Decolourisation And Biodegradaation Of Textile Reactive Azo Dyes By A Strain Of Proteus Mirabilis

Olukanni, O.D (2012)

Thesis

The discharge of coloured wastewaters into water-bodies has serious implications for the environmental and public health. Furthermore, the usual colour removal methods often result in aromatic amines which are toxic, mutagenic and carcinogenic. Developing a method that can both decolourise azo dyes and degrade the corresponding aromatic amines formed is expedient. In this study, the ability of newly isolated indigenous bacteria to decolourise textile/non-textile azo dyes and degrade the aromatic amines formed was investigated. An estimated 0.05 g of the organism was inoculated into triplicates flasks containing 100 ml of a simulated effluent prepared from reactive blue 13 (RB 13), reactive yellow 42 (RY 42) and reactive red 58 (RR 58). The effect of media, oxygen, pH and temperature on the decolourisation activity of an effective strain was also investigated. The formation of aromatic amines and the mineralization of same were investigated by subjecting the metabolites of RB 13 to UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and gas chromatography/ mass spectroscopy (GCMS) analyses. The enzyme systems responsible for the decolourisation/ biodegradation were also studied. Three of the fourteen bacterial strains isolated from various environmental sources showed >95 % decolourisation of the simulated effluent, within 24 hours. One of these effective strains, which has been identified as a stain of Proteus mirabilis using 16S rDNA, decolourised methyl red (a standard azo dye) effectively at optimal pH and temperature of 7 and 37OC respectively. The strain preferred nutrient broth to minimal media of Mills et al and 0.02 g dry mass decolourised 50 ml, 56 mg/L solution of methyl red within 6 hours under adequate oxygen supply. UV-visible analyses of aniline sulphate (an aromatic amine) and those of the metabolic products of methyl red revealed that methyl red was first converted to aromatic amines and that the aromatic amines were probably mineralized. The Fourier transform infra-red (FTIR) spectra of the metabolites of RB13, when compared to that of the dye, revealed the disappearance of certain peaks, which were noticeably those of the aromatic C – H bending around 600 – 800 cm-1. Similar results were obtained using high performance liquid chromatography (HPLC). Gas chromatography- mass spectrophotometry (GCMS) analysis of the dye metabolite showed (among other metabolites) the presence of Sodium 2(2-formyl-2hydroxyvinyl) benzoate, with a tropylium cation (m/z 91) as its base peak. This suggested the breakage of naphthalene rings in RB13, indicating mineralization of the aromatic amines. The presence of azoreductase and laccase activities suggested both symmetrical and non-symmetrical enzymatic reduction of azo bonds prior to mineralization. Based on the FTIR, GCMS and enzymatic studies a pathway for the biodegradation of RB13 was proposed. Partial characterization of the laccase revealed that it is a protein with molecular weight of about 60kD. It has an optimum temperature of about 40 °C and acts best between pH 4 and 5. In conclusion, an indigenous strain of Proteus mirabilis that can decolourise, degrade and detoxify azo dyes has been isolated. This organism is thus a candidate for ecofriendly treatment of coloured effluents.

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