pected to be mineralized during the subsequent aerobic stage. In
this context, successful mineralization of some aromatic amines
derived from anaerobic azo dye reduction has been achieved aero-
bically (Pandey et al., 2007). However, biodegradation of most aro-
matic metabolites formed from the reduction of sulphonated azo
dyes is still one of the challenging issues of ultimate removal of
these dyes from the contaminated wastewaters. Generally, miner-
alization of sulphonated aromatic amines has only been reported
for relatively simple sulphonated amino-benzene and amino-
naphthalene compounds (Tan and Field, 2000). Tan et al. (2005)
tested ten sulfonated aromatic amines for their biodegradability
and reported that only two aminobenzenesulfonic acid (ABS) iso-
mers, 2- and 4-ABS, were degraded under the aerobic condition.
This low biodegradability is because of the high hydrophilic nature
of sulfonated groups obstructing cell membrane transport (Van der
Zee and Villaverde, 2005). It has been also observed that various
sulfonated aromatic amines especially those containing a hydroxy
group in ortho-position to the amino group undergo autoxidation
in the presence of oxygen leading to the formation of more aerobi-
cally recalcitrant compounds (Kudlich et al., 1999). In view of these
facts, it is still required to research for adopting novel or modified
treatment techniques to improve biodegradation of sulfonated azo
dyes and their metabolic intermediates.
The immobilization of microorganisms on surfaces of biofilm
carriers is an increasingly used biological treatment strategy.
Lower sensitivity to toxic loads, greater catalytic stability, longer
microbial residence time, more tolerant to oligotrophic condi-
tions and lower biomass washout risk are the preferable features
of the immobilized cells in comparison to the non-immobilized
counterparts (Gómez-De Jesús et al., 2009; González et al.,
2012). Besides these advantages, introduction of biofilm carriers
can provide a suitable environment in which simultaneous aero-
bic and anoxic metabolic activity occurred (Buitrón et al., 2004).
In recent years, biofilm reactors have attracted more attention
especially for treatment of wastewaters containing bio-
recalcitrant, inhibitory and toxic compounds (Bajaj et al., 2008;
Farhadian et al., 2008). It has offered effective degradation of
several biorefractory compounds such as phenol (Bajaj et al.,
2008), chlorophenol (Gómez-De Jesús et al., 2009), phenoxy
herbicide (González et al., 2012), aniline (Delnavaz et al.,
2010), arsenic (Upadhyaya et al., 2010) and formaldehyde
(Moussavi and Heidarizad, 2010). Successful treatment of high-
strength wastewaters such as oilfield wastewater (Dong et al.,
2011) and coal gasification wastewater (Li et al., 2011) has been
also achieved using attached-growth biofilm processes.
Given the above explanation, a series of research projects have
been conducted by the authors to evaluate and develop novel
application of attached-growth biofilm processes to enhance re-
moval of azo dyes from wastewaters. The present study is follow-
ing the authors‘ earlier research work in which moving bed
sequencing batch biofilm reactor (MB-SBBR) was successfully
tested and proposed as post-treatment of anaerobically degraded
azo dye Acid Red 18 (AR18) (Jonstrup et al., 2011; Singh and Arora,
2011). The objective of this article is to investigate and compare
the performance of two fixed-bed sequencing batch biofilm reac-
tors (FB-SBBR) equipped with two different types of packing media
