Azo Dye Removal

Dye classification
• All aromatic compounds absorb
electromagnetic energy but only those
that absorb light with wavelengths in the
visible range (~350-700 nm) are
coloured.

• Dyes contain chromophores (conjugated

double bond) and auxochromes
(electron-withdrawing or
electrondonating substituents).
• Chromophores
• -C=C-, -C=N-, -C=O, -N=N-
Auxochromes
• -NH3, -COOH, -SO3H and –OH

• Based on chemical structure or chromophore,

20-30 different groups of dyes.
• Azo, anthraquinone, phthalocyanine and
triarylmethane dyes are quantitatively the most
important groups.
• Other groups are diarylmethane, azine,
oxazine, thiazine, nitro, nitroso, methine,
thiazole, indamine, indophenol, lactone,
aminoketone and hydroxyketone dyes
Without Cu
Methylene blue,
thiazine

nitroso
 Dye classification
Acid dyes
• Acid dyes are anionic
compounds that are
mainly used for dyeing
nitrogen-containing
fabrics like wool,
polyamide, silk and
modified acryl. They
bind to the cationic
NH4+ions of those
fibres.
Reactive dyes
• Reactive dyes are dyes with reactive groups
that form covalent bonds with OH-, NH-, or SH-
groups in fibres (cotton, wool, silk, nylon). The
reactive group is often a heterocyclic aromatic
ring substituted with chloride or fluoride, e.g.
dichlorotriazine.
Metal complex dyes
• Among acid and reactive dyes, many Metal
complex dyes can be found
• These are strong complexes of one metal atom
(usually chromium, copper, cobalt or nickel)
and one or two dye molecules.
Direct dyes
• Direct dyes are relatively large molecules with
high affinity for especially cellulose fibres. Van
der Waals forces make them bind to the fibre.
Direct dyes are mostly azo dyes with more than
one azo bond or phthalocyanine or oxazine
compounds.
Basic dyes
• Basic dyes are cationic compounds that are
used for dyeing acid-group containing fibres,
usually synthetic fibres like modified polyacryl.
They bind to the acid groups of the fibres. Most
basic dyes are diarylmethane, triarylmethane,
anthraquinone or azo compounds.
 Azo dye containing
wastewater
• Azo dyes are extensively used for dyeing of
cotton and constitute about 60–70% of total
dyes produced.
• About 1000 mg/l of dyes is present in a typical
dyebath. However, due to the poor exhaustion
properties of reactive dyes as much as 40% of
the initial dyes remains unfixed and ultimately
ends up in the dyebath effluent.
• In a textile industry about 40–65 l of
wastewater is generated per kg of cloth
produced.
 Azo dye

Reactive Orange 16
• Wastewater from a textile industry are
characterised by their highly visible color,
Chemical demand (COD) (800–1600
mg/l), alkaline pH (9–11) and total solids
(TS) (6000–7000 mg/l).
• Color and presence of organics in the
wastewater are of concern and are to be
reduced before disposal.
 Dye removal techniques
• Various physical, chemical and biological pre
treatment, main treatment and post treatment
techniques can be employed to remove colour
from dye containing wastewaters.
• Physicochemical techniques include membrane
filtration, coagulation/flocculation, precipitation,
flotation, adsorption, advanced oxidation
(ozonation, Fenton oxidation and photocatalytic
oxidation).
• Biological techniques include bacterial and
fungal biosorption and biodegradation in
aerobic, anaerobic, anoxic or combined
anaerobic/aerobic treatment processes
• Bioremediation can be implemented in a number of
treatment modes:
- aerobic (oxygen respiration)
- anoxic (nitrate respiration)
- anaerobic (non oxygen respiration)
- co-metabolic
• Three primary ingredients for bioremediation are:
- presence of a contaminant,
- an electron acceptor,
- presence of microorganisms that are capable of
degrading the specific contaminant.

