Isolation, Screening And Decolorization Characteristics Of Azo Dye-decolorization And Salt-tolerance Strain

Two salt-tolerance strains（named P-1and P-2, respectively）which can degradate acidred B efficiently were isolated and screened from activated sludge of a sewage treatmentunit of a dye plant. On the basis of morphological observation and physio-biochemical test,the strain P-1was identified as Pichia kudriavzevii strain by26S rDNA gene sequence andphylogeny analysis, and the strain P-2was identified as Pseudomonas cedrina subsp.Fulgida by16S rDNA gene sequence and phylogeny analysis.The effect of different salt concentration, initial substrate concentration, temperature,pH, and inoculum dosage on degradation efficiencies was investigated, and then thedegradation conditions were optimized. The results showed that, the optimal conditions fordegradation of acid red B by strain P-1were: salt concentration10%;initial substrateconcentration,100mg/L; temperature,33℃; pH,5.0; inoculum dosage,10%(V/V). Theoptimal conditions for degradation of acid red B by strain P-2were: salt concentration3%;initial substrate concentration,100mg/L; temperature,33℃; pH,5.0; inoculum dosage,10%(V/V). Under the optimal conditions, the degradation rate of acid red B reached94.29%and92.85%by the strain P-1and P-2, respectively.The degradation efficiencies of eight azo dyes including basic orange, reactive red3BE,direct orange S and others by the strain P-1and P-2were investigated. The results indicatedthat the degradation efficiencies of these azo dyes by the strain P-1and P-2were higher than70%, which showed that the strain P-1and P-2were broad-spectrum degradation microbes.Alkaline lysis was applied to extract plasmids from strain P-2. A stripe of plasmidwhich was greater than15kb was detected through the test of agarose gel electrophoresis.The transformants obtained the ability to degrade acid red B when the plasmid of strain P-2was transformed to competent cell of Escherichia coli DH10B, it can remove41.2%of acidred B in the solutions with concentration of100mg/L within48hours. But the mutation lostthe ability to degrade acid red B when its plasmid was eliminated by SDS and hightemperature. This indicated that the plasmid of strain P-2may carry the degrading gene ofacid red B. In order to further judge whether the degrading gene of strain P-2was located onthe plasmid, the universal primers of azoreductase gene were used as the primers and thegenome DNA and plasmid DNA of strain P-2were used as template, respectively, to carryon PCR amplification. The results indicated that, a specific stripe which was about500bp can be detected when plasmid DNA was used as template. After detecting the sequence ofthe PCR product, we found that this gene owned90%and89%homology compared withthe azoreductase gene of Rhodobacter sphaeroides and Bacillus sp. OY1-2. Furthermore,the beginning and ending of this gene were conservative. So this gene may be azoreductasegene by inference. On contrast, no specific stripe appeared when the genome DNA was usedas template, which further proved that the degrading gene of acid red B was located on theplasmid.