for treatment of azo dye-containing wastewater which to our
knowledge has not been previously addressed.
For this purpose, COD and dye removal efficiency as well as bio-
mass concentration were monitored. The UV–Vis spectra of the
effluent samples were compared with those of the dye and 1-naph-
thylamine-4-sulfonate (1N-4S) standard solution. The decoloriza-
tion kinetics was also studied in details and the reaction
constants were calculated. Biodegradation of 1N-4S as one of the
main sulfonated aromatic constituents of the dye was investigated
using HPLC. Furthermore, morphological observation was per-
formed and the bacterial structure of the biofilm grown on surfaces
of the two types of packing media was compared.
2. Methods
2.1. Fixed-bed sequencing batch biofilm reactor (FB-SBBR)
The experimental setup used in this study consisted of two lab-
scale integrated anaerobic/aerobic fixed-bed sequencing batch bio-
film reactors (FB-SBBR1 and FB-SBBR2). FB-SBBR1 was equipped
with volcanic pumice stones as packing media while in FB-SBBR2,
the packing material was a type of plastic media made of polyeth-
ylene (2H-BCN017KL, Germany). Schematic of the reactors and the
two types of packing media are illustrated in Fig. 1. The reactors
were made of Plexiglas having inner diameter, height and effective
volume of 22 and 55 cm and 14 L, respectively. The packing media
were confined between the reactors wall and a 316 stainless steel
cylindrical hamper.
2.2. Composition of synthetic wastewater
The composition of synthetic wastewater (SWW) was as fol-
lows: Acid Red 18 (100 mg/L), glucose (500 mg/L), lactose
(500 mg/L), Urea (40 mg/L), KH2PO4 (8 mg/L), K2HPO4 (10 mg/L)
and NaHCO3 (250 mg/L). The SWW was prepared in tap water
and the chemicals were analytical grade (Merck, Germany). The
azo dye C.I Acid Red 18 (AR18) was obtained from Alvan Sabet
Company (Tehran, Iran) and used without further purification.
General characteristics of AR18 are presented in Table 1.
2.3. Seeding and operation of the reactors
The enrichment process was initiated by using a combination of
anaerobic granular sludge taken from a full scale UASB reactor and
activated sludge taken from a municipal wastewater treatment
plant as seed in the reactors to obtain an initial mixed liquor sus-
pended solids (MLSS) concentration of about 2500 mg/L. To accli-
matize the microorganisms, the reactors were first operated with
dye-free and low-COD wastewater for 10 days. Afterwards, during
two weeks, the concentration of COD and the dye was gradually in-
creased to the final desired values in the feed solution. After the
stabilization of the feed solution, the reactors were operated for
100 days.
A 24 h operation cycle of both FB-SBBRs consisted of six succes-
sive phases including filling (15 min), anaerobic phase (14 h), aer-
obic phase (8 h), settling (1 h), draw (30 min) and idle (15 min)
which were controlled by a digital timer. The hydraulic retention
time (HRT) was kept 2 days by supplying 7 L of new synthetic
wastewater to each FB-SBBR at the beginning of each cycle. A
low speed gear motor (150 rpm) driving three paddle-shaped
impellers was used during the anaerobic reaction phase to sustain
a continuous and homogeneous supply of substrates and also pro-
vide a complete mixing condition (Fig. 1a). An electromagnetic
pump (RESUN, ACO-006, China) was also used during the aerobic
reaction time for supplying air and keeping the dissolved oxygen
concentration above 3 mg/L during the aerobic phase.
 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421 417

