ARTICLE IN PRESS

Water Research 39 (2005) 1425–1440

www.elsevier.com/locate/watres
Review
Combined anaerobic–aerobic treatment of azo dyes—A short
review of bioreactor studies
Frank P. van der Zeea,, Santiago Villaverdeb
a
Department of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b
Department of Chemical Engineering, Faculty of Sciences, University of Valladolid. Paseo Prado de la Magdalena s/n,
47005 Valladolid, Spain
Received 2 August 2004; received in revised form 23 February 2005

Abstract

The most logical concept for the removal of azo dyes in biological wastewater treatment systems is based on
anaerobic treatment, for the reductive cleavage of the dyes’ azo linkages, in combination with aerobic treatment, for the
degradation of the products from azo dye cleavage, aromatic amines. Since the 1990s, several research papers have been
published on combined, sequential or integrated, anaerobic–aerobic bioreactor treatment of azo dye-containing
wastewater.
The extent of azo dye reduction in the anaerobic phase of those bioreactor systems was generally high, albeit the
process often required long reaction times, a limitation that can easily be remedied by making use of the property of
redox mediators to speed up the process. The consequent removal of aromatic amines under aerobic conditions was less
unequivocal. Although analytical data indicate that many of the aromatic amines were removed from the wastewater,
and although the limited amount of available toxicity data all show far-reaching detoxification during aerobic
treatment, it is clear that not all aromatic amines can be completely mineralized.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Azo dyes; Aromatic amines; Biodegradation; Anaerobic–aerobic; Bioreactors

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1426

2. Combined anaerobic–aerobic treatment of azo dyes in (semi-)continuous bioreactors. . . . . . . . . . . . . . . . 1427
2.1. Anaerobic color removal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1427
2.1.1. Dye structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1427
2.1.2. Hydraulic retention time . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1432
2.1.3. Biomass concentration . . . . . . . . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1432
2.1.4. Alternative electron acceptors (and redox mediators). . . . ......... . . . . . . . . . . . . . . . 1432
2.1.5. Primary substrate concentration . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . 1432

Corresponding author. Tel.: +351 253 604 400; fax: +351 253 678 986.
E-mail address: frankvdz@deb.uminho.pt (F.P. van der Zee).

0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2005.03.007
 ARTICLE IN PRESS
1426 F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440

2.1.6. Primary substrate type . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1433

2.1.7. Dye concentration . . . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1433
2.1.8. Dye toxicity. . . . . . . . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1433
2.2. Anaerobic formation of aromatic amines due to azo dye reduction. . . . . . . . . . . . . . . . . . . . . . . . 1434
2.3. Aerobic fate of aromatic amines . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1435
3. Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1435
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1437
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... . . . . . . . . . . . . . . . . . . . . . . . 1437

1. Introduction Generally, bacterial azo dye biodegradation proceeds

Azo dyes represent the largest class of dyes applied in
textile processing. In textile dyebaths, the degree of
fixation of dyes to fabrics is never complete, resulting in
dye-containing effluents (O’Neill et al., 1999b). The
removal of dyes from these effluents is desired, not only
for aesthetic reasons, but also because many azo dyes
and their breakdown products are toxic to aquatic life
(Chung and Stevens, 1993) and mutagenic to humans
(Brown and DeVito, 1993; Weisburger, 2002). Different
physical, chemical and biological techniques can be
applied to remove dyes from wastewater (Cooper, 1993;
Southern, 1995; Vandevivere et al., 1998; Hao et al.,
2000; Robinson et al., 2001). Each technique has its
technical and economical limitations. Most physico-
chemical dye removal methods have drawbacks because
they are expensive, have limited versatility, are greatly
interfered by other wastewater constituents, and/or
generate waste products that must be handled. Alter-
natively, biological treatment may present a relatively
inexpensive way to remove dyes from wastewater.
in two stages. The first stage involves reductive cleavage
of the dyes’ azo linkages, resulting in the formation of—
generally colorless but potentially hazardous—aromatic
amines. The second stage involves degradation of the
aromatic amines. Azo dye reduction usually requires
anaerobic conditions, whereas bacterial biodegradation
of aromatic amines is an almost exclusively aerobic
process (Fig. 1). A wastewater treatment process in
which anaerobic and aerobic conditions are combined is
therefore the most logical concept for removing azo dyes
from wastewater (Field et al., 1995; Knackmuss, 1996).
Biodegradation of dyes has been the subject of a large
number of research papers and several review articles
have been published (Walker, 1970; Levine, 1991;
Chung and Cerniglia, 1992; Chung and Stevens, 1993;
Bumpus, 1995; Banat et al., 1996; Delee et al., 1998;
McMullan et al., 2001; Stolz, 2001; Pearce et al., 2003;
Forgacs et al., 2004). Especially anaerobic azo dye
reduction has been thoroughly investigated and most
researchers agree that it is a non-specific and presumably
extracellular process, in which reducing equivalents

