| As one of the triphenylmethane dyes,malachite green is widely used in the textile printing and dyeing industry,and it has long-term use as a fishery medicine in China's aquaculture industry because of its good control effect on diseases such as fish water mold..Since the 1970s,a large number of reports have confirmed that malachite green has potential teratogenic,carcinogenic and mutagenic effects.Malachite green has been listed as a banned drug in fishery drugs in China.However,due to low prices and good bactericidal effects,there are still a small number of illegal traders.In addition,malachite green has a long residual period in the natural environment and is not easily degraded.Therefore,the malachite green degradation method needs to be studied.Due to the wide range of microbial degradation,high efficiency,and the ability to repair in situ,It is an effective means to treat malachite green residues in the environment.This paper takes the triphenylmethane dye malachite green as the research object,and collects the domesticated malachite green from the sediment in a certain aquaculture farm in JiangSu.The degrading microbial population and high-throughput sequencing analyzed the microbial community fauna changes of the three generations of the microflora,obtained the main microbial information related to degradation,and purposefully screened,and cultured to obtain the target strain JW3.-6.The degradation conditions of the strain were optimized,and the degradation products of malachite green were determined.The possible degradation pathways of JW3-6 degradation of malachite green were deduced by the detected degradation products.1.After continuous passage,the concentration of malachite green in the enrichment reached 300 mg/L.The enriched cultures of each generation were named JW1,JW2 and JW3,respectively.The sludge samples were named JWY and extracted separately.The total DNA of 4 samples was amplified by the 16S rRNA V4-V5 region,and the constructed amplicon library was sequenced and OTU(Operational Taxonomic Units)species annotated using the Illumina Hiseq 2500 platform.The results showed that JW3 was at the level of the gate,and the bacteria degrading the bacteria were mainly classified into Proteobacteria,Actinobacteria,Dependentiae,and Acidobacteria.At the level,the main bacteria of the degrading bacteria were divided.It belongs to the class of Gammaproteobacteria,Alphaproteobacteria,Actinomycetes.Actinobacteria?At the genus level,the flora is mainly divided into Pseudomonas,Labrys,Acinetobacter,Achromobacter,and Azoto Mesorhizobium,Methylobacillus,Acinetobacter,Acinetobacter.Pseudomonas sp.was found to be the main dominant flora,accounting for 0.93%of the total OTUS in the initial sample JWY and 67.81%in the JW3.The original OTUs in JW3 rose to 14.6%in the original OTUs with a total OTUs ratio of 0.16%,indicating that Labrys is also the main group involved in malachite green degradation,in addition to Acinetobacter Acinetobacter,Achromobacter,and Cupriavidus,the proportion of OTUs increased gradually after subculture,which is probably related to the degradation of malachite green.2.According to the information of the strains obtained by enrichment culture high-throughput sequencing,the third-generation enriched samples were selected for dilution coating on MSM solid plates containing 100 mg/L malachite green,and the colonies with different morphology were selected for treatment.Line and purify multiple times with the same plate.Finally,13 strains with high degradation rate of malachite green were screened,among which Pseudomonas was the most efficient,and the target strain JW3-6 was cultured in a cultivable manner.The similarity with Pseudomonas veronii was 99.69%..3.Optimize the degradation conditions of JW3-6 degrading bacteria to malachite green,set different gradient temperatures(15°C,20°C,25°C,30°C,35°C,40°C,45°C),pH(4,5,6,7,8,9)),different inoculum size(1×10~7,2×10~7,3×10~7,5×10~7,8×10~7,10×10~7 cfu/mL),studied different conditions for strain JW3-6 degradation of50 mg/L malachite green The impact of capacity.The effect on the degradation efficiency of malachite green at different initial concentrations(20,50,100,150,200,250,300)mg/L.The degradation efficiency of malachite green in the 7 d was 92.8%.The optimum degradation conditions of the strain were determined by single factor test to be 30°C,pH 7,initial inoculum size 5.0×10~7 cfu/mL.This study provides excellent microbial resources for the bioremediation of malachite green pollution.4.The qualitative products of JW3-6 degradation products were analyzed by GC-MS.The products were mainly leuco malachite green,4-(dimethylamino)benzophenone,4-dimethylaminophenol,benzaldehyde,phenol and para-benzene.Diphenol.According to the degradation products,the possible degradation pathways are:malachite green first demethylation reduction reaction to form recessive malachite green conversion,then converted to4-(dimethylamino)benzophenone and 4-dimethylaminophenol 4-(Dimethylamino) benzophenone is further decomposed into benzaldehyde and 4-dimethylaminophenol.The 4-dimethylaminophenol is further decomposed into dimethylaniline and phenol,and the subsequent stepwise decomposition products finally enter the TCA cycle. |
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