Fig. 1. The laboratory scale treatment system: (a) configuration of FB-SBBRs; (b) volcanic pumice stones; (c) plastic media made of polyethylene (2H-BCN017KL, Germany).

Table 1 Methods for Examination of Water and Wastewater (APHA, 1998).

General characteristics of Acid Red 18 (AR18). The bicarbonate alkalinity (BA) was measured by titration with
Parameter Value standardized H2SO4 (0.02 N) to pH 4.5. The dissolved oxygen
Chemical formula C20H11N2Na3O10S3
(DO) concentration, pH and oxidation–reduction potential (ORP)
Molecular weight (g/mol) 604.5 were monitored regularly using WTW Electrodes connected to
COD of 1 g-AR18/l (mg/L) 597 ± 17 the SenTix probes. In the case of ORP, the values measured by
kmax (nm) 507 the electrode (ORPAg/AgCl) were modified to the standard ORP (E0)
using Eq. (1):

E0 ¼ ORPAg=AgCl þ 200 for T ¼ 35  C ð1Þ

Molecular structure
2.4. Analytical methods and procedures
The color measurement of the samples was performed spectro-
photometrically at 507 nm (the maximum absorbent wavelength
of AR18) using UV–Vis spectrophotometer (DR 4000, HACH,
USA). Before the analysis, the samples withdrawn from the reac-
tors were centrifuged at 6000 rpm for 10 min. The qualitative
information related to the decolorization of AR18 and the metabo-
lites formed during the treatment process was determined by
scanning of complete spectrum from 200 to 800 nm.
Chemical oxygen demand (COD), MLSS, mixed liquor volatile
suspended solids (MLVSS) and sludge volume index (SVI) analyses
were carried out according to the methods outlined in the Standard
In order to compare the microstructure of the biofilm grown of
surfaces of the two types of packing media used in this study, bio-
film samples were taken and examined by a digital scanning elec-
tron microscope (Philips-XL30, Holland) applying 25 kV
accelerating voltage.
HPLC analysis was performed using an Agilent 1200 chromato-
graph which was equipped with a variable wavelength detector
and connected to a 5 lm C18 column. The mobile phase was a gra-
dient started with 91% water, 5% acetonitrile and 4% methanol. The
gradient was changed linearly to 69% water, 27% acetonitrile and
4% methanol over 25 min. The detection was performed at 254 nm.
2.5. Bio-sorption test
In order to realize whether the adsorption of dye into biomass
has contributed to the decolorization or not, the methanol extrac-
tion method was used to extract the adsorbed dye from the sludge.
This method has been successfully applied by Buitrón et al., (2004).
In the present study, it was conducted by adding 10 mL of methanol
plus 40 mL of distilled water to the pellet of 50 mL of a centrifuged
418 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421
sample. The mixture was mixed thoroughly and again centrifuged.
The absorbance of the supernatant was measured at 507 nm.
2.6. Specific oxygen uptake rate (SOUR)
The activity of the suspended biomass in FB-SBBRs was calcu-
lated by measuring the specific oxygen uptake rate (SOUR) for
the mixed liquor samples taken at the end of the anaerobic phase
and before the start of the aeration phase. The test was
accomplished by sampling 100 mL of mixed liquor from the reac-
tors and immediate transferring to a vessel equipped with a DO
analyzer for determination of SOUR according to the Standard
Methods for Examination of Water and Wastewater (APHA, 1998).
2.7. Statistical analysis
Standard statistical parameters including mean and standard
deviation (for more than duplicate data points) were used. The
experimental data were also analyzed by one-way ANOVA (95%
confidence interval) using MINITAB software when significant dif-
ference analysis was required.
3. Results and discussion
3.1. Performance of FB-SBBRs in COD and dye removal
The operational data of the applied reactors are summarized in
Table 2. As it is shown, the main part of COD (more than 92%) and
dye (more than 95%) was removed during the anaerobic phase in
both FB-SBBRs. The statistical analysis showed that there was no
meaningful difference between FB-SBBR1 and FB-SBBR2 in terms
of AR18 decolorization (P = 0.891 > 0.05). However, the difference
observed between these two reactors in terms of COD removal
was statistically significant (P = 0.000). The average of COD concen-