Anaerobic Aerobic

azo dyes

R R* R R*
N N N N

4[H]
aromatic amines

R R*
NH2 + H2N

CO2+ H2O + NH3

O2
R R
NH2 NH2

autoxidation

Fig. 1. General overview of the fate of azo dyes and aromatic amines during anaerobic–aerobic treatment.
 ARTICLE IN PRESS
F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
from either biological or chemical source are transferred
to the dye.
Also biodegradation of aromatic amines has been the
subject of a large number of publications, including
papers about amine biodegradation by activated sludge
or in activated sludge systems (Baird et al., 1977; Brown
and Laboureur, 1983; Ekici et al., 2001). As summarized
in a recent review paper (Pinheiro et al., 2004), various
(substituted) aminobenzene, aminonaphthalene and
aminobenzidine compounds have been found aerobi-
cally degradable. The conversion of these compounds
generally requires enrichment of specialized aerobes.
Especially sulfonated aromatic amines, including many
of the constituent aromatic amines from water-soluble
azo dyes, are difficult to degrade. This low biodegrad-
ability is due to the hydrophilic nature of the sulfonate
group, which obstructs membrane transport. Generally,
biodegradation of sulfonated aromatic amines has only
been demonstrated for relatively simple sulfonated
aminobenzene and aminonaphthalene compounds
(Tan and Field, 2000).
Another transformation that aromatic amines may
undergo when being exposed to oxygen is autoxidation.
Especially aromatic amines with ortho-substituted hy-
droxy groups, including a large fraction of aromatic
amines from azo dyes, are susceptible to autoxidation
(Kudlich et al., 1999). Many aromatic amines, e.g.
substituted anilines, aminobenzidines and naphthyla-
mines, have been found to oxidize, initially to oligomers
and eventually to dark-colored polymers with low
solubility that are easily removed from the water phase
(Klibanov and Morris, 1981; Field et al., 1995).
However, autoxidation does not always imply a high
degree of polymerization but can be limited to dimer-
ization or to only a small oxidative change in the
molecular structure (e.g. deamination), yielding stable,
water-soluble, highly colored compounds (Kudlich
et al., 1999).
Research on aromatic amine biodegradation has been
usually conducted with relatively stable, not easily
autoxidizing aromatic amines, representing only a part
of the aromatic amines from azo dyes. In many cases,
the aromatic amines from azo dye reduction will be
highly reactive in the presence of oxygen. For example,
17 out of 20 anaerobically decolorized azo dyes
autoxidized rapidly upon exposure to air (Van der Zee
et al., 2001b). In aerobic bioreactors, autoxidation and,
possibly, reactions with compounds within the sludge
matrix will compete with biodegradation. The limited
amount of data on these chemical oxidation processes,
in combination with analytical problems, makes it
difficult to predict the fate of aromatic amines during
anaerobic–aerobic treatment of azo dyes.
Several research papers have reported the results of
combined anaerobic–aerobic bioreactor treatment of
azo dye-containing wastewaters. This review article
1427
summarizes the results of those research studies,
sketches the general trends and discusses the feasibility
of combined anaerobic–aerobic treatment for the
complete removal of azo dyes from wastewater.
2. Combined anaerobic–aerobic treatment of azo dyes in
(semi-)continuous bioreactors
An overview of the research papers reviewed is
presented in Tables 1–3. A distinction was made
between the different approaches used to obtain a
combined anaerobic–aerobic reactor system. Table 1
lists the systems based on anaerobic–aerobic treatment
in separate reactors. Table 2 lists sequencing batch
reactors (SBR) based on temporal separation of the
anaerobic and the aerobic phase. Table 3 lists the other
systems, either hybrids with aerated zones or micro-
aerobic systems based on the principle of limited oxygen
diffusion in microbial biofilms.
2.1. Anaerobic color removal
The color removal percentages listed in Tables 1–3
show that removal of color is mainly associated with the
anaerobic stage, whereas further decolorization in the
aerobic stage is usually limited to a few extra percents.
In one of the reactor studies it was shown that the color
removal by a two-stage anaerobic–aerobic treatment
process was 70% higher than that of a one-stage aerobic
treatment process (Minke and Rott, 2002). These results
are in agreement with previously published data on
recalcitrance of azo dyes in aerobic sludge environments
(Pagga and Brown, 1986; Shaul et al., 1991; Ganesh et
al., 1994; Ekici et al., 2001). It is reasonable to assume
that anaerobic color removal is mainly due to azo dye
reduction (see Section 2.2 on formation of aromatic
amines due to azo dye reduction).
The extent of azo dye color removal achieved in the
anaerobic stages of the studies listed in Tables 1–3 was
mostly higher than 70% and in several cases almost
100%. Ranges of anaerobic color removal efficiencies
refer to different circumstances tested. Very low
efficiencies usually reflect very sub-optimal conditions,
e.g. short hydraulic retention times. Only one of the azo
dyes studied, Acid Yellow 17, was hardly decolorized
(An et al., 1996) or not decolorized at all (Basibuyuk
and Forster, 1997).
From the reactor studies listed in Tables 1–3 it can be
concluded that some factors can be important for
anaerobic azo dye decolorization (Sections 2.1.1–2.1.8).
2.1.1. Dye structure
When the removal of different azo dyes was tested
under similar conditions, different color removal effi-
ciencies were achieved (Jiang and Bishop, 1994; Seshadri
 1428
Table 1
Sequential anaerobic-aerobic reactor systems treating azo dye-containing wastewater

Anaerobic Aerobic Wastewater characteristics Color removal Aromatic amines References

F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440

Typea HRT (h) Typeb HRT (h) wwc Dyed Conc. (mg/l) Substratese Anaerobic Aerobicf Recovery Removal Detect.
anaerobicg aerobich methodi

3j 36 1j 36 Sk AO10, ma 5–100 Glucose 90–100% + 25–50% Max. 100% 2 Rajaguru et al. (2000)
ABk1, da 10–100 100%
DR2, da 25–200 95–100%
DR28, da 25–200 80–100%
1 24 1 19 S h-RR141, da, mct 450 Starch and acetate 64% 11% + + 1 O’Neill et al. (2000b)

ARTICLE IN PRESS
1 24 1 19 S h-RR141, da, mct 150–750 Starch and acetate 38–59% 6.82 n.e. n.e. O’Neill et al. (2000a)
1 24–48 1 NM S h-RR141, da, mct 1500 Starch and acetate
78% 7% n.e. n.e. O’Neill et al. (1999a)
5 34–84 1 NM S h-RR141, da, mct 1500 Starch and acetate Max. 62% 7% n.e. n.e. O’Neill et al. (1999a)
2 24-Jan 4 NM S AO7, ma 5–40 ME, peptone, YE, trout chow 20–90%l 0 + n.e. n.m. Seshadri et al. (1994)
AO8, ma 5–40 0
AO10, ma 5–40 0
AR14, ma 5–40 +
2 31 4 3.1 S AO10, ma 10 ME, peptone, YE, trout chow
62% + o1% + 1-MS FitzGerald and Bishop (1995)
AR14, ma 10
90%
AR18, ma 10
90%
4 15 2 (2) 7.5 S h-RV5, ma, vs 650–1300 Acetate and YE 90–95% 
69–83%
100%m 1 Sosath and Libra (1997)
4 31 2 (2) 7.5 S h-RBk5, ma, vs 600 Acetate and YE
70% + n.e. n 4 Sosath et al. (1997), Wiesmann et al. (2002)
4 15 2 7.5 S (h-)RBk5, ma, vs 530 Acetate and YE
100%o 35%o
100%p +p 1-MS, 4 Libra et al. (2004)
1 8–20 1q 23 S AY17, ma 40 Glucose 20% +0% to 13% n.e. n.e. An et al. (1996)
(BB3, ox) 40 72%
(BR2, az) 40 78%
1 6–10 1 6.5 R/S ww from a dye-manufacturing factory, mixed with simulated municipal wastewater 70–80% +10% to 20% +r +r An et al. (1996)
4 7–8 2 4.5–5 R Textile dye wastewater with PVA and LAS as main COD 60–85% n.e. n.e. Zaoyan et al. (1992)
1 6–10 1 6 R Textile dye wastewater with PVA and LAS as main COD 90–95% + (max. 96%) n.e. n.e. Jianrong et al. (1994)
3 6 3 (2) 7.7+8.6 S AY17, ma 25 Starch and glucose 0% + n.e. n.e. Basibuyuk and Forster (1997)
BR22, ma 200 499%
1 25 1 10 S MY10, ma 100–200 Ethanol
100% 0
100%s
100%s 1 Tan et al. (2000)
3t Var. 3t 12–24 S DisB79, ma 25–150 Glucose and acetate or none max. 100% n.m.
40%u 65%v 1, 2, 3 Cruz and Buitrón (2001)
w w
3 3 S h-RR198, ma, vs+mct 5000 Starch, wax and acetate 97% 1.97 +2% to 3% + (
100%) 1 Sarsour et al. (2001)
1x 24–48 1p R Highly colored textile wastewater 70–90% +(
100%) n.e. n.e. Kuai et al. (1998)
1 15–16.5 1 55–60 S DBk38, ta 100–3200 Glucose 80–100% + 85–95%
50% 2 Sponza and Is-ik (2005)
1 86.4 1 432 S DBk38, ta 3200 Glucose 81% 13% 74% 81% 2y Is-ik and Sponza (2004c)
1 3–30 1 10–30 S RBk5, da, vs 100 Glucose 82–98% n.m. n.e. n.e. Sponza and Is-ik (2002a)
1 3–30 1 10–108 S RBk5, da, vs 100 Glucose 82–98% — n.e. n.e. 3 Sponza and Is-ik (2002b)
1 2.6–26 1 10–102 S DR28, da 100–4000 Glucose 97–100% n.m. 40–95% 80–100% 2 Is-ik and Sponza (2003)
1 3–30 1 10–108 S RBk5, da, vs 100 Glucose 87–98% 10% to 20 % n.e. n.e. Is-ik and Sponza (2004b)
1 2.5–19 1 9–67 S DR28, da 100 Glucose 92–97% 1% to 15%
1 30 1 108 R Cotton mill wastewater (CMW) 46–55% 10% to +25% + 35–90% 1,2 Is-ik and Sponza (2004a)
CMW+mixture of azo dyes (250–500lmg/l) and glucose 60–75% 1% to 15% + 40–80%
3 12–72 1 10 S RR195, ma, vs+mct 50–400 Molasses 60–100%c +Max. 15% n.e. n.e. Kapdan et al. (2003)
3z 12–72 1 10 R Textile wastewater with added glucose and nutrients 60–85% 10% n.e. n.e. Kapdan and Alparslan (2005)
1 24 5 21.5(24) S AO7, ma 60–300 Glucose+peptone 60–97% + n.e. n.e. Ong et al. (2005)
(6) (15–18) 1 15–18 R Bleaching, scouring, dyeing (+desizing) wastewater containing 10–150 mg/l dyes (50–70%) 5% to +5% n.e. n.e. Frijters et al. (2004)
1 7 80–95%
3 26–90 6 (480) R 1. Reactive dyebath waste and ww with starch & PVA 89–94% +1% to 2% n.e. n.e. Minke and Rott (2002)
2. Split flows from yarn processing 81–92% +1% to 7%

