Determination and Biodegradation of azo dyes in soils contaminated with textile dye effluent

Presented by Sabaina Tahir M.Phil 3rd

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 synthetic colorants that are extensively used in textile, food, pharmaceutical and printing industries

 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 been reported 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 to investigate the potential of aerobic bacteria to degrade azo dyes

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 layer upto 15cm Samples are to be stored in polythene bags aerobically at 4C

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 final concentration 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 and the Petri plate is covered with lid and incubated for period of 25 days.

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.

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 colonies with different morphologies were picked and purified using streak plate method

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 and agar) The maximum resistance limits (MRL) beyond which the bacterial growth is determined apparent, decolorization abilities of the bacterial strains are monitored at 37C for 8 days

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 are transferred into cryotubes with 0.5 mL broth containing 40% glycerol

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

 Development of consortium in 10 ml culture tubes containing mixture 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)

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

Thank You