Study On Isolation,Identification, Degradation Characteristics And Mechanism Of Malachite Green Degrading Bacteria

Malachite green pollution in aquatic environment leads to an increasing risk of malachite green contamination in aquatic product,causing significant threats to human health.Malachite green degradation by microorganisms has been shown of broad application prospects in aquatic environment.In this paper,strains with high-efficiency malachite green degradation ability were isolated and identified,and the degradation characteristics of strains were systematically studied.RNA-Seq was used to screen the key genes and metabolic pathways related to malachite green resistance and degradation.The preliminary mechanisms of the malachite green degradation were revealed at the transcriptome level.Recently,there is no feasible way for solving the problem of malachite green polution in aquatic environment.This study is important to provide a significant clue for selecting genes and studying mechanisms involved in malachite green degradation,and lay an important theoretical foundation for the development and utilization of malachite green degrading microorganisms and enzymes.1.KBH₄ reduction-HPLC method was applied to analyze the malachite green.At the malachite green spiked concentration ranged from 1 mg·L^-1 to 20 mg·L^-1,the recoveries of the method were 88.357%⁹4.135%with relative standard deviations of 0.035%².599%,which met the detection requirements.Two strains?WA-1 and B-20?with powerful ability of malachite green degradation were screened from the malachite green polluted sediments in aquaculture ponds.The degradation rate of WA-1 and B-20 at 20 mg·L^-11 malachite green in LB medium for24 h was 99.78%and 97.21%,respectively.After preliminary identification by API 20E identification card,VITEK 2 GN microbial identification system and 16S rRNA gene sequence,strain WA-1 wasidentified as Klebsiella sp.WA-1 and strain B-20 wasidentified as Enterobacter sp.B-20.2.Enterobacter sp.B-20 showed great degradation ability for 1⁴0 mg·L^-11 malachite green in LB medium.This strain could degrade over 92.2%malachite green at 1 mg·L^-11 and 82.4%malachite green at 5⁴0 mg·L^-11 within 12 h,and the malachite green degradation rate was over97%at 5²0 mg·L^–1 malachite green for 24 h.The malachite green degradation rate significantly increased with the increase of pH,and the maximum malachite green degradation rate was found at pH 9.Enterobacter sp.B-20 exhibited high salt tolerance and had stable malachite green degradation at 1%⁴%NaCl concentration.Enterobacter sp.B-20 showed strong metal ion tolerance and stable malachite green degradation at high concentrations of metal ions(0.1²mmol·L^-1).The metal ions including Fe³⁺at 0.1¹ mmol·L^–1,Cu²⁺at 0.1⁰.5 mmol·L^-1,Mn²⁺at0.1 mmol·L^–1 and Pb²⁺at 0.1¹ mmol·L^–1 increased the malachite green degradation rate of Enterobacter sp.B-20 significantly.3.Klebsiella sp.WA-1 could efficiently degrade malachite green in aqueous solution without any inorganic or organic nutrient substances.This strain showed great malachite green degradation ability in aqueous solution that could degrade over 94.0%malachite green at 1²0mg/L within 0.5 h,biodegrade over 94.4%malachite green at 1-40 mg/L within 12 h.The malachite green degradation rate of Klebsiella sp.WA-1 was significantly enhanced when the pH of malachite green aqueous solution increased,however,mainly resulting from the self-degradation afteraddition of NaOH solution.Klebsiella sp.WA-1 showed stable malachite green degradation at 1%⁴%NaCl concentration that the malachite green degradation rate at 5and 40 mg·L^-1 respectively reached to over 97.0%and 94.0%within 12 h.The maximum malachite green degradation rate was found at 37?,higher temperature was more suitable for the malachite green degradation than lower temperature.Klebsiella sp.WA-1 showed stable malachite green degradation at high concentrations of metal ions(0.1¹ mmol·L^-1).The metal ions including Mn²⁺at 0.1⁰.5 mmol·L^-1,Mg²⁺at 0.1¹ mmol·L^-1 and Cu²⁺at 0.1mmol·L^-1significantly improved the malachite green degradation rate of Klebsiella sp.WA-1.4.The RNA-Seq was used to study the differential gene expression of Enterobacter sp.B-20 at different concentrations of malachite green.The genes with Adjusted P-value<0.05 and|log2?FPKM foldchange?|>1 were assigned as differentially expressed genes?DEGs?and used for GO and KEGG pathway analysis.There were 115 DEGs between CK group and M5 group with80 up-regulated DEGs and 35 down-regulated DEGs in M5 group.There were 466 DEGs between CK group and M20 group with 229 up-regulated DEGs and 237 down-regulated DEGs in M20 group.The key genes and KEGG pathway related to the resistance and degradation of malachite green and its metabolites were selected.The ABC transporters for various cyclic compounds such as carbohydrates may involve in uptake of malachite green in Enterobacter sp.B-20.The multidrug efflux proteins from ABC,MFS,and RND superfamily transporters in Enterobacter sp.B-20 play an important role in the resistance of malachite green and its metabolites.The expression of genes coding for triphenylmethane reductase and genes related to aromatic compound degradation were up-regulated at different concentrations of malachite green,contributing to the degradation of malachite green and its metabolites in Enterobacter sp.B-20.