Decoloration of Industrial Azodyes using Bacteria Acinetobacter junii --Decolonization of direct red 2

1. Decolorization of Direct Red (Azo dye) by
Acinetobacter junii
INTRODUCATION:
The dying of textiles has been done for thousands of year but most of
this time the dyes have been derived from natural sources such as plants,
animals, insects and minerals. In the mid of 18th century the synthetic
organic dyes started to emerged. Synthetic dyes are poly-aromatic
molecules that give a permanent color to selected substance which may
be are textile fibers. More than one hundred thousand industrial
synthetic dyes including myriad denominations are made to known
worldwide with annual production of about 280,000 tons. These
synthetic dyes are widely used in textile, paper, food, cosmetics and
pharmaceutical industries with the textile industry as a voracious for
these dyes. Among all of the dyes which have been prepared
synthetically azo dyes are most common, constituting of 60%-70% of all
the substance used for dying. The reason behind is the stability,
cheapness of azo dyes, easy to produce, easily accessible and the variety
of colors given by these azo dyes.
The first azo dye was discovered accidently in 1856 by William Henry
Perkin when he was trying to make an anti-malarial drug. It was
originally known as aniline purple. This discovery led to the creation of
whole bunch of aniline base dyes and it induces the development of
synthetic dye industry. All these dyes have characteristics nitrogen,
nitrogen double bond (-N=N-) known as azo group. By changing the
attachments on either side of the azo group, it is possible to generate a
wide range of colors. These types of azo dyes are now having become
more popular. The reason behind the great success of azo dyes is that

2. they can be prepared easily, in a large amount, show good binding when
they once attached and provide a wide range of colors.

4. Dyes are also used in making CD/DVDs and most of the companies
prefer azo dyes. Lasers strike dyes and decompose them in specific
places. When it decomposes, it changes color and by authorizing
between changed and unchanged, it is possible to etch a binary message.
In general, azo dyes are extremely diverse family with many unique
properties but majority of them are made in use by the same process.
Azo dyes are most common synthetic dyes released into the
environment. Azo dyes are withdrawing electron groups, creating the
deficiency of electrons in the molecules and making them resistant to
degradation. All of the dyes used in a given process are unable to bind
with the fibers and resulted in 10%-50% of unused dyestuff entering the
wastewater directly. Improper textile wastewater disposal in aqueous
ecosystem cause severe environmental problem such as obstruction of
light to penetrate in the water bodies, oxygen transfer and depict severe
effects on zooplankton and phytoplankton. In addition some of the azo
dyes and their degradation intermediates are virulent causing mutation to
humans as well as to other animals. These azo dyes are major source of
pollution having adverse effects on aquatic as well as human life.

5. These azo dyes cause eutrophication in the aquatic ecosystem which
results in a great population of anaerobic bacteria, the ultimate survival
of that ecosystem, as all other living organism die due to the deficiency
of oxygen, the corollary of water pollution.
Review of Literature
Several scientific methods are practiced for the treatment of wastewater
of industries such as adsorption, coagulation, membrane filtration,
precipitation, filtration and oxidation but these methods are costly,
producing a lot of sludge which requires safe disposal. It is, therefore
necessary for the ecosystem to find out efficient and reasonable methods
for the removal of azo dyes in textile effluents and contaminated soil. As
a feasible alternative, biological process including different micro-
organisms of varying phyla gained ameliorative interest due to their cost
effectiveness, ability to produce less sludge and environment friendly
nature. Fungi decolorize the dyes mainly by adsorption but slow growth
and low decolorization efficiency limits the use of fungi for the
treatment of textile effluent. In volt-face, different subdivisions of
bacteria can achieve a higher degree of degradation and even complete
mineralization of dyes under optimum condition. The biodegradation of
azo dyes may occur either aerobically, anaerobically or by a
combination of both. Together with industrialization, awareness
towards the environmental problems arising due to effluent discharge is
of critical importance. Pollution caused by dye effluent is mainly due to
durability of the dyes in wastewater. Therefore, both color creating and
color producing industries are compelled to search for novel biochemical
or physiochemical treatments and technologies which are directed
particularly towards the decolorization of dyes from the effluents.
Various physiochemical and biochemical methods are available for this

6. purpose. A brief description of some of these methods is given in the
following table.
In advanced countries, the coagulation/flocculation is most widely used
method in textile wastewater plants. It can be used either as a pre-
treatment, post-treatment, or even as a main treatment system. This
method can efficiently remove sulphur and disperse dyes whereas acid,
reactive, direct and vat dyes presented very low coagulation/flocculation
capacity. Filtration methods such as ultra-filtration, nanofiltration and
reverse osmosis have been for water reuse and chemical recovery. In the
textile industry, these filtration methods can be used for both filtering
and recycling. The specific temperature and the chemical composition of
the wastewater determine the type and porosity of the filter paper to be
applied. The main drawbacks of this technology are the high investment
costs, the potential membrane fouling; produces secondary waste
streams which need further treatments. The adsorption methods for the
color removal are based on the high affinity of many dyes for the
adsorbent materials, found effective for a wide range of dyes. The main

