Mechanism And Application Of The Degradation Of Azo Dyes And Antibiotics By Aspergillus Fungus

Azo dyes and antibiotics are two types of emerging pollutants,their discharge and transfer in aquatic and terrestrial environments cause serious harm to environmental ecosystems.Compared with physicochemical methods,biodegradation technology shows great potential in organic wastewater treatment.Fungal-mediated biodegradation has advantages over bacteria.Fungi can secrete many non-specific oxidase to decompose stubborn organic matter.During the process of degrading azo dyes,the toxic intermediate product aniline will not be accumulated,and the degradation of antibiotics is not easy to cause the spread of resistance genes.At present,the research on the fungal degradation of azo dyes and antibiotics is mainly the aerobic action of filamentous fungi.Although filamentous fungi can secrete enzymes related to anaerobic metabolism and may have efficient anaerobic degradation capacity,few relevant studies have confirmed it,especially the research on the anaerobic degradation mechanism.In this study,Aspergillus tabacinus LZ-M was screened and obtained,which could degrade azo dyes and antibiotics under anoxic condition.It was found that it could completely mineralize the two organic compounds,indicating that it had efficient degradation ability.Therefore,on this basis,with Aspergillus tabacinus LZ-M as the research object,acid red 73（AR73）and metronidazole（MNZ）as the model pollutants,the anoxic degradation effect of LZ-M on these two organic compounds was analyzed.The degradation pathways were analyzed by metabolomics.The degradation mechanism was revealed by combining genomics,comparative transcriptomics and proteomics.The potential application of strain LZ-M in municipal wastewater treatment was explored by immobilized bioaugmentation technology.The specific research results are as follows:（1）The results of degradation experiment analysis showed:strain LZ-M grew under aerobic condition,decomposed glucose to produce CO₂ under anoxic condition,and effectively degraded different dyes and antibiotics.The degradation rates of 400mg/L AR73,400 mg/L methyl orange,200 mg/L neutral red and 30mg/L malachite green were 90.28%,86%,87%and 63.23%,respectively.The degradation rates of 5mg/L MNZ,10 mg/L furazolidone and 10 mg/L 3,5-dinitrosalicylic acid were75.02%,87.17%and 68.13%,respectively.The results showed that the degradation ability of LZ-M in anoxia was significantly better than that in aerobic culture.（2）On the result of（1）,metabolomic analysis of AR73 metabolites by LC-MS showed that AR73 was hydrolyzed to 2-hydroxy naphthalene and N-phenylnitamide,and then mineralized to CO₂.During the degradation process,the electron transfer caused carbon oxidation and nitrogen reduction,which revealed that AR73 was completely mineralized through the autoredox process.This revealed a new degradation mechanism of azo dyes for the first time.Carbon balance and electron balance analysis showed that 31.8%of the carbon in AR73 could be mineralized and converted to CO₂ by strain LZ-M under anoxic condition.（3）Comparative transcriptomic analysis of AR73 degradation showed that genes involved in aromatic degradation,glycolysis,cytochrome C and quinone oxidorereductases showed high transcription levels and significant up-regulation,which further indicated that the aromatic compounds produced by AR73 degradation were mineralized to produce CO₂ through catechol pathway and glycolysis process.However,the key decolorizing enzyme,the hydrolase gene that decomposes AR73into 2-hydroxynaphthalene and N-phenylnitamide,has not been found.At the same time,unknown genes accounted for 59.23%of the up-regulated genes,indicating the existence of a large number of proteins with unknown functions.Further cloning of these unknown genes revealed a novel hydrolyzed decolorizing protein Ord95 with significant AR73 decolorization capacity,using NADH as electron donor.Based on product identification,AR73 was cleaved by this enzyme to 2-hydroxynaphthalene and N-phenylnitamide,which was consistent with the degradation products of LZ-M.Ord95 is the key decolorization enzyme for AR73 degradation by LZ-M.Through SWISS-MODEL structural analysis,the enzyme contains a glutaredoxin domain.Point mutation assay confirmed that 38R,54R and 55R were the key active sites in the domain.（4）The pathway analysis of MNZ metabolites by LC-MS showed that-C-C-bond break,-C-N-bond break and hydroxylation occurred during the degradation of MNZ,and acetylene and urea were finally generated.The metabolomics analysis of its intracellular metabolites showed that the metabolites in the amino acid metabolism pathway were significantly up-regulated,and the transformation reactions involved in them were related to hydrolases,transaminases and oxidoreductases.（5）The results of comparative transcriptome analysis of MNZ degradation showed that transaminase,methyltransferase,monooxygenase,-C-N-lyase and hydrolase related genes were significantly up-regulated.These enzymes may be key enzymes involved in the cleavage of-C-C-,-C-N-,and hydroxylation in MNZ degradation.Proteomic analysis also revealed that these enzymes were highly expressed in LZ-M.This indicated that LZ-M secreted transaminase,methyltransferase,monooxygenase,-C-N-lyase and hydrolase to degrade MNZ through cleavage of-C-C-and-C-N-bond,and hydroxylation.（6）Further application of strain LZ-M in the anaerobic treatment of MNZ wastewater by immobilized bioaugmentation technology showed that the survival rate of LZ-M in the activated sludge system could be increased when LZ-M were fixed by sponge and then added to the anaerobic activated sludge system for bioaugmentation experiment.At the same time,using sponge as carrier,LZ-M and activated sludge could coexist in sponge pores.After the immobilized biological bioaugmentation,the system can effectively remove MNZ from wastewater,and the removal rate of COD increases by 7.12 times,and the gas production increases by 6.14 times.In addition,bioaugmentation reduced the toxic effects of MNZ on activated sludge,promoted its metabolism and growth,and protected the microbial community of activated sludge from MNZ.Through nim gene detection,it was found that MNZ could cause the increase of resistance genes in the anaerobic degradation system,while the bioaugmentation reduced the abundance of resistance genes.These results indicate that bioaugmentation with immobilized LZ-M is an effective method to protect the anaerobic activated sludge system in the presence of MNZ contamination.In this study,Aspergillus tabacinus LZ-M obtained from soil can efficiently degrade new pollutants,including a variety of azo dyes and antibiotics,and at the same time,the key degradation enzymes and genes were explored.This study provided new microbial resources and research ideas for fungal degradation of azo dyes and antibiotics.At the same time,it provides a new method for the application of fungi in the treatment of antibiotic wastewater and the treatment of resistance genes.