The discharge of highly coloured azo dyes effluent has caused serious environmental damages. In this study, bacteria isolated from Antarctica seawater were screened for their ability to decolourise azo dye Reactive Black 5 (RB5). The selected bacterium was further investigated to study its ability to decolourise RB5. The best bacteria from Antarctica seawater that had the ability to decolourise RB5 was identified using 16S rDNA sequence analysis and revealed that the bacteria 15C shared 99% homology to Klebsiella pneumoniae. Selection of the most effective bacteria was followed by its acclimatisation to decolourise higher concentrations of RB5 by growing it in successively higher concentrations of RB5. Following that, optimization of RB5 decolourisation by the selected bacteria was performed using one factor at time (OFAT) including concentration of dye, pH and temperature. The results obtained indicated the optimal condition for decolourisation of RB5 using this bacterium was at pH 10 and 37°C in 70 mg/L RB5 with 98% decolourisation within 24 h under facultative anaerobic treatment. Besides that, 70% of COD removal was achieved after 96 h of sequential anaerobic and aerobic treatment of RB5. In addition, FTIR and HPLC were used to analyze the metabolite of RB5 decolourisation. Products of RB5 decolourisation was confirmed by the presence of sulphanilic acid in HPLC analyses and the changes observed in the functional groups of the FTIR spectrum suggesting the possibility of RB5 degradation. Ammonia test and carbon dioxide test showed higher concentration of ammonia and carbon dioxide that indicates the mineralisation of product after treatment.
Azoreductases are often associated with decolourisation of non–degradable azo dyes via cleavage of azo bonds. In this study,Brevibacillus panacihumi ZBI, an azo dye–degrading bacterium which has not been reported before, was used for the decolourisation of Reactive Black 5 (RB5) dye. The highest activity of azoreductase was obtained during the end of log phase. Azoreductase produced intracellularly had the highest specific activity of 0.334 U/mg compared to the culture supernatant (extracellular), resting cell and cell debris with low enzyme activity of 0.034 U/mg, 0.010 U/mg and 0.200 U/mg respectively. The optimum assay conditions for the maximum azoreductase activity were at 37°C, pH 7, RB5 dye concentration of 100 mg/L and NADH concentration of 0.2 mM by using phosphate buffer as a medium for the enzyme reaction. Alternatively, the azoreductase assay was also carried out using ionic liquid, [emim][EtSO4] that may function to enhance the activity and stability of azoreductase. Results using phosphate buffer (pH 7) showed higher enzyme activity twice that of the ionic liquid besides enhancing the stability of enzyme. Under the optimum assay conditions up to 93 % of decolourisation was achieved after 8 h of incubation. In addition, growth of bacteria was also concurrently observed during the decolourisation of RB5.
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