a
Anaerobic reactor types: 1, upflow anaerobic sludge bed; 2, anaerobic fluidized bed; 3, anaerobic filter; 4, anaerobic rotating disc; 5, inclined tubular digester; (6, pre-acidification
tank).
b
Aerobic reactor types: 1, aerobic tank; 2, aerobic rotating disc; 3, aerobic filter; 4, swisher; 5, sequential batch reactor; 6, aerobic biodegradability tests (BOD20).
c
Wastewater type (ww): S, synthetic wastewater; R, real wastewater.
d
Dyes: First abbreviation refers to Colour Index generic names: A, acid; B, basic; D, direct; Dis, disperse; M, mordant; R, reactive; B, blue; Bk, black; O, orange; R, red; V, violet;

F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440

Y, yellow. Second abbreviation refers to amount of azo linkages: ma, monoazo; da, disazo; ta, triazo; (ox, oxazine; az, azine). Third abbreviation refers to reactive groups (reactive
dyes only): vs, vinylsulfone; mct, monochlorotriazine. The prefix ‘h-’ means hydrolyzed (reactive dyes only). Libra et al. (2004) investigated both hydrolyzed, partially hydrolyzed and
non-hydrolyzed Reactive Black 5.
e
Substrates, abbreviations: YE, yeast extract; PVA, polyvinylalcohol; LAS, linear alkyl benzene sulfonate; ME, meat extract.
f
Color removal aerobic: positive values express the additional color removal as percentage of the influent color, negative values express development of color (autoxidation) as
percentage of influent color. ‘n.m.’ not mentioned.
g
Anaerobic aromatic amine recovery: ‘+’ indicates non-quantified sign of recovery; ‘n.e.’ not evaluated.

ARTICLE IN PRESS
h
Aerobic aromatic amine removal: ‘+’ indicates non-quantified sign of removal; percentages express removal of recovered aromatic amines; ‘n.e.’ not evaluated.
i
(Main) detection method aromatic amines: 1, HPLC; 1-MS, LC–MS; 2, diazotization-based colorimetric method; 3, UV spectophotometry; 4, DOC measurements.
j
Both anaerobic and aerobic reactor inoculated with a mixture of four pseudomonads isolated from dyeing effluent-contaminated soils.
k
Nitrogen-free medium.
l
Depending on dye, dye concentration and HRT. All dyes 480% decolorization at high HRT.
m
Complete removal of the metabolites from anaerobic treatment, probably mostly due to autoxidation.
n
Presumably no removal of dye metabolites: hardly any DOC removal and only slight decrease of toxicity.
o
Data refer to fully hydrolyzed RBk5, less color removal for partially hydrolyzed RBk5.
p
Fully hydrolyzed RBk5 was completely converted in the anaerobic phase, to p-aminobenzene-2-hydroxyleethylsulfonic acid (2 mol p-ABHES per mol RBk5) and 1,2,7-triamino-
8-hydroxynaphthalene-3,6-disulfonic acid (1 mol TAHNDS per mol RBk5). In the aerobic phase, p-ABHES was mineralized while TAHNDS autoxidized to 1,2-ketimino-7-amino-
8-hydroxynaphthalene-3,6-disulfonic acid. Partially hydrolyzed RBk5 was not completely converted in the anaerobic phase. p-ABHES and TAHNDS were detected, but in relatively
small amounts. There was no removal of p-ABHES in the aerobic phase.
q
Semi-continuous system.
r
Increased BOD5/COD ratio after anaerobic treatment may point at formation of biodegradable dye metabolites.
s
Almost complete recovery of one of the dye metabolites, sulfanilic acid; partial anaerobic degradation of the other, 5-aminosalicylate. In the aerobic reactor complete
mineralization of 5-aminosalicylate; after bioaugmentation also complete mineralization of sulfanilic acid.
t
Discontinuously fed reactors.
u
Percentage expresses HPLC recovery of 2-bromo-4,6-dinitroaniline (BDNA). Additional thin layer chromatography measurements indicate anaerobic transformation BDNA.
v
Percentage based on total amine measurements (diazotization method).
w
HRT total system 96 h.
x
Sludge bed amended with granular activated carbon.
y
Additional support of aerobic AA removal from HPLC, GC–MS and nitrate analyses.
z
Inoculated with a facultative anaerobic consortium (PDW, Alcaligenes faecolis and Comamonas acidourans).

1429
 1430
Table 2
Anaerobic-aerobic sequenced batch reactor (SBR) systems treating azo dye-containing wastewater

Cycle Wastewater characteristics Color removal Aromatic amines References

Anaerobic Aerobic Total wwa Dyeb Conc. Substratesc Anaerobic Aerobicd Recovery Removal Detect.
(h) (h) time (h) (mg/l) anaerobice aerobicf meth.g

F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440

13 8 24 S h-RV5, ma, vs 60–100 Starch 30–90%h +/0 + +i 1 Lourenc- o et al. (2000)
9–12 8–12 24 S h-RV5, ma, vs 60–100 Starch 20–90%j n.m. + +i 1 Lourenc- o et al. (2001)
h-RBk5, da, vs 30 65%k n.e. n.e.
9–13 8–12 24 S h-RV5, ma, vs 100 Starch Max. 90% n.m. + +i 1 Lourenc- o et al. (2003)
10.5 10 24 S h-RV5, ma, vs 100 Starch 90–99% n.m. + n.e. 1 Albuquerque et al. (2005)
10.5–17 3.5–10 24 S AO7, ma 25 Starch 5–55% n.e.
10.5 10 24 S AO7, ma 25 Starch+lactate Max. 95%l n.e.
0–12 8–12 24 R Wool dyeing effluent with azo and anthraquinone dyes + + n.e. + 3 Cabral Gonc- alves et al. (2005)