Microbes + Electron Donor (Energy & Carbon Source) + Nutrients +

Electron Acceptor → More microbes + Oxidized End Products
Electron donor : waste contaminants as energy source
Electron acceptor: O2, NO3, SO4, CO2, organic carbon
 Aerobic and anaerobic bacteria can be identified by growing them in liquid
culture:
1: Obligate aerobic (oxygen-needing) bacteria gather at the top of the
test tube in order to absorb maximal amount of oxygen.
2: Obligate anaerobic bacteria gather at the bottom to avoid oxygen.
3: Facultative bacteria gather mostly at the top, since aerobic respiration is
the most beneficial one; but as lack of oxygen does not hurt them, they can
be found all along the test tube.
4: Microaerophiles gather at the upper part of the test tube but not at the
top. They require oxygen but at a low concentration.
5: Aerotolerant bacteria are not affected at all by oxygen, and they are
evenly spread along the test tube.
 Advance Oxidation Process
(AOP)
• Advanced chemical oxidation typically involves the use
of chemical oxidants (e.g. ozone or hydrogen
peroxide) to generate highly reactive radical
(especially hydroxyl radicals (i.e. •OH)), one of the
strongest oxidants known.
• Hydroxyl radicals are reactive and non-selective,
capable of rapidly degrading a number of organic
compounds.
• The kinetics of reaction is generally first order with
respect to the concentration of hydroxyl radicals and
to the concentration of the species to be oxidized.
• The AOPs are used to destroy the
complex refractory organic constituents
even after treatment with conventional
methods. These processes have shown
great potential in the treatment of
pollutants, either in high or in low
concentrations.
• The ultimate goal of the oxidation
process is to mineralize the organic
contaminants present in water in
carbondioxide, water and inorganic ions
through degradation reactions involving
species strongly oxidizing.
• Oxidation potential against Standard Hydrogen
Electrode of some relevant oxidants (Legrini et al.,
1993; Domènech et al., 2001).
• As can be seen from Table, hydroxyl
radicals are powerful oxidizing agents
with an oxidation potential of 2.8 V and
can react with most organic and any
inorganic solutes with high rate constants
(Zhihui et al., 2005).
 OH· Generation
• The hydroxyl radicals can be generated by
different oxidation processes:
- Ozone (O3)
- Ultraviolet (UV)
- Hydrogen peroxide combined with ultraviolet
radiation (H2O2/UV),
- Fenton reagent (Fe2+/H2O2)
- Photo-Fenton process
- O3/UV
- O3/H2O2
- Photocatalysis using zinc oxide (ZnO) but
mainly titanium dioxide (TiO2) - TiO2/UV
process
 Fenton’s reagent
• The Fenton reagent is a catalytic oxidation process which is the
combination of an oxidizing agent (hydrogen peroxide) and a catalyst
(an oxide or metal salt, usually iron) to produce hydroxyl radicals.

There are a complex series of chain reactions proceeding

simultaneously which can regenerate Fe+2
Fe3  H 2 O 2  FeOOH 2  H 
FeOOH 2  HO2  Fe 2 Fe3+ + H2O2 ----> Fe2+ + .OOH + H+

HO2  Fe3  Fe 2  O 2  H 
 System Description/Design
Parameters
• The use of Fenton’s chemistry to destroy
organic compounds in drinking water or
wastewater requires the addition of iron and
H2O2 to the source water. The dosages of
Fe(II) and H2O2 are determined based on the
organic contaminant removals required.
• The reactor must be configured to provide
adequate mixing of Fe(II) and H2O2 in order to
optimize hydroxyl radical formation and
destruction of organic contaiminats.
The procedure requires:

• adjusting the wastewater to pH 3-5;

