Accelerating Effect Of Anthraquinone Dye Intermediates In Decolorization Of Azo Dyes And Their Aerobic Degradation

The purpose of this dissertation is to investigate the characteristics of Sphingomonas xenophaga QYY with the ability to decolorize 1-aminoanthraquinone-2-sulfonic acid(ASA-2) under aerobic conditions,and analyze anthraquinone ring cleavage reaction.Decolorization of azo dyes by Strain QYY under anaerobic conditions and the final products were also studied. Based on this,effect of ASA-2 and bromoamine acid(BAA)on the decolorization of azo dyes by Strain QYY was also investigated.Mechanism of anthraquinone compounds mediated decolorization of azo dyes is proposed.The characteristics of Strain QYY growth and the possible pathway of ASA-2 decolorization in basal mineral medium were investigated.The optimal conditions for both growth and decolorization process are as follows:pH 7.0,temperature 30℃,shaking speed 100-150 r·min^-1.Decolorization efficiency of 3.3 mM ASA-2 in 120 h is over 98%.ASA-2 metabolites were identified using analytical instruments such as UV-Vis,HPLC-MS,H-NMR, etc.The main metabolites are 2-(2'-hydroxy-3'-amino-4'-sulfo- benzoyl)-benzoic acid, 2-(2'-amino-3'-sulfo-6'-hydroxy-benzoyl)-benzoic acid,o-phthalic acid and 2-amino-3-hydroxyl-benzenesulfonic acid.A possible pathway of ASA-2 decolorization by Strain QYY is proposed.The presence of the two metabolites implies the involvement of a Baeyer-Villiger type reaction.Cell extracts were obtained by sonication and used for decolorization activity assays. The results show that the ASA-2 decolorization activity in Strain QYY is induced by ASA-2. Moreover,the ASA-2 decolorization activity is only detected in the presence of NADH.The optimal temperature and pH for the crude enzyme are 50℃and 7.5,respectively.The ASA-2 decolorization enzyme(s)is very unstable and lose its activity completely after storage overnight at 4℃in phosphate buffer.5 mM metyrapone obviously inhibits the decolorization activity of the crude enzyme.So the major enzyme responsible for ASA-2 decolorization is a NADH-dependent oxygenase.Strain QYY can not only degrade ASA-2 under aerobic conditions,but also decolorize azo dyes under anaerobic conditions.The optimal conditions for decolorization process of Acid Red 3R are as follows:4 g·L^-1glucose,pH 7.0,temperature 30℃.Acid Red 3R is reduced to 4-amino-1-naphthalenesulfonic acid and 7-hydroxy-8-amino-1,3-disulfonate -naphthalene,which were identified using HPLC-MS.The comparison of the decolorization rate of four sulfonated azo dyes with similar structure is as follows:V_{Acid Red B}＞V(Acid Red GR)＞V(Acia Red 3R)＞V(amaranth).The results show that the decolorization rate of azo dyes is greatly influenced by the quantity and position of sulfonate groups in the structure of azo dyes.It also is influenced by the concentration of azo dyes.The decoIorization rates of Acid Red B increase with the increase of its concentration,when its concentration is below 1 mM.But its decolorization rates are obviously inhibited when its concentration is above 1 mM.Under anaerobic conditions,ASA-2 and BAA as redox mediators can improve the decolorizaiton rate of Acid Red 3R.When the concentrations of ASA-2 and BAA reach 0.4 mM,the decolorization rates increase 4.9-fold and 2.9-fold,respectively,compared to those lacking two compounds.The results show that ASA-2 is a more effective redox mediator than BAA.The addition of ASA-2 results in significantly increased decolorization rate of sulfonated azo dyes by Strain QYY.Moreover,ASA-2 could accelerate the decolorization of azo acid dyes tested with E.coli,and ASA-2 was also effective in combination with nonadapted activated sludge.This show that the general applicability of ASA-2 for the decolorization of sulfonated azo dyes.Five repeated experiments show that ASA-2 and BAA mediated reduction of sulfonated azo dyes and two anthraquinone compounds degradation by Strain QYY could be achieved under the optimal conditions in an anaerobic-aerobic process.The mechanism of anthraquinone compounds mediated decolorization of sulfonated azo dyes was discussed.AQS is the most effective redox mediator for Strain QYY.Reduction peak of AQS was detected using UV-Vis spectrum.AQS mediated decolorization of Acid Red B was analyzed.In this process the reduction of AQS is limit step.Cell membranes and cytoplasm extracts were separated by ultracentrifugation.The quinone mediated decolorization activities of sulfonated azo dyes were studied.PQQ-dependent D-glucose dehydrogenase can be concerned with quinone mediated decolorization of sulfonated azo dyes. FMN-dependent azo reductase and FMN-dependent AQS reductase in cytoplasm extracts were detected by nondenaturing PAGE.These reductases may be important in the decolorization of sulfonated azo dyes.