ARTICLE IN PRESS
18 5 24 S RBk5, ma, vs 20–100 Glucose and acetate 58–63% + +  1 Luangdilok and Paswad (2000), Panswad and Luangdilok (2000)
(RB19, aq, vs) 20–100 (64–32%)
(RB5, aq, mct) 20–100 (66–41%)
(RB198, ox, hh) 20–100 (—)m
18 5 24 S RBk5, da, vs 10 NB+acetate or glucose 68–72% +2–8% + n.e. 3 Panswad et al. (2001a)
63–68% +8–11%
0–8 3–11 12 S RBk5, da, vs 10–80 NB+acetate or NB+glucose 30–61% 2–17% + n.e. 3 Panswad et al. (2001b)
18.5 0.5 24 S h-RBk5, da, vs 533 Starch, PVA, CMC 86–96% + + +/0 3 Shaw et al. (2002)

a
Wastewater type (ww): S, synthetic wastewater; R, real wastewater.
b
Dyes: First abbreviation refers to Colour Index generic names: A, acid; R, reactive; B, blue; Bk, black; O, orange; V, violet. Second abbreviation refers to amount of azo linkages:
ma, monoazo; da, disazo; (aq, anthraquinone; ox, oxazine). Third abbreviation refers to reactive groups (reactive dyes only): vs, vinylsulfone; mct, monochlorotriazine; hh,
halogenohetrocyclic. The prefix ‘h-’ means hydrolyzed (reactive dyes only).
c
Substrates, abbreviations: NB, nutrient broth; PVA, polyvinylalcohol; CMC, carboxymethylcellulose.
d
Color removal aerobic: positive values express the additional color removal as percentage of the influent color, negative values express development of color (autoxidation) as
percentage of influent color. ‘n.m.’ not mentioned.
e
Anaerobic aromatic amine recovery: ‘+’ indicates non-quantified sign of recovery; ‘n.e.’ not evaluated.
f
Aerobic aromatic amine removal: ‘+’ indicates non-quantified sign of removal; ‘p’ non-quantified sign of partial removal; percentages express removal of recovered aromatic
amines; ‘n.e.’ not evaluated.
g
(Main) detection method aromatic amines: 1, HPLC; 3, UV spectophotometry.
h

90% color removal at a sludge concentration of 2.0 g VSS/l and SRT ¼ 15 days,
30% color removal at a sludge concentration of 1.2 g VSS/l and SRT ¼ 10 days.
i
No degradation of RV5’s constituent naphthalene-based amine; (bio)transformation but no mineralization of its benzene-based amine.
j

90% color removal at a sludge concentration ¼ 2.0 g VSS/l, SRT ¼ 15 days and feed dye concentration ¼ 60 mg/l,
20% color removal at a sludge concentration ¼ 1.2 g VSS/l,
SRT ¼ 10 days and feed dye concentration ¼ 100 mg/l.
k
No effect of changing the SRT.
l
Highest color removal achieved with addition of anthraquinone-2,6-disulfonate.
m
Could not be quantified.
Table 3
Integrated anaerobic-aerobic reactor systems treating azo dye-containing wastewater

F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440

System Wastewater characteristics Color removal Aromatic amines References

Reactor typea Total wwb Dyec Conc. Substratesd Anaerobic Aerobic Recovery Removal Detect.
time (h) (mg/l) anaerobice aerobicf meth.g

EGSB with oxygenation of recycled effluent 36–43 S MY10, ma 59–65 Ethanol
100% +i +i 1 Tan et al. (1999), Tan (2001)
26–34 4-PAP, ma 50 o100%h +i +i 1

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UASB with aerated upper part 1–100 S DY26, da 300 Ethanol 40–70% 9.8 +j +j 3 Kalyuzhnyi and Sklyar (2000)
RAD 0.16–3 S AO7, ma 0–22 ME, peptone, YE, trout chow 18–97%k + + 1,5 Harmer and Bishop (1992)
RAD 2 S AO8, ma n.m. ME, peptone, YE, trout chow 20–90%k n.e. n.e. Jiang and Bishop (1994)
AO10, ma Max. 60%
AR14, ma Max. 60%
Baffled reactor with anaerobic and aerobic compartments 48+18 S h-RBk5, da, vs 500 Starch, PVA, CMC 84–88% + +l 3 Gottlieb et al. (2003)

a
Reactor types: EGSB, expanded granular sludge bed; UASB, upflow anaerobic sludge blanket; RAD, rotating annular drum.
b
Wastewater type (ww): S, synthetic wastewater.
c
Dyes: 4-PAP is 4-phenylazophenol. For the other dyes, the first abbreviation refers to Colour Index generic names: A, acid; D, direct; M, mordant; R, reactive; Bk, black; O,
orange; R, red; Y, yellow. The second abbreviation refers to the amount of azo linkages: ma, monoazo; da, disazo. The third abbreviation refers to the reactive groups (reactive dyes
only): vs, vinylsulfone. The prefix ‘h-’ means hydrolyzed (reactive dyes only).
d
Substrates, abbreviations: YE, yeast extract; PVA, polyvinylalcohol; ME, meat extract; CMC, carboxymethylcellulose.
e
Anaerobic aromatic amine recovery: ‘+’ indicates non-quantified sign of recovery; ‘n.e.’ not evaluated.
f
Aerobic aromatic amine removal: ‘+’ indicates non-quantified sign of removal; percentages express removal of recovered aromatic amines; ‘n.e.’ not evaluated.
g
(Main) detection method aromatic amines: 1, HPLC; 3, UV spectophotometry; 5, GC–MS.
h
Residual color due to autoxidation of 4-aminophenol (one of 4-PAP’s constituent aromatic amines).
i
Aromatic amines from MY10: almost complete recovery of sulfanilic acid, partial anaerobic degradation of 5-aminosalicylate; aromatic amines from 4-PAP: complete
mineralization of aniline, autoxidation of 4-aminophenol.
j
One of the dye’s aromatic amine (5-aminosalycilate) was partially degraded in the anaerobic part and underwent autoxidation in the aerobic part.
k
At high oxygen/low COD flux, dye removal probably (partly) due to aerobic degradation.
l
Decrease of toxicity after addition of adapted biomass may indicate biological degradation of aromatic amines.

