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Presented by
Sabaina TahirM.Phil 3rd
Determination and Biodegradation of
azo dyes in soils contaminated with
textile dye effluent
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Azo Dyes
Azo compounds, i.e. molecules with one or more azo
(N=N) bridges linking substituted aromatic structures
They are generally aromatic hydrocarbons, derivatives of
benzene, toluene, naphthalene, phenol and aniline
Azo dyes are the most important group of syntheticcolorants that are extensively used in textile, food,
pharmaceutical and printing industries
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Physicochemical processes for biodegradation have
limitations and also produce toxic by products Biodegradation using microorganisms is gaining
importance as it is cost effective, environmentally
friendly, and produces less sludge
Different taxonomic groups of bacteria have beenreported for their ability to decolorize azo dyes
Aerobic degradation of azo dyes has been reported by
many investigators
This work has also been undertaken in order toinvestigate the potential of aerobic bacteria to degrade
azo dyes
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Sampling
Soils affected by azo-dye contamination around textile
industries are to be selected for sampling
Soil samples are to be collected from the areas of
Rawalpindi and Gujranwala
Scalpel will be used for this purpose by removing soil layerupto 15cm
Samples are to be stored in polythene bags aerobically at
4C
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Enrichment culture techniques
Enrichment techniques is carried out by taking clay loam
soil in a petri dish and calculated quantity of textile
effluent like azo dyes is added so that, the finalconcentration is around 500 ppm of soil.
Enough quantity of water is added to the petri dish so
that soil becomes moist, stirred well with glass rod andthe Petri plate is covered with lid and incubated for period
of 25 days.
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A solution will be prepared by taking five
grams of enriched soil samples and
transferred to a 250 mL conical flask
containing 100 mL of sterile distilled water,stirred well and the supernatant was diluted
up to five dilutions through a serial
dilution method.
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Isolation
For the dilution plate method 10 g of sample is transferred in 90
mL of 0.85% saline water
Pasteurization at 80C for 10 min
1 mL aliquot from each of the samples is transferred in 9 mL of
0.85% saline water and preparation of 6 fold dilutions as done
1 mL of dilutions was plated on nutrient agar plates/ mineral salt
medium and incubated for 48 - 72 h at 50C.
The plates were covered with aluminium foil and single colonieswith different morphologies were picked and purified using streak
plate method
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Screening
The bacterial isolates are to be sequentially
adapted to higher concentration of azo
dyes by repeated sub culturing on solid
culture media (Mineral salt medium andagar)
The maximum resistance limits (MRL)
beyond which the bacterial growth isdetermined apparent, decolorization
abilities of the bacterial strains are
monitored at 37C for 8 days
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Preservation of isolates
Glycerol stocks are prepared and stored at -
80C for long term preservation
Pure cultures strains are incubated at 50C
for 48 h in isolation broth
Then 0.5 mL of each of the cultures aretransferred into cryotubes with 0.5 mL broth
containing 40% glycerol
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Degradation studies
The isolates obtained are to be placed on Mineral Salt
Medium with different conditions
Growth of isolates is to be observed by incubation at
different temperatures, providing varying pH and with
different substrate concentrations
In all above cases growth of bacterial strains is to be
determined with an interval of 4 days
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Development of consortium in 10 ml culture tubes containingmixture of reactive azo dyes, 5 ml nutrient broth and a loop ful
of each microbial culture (incubated for 24 hrs)
5 ml of each bacterial strain culture is to be added to 250 mL
Erlenmeyer flask containing 100 mL of textile effluent The flask was further incubated to observe the time required for
decolorization
3ml of aliquots of the culture media are to be withdrawn at
different time intervals, centrifuged at 15 min to separate the
bacterial cell mass
Decolorization of the textile effluent was analyzed using a
UV/Vis spectrophotometer (deminishing of peaks in visible
region)
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Morphological characters of the isolates were
determined by gram staining, spore staining and
motility testing
16S rRNA sequencing is to be performed to find out
the nucleotide sequence and it is helpful for
determining the genus of a particular specie
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Thank You