tration in the effluent of FB-SBBR1 and FB-SBBR2 was obtained
56.2 mg/L and 40.2 mg/L, respectively. According to the UV–Vis
analysis (Section 3.2) and also HPLC test (Section 3.4), the lower
COD concentration in the effluent of FB-SBBR2 compared to that
of FB-SBBR1 is due to better performance of FB-SBBR2 in the re-
moval of aromatic compounds released after anaerobic decoloriza-
tion of AR18.
The variation of COD and AR18 concentration after the end of
both anaerobic and aerobic phases in FB-SBBRs as well as their re-
moval efficiency during the whole operation period are shown in
Fig. 2.
According to Fig. 2a and b, both reactors reached the steady
state condition in terms of COD removal after about 60 days. How-
ever, as clear in Fig. 2c and d, the constant decolorization was
achieved after about 30 days. Therefore, since the dye removal
efficiency was reached its constant level much earlier than COD
Table 2
The operational data of the operated reactors.
Experimental data FB-SBBR1
End of anaerobic phase End of aerobic phase
COD concentration (mg/L) 74.2 ± 5.9 56.2 ± 2.8
COD removal (%) 92.9 ± 0.6 94.6 ± 0.3
AR18 concentration (mg/L) 3.1 ± 0.6 3.4 ± 0.5
AR18 decolorization (%) 96.9 ± 0.6 96.6 ± 0.5
MLSS (mg/L) – 3494 ± 130
MLVSS (mg/L) – 2652 ± 116
MLVSS/MLSS (%) – 75.9 ± 2.6
Standard ORP (mV) 206 ± 19 253 ± 15
pH – 8.5 ± 0.2
BAa (mg CaCO3/l) – 2205 ± 129
a
BA: Bicarbonate alkalinity.
removal efficiency, it can be concluded that the production of
reducing equivalents was not a limiting factor for the anaerobic
decolorization of AR18. The great contribution of anaerobic phase
in the removal of COD and also better performance of FB-SBBR2
in COD removal in comparison with that of FB-SBBR1 are obvious
in Fig. 2a and b. In addition to the high capacity of the anaerobic
phase in terms of COD removal, it is evident from Fig. 2c and d that
the main part of the dye decolorization (more than 95%) was also
achieved anaerobically.
3.2. UV–Vis analysis
The UV–Vis spectrum of the standard solution and the samples
withdrawn from the reactors is presented in Fig. S1 (Supplemen-
tary data). As shown in Fig. S1a, while the absorbance peak at
507 nm was almost completely removed in both reactors, the
UV-region absorbance area was increased after the anaerobic reac-
tion. This indicates that the azo bond reduction through anaerobic
reaction phase was the main mechanism for AR18 decolorization
and the contribution of the physical adsorption in decolorization
was negligible. This result was also proved by the bio-sorption test
which proved that less than 3% of the dye was absorbed into the
biomass.
According to Fig. S1b, the UV–Vis spectrum of the standard
solution containing 1-naphthylamine-4-sulfonate (1N-4S) exhib-
ited a strong absorbance peak at 320 nm. The appearance of a
peak at 320 nm in the spectrum of the samples taken after the
end of the anaerobic phase (Fig. S1a) proved the formation of
1N-4S during the anaerobic degradation of AR18. It is clear that
after the aerobic phase, this peak (320 nm) was reduced and
shifted to the lower wavelength (275 nm). It is also clear that
the UV-region absorbance area was significantly decreased after
the aerobic phase indicating the degradation of the aromatic
metabolites formed during the anaerobic decolorization of AR18.
In agreement with these results, Buitrón et al. reported the
appearance of two peaks (between 300 and 370 nm) at the end
of the anaerobic degradation of azo dye Acid Orange 7. It was
reported that at the end of the aerobic stage, these peaks were re-
moved and a new peak at lower wavelength (250 nm) was de-
tected (Buitrón et al., 2006).
3.3. Decolorization kinetics study
Eq. (2) shows the general kinetic model of the dye decoloriza-
tion (Yu et al., 2001).
dC t m
¼ kM C nt ð2Þ
dt
where t is the time (h), M and m are the biomass concentration and its
partial reaction order, respectively, C and n are the dye concentration
FB-SBBR2
End of anaerobic phase End of aerobic phase
51.8 ± 2.3 40.2 ± 2.2
95.0 ± 0.2 96.1 ± 0.2
2.9 ± 0.7 3.5 ± 0.5
97.1 ± 0.7 96.5 ± 0.5
– 3624 ± 125
– 2790 ± 104
– 77.0 ± 1.2
188 ± 24 282 ± 17
– 8.3 ± 0.1
– 1986 ± 107
 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421 419