1431
 ARTICLE IN PRESS
1432 F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
et al., 1994; FitzGerald and Bishop, 1995; Rajaguru
et al., 2000; Lourenc- o et al., 2001; Is-ik and Sponza,
2004b; Albuquerque et al., 2005).
2.1.2. Hydraulic retention time
Several studies report a positive relation between the
hydraulic retention time of the anaerobic stage and the
color removal efficiency (Seshadri et al., 1994; An et al.,
1996; Panswad et al., 2001a; Panswad et al., 2001b;
Kapdan et al., 2003; Is-ik and Sponza, 2004b; Albu-
querque et al., 2005).
2.1.3. Biomass concentration
Lowering the biomass concentration from 2 to 1.2 g
VSS/l and the solids retention time from 15 to 10 days in
a dye-treating SBR resulted in a considerable decrease,
from
90% to
30%, of the color removal efficiency
(Lourenc- o et al., 2000). Systems with a higher biomass
retention capacity (e.g. upflow anaerobic reactors) might
therefore be better suited for azo dye decolorization
than systems with a lower biomass retention capacity
(e.g. SBR systems). The higher Reactive Black 5 color
removal efficiency of an upflow anaerobic sludge
blanket (UASB) reactor (Sponza and Is-ik, 2002a) in
comparison to those of the anaerobic phases of SBR
systems operated with similar dye concentrations and
reaction times (Luangdilok and Paswad, 2000; Panswad
and Luangdilok, 2000; Lourenc- o et al., 2001; Panswad
et al., 2001a) supports this hypothesis.
2.1.4. Alternative electron acceptors (and redox
mediators)
Reduction of azo dyes is an oxidation–reduction
reaction in which the dye acts as an electron acceptor.
The presence of an alternative electron acceptor may
compete with the azo dye for reducing equivalents. The
presence of nitrate, a normal constituent of textile-
processing wastewater, was shown to slow down
decolorization (Lourenc- o et al., 2000; Panswad and
Luangdilok, 2000). Those results are agreement with
previously published data from batch experiments to
azo dye decolorization in the presence of nitrate (Carliell
et al., 1995; Carliell et al., 1998) and nitrite (Wuhrmann
et al., 1980).
In contrast, the presence of sulfate, also a normal
constituent of textile-processing wastewater, was not
found to significantly affect dye decolorization (Pans-
wad and Luangdilok, 2000; Albuquerque et al., 2005),
which is in agreement with previously published data
from batch experiments to azo dye decolorization in the
presence of sulfate (Carliell et al., 1995; Carliell et al.,
1998; Van der Zee et al., 2003a). As sulfate can be
biologically reduced to sulfide, a well-known bulk
reductant of azo dyes, it has been suggested that its
presence may rather stimulate than competitively sup-
press azo dye decolorization. Support for this hypothesis
was reported in the SBR study by Albuquerque et al.
(2005), who observed a sharp decline of the reactor’s
anaerobic azo dye decolorization efficiency when selec-
tively inhibiting its sulfate-reducing activity by the
addition of molybdate. However, since a sulfate-free
control experiment had not been included, it cannot be
ruled out that the effect of molybdate was merely due to
mechanisms other than sulfidogenesis inhibition.
In the same SBR study, the effect of ferric iron on the
decolorization of the monoazo dye Acid Orange 7
(AO7) was tested. Like sulfate, ferric iron has the
ambiguous property of being on the hand a competing
electron acceptor and on the other hand the precursor of
a bulk reductant (i.e. ferrous iron) for azo dye reduction.
The results showed a slightly stimulatory effect of ferric
iron, added at a substoichiometric molar Fe(III)/AO7-
ratio of 0.5, on the reactor’s color removal efficiency.
This indicates that the role of ferrous iron as electron
source for azo dye reduction is more important than the
role of ferric iron as competing electron acceptor and it
suggests that the redox couple Fe(III)/Fe(II) may act as
an electron shuttle for transferring reducing equivalents
from biological oxidation of primary substrate to the
electron accepting azo dye. However, the observed effect
of ferric iron on azo dye decolorization was very weak in
comparison to the effect of two apparently much more
powerful redox mediators, the quinone moieties, 1,4-
naphthoquinone (menadione) and anthraquinone-2,6-
disulfonic acid (AQDS), that were also tested in the SBR
system operated by Albuquerque et al. (2005). In Section
3, we will return to the concept of redox mediation and
its application in anaerobic bioreactors.
2.1.5. Primary substrate concentration
The presence of an electron donor is a prerequisite for
azo dye reduction. In theory, the required amount of
electron-donating primary substrate is low, four redu-
cing equivalents per azo linkage, i.e. 32 mg COD per
mmol monoazo dye. Competition for reducing equiva-
lents by other reactions will increase the required
amount of primary substrate. Moreover, the rather slow
process of azo dye reduction may benefit kinetically by a
higher primary substrate concentration. In the studies
listed, the electron-donating primary substrate (biode-
gradable COD) was usually in large excess of the
stoichiometric quantity needed for azo dye reduction.
Only in one of the studies (Cruz and Buitrón, 2001) has
the reactor been operated for a while under clear
substoichiometric conditions, when the reactor influent
contained no other electron donor than the dye’s
dispersing agent. Consequently, the reactor’s anaerobic
color removal efficiency dropped. Substoichiometric
conditions might also explain the low color removal
efficiency before raising the level of primary substrate in
another reactor system (Sosath and Libra, 1997). Two
other studies report sub-optimal primary substrate levels
 ARTICLE IN PRESS
F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
in large excess of the stoichiometric level. Kapdan et al.
(2003), investigating the decolorization of Reactive Red
195 at a concentration of 100 mg/l (
0.1–0.2 mM,
assuming that the dye’s unknown molecular weight lies
between 500 and 1000 g/mol), report declining color
removal efficiencies at substrate concentrations below
3000 mg/l, which is in large excess of the theoretically
necessary 3.2–6.4 mg/l. Likewise, O’Neill et al. (2000a)
reported improved color removal efficiencies when
doubling the primary substrate concentration, even
though the original primary substrate concentration
was already 60–300 times in excess of the stoichiometric
amount.
2.1.6. Primary substrate type
The electron-donating primary substrates used in the
reactor studies varied from simple substrates like
acetate, ethanol and glucose to more complex ones,
including relevant constituents of textile-processing
wastewaters like starch, polyvinylalchol (PVA) and
carboxymethylcellulose (CMC). In all cases, azo dye
decolorization occurred, which suggests that the process
is relatively non-specific with respect to its electron
donor. Yet, some primary substrates may be better
suitable for delivering reducing equivalents to azo dyes,
either because of the substrate itself or because of the
microorganisms involved. Three of the studies listed in
Table 2 report substrate effects. In one of those SBR
studies, the anaerobic removal of AO7 color, which was
very poor when a starch derivative was used as main
COD source, greatly raised when lactate was added to
the reactor influent (Albuquerque et al., 2005). Another
SBR study reported considerably higher anaerobic color
removal efficiencies with acetate and nutrient broth than
with glucose, an effect that was subscribed to a better
azo dye-reducing capacity of polyphosphate-accumulat-
ing microorganisms versus that of glycogen-accumulat-
ing microorganisms (Panswad et al., 2001a). In a
consequent study by the same research group, the
substrate effect, now comparing acetate and glucose
both in the presence of nutrient broth, was less
pronounced (Panswad et al., 2001b). Therefore, the
substrate effect may have been at least partly due to
(compounds present in the) nutrient broth and does not
necessarily relate completely to the microbial composi-
tion of the biomass.
