Dissection Of The Mechanism Of A Thermophilic Microbiome For Effective Azo Dye Decomposition

In this study,a previously developed thermophilic microbiota that can effectively decolor and degrade azo dyes was used as the research target,and the effects of environmental factors on the decolorization and degradation of the thermophilic microbiota were investigated.Furthermore,Ultraviolet and visible spectrophotometry?UV-Vis?,Fourier transform infrared spectroscopy?FTIR?,as well as High performance liquid chromatography-tandem mass spectrometry?HPLC-MS/MS?was used to analyze the intermediate of the degradation of Direct Black G?DBG?,and a possible degradation pathway of DBG was deduced.Besides,the dilution-to-extinction approach was performed,and the critical ratio of decolorization and degradation of azo dye by the thermophilic microbiota was determined according to the situation of decolorization and the key indicators in the degradation process of azo dyes under different dilution ratios.On this basis,Polymerase chain reaction-denaturing gradient gel electrophoresis?PCR-DGGE?and High-throughput sequencing technologies?HTST?were combined to sequence the samples under different dilution ratios to clarify the microbial diversity and key functional bacteria of the thermophilic microbiota.Finally,the metagenomic sequencing was used to analyze the differential gene between the before and after the critical ratio of decolorization and degradation of azo dye by the thermophilic microbiota,and the metabolism and signal transduction pathways involved in the degradation of dyes were clarified.Which provides a theoretical basis for the efficient biological treatment of azo dye wastewater.The main conclusions of this study are as follows:?1?Degradation characteristics study of the thermophilic microflora show that the best conditions of decolorization and degradation were as follows:temperature of 55?,the initial pH 8.0.The decolorization ratio could reach over 96%within 24 h of incubation when the concentration of DBG was maintained at the range of 200-600 mg·L^-1.In addition,it was observed over 88%of decolorization efficiency was observed after 48 h of incubation at high salt concentration?5%?.Besides,the thermophilic microflora reveals great potential in decolorizing dye wastewater containing various azo dyes,such as,Methyl Orange,Congo Red,DBG,Direct Black 38.?2?In this study,UV-Vis,FTIR,as well as HPLC-MS/MS was used to analyze the intermediate of the degradation of DBG.The results showed that-N=N-of DBG could be broken and the dye can be further degraded into small molecule compound by the thermophilic microflora,and some of these intermediates were identified,such as,2,7,8-triaminonaphthalen-1-ol,m-phenylenediamine,aniline,phthalicacid,4-hydroxy-2-oxovaleric acid.Accordingly,it is speculated that the first step in degradation of DBG by the thermophilic microflora was asymmetric cleavage of azo bond,which lead to formation of intermediate.Because of the instability of sulfonate group at high temperature,the desulphonation lead to the formation of 2,7,8-triaminonaphthalen-1-ol.Additionally,the initial degradation products also including m-phenylenediamine and aniline in the biodegradation of DBG by this microflora.The presence of these products demonstrated the azo bonds of DBG was broken.Besides,additional metabolites were also identified as phthalic acid and 4-hydroxy-2-oxovaleric acid.However,they can be further degraded into smaller compounds,and may eventually be mineralized to H₂O and CO₂.Besides,the above phytotoxicity analysis indicated the microflora can convert toxic dye DBG into low toxicity metabolites.?3?Dilution-to-extinction approach combined with PCR-DGGE and HTST was utilized to clarify the microbial diversity and key functional bacteria of the microbiota.It was found the strains belonging to Tepidiphilus sp.were identified as the most key functional microbes of this microbiota for effective azo dyes decolorization.Besides,the synergistic action of other microbes,such as Anoxybacillus sp.,Caloramator sp.,Clostridium sp.,Bacillus sp.was believed can further promote the decolorization process.?4?According to the analysis of the metagenomic sequencing of group A and B(A:solution of decolored dye in 10^-7;B:solution of undecolored dye in 10^-9),it can be concluded that the degradation of chlorocyclohexane and chlorobenzene,degradation of toluene,degradation of benzoic acid,degradation of aromatic compounds,degradation of xylene,and degradation of dioxin among the metabolic pathways with the most significant gene enrichment,were significantly different between groups A and B,and most of them were involved in the degradation of benzene-containing substances,suggesting that these metabolic pathways may have an important relationship with the decolorization and degradation of azo dyes.The genes involved in the important metabolic pathway of the degradation and decolorization of azo dye were annotated to KO including K16243,K16244,K16242,K16245,K16246,K00446,K16249,K10217,K10216,K18365,K01617,K01821,K00074,K00626,K18366 and K18364.According to the analysis,it was speculated that the gene corresponding to the gene entry?KO?associated with the significantly different metabolic pathways in group A and group B might be the key differential gene for the degradation of azo dyes in the thermophilic microflora.Besides,Fluorescence quantitative PCR?qPCR?was used to detect the differentially expressed genes in the results of the macronomic analysis,so as to further verify the results of macronomic sequencing.