BIOREMEDIATION

AND BIODEGRADATION
Lecture Week 4
Biodegradation of Azo Dyes
By Puan Norhayati Abdullah

Biodegradation of Azo Dyes

Abstract An aerobic bacterial consortium consisting of two

isolated strains (BF1, BF2) and a strain of Pseudomonas
putida(MTCC1194) was developed for the aerobic degradation of
a mixture of textile azodyes and individual azodyes at alkaline pH
(910.5) and salinity (0.93.68 g/l) at ambient temperature (28 2
C). The degradation efficiency of the strains in different media
(mineral media and in the Simulated textile effluent (STE)) and at
different dye concentrations were studied. The presence of a H2O2
independent oxidase laccase (26.5 IU/ml) was found in the
culture filtrate of the organism BF2. The analysis of the degraded
products by TLC and HPLC, after the microbial treatment of the
dyes showed the absence of amines and the presence of low
molecular weight oxidative degradation products. The enzymes
present in the crude supernatant was found to be reusable for the
dye degradation.

History of dyes

Since prehistoric period, people have been

using colourants to dye furs, textiles and other
objects.
These natural substances were mainly obtained
from plants, vegetables or animals.
Cave-drawing such as those in the Chauvet
Cave in France and Altamira Cave in Spain
showed the evidences of dyes application.

Yarn drying after being dyed in early American tradition,

at Conner Prairie living history museum in Fishers, Indiana.

Some 35,000 years ago

Horses

Fighting Rhinos

Archaeological evidence shows that,

particularly in India and the Middle East,
dyeing has been carried out for over 5000
years. The dyes were obtained from animal,
vegetable or mineral origin, with no or very
little processing.

By far the greatest source of dyes has been

from the plant kingdom, notably roots, berries,
bark, leaves and wood, but only a few have
ever been used on a commercial scale.

In 1856, synthetic dyes manufacturing started.

Mauveine, also known as aniline purple and
Perkins mauve, was the first synthetic dye
produced. It was originally manufactured under
the name Tyrian Purple.

Tyrian purple was discovered by an 18-year old

William Henry Perkin, a talented English
chemist, who worked at Royal College of
Chemistry in London. Perkin patented his
discovery and commercialized it.

Later on, a lot of synthetic new dyes started to

appear in the market. Until now, almost all of
the dyes appear in the market are synthetic
dyes.

Perkins letter with an

Example of Tyrian Purple
dye on silk

Introduction to Dye

A dye is a colored substance that has an affinity to the

substrate to which it is being applied. The dye is
generally applied in an aqueous solution, and may
require a mordant to improve the fastness of the dye
on the fiber.
Chemical affinity is defined as electronic properties
by which dissimilar chemical species are capable of
forming chemical compounds. It can also refer to the
tendency of an atom or compound to combine by
chemical reaction with atoms or compounds of unlike
composition

Both dyes and pigments appear to be colored

because they absorb some wavelengths of light
preferentially. In contrast with a dye, a pigment
generally is insoluble, and has no affinity for
the substrate.

Classification of dyes

Dyes are characterized by their ability to absorb

visible light (400-700 nm).
Solids, liquids or gases can absorb light completely,
partially or not at all.
The part that is not absorbed can be reflected at the
surface of solids, liquids or transmitted through gases.
The reflected light reaches the retina in human eye
and goes through a series of photochemical reaction.

Dyes contain chromophores and auxochromes

Chromophores refer to functional groups which
are unsaturated and they cause a compound to
become coloured. Examples of chromophores
are N=N-, -C=C-, -C=N-and -C=O
Auxochromes are saturated functional groups
and the presence of these groups in a
compound greatly increases the color-yielding
power of the compound.
Such as -NH3, -COOH, -OH and NHR.

The huge number of commercial dyes is

classified in terms of structure, colour and
application according to Colour Index (C. I).

Society of Dyers and Colourists and the

American Association of Textile Chemists and
Colourists are societies which are responsible
to revise Colour Index every three months.

Acid dyes are anionic compounds that are used

for dyeing nitrogen-containing fibers such as
silk (contains about 250 mmol/kg of
ammonium groups), wool (contains about 850
mmol/kg of ammonium groups) and synthetic
polyamides (contains about 30-50 mmol/kg of
amino groups).

The name of acid dyes refers to the dyeing

process, which means these dyes are applied to
wool, silk and polyamides using from neutral
to acidic solutions (pH 2-6). Most synthetic
food colours falls in this category.

