| Azo dye wastewater has the characteristics of high salt and high toxicity,which seriously endangers water and soil.The carcinogenic compounds could induce cancer.At present,biodegradation has become the main research method of domestic and foreign scholars.Screening microorganisms that can degrade azo dyes efficiently and applying them to azo dye wastewater treatment has become one of the current research hotspots.Because the degradation efficiency of single strain was low and incomplete,it was easy to form secondary pollution,and mixed bacteria could overcome these shortcomings.The study on the degradation of azo dyes by halophilic/halotolerant microflora has positive significance for improving the biological treatment efficiency of azo dye wastewater in the future.The halophilic/halotolerant microflora,with the remarkable ability to decolorize the Direct Black（DBG）,was isolated from the Yanjing,Millennium Ancient Salt Village,Yanjing Town,Mangkang County,Tibet Autonomous Region,China.The synergistic degradation of various enzymes in the process of biodegradation of azo dye direct black G（DBG）by the halophilic/halotolerant microflora was explored.Futhermore,Ultraviolet and visible spectrophotometry（UV-Vis）,Fourier transform infrared spectroscopy（FTIR）and High performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）were used to analyze the intermediate of the degradation of Direct Black G（DBG）,and a possible degradation pathway of DBG was deduced.The microbial diversity of halophilic/halotolerant microflora under different NaCl concentrations were analyzed by high-throughput sequencing technology,Finally,the degradation liquid after dialysis and desalination was used for plant toxicological experiments,which laid a theoretical foundation for the study of halophilic/halotolerant microflora treatment of azo dye wastewater.The main conclusions of this study as follows:（1）The study results of degradation characteristics of the halophilic/halotolerant microflora showed that the best conditions of decolorization and degradation were as follows:Under the conditions of 40°C,p H 7.0 and static culture,the composite bacteria had the highest decolorization and degradation efficiency for DBG.When the concentration of DBG was 200～600 mg/L,the good degradation efficiency was showed by the composite bacteria,and the degradation rate could reach more than 70%after 12 h of culture.After 24 h,azo dyes were completely degraded.Under the optimal conditions,600 mg/L Congo red,DBG,amaranth red,methyl red and methyl orange could be completely decolorized at 24 h,8 h,8h,12 h and 12 h,respectively.The decolorization rate of azo dyes with 0 g/L～60 g/L NaCl for 24 h was above 95%,while the decolorization rate was decreased after 12 h with 40 g/L NaCl.（2）When the concentration of DBG was 600 mg/L,the activities of NADH-DCIP,laccase and azo reductase increased significantly,which proved that NADH-DCIP,laccase and azo reductase were primary reductase and oxidase in the degradation process.Through the study of the degradation of DBG by the complex bacteria with different concentrations of NaCl,the results were shown that the bacteria could not only tolerate the change of salt concentration of 0～80 g/L,but also the decolorization rate reached more than 95%after24 h of degradation of azo dyes with salt concentration of 0 g/L～60 g/L,indicating that the bacteria had strong degradation ability for DBG.At high salt concentration（60 g/L NaCl）,NADH-DCIP reductase was mainly used for the decolorization of azo dyes,however,at low salt concentration（≤40 g/L NaCl）azo reductase began to work,manganese peroxidase and lignin peroxidase could participate in the degradation of DBG.（3）In this study,UV-Vis,FTIR and HPLC-MS/MS were used to analyze the intermediate of the degradation of DBG.The results showed that the intermediate products of azo dye DBG degradation were 2,7,8-triaminonaphthalene-1-ol,2,4-triol,catechol,p-phenylenediamine,phthalic acid,4-hydroxy-2-oxopentanoic acid,aniline,succinic acid,and so on.Therefore,the possible biodegradation pathway of azo dyes was deduced as follow:under the catalysis of NADH-DCIP,the azo bond was broken firstly to produce aromatic amine compound 2,7,8-triaminonaphthen-1-ol and p-diphenylamine,which were then oxidized by manganese peroxidase and laccase to form cis-9-octadecenoic acid or 4-hydroxy-2-oxopentanoic acid.The degradation product could enter the TCA cycle and finally be metabolized to CO2 and H2O.（4）The high-throughput sequencing was used to analysis the microbial diversity of halophilic/salt-tolerant microbial communities.The results showed that when the NaCl concentration in the medium was 20 g/L,the composite flora was mainly composed of five genera:Enterococcus,unclassified＿f＿Enterobacteriaceae,Staphylococcus,Bacillus and Kosakonia.The most abundant were Enterococcus and Enterobacteriaceae,accounting for more than 90 percent.When the NaCl concentration increased to 40 g/L,the composite flora was changed greatly:Bacillus occupied an absolute advantage,while Enterococcus and Enterobacteriaceae were almost disappeared in the 40～80 g/L three groups.With the NaCl concentration was continuous increased,the degradation efficiency decreased,indicating that Bacillus played a key role in the degradation of DBG,while Enterococcus,Enterobacteriaceae and Staphylocccus played a certain role in promoting the degradation of DBG.（5）The results of seed toxicity test showed that when the seeds were treated with 600mg/L DBG,the germination rates of rice,maize and wheat could only reach 56.3%,47.4%and 42.5%,respectively.However,after dialysis and desalination of the supernatant after DBG degradation by the microbial flora,the germination rate of seeds could reach to 100%,indicating that the toxicity of DBG degradation by the halophilic/salt-tolerant composite flora was significantly reduced.Currently,the treatment of azo dye wastewater-biological method has become the focus of research at home and abroad,and the research on the degradation of salt-tolerant biological communities is relatively limited.So far,there were few reports on the degradation and decolorization of azo dyes by the halophilic/halotolerant microflora.This study would be great significance for the treatment of high salt and high toxicity dye wastewater. |
