Decolorizing Research Of A Shewanella Putrefaciens Strain Against Reactive Brilliant Red X-3B And Reactive Brilliant Blue KN-R

The traditional biological treatment based on bacteria system could not decolorize non-azo dyes, and decolorize the azo dyes under anaerobic conditions, but hardly under aerobic conditions. In our previous study, a strain which could decolorize azo dyes under both anaerobic and aerobic conditions and also could decolorize anthraquinone dyes under anoxic condition was screened and identified as a Shewanella putrefaciens strain, named S. putrefaciens 4H. In this paper, we studied the decolorizing characteristic and mechanism of strain 4H against reactive brilliant red X-3B, a typitical azo dye, and reactive brilliant blue KN-R, a typitical anthraquinone dye, to analysis the application potential of strain 4H in the biological treatment against dyes pollution.Firstly, the decolorizing characteristics of S. putrefaciens 4H against reactive brilliant red X-3B and reactive brilliant blue KN-R were studied. When cultures in LB which contained 50mg/L dyes, strain 4H showed the best performance of decolorization efficiencies and growth mass under the condition of 37℃, initial pH=8 and static cultivation, and the decolorization efficiencies was positively associated with bacteria growth mass. However, the aeration rates influenced the biomass and decolorization efficiencies greatly, the increasing aeration rates could result in the increasing biomass but result in decresing decolorization efficiencies for both two dyes.Then, we found that S. putrefaciens 4H decolorized reactive brilliant red X-3B by its extracellular enzyme. By PCR, the azo reductase coding gene was amplified in strain 4H. The sequence of PCR product was sequenced and showed high nucleotide identities(over 99%) with all azo reductase coding genes of S. putrefaciens strains in GenBank. The extracellular enzyme of the strain was extracted by ammonium sulfate precipitation and purified by affinity column. By SDS-PAGE analysis, the extracellular enzyme was identified as a 22 KDa protein, just like azo reductase. During this process, we found that the decolorizing activity of the extracellular enzyme against reactive brilliant red X-3B was lost after the dialysis, and the activity could be recovered again after the addition of coenzyme NADH, thus we concluded that this enzyme was a holoenzyme which was composed of enzyme protein and coenzyme. The best decolorization activity of the enzyme against 10mg/L reactive brilliant red X-3B was found under the condition of 50℃, 2mmol/L coenzyme NADH, initial pH=7, and in anaerobic conditions. The addition of coenzyme FMN alone could not influence the extracellular enzyme’s activity of azo reduction, but the addition of FMN and NADH both showed better activity than the addition of NADH alone. The synergistic effect between FMN and NADH on the extracellular enzyme’s azo reduction activity was directly proportion to the concentration of coenzyme FMN.Finally, the decolorization path of S. putrefaciens 4H against reactive brilliant blue KN-R was found that the dye molecules were adsorbed firstly and then were degradated by the cells. Growth factors, such as amino acids and vitamins, influenced the absorption efficiency greatly. Eight growth factors had been found that they could enhance the cells’ adsorption efficiency, the sequence of them is: cysteine > threonine > ascorbic acid(vitamin C) > alanine > > tryptophan asparagine > leucine > glutamine.The results showed that S. putrefaciens strain 4H has great potential of application in biological treatment against dyes pollution.