• adding the iron catalyst (as a
solution of FeSO4);
• adding slowly the H2O2.
• If the pH is too high, the iron
precipitates as Fe(OH)3 and
catalytically decomposes the H2O2
to oxygen
 Degradation of azo dye Amido black
10B in aqueous solution by Fenton
oxidation process (Sun et al., )
• Nowadays, various chemical and physical
such as elimination by adsorption onto
activated carbon, coagulation by a chemical
agent, ozone oxidation, electrochemical
method, etc. are applied for the treatment of
dye waste effluents.
• Nevertheless, these methods are usually non-
destructive, inefficient, costly and resulted in
the production of secondary waste products.
Therefore, purification of azo dye wastewater is
becoming a matter of great concern and it is
necessary to develop novel and cost-effective
technologies to treat azo dye wastewater.
• O3/H2O2, O3/UV, H2O2/UV, TiO2/air/UV,
Fe(II)/H2O2 (Fenton reagent), Fe(III)/H2O2,
( Fenton-like reaction), an oxidant (H2O2, O3)
and ultrasonic irradiation are the main types of
AOPs that have been suggested in recent years.
• Fenton reagent is particularly attractive
because of the low costs, the lack of toxicity of
the reagents (i.e., Fe(II) and H2O2)and the
simplicity of the technology.
• The aim of the present work is to investigate
the influence of various parameters on the
degradation of Amido black 10B, in aqueous
solution by the AOPs. The effects of pH,
dosages of hydrogen peroxide and ferrous, the
concentration of Amido black 10B and the
temperature were examined.
 Materials and methods

 Molecular structure of Amido black 10B

pH meter
- an appropriate amount of stock dye solution -
- addition of ferrous ion
- dilution with deionized water to 200 mL
thermostat - adjust pH to the desired level using dilute
water bath sulfuric acid and sodium hydroxide
- adding hydrogen peroxide to the beaker
- collect sample periodically for UV-Vis analysis
stirer
500 ml beaker
 Analytical methods
• The UV–vis spectra of dye were recorded
from 200 to 800 nm using a UV–vis
spectrophotometer
• The maximum absorbance wavelength
(λmax) of Amido black 10B could be found
at 618 nm from the spectra.
• Concentration of the dye in the reaction
mixture at different reaction times was
determined by measuring the absorption
intensity at λmax = 618 nm and from a
calibration curve.
 Results and discussion
- The pH of the solution is an
• Effect of pH important parameter for fenton
oxidation process which controls the
production rate of hydroxyl radical and
the concentration of Fe2+.

-The optimal solution pH was observed

at about 3.50.

- At low pH (below 3.00),

-At pH > 4.00,
formation of ferric hydroxide
complexes leading to a reduction of
OH radical.
Effect of the initial H2O2 concentration
From 0.1 to 1mM H2O2, the
degradation of Amido black
10B increase - increasing of
OH radical obtained from the
decomposition of increasing
hydrogen peroxide.

From 2 to 4 mM H2O2, the

degradation rate of Amido
black 10B reduced – the very
reactive OH radical could be
consumed by H2O2 and
results in the generation of
less reactive OOH
radical
• Effect of the initial Fe2+ concentration
At a low [Fe2+]0 (0.01 mM), the
degradation efficiency was
86.92% after the 60 min reaction
time.

Both degradation efficiency and

degradation rate were increased
with increase of [Fe2+]0, the
degradation efficiency being
97.35%, 98.57% and 98.89%
after the 30 min reaction time with
[Fe2+]0 of 0.025 mM, 0.05 mM and
0.10 mM, respectively.

This is because more OH radicals

are produced with the increase of
[Fe2+]0 according to Eq. (1).
• Effect of temperature

- The temperature exerts a

strong effect on the
degradation rate of Amido black
10B and the degradation was
accelerated by a rise in
temperature.

- This is because higher

temperature increased the
reaction rate between hydrogen
peroxide and any form of
ferrous/ferric iron (chelated or
not), thus increasing the rate of
generation of oxidizing species
such as OH radical.
Spectral changes of Amido black 10B during
Fenton oxidation process
The adsorption peak at 618 nm
diminished very fast and nearly
318 nm completely disappeared under
napthalene 618 nm 60 min - This indicated a rapid
–N=N- degradation of Amido black 10B, a
complete discolouration of 50 mg/L
Amido black 10B can be achieved in
60 min.
226 nm
The ultraviolet band at 318 nm was
Benzene
also observed to gradually diminish
but at a lower rate than that of
visible band, which indicated the
destruction of the naphthalene
rings.