(a) FB-SBBR1: End of anaeobic phase (c) 30

300 FB-SBBR1: End of aerobic phase

25
Effluent COD (mg/l) FB-SBBR2: End of anaerobic phase

Effluent dye (mg/l)

250 FB-SBBR2: End of aerobic phase FB-SBBR1
20
FB-SBBR2
200
15
150
10
100

5
50
10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100
Time (day) Time (day)

(b) (d)
95 95

Decolorization efficiency (%)

COD removal efficiency (%)

90
90

85
FB-SBBR1
85
FB-SBBR2
80
FB-SBBR1: End of anaeobic phase
80
75 FB-SBBR1: End of aerobic phase
FB-SBBR2: End of anaerobic phase 75
70
FB-SBBR2: End of aerobic phase

65 70
10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100
Time (day) Time (day)

Fig. 2. Performance of FB-SBBRs in COD and dye removal: (a) effluent COD concentration; (b) COD removal efficiency; (c) effluent dye concentration; (d) decolorization
efficiency.

and its partial reaction order, respectively and k is the specific decol-
orization rate. As described by other researchers, if cell growth and
death are being assumed negligible during the decolorization reac-
tion cycle (Mm = constant), Eq. (3) can be applied as simplified form
of Eq. (2) (Dafale et al., 2008; Lourenço et al., 2006):
 1n
dC n C ð1  nÞk
¼ kC ) ¼ 1  ð1nÞ t ðn–1Þ
dt C0 C0
In order to find out the most appropriate decolorization kinetic
model, the residual dye concentration in the reactors was moni-
tored throughout three anaerobic phases at 80th, 92th and 96th
day of the reactors operation (three repetitions). The decoloriza-
tion constants were calculated for zero, first and second reaction
order. Time-course profile of the anaerobic AR18 decolorization
and the average values of zero, first and second specific decoloriza-
tion rate constants are presented in Fig. 3 and Table 3, respectively.
According to the obtained data, AR18 decolorization in both
reactors followed first-order kinetic with respect to the dye con-
centration. This finding is in agreement with those reported by sev-
eral researchers in which first-order reaction model was observed
for the anaerobic decolorization of most mono azo dyes (Farabegoli
et al., 2010; Hosseini Koupaie et al., 2012; Van der Zee et al., 2001).
It is noteworthy that there was no statistically significant differ-
ence between two reactors in the case of specific decolorization
rate (P = 0.269 > 0.05). Therefore, in this study neither decoloriza-
tion efficiency nor decolorization rate was affected by the use of
two different packing media.
3.4. HPLC analysis
The HPLC chromatogram of the standard solution and the sam-
ples taken from FB-SBBRs are shown in Fig. S2 (Supplementary
data). The standard solution contained 100 mg/L of AR18 and
100 mg/L of 1-naphthylamine-4-sulfonate (1N-4S). As shown in
Fig. S2a, two peaks were detected in the chromatogram of the stan-
ð3Þ dard solution at retention times (RT) of 6.8 and 16.6 min related to
1N-4S and AR18, respectively.
According to Fig. S2, a peak at the retention time of 6.8 min is
observed in Fig. S2(b-1) and S2(c-1), the HPLC chromatogram of
the samples taken after the anaerobic phase from FB-SBBR1 and
FB-SBBR2, respectively. Therefore, 1N-4S was detected as one of
the anaerobically formed aromatic amines. This result confirmed
the outcome of the UV–Vis analysis in which the formation of
1N-4S was proved (Fig. S1). The HPLC analysis also confirms that
after the aerobic phase, the chromatographic peak area related to
1N-4S was significantly decreased indicating further biodegrada-
tion of this aromatic compound.
The concentration of 1N-4S at the end of the anaerobic and
aerobic phases as well as its aerobic removal efficiency is listed
in Table 4. In contrast with most of researches in which difficulties
with aerobic removal of sulfonated aromatic amines were ob-
served (Pandey et al., 2007; Pinheiro et al., 2004; Van der Zee
and Villaverde, 2005), in this study more than 63.7% and 71.3% of
1N-4S formed through the anaerobic reaction phase was removed
in the subsequent aerobic reaction phase in FB-SBBR1 and FB-
SBBR2, respectively.
420 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421

First repetition- 80th day First repetition- 80th day

45
45 Second repetition- 92th day Second repetition- 92th day
40
40 Third repetition- 96th day Third repetition- 96th day
Residual dye (mg/l)

Residual dye (mg/l)

35
35
C=48.03exp(-0.3419t) 30 C=44.18exp(-0.3093t)
30
25 25

20 C=43.88exp(-0.384t) 20 C=45.53exp(-0.3483t)

15 15
10 10
C=44.82exp(-0.3594t)
5 C=46.81exp(-0.3661t)
5

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (h) Time (h)
(a) (b)
Fig. 3. Time-course profile of anaerobic AR18 decolorization in (a) FB-SBBR1 and (b) FB-SBBR2.

Table 3 Another observation should be mentioned is that after the aer-

The average values of zero, first and second specific decolorization rate constants. obic phase, all the chromatographic peak area decreased and
Order of kinetic Zero-order First-order Second-order shifted to the lower retention times (Fig. S2(b-2) and (c-2)). As re-
FB-SBBR1 k0 = 5.217 ± 0.383 k1 = 0.364 ± 0.021 k2 = 0.040 ± 0.004
ported by other researchers, this phenomenon confirms the forma-
R2 = 0.883 R2 = 0.997 R2 = 0.897 tion of less aromatic and more polar compounds during the aerobic
FB-SBBR2 k0 = 5.387±.822 k1 = 0.339 ± 0.026 k2 = 0.039 ± 0.008 reaction phase (O’Neill et al., 2000; Supaka et al., 2004).
R2 = 0.898 R2 = 0.997 R2 = 0.855

