| Azo dye,one of the largest industrial synthetic dyes,has been widely used in printing and dyeing,textile,leather,and food processing industries.Though it brings convenience to people's lives,azo dye also produces a huge amount of highly toxic wastewater and causes a serious threat to human health and the ecological environment,due to the lack of effective treatment measures.However,the biological process,an environmentally friendly method,has been widely used in the treatment of azo dye wastewater because of its advantages of simple operation,complete degradation,no secondary pollution,and low cost.Therefore,using biological process has become a research hotspot in the treatment of azo dye wastewater in recent years.In this study,the azo dye Direct Black G was selected as the research object,and a thermophilic strain Anoxybacillus sp.PDR2 with high degradability was screened and isolated.The influence of external factors on the decolorization and degradation of the strain was investigated,and the intermediate metabolites of dye were identified.Then,the possible degradation pathways were speculated,and verified the detoxification of dye after degradation.Besides,to further explore the key functional genes and their mechanisms for the efficient degradation of dye by strain PDR2,genome sequencing technology was used to excavate the full genetic background of strain PDR2 while its efficient degradation of azo dyes and its adaption of wastewater,and the gene clusters for the degradation of azo dyes by strain PDR2 were speculated.Subsequently,transcriptome sequencing technology was used to analyze the differentially expressed genes of strain PDR2 under the stress of azo dye Direct Black G,and combined with RT-q PCR technology,the key functional genes of degradation of Direct Black G were deduced,which preliminarily reveals the degradation mechanism of strain PDR2 to Direct Black G.The main research results are as follows:(1)A strain with high degradation ability to Direct Black G was screened and purified from the soil that has been polluted by azo dye for a long time.Through morphological observation and biochemical identification showed that the rod-shaped strain was Grampositive strain,and it was found that the bacterial colony was round,gray,translucent,thick,and moist,with neat edges.The 16 S r RNA sequencing confirmed that the strain was identified as a Anoxybacillus genus and named Anoxybacillus sp.PDR2(KP221933).(2)Through optimization experiments on decolorization and degradation conditions of the strain,the results showed that stain PDR2 had the best decolorization effect on Direct Black G under the conditions of glucose as carbon source,beef extract as nitrogen source,p H=7.0,and temperature at 55 ?,which allowed the decolorization rate of Direct Black G reached more than 95% after 48 h culture.The strain had a strong tolerance to high concentration of Direct Black G.When the initial concentration of Direct Black G achieved even 600 mg/L,the decolorization rate could still reach 82.12%.Besides,the strain had high decolorization and degradation ability for azo dyes with different structures(Amaranth,Methyl Orange,Congo Red,Direct Black 38 and Direct Black G),whose decolorization rate reached 95.87-99.18% after 48 h culture,showing good degradation broadness.(3)The intermediate metabolites of Direct Black G were detected by Ultraviolet-visible spectroscopy(UV-Vis),Fourier infrared spectroscopy(FTIR),and Liquid-mass spectrometry(LC-MS).The results showed that its degradation products mainly include 2,7,8-Triaminonaphthalen-1-ol,p-phenylenediamine,1,7-naphthalenediamine,2-naphthylamine,phthalic anhydride,and 4-aminobenzoic acid.According to the degradation products,the possible degradation pathways of Direct Black G are inferred: first,the azo double bonds of Direct Black G are cleaved to form intermediate metabolites 2,7,8-triaminonaphthalen-1-ol,p-phenylenediamine and 1,2,4-triaminobenzene,and then the intermediate metabolite 1,2,4-triaminobenzene is deaminated to p-phenylenediamine,while another intermediate metabolite 2,7,8-triaminonaphthalen-1-ol removes part of the amino group and hydroxyl group to form 1,7-naphthalenediamine,which further occurs ringopening reaction and finally generates 4-aminobenzoic acid.Besides,the intermediate metabolite 1,7-naphthalenediamine can also be deaminated to produce 2-naphthalenediamine,which will be further oxidized to form phthalic acid and eventually dehydrated to form phthalic anhydride.In addition,through the results of the germination condition of green beans and rice seed after being treated with Direct Black G and its degradation products,the measurement of activity of antioxidant enzymes(SOD,CAT,POD,PPO,APX)in root cells of the two plants and the analysis of DNA damage of human liver cancer cells Hep G2,it was found that the biotoxicity of degraded Direct Black G was significantly lower than that of undegraded Direct Black G,which indicated that strain PDR2 had a better detoxification effect on Direct Black G.