The Decolorizing Characteristics And Reduction Mechanism Studies Of Azo Dyes By Bacteria

Azo dyes are the largest and most versatile class of dyes. During the dyeing process, upto15%of the dyestuffs are discharged into the environment. These dyes are toxic and threatening people's health seriously. Thus, industrial wastewater containing azo dyes must be treated before it is released into the environment. Many new processes for azo dye decolorization have been developed. One promising strategy is to use microbes to decolorize azo dyes. The biodegradation of azo dyes is considered to be an environmentally friendly option.The purpose of this study was to isolate azo dye degrading bacteria from dye-contaminating environment. The decolorizing characteristics of azo dye were investigated and effects of various factors on the decoloration were studied, which offered the technology parameters for the industrial application of related strains. In addition, the mechanism of azo dye decolorization by bacteria was explored through azoreductase and redox mediator studies. Moreover, two environmentally friendly consortia which could decolorize azo dyes under aerobic and anaerobic conditions, respectively, were enriched. Similarities and differences of microbial stuctures between the two consortia were compared by using PCR-DGGE method. The main results are as follows:(1) In this study, we isolated and characterized a new strain of Klebsiella sp. Y3, that was capable of decolorizing azo dyes under anaerobic conditions. The strain Y3could decolorize100mg/L Methyl Red completely within16h, indicating that it had high ablility to decolorize azo dyes. The effects of physico-chemical parameters, including the temperature, pH and salinity of the culture medium on the Methyl Red degradation by the strain were determined. The results indicated that strain Y3exhibited a good decolorization ability in the ranges of pH from4-9, temperature from25-45℃and salinity from1%-4%. A50%Methyl Red decolorization was observed within64h without an additional carbon source. The strain could decolorize a broad spectrum of azo dyes with different structures. Strain Y3could tolerate and degrade azo dyes at high concentrations. An almost complete mineralization of Methyl Red and Congo Red at the concentration of800mg/L was observed within48h. The phytotoxicity tests showed that the degradation products were less toxic to plant seeds compared with the control Methyl Red, indicating that Methyl Red could be detoxicated by strain Y3. UV-visible analysis suggested that all the azo dyes were decolorized completely. However, the appearance of the new peak in the UV spectra indicated the formation of other metabolites.(2) Using the similar method with anaerobic bacteria isolation, we found a new strain which could degrade azo dyes effectively under aerobic conditions. Identification of this isolate by16S rDNA technique revealed that the strain clustered within Escherichia coli. Various environmental factors influencing dye degradation were investigated, the results showed that the strain could decolorize different azo dyes effectively at high salt concentration and over a wide range of pH. This degradation potential increased the applicability of this strain for the azo dye removal.An oxygen-insensitive intracellular enzyme that was responsible for the decolorization of azo dyes was purified from strain CD-2by ion-exchange and molecular exclusion chromatography. Protein identification indicated that the enzyme had high sequence homology with Escherichia coli K12quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. According to these, we concluded that several kinds of enzymes which played important roles in bacterial cells could also reduce azo compounds as their secondary activities.The purified enzyme could efficiently decolorize Methyl Red outside cells and was relatively stable over wide ranges of pH, temperature and salinity. Therefore, this enzyme might have great potential for industrial use. The HPLC analysis showed that the purified enzyme could catalyze the reductive cleavage of the azo bond of Methyl Red in the presence of NADH as electron donor. The metabolites of Methyl Red might be degraded by other enzymes.(3) In present study, quinone-mediated decolorization of different azo dyes by strain CD-2under aerobic conditions was investigated. The results showed that reduction rates of different azo dyes by strain CD-2were greatly increased in the presence of quinone compouds as redox mediators. Based on these, we concluded that the aerobic dye decolorization reaction was unspecific when the system contained redox mediators. The quinone compouds could accelerate electron transmembrane reaction. Thus, the decolorization rate was enhanced.Although the whole cells incubated with quinones could significantly increase the rate of decolorization of azo dyes, the quinone compounds did not promote azoreductase activity directly. It was likely because that the reduction of azo dyes was limited by the permeation of the dyes through the cell membrane, and when the decolorization system contained redox mediators, the quinones were enzymatically reduced first, and the azo dyes were subsequently reduced in a purely chemical redox reaction. However, when the cell membrane was broken up, the crude cell extracts could reduce azo dyes directly outside the cell and it did not need quinones to transfer H ions through the cell membrane.(4) In this study, two consortia which could decolorize different azo dyes under aerobic and anaerobic conditions, respectively, were finally enriched. Both of the two consortia could decolorize different azo dyes effectively in a short time, and tolerate Methyl Red with high concentrations. Azo dye decolorization rate was significantly higher with the use of consortia compared to that with the use of individual strains. Thirteen strains were isolated from the two consortia and each isolate was identified by16S rDNA sequencing. The results revealed that all of the isolated strains were facultative anaerobic bacteria of the family Enterobacteriaceae. To provide further insight into the microbial diversity of the bacteria consortia under aerobic and anaerobic conditions, PCR-DGGE analysis was performed. PCR-DGGE profiles revealed that the microbial community changed significantly with varying initial concentrations of Methyl Red. Phylogenetic analysis indicated that microbial populations in the aerobic compartment belong to Klebsiella, Buttiauxella and Bacillus, whereas Klebsiella, Escherichia, Bacillus and Clostridium were present in the anaerobic compartment.