Screening And Identification Of Moderately Salt-Tolerant Bacteria And Degradation Of Triphenylmethane Dyes

The dye wastewater of trimethylbenzene is a typical wastewater of chemical industry.Generally,the discharged wastewater has the characteristics of bright color and high salinity,such as the typical trimethylolane dye-malachite green dye.It was used as fish water antimicrobial agents at times.Later,it was found that the dye had obvious"trisomy"effect.Therefore,people began to pay attention to the environmental problems caused by the discharge of such waste water.More and more scholars began to use microorganisms to degrade dye wastewater.The microorganism which living in the high salt environment has its unique physiological metabolic mode.As a result,the microorganism can adapt to the environment of the high salt dye wastewater and avoid to achieve the purpose of salt tolerance by acclimating the strain specially.In this paper,a salt-or salt-tolerant strain was isolated and screened from the salt field of zhoushan shuangfeng salt works.Then,a salt-tolerant strain with good decolorization ability to malachite green was selected,we study its physiological and biochemical characteristics,decolorization conditions and so on.Finally,through the whole genome sequencing analysis,the basic information of the genome of the bacteria was preliminarily understood from the genetic perspective.The main research contents and results are as follows:（1）Eight strains of halophilic（resistant）bacteria were isolated and purified on the solid medium of high salt ATCC213 by using the traditional coating separation and marking purification methods.According to the analysis of 16s rDNA sequencing,the eight strains of bacteria are mainly Staphylococcus,Bacillus,Halomonas,Salinivibrio,Halobacterillus and Pontibacillus.（2）Through malachite green decolorization experiment,it was found that strain A4 had excellent decolorization performance.After morphological observation and phylogenetic analysis of 16s DNA sequence,it was identified as Staphylococcus saprophyticus JJ-1.The determination of the salt concentration growth of strain S.saprophyticus JJ-1 showed that the strain JJ-1 could grow at the concentration of sodium chloride from 0 to 150 g/L,and the optimal concentration of sodium chloride was 0 g/L.Therefore,S.saprophyticus JJ-1 is a typical extreme salt-tolerant bacterium.（3）The effects of malachite green concentration,decolorization pH,sodium chloride concentration and oxygen on dye decolorization were studied,and the decolorization products were analyzed by uv-visible absorption spectroscopy,HPLC and gc-ms to reveal the degradation products of malachite green.A preliminary study of decolorization conditions showed that S.saprophyticus JJ-1 could decolorize 200mg/L malachite green as high as 95%within 4 hours.At low concentration of malachite green（<50mg/L）,the bacteria could rapidly decolorize within 1 h.Under aerobic and anaerobic conditions,the decolorization rate of the bacteria was basically the same.Under the condition of 15%NaCl,200 mg/L malachite green,the decolorization rate after 5 h was 94%.When the amount of inoculated bacteria OD₆₀₀ was 0.5,200mg/L malachite green was completely decolorized in 2 hours.The suitable pH of decolorization was 7-8,the culture time was 4h and the decolorization rate reached95%.The main degradation product of malachite green is 4-（dimethylamino）diphenylketone.（4）Whole genome sequencing showed that the genome of S.saprophyticus JJ-1was composed of four contig,with a size of 2,638,586 bp,GC content of 32.31%and predicted encoding genes of 2,546.Genomic analysis revealed 1 CRISPR system and3 gene islands.