(4)Based on genomic techniques,all the genetic information of strain PDR2 degrading azo dyes and its adaptive to complex wastewater environment were excavated.It was found that the genome of strain PDR2 was 3791144 bp.The GC content was 42.48%,and there were 4098 protein-coding genes,accounting for 84.14% of the genome.Strain PDR2 contained genes encoding cytochrome P450,the degradation of aromatic compounds,benzene and various heterogeneous pollutants,electron transfer system,which may be involved in the degradation of azo dye.In addition,strain PDR2 contained genes encoding chemotactic,motor,heat/cold shock proteins,two-component system,sigma factor,transposase,and integrase,which may be the basis for strain PDR2 to adapt to complex azo dye wastewater environment.Moreover,based on the reported key degrading enzymes,three gene clusters of azo dye degrading were deduced.Among them,the size of gene cluster A was 11265 bp,composed of 9 genes arranged together.There are seven genes mainly responsible for improving the basic metabolism of the strain,providing a large amount of energy,reducing power and electrons for the strain to degrade azo dyes.The gene encoding FMN-dependent NADH-azo reductase(GM001587)took charge of destroying the azo dye chromogenic group(-N=N-),while the gene encoding Methyl-accepting chemotaxis protein(GM001596)was responsible for enhancing the strain's adaptability to the complex azo dye wastewater environment.The size of gene cluster B was 2511 bp,which was composed of four genes arranged together.Among them,the genes encoding FMN-dependent NADH-azo reductase(GM00710)and catechol-2,3-dioxygenase(GM00734)were mainly responsible for breaking nitrogen-nitrogen double bond and destroying benzene ring structure.The gene encoding NAD(P)H: quinone oxidoreductase(GM000693)was responsible for improving the activity of TCA cycle and glycolysis,providing reduction equivalents for the degradation of dyes,and the gene encoding Flavodoxin(GM000735)was responsible for improving the activity of electron transport system,providing a large number of electrons for the process.The size of gene cluster C was 10278 bp and consisted of 11 genes,including a series gene encoding NADH ubiquinone oxidoreductase and NADH dehydrogenase,whose main functions were providing energy and electrons for the degradation of azo dyes.(5)Based on transcriptome techniques,the gene expression of strain PDR2 under Direct Black G stress and normal glucose culture was further investigated.The results showed that the expression of 226 differential genes was up-regulated,and 183 genes were downregulated.In addition,processes of basal metabolism in the strain were all increased,which provided a large amount of energy,reducing power and electrons for the degradation process,and enhanced the capacity of the strain to the environment of complex azo dye wastewater.Through functional annotation analysis of differential genes and RT-q PCR verification,it was found that the relative expression levels of genes encoding NAD(P)H-flavin reductase,2Fe-2S ferredoxin,and NAD(P)-dependent ethanol dehydrogenase under Direct Black G stress were significantly higher than those in normal glucose culture,so they are highly likely to be involved in the degradation of Direct Black G,and it is believed that the degradation is closely related to the electron transport chain.By the above results,the degradation mechanisms of azo dye Direct Black G by strain PDR2 are preliminarily speculated: the upregulated expression of pyruvate kinase gene(GM003276)enhances the activity of glycolytic metabolism.Then,pyruvate is further converted into a large amount of acetyl Co A under aerobic condition,which accelerates the metabolism of the TCA cycle and produces a large amount of energy.Besides,a large amount of NADH: quinone oxidoreductase also improves the activity of the TCA cycle and glycolytic metabolism,producing a large amount of reducing equivalents,which are further used to promote the activity of the electron transport chain system and generate more electrons and energy.Finally,these electrons are transferred outside the cell and directly accepted by Direct Black G to achieve the processes of reduction and degradation.In this study,through the analysis of the degradation characteristics of strain PDR2,it was found that strain PDR2 was a strain with great application potential,and its degradation pathway,functional genes and degradation mechanism were preliminarily explored,in order to provide a good research basis for in-depth analysis of its molecular degradation mechanism and bioremediation field. |
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