Direct dyes are compounds that have a high affinity

for cellulose fibers.

Direct or substantive dyeing is normally carried out in

a neutral or slightly alkaline dyebath, at or near
boiling point, with the addition of either sodium
chloride (NaCl) or sodium sulfate (Na2SO4). Direct
dyes are used on cotton, paper, leather, wool, silk and
nylon. They are also used as pH indicators and as
biological stains.

During the dyeing process, van der Waals forces are

involved - cause the sorption of dyes to cellulose
fibers.

Reactive dyes refer to the coloured

compounds with reactive group that form a
covalent bonds with OH-, NH- or SH-groups
in fibers.
The reactive group is usually with two labile
chloride or fluoride.

Metal complex dyes are dye compounds with

metal atom. Usually metal atom that involved
is chromium, copper or cobalt.

They are normally 1:1 complexes (1 metal

atom with 1 dye molecule) or 1:2 complexes (1
metal atom with 2 dye molecules).

The addition of metal atom improves the

fastness properties of the dye compounds.

Basic dye is a class of dyes that contain basic

groups in their chemical structure. They are
used for dyeing acid-containing fibers.

Basic dyes are mainly used in paper industry,

printing industry, cosmetics industry and food
industry.

Mordant dyes are dyes that naturally have no affinity to

the fibers. Thus, mordant, a chemical compound, is
needed to bind with fibers during the dyeing process.

Most of these dyes contain sulphonic acid groups and

metal atom such as chromium, cobalt or nickel.

Dyes attachment to the fibers depends on the formation

of metal-fiber bonds, and thus increases the strength of
fastness properties.

Mordant dyes are used for wool, silk, nylon and leather.

Most of the disperse dyes are insoluble in

water. They can be applied to cellulose acetate
fibers and polyester fibers.

The dyeing process for disperse dyes needs the

help of chemical softeners or high temperature
(>120oC) and thus allows dyes to penetrate the
fibers.

Sulfur dyes are dyes that have S-containing

and water-insoluble compounds.

They are produced by treating aromatics,

phenol and amino phenols with sodium
polysulfide or sulfur or both.

These dyes are mainly used for cellulose fibers

(Abrahart, 1977).

Fixation of Dyes On Its Fiber

DYE CLASS

Acid
Basic
Direct
Disperse
Metal-complex
Reactive
Sulfur

DEGREE OF
FIXATION (%)

LOSS TO EFFLUENT
(%)

80-95
95-100
70-95
90-100
90-98
50-90
60-90

5-20
0-5
5-30
0-10
2-10
10-50
10-40

Azo Dyes

There are more than 100,000 commercially

available dyes whilst over 7 x 105 metric tons
of dyestuffs are produced annually.

Amongst the synthetic dyes, azo dyes are the

largest group and it is estimated more than half
of the annually produced amounts of dyes (for
the year 1994 worldwide azo dyes production
as 1 million tons) are azo dyes.

Azo dyes are basically characterized by the

presence of one or more azo groups (-N = N-).

They are usually complex aromatic compounds

which are chemically stable and more difficult to
biodegrade in nature.

Hence, during the synthesis of azo dyes and dyeing

process in industries, dyes that are lost to the
industries wastewater remain recalcitrant.

Consequently affect photosynthetic activity in

aquatic life due to the reduced light penetration.

Aryl azo compounds have vivid colours,

especially reds, oranges, and yellows.
Therefore, they are used as dyes, azo dyes for
example Disperse Orange 1.

Some azo compounds, e.g. methyl orange, are

used as acid-base indicators due to the different
colours of their acid and salt forms. The
development of azo dyes was an important step
in the development of the chemical industry.

Disperse Orange 1 (4anilino-4'-nitroazobenzene)

is a dye of the azobenzene
class. Disperse Orange 1
contains approximately
25% dye by weight, with the
remaining mass consisting
of NaCl and other salts.

Several of the azo dyes are mutagenic and

carcinogenic including 4-phenylazoaniline and
N-methyl and N,N-dimetyhl-4phenylazoaniline (Sparado, 1992).

Their chemical structures are based on

azobenzene and the azo naphthol derivatives.

e.g. Amaranth = Acid Red 27 = FD&C Red 2

Hazard of Azo Dyes

Azo dyes can be reduced by azoreductases in

liver cells, skin surface microflora, kidney cells
and intestinal bacteria and thus aromatic amines
(aryl amines) are released.