It could be found that the

discolouration of Amido black 10B is
a fast, but the destruction of the
aromatic rings is difficult.
 Conclusion
• It has been found that the solution pH, the
initial H2O2 concentration, the initial Fe2+
concentration and the temperature are the
main factors that have strong influences on the
degradation of Amido black 10B by Fenton
oxidation process.

• The UV–vis spectral changes of Amido black

10B in aqueous solution during fenton
treatment process showed that it was easier to
destruct the –N=N– group than to destruct the
aromatic rings of Amido black 10B.
 Ozone
• Ozone is a very powerful oxidizing
agent, which is able to participate in
a great number of reactions with
organic and inorganic compounds.
• Among the most common oxidizing
agents, it is only surpassed in
oxidant power by fluorine and
hydroxyl radicals.
 Ozone generation
• Ozone generators can create ozone artificially
by means of extremely high voltages or by
means of UV-light. Both methods involve the
decomposition of the oxygen molecule. This
causes oxygen radical formation. These oxygen
radicals can bind to oxygen molecules, forming
ozone (O3).

2 O3 → 3 O2
 Ozone (O3)
• When O3 is added to water, it participates in a
complex chain of reactions that result in the
formation of radicals such as the hydroxyl
radical (•OH) and the superoxide radical (O2•)
• In an ozonation process two possible
pathways have to be considered:
- the direct pathway through the reactions
with molecular ozone, and
- the radical pathway through the reactions of
hydroxyl radicals generated in the ozone
decomposition and the dissolved compounds.
 Direct reactions
• When the direct ozonation takes place, ozone is the
main oxidizing agent of the process.

• An ozone molecule can undergo a 1-3 dipolar cyclo

addition with unsatureted compounds (double or
tripple bonds). This leads to the formation of a
compound called ‘ozonide’
 Indirect reactions
• ozone decomposes in water to form •OH, which
are stronger oxidizing agents than ozone itself,
thus inducing the so-called indirect ozonation.

• Ozone decomposition in water can be initiated

by the hydroxyl anion, HO-, and thus indirect
ozone oxidation is favored at alkaline pH
conditions.
Indirect reactions
 Ozone (O3)
• Ozonation can only be considered as an
Advanced Oxidation Process when the OH·
radicals are the oxidizing agents of the
process.
• Due to the different oxidation power
between molecular ozone and hydroxyl
radical, the rate of the attack by indirect
ozonation (i.e. OH·) is typically 106 to 109
times faster than the corresponding
reaction rate for direct ozonation (i.e. O3)
Ozone (O3)

pH<4 direct ozonation

pH= 4-9 both are present,
pH > 9 indirect ozonation
 System Description
• Since O3 decomposes rapidly, it is typically
produced on-site using a generator fed with dried
compressed air or oxygen
• For AOPs, O3 gas is fed through spargers,
porous piping or plates at dosages equivalent to 1
to 2 mg/L ozone per mg/L DOC
• In a conventional ozone reactor, ozone is
bubbled through the base of the reactor and
allowed to diffuse through the reactor until it
either escapes through the top or is completely
reacted.
A schematic of a conventional (continuously stirred
tank reactor) H2O2/O3 system equipped with UV
lamps
 Biological systems for azo
dye removal
• As dyes are designed to be stable
and long-lasting colorants, they are
usually not easily biodegraded.

