| Azo dyes are a major class of synthetic dyes and are being extensively discharged in high concentrations in the effluents of textile, food, paper printing, and cosmetic industries. Water contamination by azo dyes is not only arousing esthetic problems but also is causing toxic impacts on aquatic lives and even threatening human health. Biological processes have been recognized as an inexpensive and environmentally friendly way to treat azo-dye-rich wastewaters. The main focus of this research is the use of methyl orange (MO) as a model dye to carry out a detailed study on the anaerobic biodegradation of azo dye.The biological decolorization of methyl orange (MO), a typical azo dye, was investigated in anaerobic sequenced batch reactors (ASBRs). These reactors were adapted to an MO loading rate of up to 1.5 g 1-1 day-1 with complete dye decolorization achieved in each cycle. The anaerobic sludge was found to have a saturated MO adsorption capacity of 36±1 mg g MLSS-1. UV/visible spectrophotometer and high-performance liquid chromatography analytical results indicated that the MO adsorption and decolorization occurred simultaneously. In light of the considerable adsorption ability of anaerobic sludge, a modified two-stage pseudo-first order kinetics that takes into account adsorption and decolorization were established to describe the MO removal process.Performance of MO decolorization under different substrate and salt (NaCl) concentrations were also studied. A high salt concentration was found to pose negative effects on MO decolorization. A Monod equation was introduced to describe the inhibition effects of salt on the biodegradation process, since a first-order kinetic was not able to reflect the inhibition type and level. The calculated parameter Rmax (maximum decolorization rate) decreased from 92.6 to 17.2 mg 1-1 h-1, when salt concentration increased from 0 to 40 g 1-1, while the Ks (half saturation concentration) value varied only slightly and non-systematically. The negative correlation between Rmax and salt concentration indicated that salt inhibition is of noncompetitive nature, and the inhibition constant K1 was calculated to be 3.67 g-NaCl 1-1In order to offer a better insight into the decolorization mechanisms of azo dyes, pure microbial cultures have also been used to explore the MO decolorization process. Specifically, a facultative anaerobic bacteria strain GS-4-08 was isolated from an ASBR and used for MO decolorization. This bacterium strain was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA genes analysis. It exhibited good ability in simultaneous azo dye decolorization and hydrogen production in the presence of an appropriate electron donor (ED). The optimum pH, temperature and ED type and concentration for MO decolorization were 7,35℃and 20 mM of sucrose, respectively. This strain was able to efficiently convert ED into H2 and other valuable products (e.g., ethanol and acetate) during MO decolorization even at a high MO concentration of 0.5 mM, indicating a high MO tolerability. Hence, the use of this bacterium strain for MO decolorization not only avoids the inhibition of accumulated volatile fatty acids (VFAs) during MO decolorization, but also enables recovery of energy at the same time.Furthermore, a possible co-metabolism mechanism of MO decolorization and H2 production by the genus GS-4-08 was proposed based on the analysis of the intermediate and end products. A membrane-associated pyruvate dehydrogenase is likely to be the primary enzyme responsible for NADH2 generation. The hydrogenase and some other dehydrogenases involved in this system are mainly for hydrogen production, while the membrane-bound putative azoreductase functions as the terminal reductase. NADH2 produced during glucose hydrolysis and the primary dehydrogenases that serve as electron donors are transported through the electron transport chain to the azoreductase, driving the cleavage of azo bonds. This possible mechanism of electron transfer explains why higher decolorization rate was obtained when sugars and pyruvate were used as substrates than when other end products of glycolysis (e.g., ethanol, acetate, formate and hydrogen) were utilized.
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