7. criteria for the selection of an adsorbent should be based on the
characteristics such as, high affinity, capacity for the target compounds
and the possibility of adsorbent regeneration. Activated carbon is one of
the most common adsorbent and found very effective for the various
types of dyes, but due to high cost, it is not used conventionally. Ion-
exchange and electro-kinetic coagulation was also found effective but
due to their high sludge properties and ineffective to high diversity of
dyes it became economically unfeasible hence not accepted widely.
Moreover, chemical oxidation methods enable destruction or
decomposition of dye molecules. In which various oxidizing agents such
as ozone, hydrogen peroxide and permanganate were used. Modification
in the chemical composition of a compound or a group of compounds
(for example, dyes), takes place in the presence of these oxidizing
agents, by which the dye molecule becomes susceptible to the
degradation. Ozonation found to be effective due to its high reactivity
with many azo dyes (by breaking azo bond), application in gaseous state,
no alteration of the reaction volume and providing good color removal
efficiencies. However, it has limitations to disperse dyes and those
insoluble in water, short life time, low COD removal, as well as the high
cost of ozone. Electrochemical oxidation found to be very effective in
which destruction of organic compounds resulted into non-hazardous
products but high cost of the electricity limits the process. Thus majority
of color removal techniques work either by concentrating the color into
sludge, or by the complete destruction of the colored molecule.
According to Integrated Pollution Control (IPC) regulations,
decolorization systems involving destruction technologies will persist, as
the transferal of pollution from one part of the environment to another
need to prevent. Thus implementation of physical/chemical methods
have inherent drawbacks, of being economically unfeasible (more
energy and chemicals), unable to complete removal of the recalcitrant

8. azo dyes and/or their organic metabolites because of the color fastness,
stability and resistance of azo dyes to degradation, generating a
significant amount of sludge that may cause secondary pollution
problems, substantially increases the cost of these treatment methods
and involving complicated procedures.
The removal of waste substances from the environment by using living
organisms is major approach in the environmental sciences.
Bioremediation is recently developed technique but accrued at
ameliorative rate in these days. In this approach, the microorganisms can
habituate themselves to toxic substances and are responsible for
conversion of various carcinogenic chemicals into less harmful forms.
Many reports suggest the degradation of complex organic dyes that may
be dyes, through a mechanism regulated by biological enzymes present
in microbes.

9. Hfh
The use of mechanism regulated by enzymes for the complete
degradation and decolorization of azo dyes has many benefits: 1) eco-
friendly nature 2) cheapness 3) ability to produce less sludge 4) non-
toxic end products 5) can also reduce the use of huge amount of water as
compared to physiochemical methods.

10. The adaptability and the activity of selected microorganisms also
influence the effectiveness of microbial decolorization of various dyes.
Many a number of species has been scrutinized for the decolorization
and mineralization of several dyes, and their number is steadily
enhancing in the recent past. Many a variety of organisms are enable to
decolorize many a number of dyes including many a number of
microorganisms such as bacteria, plants, actinomycetes, algae, yeast and
fungi under specific conditions.
The ability of fungi to rapidly adapt their metabolism to varying carbon
and nitrogen sources is an integrated aspect for their survival. This
metabolic activity achieved through the production of a large set of intra
and extracellular enzymes, able to degrade complex various kinds of
organic pollutants. In addition to the production and secretion of number
of enzymes, fungi can secrete a great diversity of primary and secondary
metabolites (e.g. antibiotics) and perform many different complex
conversions such as hydroxylation of complex polyaromatic
hydrocarbons, organic waste, dye effluents and steroid compounds.
Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents. The ability of these fungi to degrade such