• Biological dye removal techniques

are based on microbial
biotransformation of dyes.
 Azo dyes

Chemical structures that after metabolic activation of azo

dyes show toxic
 Biological systems

fixed activated sludge

 Biodegradation of azo dyes
and aromatic amines
• The mineralization of azo dyes requires a
integrated or sequential anaerobic and aerobic
step
• The first step in the biodegradation of azo dyes
concerns the azo dye reduction that readily
proceeds under anaerobic conditions and
results in the formation of aromatic amines
• Anaerobic consortia generally do not degrade
the aromatic amines but most of the aromatic
amines are readily biodegraded under aerobic
conditions
• A precondition for the reduction of azo dyes is the
presence and availability of a co-substrate, because it
acts as an electron donor for the azo dye reduction.
• Many different co-substrates were found to suite as
electron donor, like glucose, hydrolyzed starch,
tapioca, yeast extract, a mixture of acetate, butyrate
and propionate and the azo dye reduction product 5-
ASA as well

Co-subtrate Remazol Black B, Nigam et al. 1996

decolorization
Glucose 82%
Glyserol and 71%
lactose
Starch 51%
• Direct enzymatic azo dye reduction

• According to the first mechanism of biological

azo dye reduction, enzymes transfer the
reducing equivalents originating from the
oxidation of organic substrates to the azo dyes.
 Indirect (mediated) biological azo dye
reduction
• According to the second mechanism of biological
azo dye reduction, azo dyes are indirectly reduced
by enzymatically reduced electron carriers.
• Early research has hypothesised that reduced
flavins (FADH2, FMNH2, riboflavin) generated by
flavin dependent reductases can reduce azo dyes in
a non-specific chemical reaction.
Mechanism of azo dye
reduction
 The aerobic biodegradation of
(sulfonated) aromatic amines
• The aerobic biodegradation of many aromatic amines has
been extensively studied. Many of these compounds were
found to be degraded under aerobic conditions e.g.
compounds like aniline, carboxylated aromatic amines,
chlorinated aromatic amines and (substituted) benzidines.

• However, a group of aromatic amines that remain difficult

to degrade are the sulfonated aromatic amines.
Sulfonated aromatic amines
 Granular activated carbon-biofilm
configured sequencing batch reactor
treatment of C.I. Acid Orange 7
SA Ong, E. Toorisaka, M. Hirata, T. Hano
(2008, Dyes and Pigments)
• The only disadvantage of the anaerobic biological
techniques, using conventional methods (e.g.
stirred tank reactors), is the need for long hydraulic
residence times due to the low growth yields of the
anaerobic bacteria; usually, less than 10% of the
substrate carbon can be incorporated into cell
matter, as opposed to around 50% with aerobic
bacteria.

• The above disadvantage can be overcome by the

utilization of methods such as immobilization
techniques which could retain high densities of
specialized microorganisms
• There are two types of immobilization
technique: attachment and incapsulation.

• The objective of this study is to investigate

the feasibility of using GAC-biofilm configured
sequencing batch reactor (SBCR) in color and
organic substrates removals with and without
co-substrates.

• Besides, the color removal by adsorption and

biodegradation processes by the use of virgin
GAC, living biofilm-GAC and dead biofilm-
GAC was compared.
 Materials and methods
• GAC-biofilm sequencing batch reactor
(SBCR) - filled with 2 l of AO7-containing
wastewater daily
-FILL, REACT, DRAW and IDLE
periods in the time ratio of
3:20:0.45:0.15 for a cycle time of 24
h
-temperature of SBCR system was
maintained at 25 C.
(2.3 l of - During FILL and REACT periods, the
GAC- partially treated AO7-containing
biofilm) wastewater, which flows underneath
from GAC compartment
to MP compartment will be recycled
to the GAC compartment with a flow
rate of 20 ml/min
Materials and methods
Chemicals and analytical methods
• C.I. Acid Orange 7

