الفهرس 
Title : A Study on a Novel Azo Dye Degrading Enzyme(s) from Waste Water Systems; Isolation and characterization of the Enzyme(s) for Industrial Applications
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Abstract
Contamination of the aquatic ecosystems with chemicals, especially azo dyes, are known worldwide, and their toxicity are reported. The study was designed to solve the problem of contaminated effluents from industrial textile and tanning wastewater. Direct red 81 (DR81) was chosen as an example of diazo dye, the major contaminant in textile and tanning industries for decolorization as it is a sulphonated sodium salt and very water-soluble dye.Forty-one decolorizing mixed cultures were obtained from 67 samples.The obtained data showed that supplementation of MSM medium with yeast extract (0.1%) was important for DR81 decolorization as it provides cofactors for decolorization (NADH, FAD).Regarding the extent of decolorization, three mixed cultures 27W, 28W and 34W were able to decolorize DR81 in medium (MSM-Y), and real effluents (SWW and NSWW), showing ≥ 80% decolorization within 8 h of incubation.
Real wastewater analysis after decolorization revealed sharp decrease in COD and BOD denoting decrease in toxic chemicals load in water. Also, ammonium, nitrates, total nitrogen decreased denoting decreased organic substances load. However, the chosen mixed cultures decolorization was due to enzymatic action but not adsorption. In addition, increasing the initial dye concentration to 200 mg/l increased the decolorization,whileincreasing the dye concentration to 400 and 600 mg/l decreased the extent of decolorization due to possible toxic effects of the dye on bacterial growth. Regarding the factors affecting DR81 decolorization, glucose, sucrose and lactose increased decolorization due to enhancement of microbial growth. In addition, when the yeast extract concentration was increased up to 1%, it resulted in increased decolorization. Moreover, the selected mixed cultures showed marked tolerance to acidic and alkaline conditions, 5% salinity and high temperatures up to 60˚C.
Screening for degradative enzymes showed strong induction of azoreductase activity along with lower activities of oxidative enzymes.Moreover, crude azoreductase enzyme from the chosen mixed cultures showed optimum activity at 70˚C and pH 6 and stability up to 50˚C with wide pH stability (4-9).In addition, azoreductase (AzoR) gene detection in the total DNA isolated from the chosen mixed cultures showed homology between the genus Achromobacter and Alcaligenes and homology between the Pseudomonas monteili, Pseudomonas taiwanensis and Pseudomonas putida.Azoreductase (AzoR) gene detection in the total DNA isolated from the chosen mixed cultures showed a possible new azoreductase member in Pseudochrobactrum sp. deposited into GenBank with accession number MZ702782. Moreover, mixed cultures were superior to single isolates in DR81 decolorization.
To prove DR81 dye degradation, the degradative metabolites were extracted and identified. UV-Visiblespectral scan analysis of decolorized culture supernatants showed disappearance of both peaks in visible and UV regions indicating possible decolorization and degradation.Moreover, functional group analysis using FTIR showed the disappearance of characteristic bands for azo bond indicating DR81 degradation.Moreover, chromatographic techniques analyses suggested the possible DR81 biodegradation. HPLC-UV for the parent DR81 dye and its metabolites taken at different time intervals showed change in the peak at Rt 12.7 minfor DR81 to other peaks with different retention times indicating possible dye degradation.
Analysis of metabolites by mass spectrometry in TLC-MS and HPLC-MS showed that the main degradative pathway was through symmetric cleavage by azoreductase and asymmetric reductive cleavage.However, oxidative enzymes might have played synergistic role with azoreductase in DR81 degradation.Moreover, toxicity analysis for the decolorized supernatants showed less-toxic products than the parent DR81 dye. In addition, the MSM-Y medium showed toxicity at 1:1 dilution in both phytotoxixity and acute toxicity.To conclude, this study demonstrated mixed bacterial cultures that would be useful agents for detoxifying DR81, which can be safely implemented in industrial biodegradative applications. Industrial application of the study results should prove useful in cost and time-efficient biological treatment of complex tanning and textile effluents that have high loads of organic matter, high temperature, salinity, and pH.