Walker (1970) pointed out that the metabolism

of azo dyes in mammal (dog) occurred when
the result showed the product of azo dye
(sulphanilic acid) was found in the urine of
dogs after they consumed Orange I.

A study carried out by Hildenbrand et al. (1999) on

reduction of azo dyes in cell cultures showed that the
addition of a dye, Resacor Blue 2F, to kidney cells and
liver cells produced a carcinogenic aromatic amine
3,3-Dimethoxybenzidine.

In an international conference held in

Wurzburg, Germany in October 1992, an
incident was reported where 15 workers were
working in distilling 2-naphthylamine plant.

Long term of exposure to 2-naphthylamine

caused all of these 15 workers to develop
bladder cancer.

Over the years epidemiological studies have

shown the evidence that long-term
occupational exposure to certain aromatic
amines such as benzidine, 4-aminobiphenyl
and 2-naphthylamine, that are used in dye
industries increases the risk of developing
cancer.

Dye-degrading microorganisms

The efforts of isolating dye-degrading bacteria

started in the 1970s
Bacillus subtilis (1977)
Aeromonas hydrophila (1978)
Bacillus cereus (1980)

Dye-degrading bacterial cultures

Aeromonas hydrophila
Bacillus benzenovorans
Bacillus cereus
Bacillus subtilis
Citrobacter sp.
Echerichia coli
Klebsiella pneumoniae
Proteus mirabilis
Pseudomonas luteola

various azo
Tectilon Blue 4R-01
Azobenzene
p-aminoazobenzene
Brilliant Green
various azo
Methyl Red
Deep Red
Red G

Azo Dyes Removal Techniques

A wide range of dye removal techniques have

been studied and developed to remove dyes
from the wastewater before discharging to the
environment.

These techniques involved adsorption,

coagulation-flocculation, oxidation, membrane
filtration and microbial degradation.

Table: Current physical and chemical technologies for colour removal

Physical and/or
chemical methods

Advantages

Disadvantages

Adsorption

Good removal of a
wide variety of dyes

Absorbent requires regeneration

or disposal

Membrane
technologies

Removes all dye types

Concentrated sludge
production

Coagulation/
flocculation

Economically feasible

High sludge production

Oxidation

Rapid process

High energy costs

Biological treatment is advantageous over

the physical-chemical techniques as it is
inexpensive and more environmental
friendly.

However, conventional biological

treatment techniques involving the use of
activated sludge proved to be ineffective in
terms of removing colour in the dyecontaining wastewater.

Thus, since the 1990s, numerous of research

papers have been published on combined
anaerobic-aerobic treatment of dyecontaining wastewater.

The anaerobic-aerobic treatment studies

showed that a generally high colour removal
can be achieved and some of the studies
showed the evidence of mineralization of the
dye compounds.

Biodegradation of azo dye occurs in two

stages.
first stage --reduction of azo linkage
resulting in the formation of aromatic
amines
second stage --degradation of aromatic
amines

First stage: reduction of azo bond

Second stage: degradation of aromatic amines

TCA

Degradation Potential Aerobic Condition

Azo dyes seldom degrade under aerobic

conditions. The only aerobic degradation has
been attributed to the fungus Phanerochaete
chrysosporium and the action of Pseudomonas
strains adapted to growth on carboxylated azo
dyes (Pasti-Grigsby, 1992).

Phanerochaete chrysosporium is reported to

degrade three azo dyes, congo red, orange II
and tropaeolin O (Cripps, 1990).

Degradation Potential Anaerobic Condition

Many bacteria can reduce azo compounds to

amines through cometabolism.

These microorganisms include Clostridium

spp., Bacillus subtilis, Streptococcus spp., etc.

These aromatic amines however, can be highly

toxic and carcinogenic.

Combined Metabolism

It was first reported that mineralization of azo

dye was carried out through a combined mode
of anaerobic-aerobic process.
Example:

Sulfonated azo dye mordant yellow 3 (MY 3) has

been mineralized by this dual mode
Consists of facultative organisms since the
alternating presence and absence of oxygen cause
no toxic effects.

Reduction of azo linkage under anaerobic

conditions formed 6-aminonaphthalene-2sulfonic acid (6A2NS).

Two organisms under aerobic metabolism use

6A2NS as source for carbon and energy.

The addition of 1-butanol caused a threefold

enhancement in the degradation of MY3.