2.1.7. Dye concentration
Another factor that may play a role in the color
removal process is the dye concentration. As seen in
Tables 1–3, large variations in dye concentrations have
been applied in the reactor studies. In several cases, the
applied dye concentrations largely exceed the
10–250 mg/l range of normal concentrations in dyehouse
effluents (O’Neill et al., 1999b). High dye concentrations
may negatively affect the anaerobic color removal
1433
efficiency, either by exceeding the reactor’s biological
azo dye reduction capacity or by causing toxicity to the
anaerobic biomass. Investigations with different dye
concentrations usually reported higher net color re-
moval efficiencies at lower dye concentrations (Seshadri
et al., 1994; Luangdilok and Paswad, 2000; O’Neill et
al., 2000a; Rajaguru et al., 2000; Cruz and Buitrón,
2001; Kapdan and Oztekin, 2003; Sponza and Is-ik,
2005), even though the amount of dye reduced per unit
of time may increase with increasing dye concentrations,
e.g. as reported by Cruz and Buitrón (2001).
2.1.8. Dye toxicity
Azo dye toxicity to anaerobic biomass has been
reported in some of the reactor studies. This toxicity was
associated to high dye concentrations, the presence of
heavy metals (metal-complex dyes) and/or the presence
of non-hydrolyzed reactive groups (reactive dyes). The
benzidine azo dye Direct Black 38, when tested at a very
high influent concentration (3200 mg/l), was found to
lower the anaerobic COD removal efficiency, whereas
concentrations up to 1600 mg/l did not seriously inhibit
the process (Sponza and Is-ik, 2005). Likewise, the Cu-
complex (pre-hydrolyzed) dye Reactive Violet 5 was
reported to inhibit anaerobic COD removal in a reactor
system that treated the dye at a concentration of 650 mg/
l (Sosath and Libra, 1997), whereas in other reactor
studies with the same dye at lower concentrations
(60–100 mg/l), no anaerobic toxicity was observed
(Lourenc- o et al., 2000; Lourenc- o et al., 2001; Lourenc-
o et al., 2003). Anaerobic batch toxicity assays usually
do not reveal severe inhibition of methanogenesis at dye
concentrations below 100 mg/l (Carliell et al., 1995;
Beydilli et al., 1998), although nitro-substituted azo dyes
may be more toxic (Donlon et al., 1997). Furthermore,
concerning the toxicity of reactive azo dyes to anaerobic
biomass, hydrolysis of the dyes’ reactive groups
(vinylsulfone or triazyl) appears to be very important.
A partially hydrolyzed vinylsulfone reactive dye, Re-
active Black 5, was found to almost completely suppress
a bioreactor’s methanogenic and sulfate-reducing activ-
ity, whereas the same bioreactor had been treating fully
hydrolyzed Reactive Black 5 without significant inhibi-
tion (Libra et al., 2004). Similarly, bioreactor treatment
of a non-hydrolyzed triazyl reactive dye, Reactive Red 2,
resulted in almost complete inhibition of methanogenic
activity, a phenomenon that, as batch toxicity studies
with both the non-hydrolyzed and the pre-hydrolyzed
dye indicated, could be subscribed to toxicity of the
dye’s non-hydrolyzed triazyl groups (Van der Zee et al.,
2001a). In general, because of the usually high tempera-
ture and high alkalinity in dyebaths, significant hydro-
lysis of reactive dyes can be expected, which reduces the
risk of toxic non-hydrolyzed (unreacted) dyes in dyebath
wastewater. However, as pointed out by Libra et al.
(2004) referring to vinylsulfone reactive dyes, the
 ARTICLE IN PRESS
1434 F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
wastewater from cold pad dyeing processes may still
contain a relatively high fraction of unreacted dye, thus
presenting a potential toxicity risk for anaerobic
treatment systems. A possible way to overcome this risk
is making use of the ability of activated carbon to
adsorb inhibitory pollutants. Implementation of a
contact-sorption layer with granular activated carbon
at the bottom of a UASB reactor has been proven an
effective and relatively easy way to protect the anaerobic
biomass against the (not necessarily dye-related) toxicity
of a textile wastewater (Kuai et al., 1998).
2.2. Anaerobic formation of aromatic amines due to azo
dye reduction
As has been demonstrated in many research papers,
e.g. those summarized by Field et al. (1995) and Pinheiro
et al. (2004), azo dye decolorization due to anaerobic
reduction results in the formation of anaerobic amines.
In several of the reactor studies listed in Tables 1–3,
attention has been paid to aromatic amine formation, all
of them providing evidence for the formation of
aromatic amines under anaerobic conditions, indicating
azo dye reduction. Some of the evidence was based on
quantitative detection of individual compounds (refer-
ences cited below). However, as this is only possible with
the use of sophisticated analytic techniques like LC–MS
or, if standards of the expected amines are available,
with HPLC, most of the evidence was based on
quantitative detection of total aromatic amines as a
bulk parameter using a diazotization-based method
(Rajaguru et al., 2000; Cruz and Buitrón, 2001; Is-ik
and Sponza, 2003; Is-ik and Sponza, 2004c; Sponza and
Is-ik, 2005), based on the appearance of non-identified
peaks in HPLC chromatograms (Lourenc- o et al., 2000;
O’Neill et al., 2000b; Lourenc- o et al., 2001; Sarsour
et al., 2001; Lourenc- o et al., 2003; Albuquerque et al.,
2005) or based on evaluation of UV–vis spectra
(Kalyuzhnyi and Sklyar, 2000; Cruz and Buitrón,
2001; Panswad et al., 2001a; Panswad et al., 2001b;
Shaw et al., 2002; Sponza and Is-ik, 2002b; Gottlieb
et al., 2003; Is-ik and Sponza, 2004c; Cabral Gonc- alves
et al., 2005). Moreover, the often observed increase of
toxicity after anaerobic treatment of azo dyes (Sosath
and Libra, 1997; O’Neill et al., 2000b; Cruz and Buitrón,
2001; Gottlieb et al., 2003; Is-ik and Sponza, 2004c; Libra
et al., 2004; Frijters et al., 2004) can be interpreted as
indirect evidence for the formation of aromatic amines.
Tables 1–3 cite the percentages of aromatic amine
recovery reported by the bioreactor studies that used
quantitative detection methods. It is shown that these
percentages cover the entire range from hardly any to
complete recovery. Complete recovery of the expected
amines from hydrolyzed Reactive Black 5 reduction (in
the expected stoichiometric ratio of 2 mol p-aminoben-
zene-2-hydroxylethylsulfonic acid and 1 mol 1,2,7-tria-
mino-8-hydroxynaphthalene-3,6-sulfonic acid per mol
hydrolyzed Reactive Black 5) could be demonstrated by
LC–MS analysis (Libra et al., 2004). Also sulfanilic acid,
one of the constituent aromatic amines from Mordant
Yellow 10, was almost completely recovered (Tan et al.,
1999; Tan et al., 2000). All other reported recovery
percentages were lower. In many cases, the incomplete
recovery was most probably due to chemical instability
of the aromatic amines upon exposure to oxygen during
sampling and analysis (see Section 2.3, aerobic fate of
aromatic amines). In some cases, low recovery percen-
tages could be attributed to further anaerobic (bio)-
transformations. The low (40–80%) recovery of 5-
aminosalicylate (5-ASA) from the reduction of Mordant
Yellow 10 (Tan et al., 1999; Tan et al., 2000) was
probably at least partly due to anaerobic biodegrada-
tion. It has been established that 5-ASA is an excep-
tional aromatic amine that can be completely
biomineralized under anaerobic conditions (Razo Flores
et al., 1997). More evidence for anaerobic biodegrada-
tion of 5-ASA, i.e. the decrease of UV absorbance and
the recovery of ammonium, was reported in another
bioreactor study (Kalyuzhnyi and Sklyar, 2000). Anae-
robic biodegradation of aromatic amines has also been
suggested as an explanation for the very low (o1%)
recovery levels (LC–MS analysis) of the expected amines
from three monazo dyes (FitzGerald and Bishop, 1995)
but this does not seem very likely, since even the simplest
structure involved, aniline from the reduction Acid
Orange 10, was repeatedly found recalcitrant to
anaerobic degradation in long-term experiments (Kuhn
and Suflita, 1989; De et al., 1994; Razo-Flores et al.,
1996).
