Research - MS (Thesis)

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Title: Effect of Physical Parameters on the Decolorization of Textile Dye by Bacteria Recovered From Textile Effluent

Author: H. M. Abdullah Al Masud

Supervisor: Md. Abul Manchur and Monzur Morshed Ahmed

Co-Supervisor: Md. Zobaidul Alam

Research Field: Environmental Microbiology

Batch: 11th

Session: 2010-2011


Environmental pollution, caused by textile effluents in Bangladesh, is increasing with the increase of textile production day by day. Textile effluents contain azo-dyes which are xenobiotics and cause serious harm to the environment as well as living beings. Microorganisms have widespread ability to transform and degrade the harmful components of textile dyes.
In our present study we used six locally isolated bacteria with a view to developing an effective biological effluent treatment process. The experimental organisms were previously isolated from textile effluents on the basis of their decolorization potential. In the present investigation we identified these bacteria based on their morphological, physiological and biochemical characteristics, as described in Bergey’s Manual of Determinative Bacteriology (8th edition). The organisms were Gram positive, spore formers, moderately halo-tolerant. Three of them produced parasporal bodies. After detail characterization the bacterial isolates were identified as Bacillus thuringiensis (Isolate A2, B6, C6), Bacillus badius (Isolate B5, B9) and Bacillus aneurinilyticus (Isolate C2). These strains were investigated for the presence of plasmid but no isolates were found to harbor plasmid DNA.
Using these six isolates the effect of temperature, pH, inoculum concentration and effect of different consortia on decolorization of commonly used textile dyes were studied. For this purpose, a synthetic media supplemented with textile dye was developed. We randomly selected four commercial textile dyes namely Reactive Yellow 18, Novacron Orange FN-R, Novacron Red FN-R and Terasil Green.The percentage of decolorization was determined by UV-Spectrophotometer. Almost all dyes were decolorized by the experimental organisms under controlled conditions. The temperature range for the decolorization was 20˚C to 40 ̊ C and the optimum temperature was found to be 35˚C. The percentage removal of dye slightly decreased with further increase in temperature upto 400C. Decolorization was observed to start within 18 hours incubation and the highest percentage of decolorization was found after 42 hours. Decolorization rate was slow at both low and high pH. The highest percentage of decolorization, irrespective of dyes, was at pH 7. The effect of inoculum concentration was studied by developing different concentrations of inoculums of experimental organisms. At 5% concentration, decolorization rate was slow and the rate was gradually increased while the percentage was increased to 10%, 15% and 20%. No significant difference was observed among 10%, 15% and 20% concentrations. To develop effective consortia instead of single isolate, the organisms were mixed together and inoculated in dye containing culture broth. A total of 14 consortia was designed and used to decolorize the dyes at 37˚C, pH 7 within 48 hours. Different consortia showed different activity for the removal of dye from culture broth. A significant phenomenon was observed-only those consortia which contained isolate C6 were the best degraders. Among the isolates tested, C6 (Bacillus thuringiensis) was found to completely (100%) decolorize all the experimental dyes except Novacron Red FN-R after 48 hours incubation period at 35˚C at neutral pH.
To determine the molecular weight of the extracellular enzymes of the experimental organisms, SDS-PAGE was performed for all isolates. A distinct 65KDa protein band was found for all of the isolates.

To disclose the possible mechanism of the dye decolorization, we analyzed the products of biotransformation of the textile dye. Reactive Yellow 18 [Ergotaman-3′, 6′, 18-trione, 9, 10 - dihydro-12′- hydroxy-2′- methyl-5′- (phynylmethyl)-, (5′.alpha.,10.alpha) with molecular mass 583] by GC-MS analysis. The isolate C6 (Bacillus thuringiensis) was used as inoculum in this stage. Three intermediates, Pyrrolo [1,2-a] pyrazine -1, 4 -dione, hexahydro-3-(phenylmethyl), Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(2-methyl-propyl), 3-pyrrolidin-2-yl-propionic acid with a molecular mass of 244, 210 and 143 were deduced with the corresponding mass spectrum at GC retention times of 25.139, 19.565 and 18.326 min respectively.
From our study it may be suggested that under the controlled conditions bacterial populations present in textile effluent have the capability to degrade dye contaminants of the effluents. These bacterial isolates can be used as biological agents to design an effective biological effluent treatment plant.