Isolation And Identification Of A Strain Degrading Crystal Violet,the Characteristics Of Degradation And The Research Of Degradation Mechanism

Crystal violet is a kind of synthetic triphenylmethane dyes,and it is commonly used in textile, printing,dyeing and printing ink industry, environmental protection,mining, steel, medicine and the daily chemical analysis.Because crystal violet contains complex structure of aromatic nucleus, it has the characteristics of high chemical stability and low solvability, thus it can accumulate in the waste water and leads to serious surface water and groundwater pollution.Environmental toxicology showed that the crystal violet has side effects of toxic,,carcinogenic, teratogenic and mutagenic.Water pollution and the safety of aquatic products caused by crystal violet has caused wide attention and urgent needs to solve. advantages Biodegradation,with the advantages of efficient, safe, low cost and no secondary pollution,is one of the effective ways to remove pollution of the environment.So it has been an important research item for us to isolate microbial resource of high efficiency crystal violet-degrading, explore the role of the degradation mechanismn, and give full play to their degradation ability to remove the environment pollution. It has important theoretical significance and application value. This research aimed at isolating the strain that can efficiently degrade crystal violet, studying their degrading characterizes of crystal violet in different environments, exploring the strain degradation mechanism of crystal violet and providing the theory base for the bioremediation of environment pollution with crystal violet.The aim of the paper was to lay the foundation for degradation theory of triphenlmethane dye crystal violet by bacteria, and provided the guidance for application of a strain removing crystal violet, who belonged to Sphingomonas sp. In this paper, we stuied systematically the physiological and biochemical characteristics,degradation characteristics of the strain, and explored the mechanism of degrading crystal violet of the strain. The strain was isolated from the soil which had been contaminated by dyes for many years, and was named as SJ-1. Strain SJ-1 was identified preliminarily as Sphingomonas sp. according to its morphological observation, physiological biochemical test, comparison sequences of 16 S rRNA and phylogenetic analysis.The paper explored The optimal growth conditions of the strain SJ-1, the optimal growth temperature and initial pH of the strain SJ-1 were 30℃and 7.0,respectively.With the increase of ventilation, the growth of the strains was on the rise.In the selected several kinds of carbon source, glucose was the best carbon source.when using glucose as carbon source and organic nitrogen as nitrogen source, peptone was best nitrogen source;when using glucose as carbon source and Inorganic nitrogen as nitrogen source, ammonium nitrate was best nitrogen source.In the medium of LB,the growth of the strain SJ-1 in 0-6 h was the lag phase, the exponential phase was in6-24 h, the stationary phase was in 24 h later.The degrading properties of the strain SJ-1 for crystal violet were also studied.The strain SJ-1 could degrade 200 mg/Lcrystal violet in 3d. Our experimental results show that the temperature needed to remove strain SJ-1from the crystal violet is25-30℃, with the suitable pH being6.0-8.0. What is more, in the aerobic condition, it is preferable to decolorize the crystal violet so as to get strain SJ-1, though it may have some effects on the degradation power of strain SJ-1 crystal violet in the anoxic condition, which can also be degraded efficiently in the anoxic conditions. However, it is surprised for us to find that the degradation rate of the crystal violet remains still over 40% in a period of 4 h for the experimental groups, whose liquid volume is only over 125 mL.In the characterization study of degradation enzyme of crystal violet, enzymatic reaction system was established: The total volume was 5 mL, PBS 4.85 m L( 0.1mol/L,pH=7.0), crystal violet 50 μL(mass concentration was 200 mg/L), enzyme 100μL, reaction 1 min, terminated reaction with ice. Crystal violet residues were extracted with acetone, enzyme activity was determined by the reduction of crystal violet. An enzyme activity unit refered to the needed enzyme of degrading 1 μmol crystal violet within 1 min. Studies had shown that below 60 ℃ the enzyme activity of it loss slower,pH5.0-8.0 the enzyme activity maintain high, Methanol and isoamyl alcohol in less influence of the activity, while methylene chloride and acetone had a greater impact on the activity. There were 10 kinds of metal ions,such as Zn2+、Ca2+、Fe2+、Cu2+、Co2+,Fe2+had promoting effect on degradation of crystal violet, and other metal ions had a different degree of inhibition effect on degradation of crystal violet. Enzyme induction and localization experiments show that, the triphenylmethane reductase in SJ-1belonged to constitutive enzyme, and it located out the cell membrane.The primers were constructed based on the published sequences of thetriphenylmethane reductase gene. The triphenylmethane reductase gene was cloned by PCR from SJ-1. the base number is 864 bp, encoding 288 amino acid sequence. Inlevel of the gene, the comparison SJ-1 gene tmr2 sequences with the relative sequences in GeneBank revealed that eighteen base points of triphenylmethane reductase gene are the different among eight degrading strains. the similarity between them were 98% to100%; In the amino acid level, ten points of triphenylmethane reductase are different in eight degrading strains, Their similarity are 98% to 99%. According to differences in genes and amino acids can be divided eight strains into A, B and C,there subtypes.Three metabolites(recessive crystal violet, leuco malachite green and phenol) of methamidophos were identified, and the likely methamidophos biochemical degradation pathway of strain SJ-1was proposed. The first step is probably the crystal violet reductive metabolism into fat-soluble recessive crystal violetThe first step involved in crystal violet degradation is that crystal violet was reduced into:Leucine Crystal Violet, Then, leuco malachite green was produced by deamination,. Finally, leuco malachite green was likely transformed into phenol.