The low (
40%) recovery of 2-bromo-4,6-dinitroani-
line (BDNA) from the reduction of Disperse Blue 79
(Cruz and Buitrón, 2001) was probably due to a minor
anaerobic transformation, the reduction of one or both
of its nitro groups. This was supported by the results of
thin layer chromatography analysis, which revealed the
presence of three aromatic compounds, in agreement
with the earlier reported data about chemical reduction
of Dispersed Blue 79 (Weber and Adams, 1995).
If standards of the expected aromatic amines are not
available, HPLC peaks cannot be identified and
quantified directly but the chromatograms can be useful
to detect formation of cleavage products. Three
significant peaks appeared in the HPLC chromatograms
of samples taken during the anaerobic phase of an SBR
treating the monoazo dye, hydrolyzed Reactive Violet 5.
By comparison of the chromatograms with those of
structurally related amines, it could be reasonably
assumed that two of these peaks corresponded to the
expected amines, respectively, a benzene-based and a
naphthalene-based amine. The third peak probably
corresponded to a metabolite that was in equilibrium
with the benzene-based amine (Lourenc- o et al., 2000;
 ARTICLE IN PRESS
F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
Lourenc- o et al., 2001; Lourenc- o et al., 2003). Appar-
ently, the anaerobic (bio)transformation of hydrolyzed
Reactive Violet 5 had not been limited to solely the
cleavage of the azo linkage. A similar observation was
made during treatment of another monoazo reactive
dye, hydrolyzed Reactive Red 198: HPLC chromato-
grams of anaerobic effluent samples showed four main
peaks, in interesting contrast to chromatograms of
chemically reduced dye samples, which only showed
(the expected number of) two peaks (Sarsour et al.,
2001).
2.3. Aerobic fate of aromatic amines
Several of the studies listed in Tables 1–3 reveal
evidence for partial or complete removal of many
aromatic amines in the aerobic stage. The decrease or
disappearance of the, sometimes unidentified, peaks in
HPLC chromatograms (Harmer and Bishop, 1992;
Jiang and Bishop, 1994; FitzGerald and Bishop, 1995;
Sosath and Libra, 1997; Kalyuzhnyi and Sklyar, 2000;
Lourenc- o et al., 2000; O’Neill et al., 2000b; Tan et al.,
2000; Sarsour et al., 2001; Lourenc- o et al., 2003; Is-ik and
Sponza, 2004c; Is-ik and Sponza, 2004a; Libra et al.,
2004), the decrease or disappearance of aromatic amines
as detected with a diazotization-based method (Raja-
guru et al., 2000; Is-ik and Sponza, 2004c; Is-ik and
Sponza, 2004a; Sponza and Is-ik, 2005), as well as the
decrease of UV absorbance (Cruz and Buitrón, 2001;
Shaw et al., 2002; Is-ik and Sponza, 2004c) all indicate
removal of aromatic amines. Moreover, the large
decreases of toxicity (mostly suppression of bacterial
luminescence or inhibition of respiration) between the
effluent of the anaerobic stage and the effluent of the
anaerobic stage (Sosath and Libra, 1997; O’Neill et al.,
2000b; Is-ik and Sponza, 2004c; Libra et al., 2004;
Frijters et al., 2004) or between the effluent of a
completely anaerobic reactor and the effluent of a
combined anaerobic–aerobic reactor (Gottlieb et al.,
2003) provide indirect evidence for the removal of
aromatic amines.
The results taken as a whole suggest that many of the
aromatic amines from anaerobic cleavage of azo dyes
were removed in the consequent aerobic stage. However,
some aromatic amines may not be removed. Especially
cleavage products from the reactive azo dyes Reactive
Black 5 and Reactive Violet 5 were often reported not to
be removed aerobically (Sosath et al., 1997; Lourenc- o et
al., 2000; Luangdilok and Paswad, 2000; Panswad and
Luangdilok, 2000; Lourenc- o et al., 2001; Shaw et al.,
2002; Lourenc- o et al., 2003; Libra et al., 2004). Also a
relatively large fraction (
50%) of the aromatic amines
from the benzidine-based dye Direct Black 38 resisted
removal in the aerobic stage (Sponza and Is-ik, 2005).
Most of the studies reporting aromatic amine removal
do not reveal the underlying mechanism. As aerobic
1435
biodegradation of aromatic amines requires specific
microorganisms, the type of biomass may play a role.
At least in one laboratory reactor study, the degradation
of an aromatic amine, sulfanilic acid, could only be
achieved after bioaugmentation with a proper bacterial
culture (Tan et al., 2000). Also the observation that
introduction of biomass from a textile waste-treating
water works decreased the toxicity of the effluent from
an azo dye-treating baffled reactor (Gottlieb et al., 2003)
suggests the involvement of specific bacteria.
In many of the studies reporting aromatic amine
removal, it is not clear whether the removal is due to
biodegradation, adsorption or chemical reactions. Re-
markably, although autoxidation of aromatic amines
during aerobic treatment, as suggested by an increase of
color, has been observed in some studies (Sosath and
Libra, 1997; Kalyuzhnyi and Sklyar, 2000; Cruz and
Buitrón, 2001; Tan, 2001; Is-ik and Sponza, 2004b; Libra
et al., 2004), a slight decrease of the color was much
more often observed (Jianrong et al., 1994; An et al.,
1996; Basibuyuk and Forster, 1997; Sosath et al., 1997;
Kuai et al., 1998; Luangdilok and Paswad, 2000; O’Neill
et al., 2000b; Panswad and Luangdilok, 2000; Rajaguru
et al., 2000; Panswad et al., 2001a; Sarsour et al., 2001;
Shaw et al., 2002; Kapdan et al., 2003; Is-ik and Sponza,
2004c; Sponza and Is-ik, 2004; Kapdan and Alparslan,
2005; Ong et al., 2005). Since many of the azo dyes
treated in these studies yield aromatic amines that are
expected to autoxidize, the latter observation suggests,
in several cases, removal of these compounds or their
autoxidation products from the water phase.
3. Discussion
According to the concept of combined anaerobi-
c–aerobic treatment, azo dyes should be removed from
the water phase by (anaerobic) reduction followed by
(aerobic) oxidation of the dyes’ constituent aromatic
amines. The anaerobic–aerobic reactor studies reviewed
in this paper show that a generally high extent of color
removal can be obtained, and several studies further-
more provide evidence for removal of aromatic amines.
Combined anaerobic–aerobic treatment therefore holds
promise as a method to completely remove azo dyes
from wastewater. However, the results of the reactor
studies reveal some possible limitations, both with
respect to azo dye reduction and with respect to the
fate of aromatic amines.
The long retention times often applied in the
anaerobic phase of the reactor studies indicate that the
reduction of many azo dyes is a relatively slow process.
The generally observed increase of anaerobic color
removal efficiencies at higher hydraulic retention times,
higher biomass concentrations, higher primary substrate
concentrations and lower dye concentrations, all point
 ARTICLE IN PRESS
1436 F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
at a bottleneck in the transfer of reducing equivalents as
the time-determining factor for anaerobic azo dye
reduction. This restriction, however, can easily be
overcome, by making use of the property of redox-
mediating compounds to shuttle reducing equivalents
from the electron-donating primary substrate to the
electron-accepting dye, thereby increasing the rates of
azo dye reduction (Field et al., 2000). Flavin enzyme
cofactors are known as effective redox mediators for azo
dyes reduction (Gingell and Walker, 1971; Chung et al.,
1978; Fujita and Peisach, 1982; Russ et al., 2000), and
also several quinone compounds have been shown to
accelerate azo dye reduction in biological systems, by
anaerobically incubated pure bacterial cultures (Keck
et al., 1997; Kudlich et al., 1997; Rau et al., 2002; Rau
and Stolz, 2003), as well as by anaerobic sludge (Van der
Zee et al., 2001a; Dos Santos et al., 2003; Van der Zee
et al., 2003a; Dos Santos et al., 2004a; Dos Santos et al.,
2004b). Continuous dosing of soluble quinones at
catalytic concentrations has been demonstrated to
strongly increase the azo dye reduction efficiencies of
lab-scale anaerobic bioreactors (Cervantes et al., 2001;