• The AO7 concentration was estimated from the

standard curve of dye concentration versus
optical density at its maximum absorption
wavelength (480 nm) using a UV-vis
spectrophotometer.
• The chemical oxygen demand (COD) and
suspended solids (SS) were determined
according to Standard Methods. In the
measurements of dissolved COD and color,
samples were prepared by filtering through a
membrane filter of 0.45 mm.
Results and discussion
Results and discussion
-The maximum adsorption capacity of
virgin GAC was about 120 mg/g
according to Langmuir isotherm
model.
- Spent GAC exhibited about 4e8
mg/g of maximum adsorption capacity
based on Langmuir isotherm model.
 Conclusions
• In the phases (1-5) without the addition of external
carbon sources, 100% color removal was achieved
in the first 2 phases and this then slightly dropped in
the following phases(3-5).
• The addition of external carbon sources (phase 7)
improved the decolorization rate but deteriorated
COD removal efficiency.
• The increase of DO by aeration in the MP
compartment had resulted in the enhancement of
the efficiency of COD removal up to 80%.
• The mix of anaerobic and aerobic microbes at
different levels in the bioreactor played an important
role in the simultaneous removal of both color and
organic matter
 AOP + Biological
Systems
Treatment of containing azo
reactive brilliant red X-3B using
sequential ozonation and upflow
aerated filter process
(Lu et. al., JHM)

• Textile wastewater is not proper to use

anaerobic process because the breakdown of
azo dye leads to the formation of aromatic
amines, which may be more toxic than the dye
molecules themselves
• Ozonation is a potential process for
decolorization of dye since the
chromophore groups with conjugated
double bonds, which are responsible for
color, can be broken down by either
directly or indirectly forming smaller
molecules, which can be removed by
biological treatment, thereby decreasing
the color of the effluents.
• It was found that refractory organic
pollutants could become biodegradable
after appropriate chemical oxidation.
• The combination of chemical oxidation and
biodegradation has a great advantage over
either of the two treatments alone in the
remediation of organic contaminants.
• To enhance biodegradability of azo dye brilliant
red X-3B containing wastewater pre-ozonation
was used.
• The objective of the work was to investigate
the decolorization and degradation of azo
brilliant red X-3B using sequential ozonation
and UBAF process. The wastewater containing
azo dye X-3B was pretreated by oxidation,
which increased the biodegradability of the
and most of the organic matter was removed in
the next step by UBAF process.
Materials and Methods

Structure of reactive brilliant red X-3B(536nm)

Simulated wastewater quality

 Experimental setup

Schematic diagram of ozone experiment system

 Experimental setup

Experimental setup for upflow biological

aerated filter process
 Results and Discussion
• Effect of pH on decolorization efficiency by
ozone

Decolorization efficiency as a function of reaction time at different pH

 Results and Discussion
• Effect of ozone dose on decolorization efficiency

Decoloration efficiency as a function of reaction time at different ozone

doses (conditions: pH 11; dye concentration, 50 mg/L; T 25 ◦C).
 Results and Discussion
• Effect of oxidation on COD removal

COD removal efficiency as a function of time at different ozone doses

(conditions:pH 11; dye concentration, 50 mg/L; T 25 ◦C)
 Results and Discussion
• Effect of ozone oxidation on biodegradability

Effect of ozonation time on the biodegradability of simulated

wastewater (conditions: initial pH 11; initial concentration of the
dye, 50 mg/L; ozone dose, 11.08 g/L)
 Results and Discussion

The UV–vis absorption spectra of X-3B before and after ozonation

(conditions: initial pH 11; initial concentration of the dye, 50 mg/L;
ozone dosage, 11.08 g/L)
 Results and Discussion
• Wastewater treatment by ozonizing-
upflow biological aerated filter process

Effect of ozone dosage in wastewater treatment using sequential

ozonation and UBAF process
 Conclusions
• Ozonation is highly efficient in the
decolorization of textile wastewater
containing azo dye X-3B, but less efficient
in terms of COD
• The subsequent UBAF process can greatly
reduce COD of wastewater treated by ozone
pre-oxidation.
• Ozone pre-oxidation process can improve
the biodegradability of wastewater
containing azo dye reactive brilliant red X-
3B significantly, which increased the value
of BOD5/COD from 0.102 to 0.406.