Biodegradation Of Lawsone By Pseudomonas LH-3 And Its Immobilization

2-hydroxy-1,4-naphthoquinone(lawsone)is widely used as medicine materials,rubber preservatives,herbicide raw materials and intermediate in the producing pesticide and dye.During its production and useage,it can get into water and soil through animal excretion and wastewater discharge.Because of its broad-spectrum cytotoxicity,it causes harm to animals and plants growth.Moreover,it pollutes the aquatic environment and can potentially threaten human health via food chain.In this study,a highly effective lawsone-degrading strain,LH-3,was isolated from the acclimated activated sludge in a pesticide plant in Jiangsu.It can use lawsone as the sole carbon source for growth.Based on morphological analysis,physiological and biochemical characteristics and 16S rRNA gene sequence analysis,strain LH-3 was identified.The growth and degradation efficiency were systematically charactered as well as its degradation dynamics.Furthermore,the degradation pathway of LH-3 was also studied by LC-MS.The strain LH-3 was immobilized in sodium alginate beads in which biochar(Using masson pine tree trunk as the feedstock to be pyrolyzed at 700?)was added to enhance the removal efficiency.The morphological analysis of the joint immobilized beads was conducted.The degradation behavior of the immobilized bacteria was tentatively investigated along with control assays in terms of different environmental conditions.In addition,the reuse ability was comparatively investigated.Finally,the operation performance of sequencing batch reactor(SBR)with immobilized strain was systematically evaluated.Strain LH-3 was preliminary identified as Pseudomonas taiwanensis.When cultured in LB liquid medium,the logarithmic period of strain LH-3 was between 4-12 h.After 12 h,the growth of strain LH-3 became stable,the growth rate of strain was slower,and the biomass began to decrease.Strain LH-3 had a better growth between pH 7.0-9.0 and temperature 25-35 ?.The most optimal growth condition was pH=7.0-9.0 and 30?.Ventilation condition can affect the growth of the strain,larger dissolved oxygen in miduim was more benefit for LH-3 growth.The strain could highly utilize glucose as the carbon sources and the followed is fructose.Additionly,the strain could highly utilize organic nitrogenas the nitrogen source,especially that it had a highest utilization of peptone than others.Environmental conditions such as pH,temperature,concentration of NaCl and initial concentration of lawsone had effects on lawsone degradation by strain LH-3.Strain LH-3 could degrade 200 mg·L-1 lawsone within 9 h.The optimal degradation conditions for strain LH-3 to degrade lawsone was temperature25-30 ?.pH 7.0-9.0,and inoculation rate at 1%.Strain LH-3 can effectively degrade lawsone under various initial concentration from 50 to 500 mg·L-1.However,when the initial concentration of lawsone increased to 600 mg·L-1,the growth of the strain was significantly inhibited.The biodegradation kinetics of lawsone by strain LH-3 was fitted via the Monod model and kinetic parameters were obtained:Vmax=179.9 hr-1,Ks=173.5 mg·L-1.The metabolites of lawsone degradated by LH-3 were detected by LC/MS,three new metabolites had been detected,and they were identified as catechol,2-hydroxy-4-oxo-chroman-2-carboxylic acid and salicylic acid.According to the identified metabolites,a tentative metabolic pathway was proposed.Lawsone was initially reduced to 1,2,4-trihydroxynaphthalene,which was unfortunately not detected.Noting that,the hydroxyl could increase the electron cloud density of naphthalene ring.As a result,the intermediate product 1,2,4-trihydroxynaphthalene was relatively unstable.That was probably why it was not detected in this study.Then,the fission of the ring 1,2,4-trihydroxynaphthalene was occurred,leading to the next intermediate product,4-hydroxy-4-(2-hydroxy-phenyl)-2-oxo-but-3-enoic acid.The possible intermediate product 4-hydroxy-4-(2-hydroxy-phenyl)-2-oxo-but-3-enoic acid quickly converted into 2-hydroxy-4-oxo-chroman-2-carboxylic acid for keto-enol tautomerism reaction Subsequently,salicylic acid was produced by the removal of side chain.After that,salicylic acid was further degraded to catechol,which was cleaved in the ortho-position by catechol 1,2-dioxygenase.Finally,the resulting cis-muconate acid was mineralized through the 3-oxoadipate pathway.BET results showed that the specific surface area of joint immobilization beads and SA+bacteria beads were 22.26 m2·g-1 and 4.02 m2·g-1,respectively.The adsorption-degradation performance of joint immobilization strains favored the degradation process.The joint immobilized bacteria showed better abilities than free bacteria in adapting to a wide range of temperature,pH,initial concentration of lawsone and heavy metal.Moreover,joint immobilized strains can be successfully reused for more than 9 times.In laboratory-scale SBR,the joint immobilized strain can degrade lawsone in a stable way.High-throughput sequencing data revealed that the bacterial richness and diversity was reduced by both bioaugmented treatments.The degrading bacteria Pseudomonas sp.played a significant role in the treatments.