3.5. Biofilm morphology

Table 4
Concentration of 1-naphthylamine-4-sulfonate (1N-4S) at the end of the anaerobic
and aerobic phases and its removal efficiency.
Reactor End of anaerobic End of aerobic Aerobic
phase (mg/L) phase (mg/L) removal
efficiency (%)
FB-SBBR1 11.10 4.03 63.7
FB-SBBR2 5.23 1.50 71.3
According to the stoichiometric calculations, 96% decolorization
of AR18 will theoretically lead to the accumulation of 38.9 mg/L of
1N-4S in FB-SBBRs. According to this and also considering the con-
centration of 1N-4S in the effluent of the reactors, it can be con-
cluded that FB-SBBR1 and 2 were capable to remove more than
89% and 96% of the anaerobically released 1N-4S. Evaluation of
overall chromatographic peak area also revealed that more than
43.2% and 51.1% of the dye total metabolites remaining at the
end of the anaerobic phase were removed during the aerobic phase
in FB-SBBR1 and FB-SBBR2, respectively.
As clear from Fig. S2 and also based on the data presented in
Table 4, FB-SBBR2 equipped with polyethylene media performed
better in removal of aromatic metabolites than FB-SBBR1 equipped
with volcanic pumice stones. It should be noted that since the
SOUR values were not significantly different for the mixed liquor
sampled from FB-SBBR1 and FB-SBBR2, it can be concluded that
the activity of the suspended biomass in both reactors was the
same. Therefore, the observed difference between these two reac-
tors in terms of the removal of aromatic metabolites is most likely
due to the difference between the capability of the biofilm grown
on surfaces of their packing media. It is noteworthy that, the differ-
ence between the dominant species of the microorganisms existing
in the biofilm attached to the pumice stones (spherical and rod
shaped bacteria) and those grown on the polyethylene media sur-
faces (bacteria with fluffy structure) was proved by the SEM anal-
ysis (Fig. S3) (Supplementary data).
The optical and SEM photographs of the 75-day old biofilm
grown on surfaces of the packing media are shown in Fig. S3
Total
(Supplementary data). As seen in Fig. S3a and d, almost all pores
removal of the pumice stones used in FB-SBBR1 and most surfaces of the
efficiency (%) polyethylene media used in FB-SBBR2 were covered by a layer of
89.6 attached-growth biofilm. SEM analysis revealed that spherical
96.1 and rod shaped bacteria were the dominant species of bacteria in
the biofilm grown of surfaces of the pumice stones (Fig. S3b and
S3c). However, the biofilm grown on surfaces of the polyethylene
media had a fluffy structure (Fig. S3e and S3f). Considering the
visual observation, it was also found that the shallow layer of the
biofilm directly exposed to oxygen during the aerobic phase had
a brown color (Fig. S3a and S3d), whereas the color of the deeper
zones of the biofilm was dark black (not shown).
The microstructure of the biofilm grown on surfaces of the poly-
ethylene media (used as fixed-bed in the present study) is interest-
ingly different from that of grown on surfaces of this type of media
when they were used as moving carriers. The biofilm attached to
the moving carriers exhibited a highly porous structure (Hosseini
Koupaie et al., 2011) in which a large number of filamentous bac-
teria existed (Hosseini Koupaie et al., 2012). However, the biofilm
attached to the fixed media had a compact structure in which no
filamentous bacteria observed (Fig. S3e and S4f).
4. Conclusions
This research demonstrated high capability of the developed
process for elimination of both COD and dye. Removal of more than
92% COD and 95% AR18 was achieved using integrated anaerobic/
aerobic FB-SBBR. The reactor equipped with polyethylene media
offered higher removal of COD and also the dye aromatic metabo-
lites in comparison to the reactors equipped with volcanic pumice
stones. More than 89.6% and 96.1% of anaerobically formed 1N-4S
were removed using FB-SBBR1 and FB-SBBR2, respectively. Accord-
ing to this investigation, integrated anaerobic/aerobic FB-SBBR
 E. Hosseini Koupaie et al. / Bioresource Technology 127 (2013) 415–421
especially that equipped with polyethylene media can be sug-
gested as an efficient strategy for biodegradation of azo dyes.
Acknowledgements
The authors wish to thanks the Amirkabir University of Tech-
nology for providing fund and support. In addition the authors
would like to express their gratitude to Ms. Lida Ezzedinloo (envi-
ronmental laboratory assistant) and Mr. Mohammad Hakimelahi
(environmental engineering M.Sc. student) for their help during
the experiments.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2012.
10.003.
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