11. a range of organic compounds results from the relatively non-specific
nature of their ligninolytic enzymes, such as lignin peroxidase and
manganese peroxidase. Fungal degradation of aromatic structures is a
secondary metabolic event that starts when nutrients (C, N and S)
become limiting. Therefore, while the enzymes are optimally expressed
under starving conditions, supplementation of energy substrates and
nutrients are necessary for the propagation of the cultures. However,
application of fungi for the removal of dyes from textile wastewater
have some problems with its use like long growth cycle and requiring
nitrogen limiting conditions, hence the enzyme production may be
unreliable, long hydraulic retention time for complete decolorization still
limit the performance of the fungal decolorization system as well as
preservation in bioreactors will be a matter of concern.
Phytoremediation is considered as feasible approach for the remediation
of soils and groundwater contaminated with heavy metals and organic
pollutants. Recently some studies describe the use of plants for the dye
removal from wastewaters. However, in large scale application of
phytoremediation presently faces a number of obstacles including the
level of pollutants tolerated by the plant, the bioavailable fraction of the
contaminants and evapotranspiration of volatile organic pollutants as
well as requiring big areas to implant the treatment.
Generally the decolorization of azo dyes has been done under
conventional anaerobic and aerobic conditions by different groups of the
bacteria. The mechanism of microbial degradation of azo dyes involves
the reductive cleavage of azo bonds (-N=N-) with the help of azo-
reductase under anaerobic conditions resulted into the formation of
colorless solutions containing potentially hazardous-aromatic amines.
The resulting intermediate metabolites are further degraded aerobically
or anaerobically. Many recent researches focus on utilization of

12. microbial biocatalyst to reduce the dye from the effluent. Decolorization
done by bacteria is much faster than that done by fungal system.
Materials & Methods
First of all, inoculum was prepared for the
bacterial strain, Acinetobector Junii. The composition of LB media for
100mL is given by:
Sr.No Substance Amount
1 NaCl 1g
2 Trypton 1g
3 Yeast extract 0.5g
4 pH 7
The bacterial strain of acinetobacter junii is put into broth medium to
check its growth on solid medium. Its growth has been checked after 24
hours at room temperature. The composition of nutrient broth media for
1000mL is given by:
Sr.No Substance Amount
1 Nutrient Broth 13g
2 Agar 18g
3 pH 7
In order to check the growth of bacterial strain in liquid medium, it is put
into the mineral salt medium (MSM). The preparation of mineral salt
medium by using different substances is done by:
Sr.No Substance Amount
1 K2HPO4 0.865g
2 KH2PO4 0.34g
3 MgSO4 0.25g

13. 4 FeSO4 0.015g
5 NaCl 2g
6 CaCl2 0.01g
7 Yeast extract 1.5g
The must be equal to 7(neutral). The bacterial strain is added in this
medium and incubated it for 18-24 hours at 37C aerobically.
Preparation of azo dye solution: Direct red azo dye solution was
prepared and poured into beaker. The bacterial strain is added in azo dye
solution by 10% v/v ratio, by adding 10mL of strain in 100mL of azo
dye solution. The system is again incubated for 24 hours.
Optical Density Calculation: The optical density of the strain is
calculated after different time intervals. The optical density of control is
1.005 at 500nm while after 20 hours of inoculation of acinetobacter junii
the optical density is 0.424 that is about 57.81%, after 25 hours of
inoculation the optical density at 500nm is 0.345 that is about 65.67%,
and after 48 hours of inoculation it is 0.091 that is 90.94%.
Sr.No Time Optical Density Percentage
1 20 h 0.424 57.81%
2 25 h 0.345 65.67%
3 48 h 0.091 90.94%
The activity of the bacteria is also shown by the graph.
Crude Enzyme Preparation: Culture was harvested after 48 hours when
the maximum decolorization has been shown by it. Culture was poured
in 50 mL falcons and centrifuged at 4oC for 30 minutes at 10,000 rpm.
All the pallets were separated from supernatant. Supernatant was used as

14. extracellular enzyme. Pallet was suspended in 0.1M potassium
phosphate buffer for overnight (10mL buffer in each falcon). Next day
lysozymes were added (1mg/mL). The amount of DNase that was also
added in it is 10սl/ml solution. Samples were incubated at 370C for 20
minutes. The lysate was clarified by centrifugation at 10,000 rpm for 30
minutes at 40C and the pallets were discarded.
Enzyme Assay: The optical density of substrate blank and enzyme blank
at 500nm is 0.0 and 0.601 respectively, while that for extracellular and
intracellular is 0.203 and 0.235 respectively.
Sr.No System Optical Density
1 Extracellular 0.203
2 Intracellular 0.235
3 Substrate blank 0.601
Protein Estimation: Protein estimation is done by Bradford method
which involves the reaction of 1ml Bradford reagent and 100սl enzyme
sample. Optical density of reagent blank at 595nm is 0.727 while that of
extracellular and intracellular are 0.795 and 0.886 respectively.
Conclusion
The azo dyes can be successfully decolorized by azo reductase
enzyme. Different factors also affect the maximum efficiency such as
pH, temperature and concentration. Direct red is almost completely
decolorized in two days. This all mechanism is done at low cost,
producing little amount of sludge. However, future work on

15. identification of genes can be helpful in enhancing the decolorization of
azo dyes.
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