Isolation And Identification Of Dipropyl Phthalate Degradation Strain And Its Interaction Mechanism With Cooperator Strain Under Heavy Metal Stress

Dipropyl phthalate?DPr P?is a kind of the phthalates esters?PAEs?,which is an important organic compound used as plasticizers to enhance its plasticity and versatility.Meanwhile,DPr P,a typical environmental endocrine disruptor,is a refractory compound with a long residual period,which often forms PAEs-Cd composite pollution with heavy metal cadmium in the environment,has caused harm to the environment and organisms.Microorganisms have gradually become the main force in the remediation of heavy metal and organic pollutants,and play a major role in the reduction and fixation of heavy metal and the transformation and degradation of organic pollutants.In order to elucidate the response mechanism of microorganisms to PAEs-Cd composite pollution at the ecological and molecular levels,in this study,non-degradating strain,had collaboration function for degradating strain to degrade DPr P,was obtained via constructing artificial bacterial consortium under Cd²⁺ stressful condition,and the interaction mechanism between the degrading strain and the cooperator was explored at the level of phenotype and transcriptome.The results are as follows:?1?Strain ZM05 with highly efficient degradation ability of DPr P and various short chain PAEs was isolated from a long-term agricultural film-covered soil.It was identified as Arthrobacter nicotianae ZM05 based on morphology and 16 S r RNA characterization,and the intermediate metabolites were detected by HPLCMS.According to the aerobic general pathway of PAEs degradation,the possible metabolic pathway for DPr P degradation was proposed,i.e.DPr P was converted into mono-propyl/ethyl phthalate?MPP/MEP?and di-ethyl/methyl phthalate?DEP/DMP?through three routes,hydrolysis,?-oxidation and deesterification,by ZM05.After that,the jointly product mono-methyl phthalate?MMP?was produced and then MMP was hydrolyzed to form a typical intermediate product phthalic acid?PA?.PA is converted to protocatechuic acid?PCA?,and finally to TCA cycle by PCA pathway.?2?It was found that its growth lag period was prolonged and the degradation efficiency of DPr P was inhibited to a huge extend under heavy metal?cadmium ions?stressful condition.Strain ZM05 co-cultured with three non-degrading bacteria from the same soil sample,and the cooperator Acinetobacter junii ZM06 with the strongest collaboration fuction was obtained.The interaction mechanism between ZM05 and ZM06 was studied based on the cell surface morphology,concentration of free cadmium ions,biomass and DPr P degradation rate.Cd²⁺ was absorbed to form the "micro-sediment" on the bacterial cell surface in the co-culture system,thus alleviating the stress of heavy metals.ZM06 might ameliorate the stressful condition through sacrificing itself and helping ZM06 to recover the growth and degradation ability.Additionally,strain ZM06 could utilize the intermediate products that metabolized by strain ZM05,such as MEP,MMP,PA and PCA,which may accelerate the substrate consumption and enhance the degradation efficiency.?3?Through transcriptomic analysis with the whole genome sequence of strain ZM05 and ZM06 as the reference genomes,we found that there were 596,1731 and 959 of differential expression genes in monoculture of strain ZM05 with DPr P?group DP vs CK?,monoculture of strain ZM05 with DPr P and additional 0.8 m M of Cd²⁺?group CD vs DP?,and coculture of strains ZM05 and ZM06 with DPr P and additional 0.8 m M of Cd²⁺?group BU vs CD?,respectively.Most of the differential expression genes mainly refer to a seires of processes such as the quorum sensing and the metabolism and transport of carbohydrate,amino acid and energy.DPr P and Cd²⁺ had toxic effects on the strain ZM05 in mono-culture system,seperately.The presence of DPr P and Cd²⁺ aroused the stress mechanism and accelerated basic metabolic function to resist adversity and ameliorate its growth and degradation ability.Totally,962 genes were downregulated under the stress of Cd²⁺,accounting for 55.6% of the total number of differential genes in this group.The stresses by Cd²⁺ slowed down the growth rate of ZM05 and the degradation ability of ZM05 on DPr P,whereas cooperator ZM06 lived peacefully with ZM05,and alleviated the adversity effectively to reduce 71.5% of down-regulated differential genes.With assist of cooperator ZM06,ZM05 restored to normal growth rate and degradation ability by the restoration of expression levels on basal metabolic function-related genes.Meanwhile,when cocultured with ZM06,more degrade-related signal and metabolism pathways were activated in ZM05 to promote DPr P degradation,which indicated ZM06 could alleviate the stress of Cd²⁺ on the strain ZM05 and improve the degradation ability of ZM05 on DPr P.In return,ZM05 provides basic nutrients for ZM06.In summary,this study not only enriched the resource library of PAEs-degrading bacteria,but also illustrated the interaction mechanism between Arthrobacter nicotianae ZM05 and Acinetobacter junii ZM06 for DPr P biodegradation under Cd²⁺ stressful condition at the transcription level,which showed a miniature of the soil microorganisms cooperatively degradating refractory pollutants,and provided a new angle of view on the remediation of the PAEs-Cd contaminated soil.