Van der Zee et al., 2001a; Dos Santos et al., 2003; Dos
Santos et al., 2004a; Dos Santos et al., 2005) and of a
lab-scale SBR (Albuquerque et al., 2005). Moreover, as
an alternative, also activated carbon, incorporated in the
sludge bed, has been shown effective as an immobilized,
biologically regenerable, redox mediator to accelerate
azo dye reduction (Van der Zee et al., 2003b).
The results taken as a whole suggest that obtaining
complete reduction of azo dyes at a reasonable time-
scale will not have to be a problem, provided that the
prerequisites for azo dye reduction are met, i.e.
anaerobic conditions (absence or limitation of compet-
ing electron acceptors like oxygen, nitrate and nitrite),
availability of an electron donor and the absence of
toxicity towards the anaerobic biomass. Regarding the
latter, as far as dyes are concerned, especially non-
hydrolyzed (unreacted) reactive dyes pose a risk. Prior
to anaerobic treatment of wastewaters containing
unreacted dyes, i.e. in particular wastewaters from low
temperature dyeing processes, a hydrolysis step may be
required.
The second limitation with respect to applying
combined anaerobic–aerobic treatment for the removal
of azo dyes from wastewater concerns the lack of
knowledge on the aerobic fate of the aromatic amines,
i.e. the extent to which aromatic amines will undergo
biological or chemical transformations or remain
unattached in the water phase, the competition between
biodegradation and autoxidation and the properties of
recalcitrant dye residues. The results of reactor studies
generally present evidence that many aromatic amines
from azo dye cleavage are removed from the water phase
under aerobic conditions. However, it is also shown that
anaerobic–aerobic bioreactor treatment of at least some
dyes, e.g. Reactive Black 5 and Reactive Violet 5, leaves
a residual fraction of water-soluble recalcitrant com-
pounds in the reactor’s final effluent. The specific
molecular structure of these recalcitrant dye residues
has so far been determined in only one case, the
autoxidation product 1,2-ketimino-7-amino-8-hydroxy-
naphthalene-3,6-disulfonic acid of one of the aromatic
amines (1,2,7-triamino-8-hydroxynaphthalene-3,6-disul-
fonic acid) from hydrolyzed Reactive Black 5 (Libra
et al., 2004). This highly colored compound resisted
further (bio)transformation during 96 days of reactor
operation, even though the results of batch incubation
studies with activated sludge had shown conversion of
the compound to an unknown colorless product
(Kudlich et al., 1999).
Also the recalcitrant dye residue resulting from
anaerobic–aerobic treatment of hydrolyzed Reactive
Violet 5 has been associated to its naphthalene amine
(Sosath and Libra, 1997; Lourenc- o et al., 2000; Lourenc-
o et al., 2003) and the same might have been the case for
that of other dyes. These observations present a
limitation of biological treatment systems: in several
cases it will not be possible to achieve complete removal
of dye residues, nor of color, from the wastewater.
Nevertheless, the often-observed further color removal
in the aerobic phase of the bioreactor studies, in contrast
to the expectation that biorecalcitrant aromatic amines
will tend to autoxidize forming colored products,
suggests that many aromatic amines can in fact be
removed from the water phase. In this respect, it will be
interesting to know more about the competition between
biodegradation and autoxidation. In recent studies with
an aerobic biofilm reactor treating Acid Orange 7,
complete biodegradation of the dye’s cleavage products
was demonstrated, including biomineralization of the
aromatic amine 1-amino-2-naphthol, which is known to
easily autoxidize (Coughlin et al., 2002; Coughlin et al.,
2003). This presents a case in which bacteria were
successful in competing with chemical (auto)oxidation,
but it is not known whether it represents a general trend
or merely an exception.
The many uncertainties about the aerobic fate of
aromatic amines reveal the need to obtain better insight
in the matter. Future research should focus on (i) the
detection of aromatic amines and their degradation and
autoxidation products, (ii) assessment of the degree of
mineralization and (iii) determination of the size and
nature (structure and hazardousness) of recalcitrant dye
residues. The available techniques for aromatic amine
analysis, as thoroughly discussed in a recent review
paper (Pinheiro et al., 2004), range from sophisticated
(and expensive) techniques for specific compound
identification to relatively simple (and cheap) semi-
quantitative techniques. Regarding the latter, the devel-
opment of a method based on direct UV–vis spectrum
analysis has been proposed. In this method, the
 ARTICLE IN PRESS
F.P. van der Zee, S. Villaverde / Water Research 39 (2005) 1425–1440
absorbance, monitored in a near-UV range
(260–300 nm) where neither textile auxiliaries present
in the wastewater nor residual dye color interfere, should
be corrected by taking into account the near-UV
absorbance of residual dyes (Pinheiro et al., 2004).
Applying this method will without doubt deliver
valuable information. However, the correction for
residual dye absorbance, which is based on an indepen-
dently assessed ratio between color absorbance and
near-UV absorbance of azo dyes, presents a weak point
of the proposed method, since it will be seriously
interfered if the wastewater also contains autoxidation
products, i.e. colored aromatic compounds with absor-
bance over the entire UV–vis spectrum.
Probably even more important than the exact nature
of recalcitrant dye residues in the final effluent of
anaerobic–aerobic biological treatment systems is its
potential hazardousness. The reactor studies that
evaluated toxicity all showed promising results, i.e. far-
reaching decrease or even complete loss of toxicity in the
aerobic phase. However, the amount of data is limited
and most data concern direct toxicity, usually biolumi-
nescence suppression or respiration inhibition, in con-
trast to genotoxicity or mutagenicity. Only one paper
also evaluated genotoxicity during anaerobic–aerobic
treatment of an azo dye. The dye, hydrolyzed Reactive
Black 5, was found weakly genotoxic, both before and
after reduction, but no genotoxicity could be detected in
the effluent of an anaerobic–aerobic baffled reactor that
treated the dye (Gottlieb et al., 2003). The results of this
study also showed severe genotoxicity of (1-amino-2-
naphthol from) reduced Acid Orange 7, in contrast to no
genotoxicity of 1-amino-2-naphthol-6-sulfonate, which
is in agreement with the lower level or absence
of genotoxicity/mutagenicity/carcinogenicity of sulfo-
nated aromatic amines in comparison to some of
their non-sulfonated analogues (Chung and Cerniglia,
1992; Jung et al., 1992). This is something highly relevant
for the toxicity of textile-processing wastewaters, since
most water-soluble dyestuffs used for textile dyeing
contain one or more sulfonate groups. Furthermore, it
should be noted that over the last decade, azo dyes that
could breakdown to carcinogenic aromatic amines have
been largely phased out in Europe (Pinheiro et al., 2004).
Although it may thus be expected that the fraction of
recalcitrant azo dye residues in the final effluent of
anaerobic–aerobic biological treatment systems would
not pose a hazard, careful evaluation of its (geno)toxicity
still remains recommendable.
Combining compound analysis with (geno)toxicity
measurements will deliver insight in the size, composi-
tion, and potential harm of the recalcitrant fraction,
thereby providing the information needed to judge
whether discharge can be allowed or whether tertiary
treatment (e.g. advanced oxidation, adsorption, coagu-
lation) will have to be applied.
1437
Acknowledgments
The authors thank the EU Project IHP Program
Contract HPMD-CT-2001-00106, the Spanish Ministry
of Science and Technology Project PPQ2003-09044 and
FCT (Fundac- ão para a Ciência e a Tecnologia) Portugal
for financial support.
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