Preliminary Study On The Degrading Charicteristics And Mechanisms Of Phthalic Acid Esters-degrading Bacteria

Phthalic acid esters(PAEs)are widely used as plasticizers to improve the flexibility and transparency of plastics.PAEs are not chemically bound to the polymer and tend to release into environment with time and use.They have become widespread contaminants in sediment,natural water,wastewater,and soils because of the extensive use of plastics.Dimethyl Phthalate(DMP),Diethyl Phthalate(DEP),Dibutyl Phthalate(DBP)and Di-(2-ethylhexyl)phthalate(DEHP)are the most frequently detected PAEs in the environment,among which DEHP is the pollutant of high concentration because of its low water solubility and complex molecular structure.PAEs are hepatotoxic,teratogenic and carcinogenic by nature,and have been proved to be potential estrogenic endocrine disruptors.PAEs can be degraded by hydrolysis,photolysis and biodegradation,while microbial biodegradation is considered to be the major way to remove PAEs from environment.Many PAEs-degrading bacteria have been isolated and reported.However,bacteria of high degrading efficiency are lack and the bacterial degradation mechanisms need further investigation.In this study a bacterial consortium being able to degrading PAEs was obtained by enrichment cultivation,several bacteria of high degrading efficiencies were isolated and the degrading characteristics and mechanisms of the consortium and isolated bacteria were explored.The main research contents and results of this study are as follows.(1)Enrichment of a DEHP-degrading bacterial consortium and studies on its degrading characteristics and metagenomeA microbial consortium,designated as SD-1,being able to mineralize di-2-ethylhexyl phthalate(DEHP)was enriched from activated sludge.SD-1 was in a flocculent precipitate form and could grow with DEHP as sole carbon and energy sources.SD-1 mineralized about 90% of DEHP(ca.1000mg/L)in 72 h.The degrading ability of SD-1 was stable during more than 2 years' subculture.The optimal DEHP degrading conditions of SD-1 were 25-35°C and pH7-10.The degradation rate was not significantly affected by different organic and inorganic nitrogen and carbon sources.SD-1 degraded DBP,DEP and DMP effecitively,with decreasing rate as the length of alkyl side-chains getting shorter(DEHP>DBP> DEP>DMP).No significant accumulation of intermediate products was detected in the medium.The microbial community of the consortium was analyzed using MiSeq Illumina platforms,which demonstrates that Pseudomonas spp was the dominant bacteria through the whole growth phase.The metagenome of SD-1 consortium was sequenced using MiSeq Illumina 2000 platforms,and about 14 Gb DNA data was abtained.Based on assemble result of metagenomics sequences,seven genome drafts at genus level were abtained.Functional genes related to DEHP degradation,including carboxylic hydrolase,pht gene cluster and pca gene clusters were found in the sequences.(2)Isolation and characterization of DEHP-and its intermedeates-degrading bacterial strains from SD-1Several bacterial strains capable of utilizing DEHP as solo carbon resource were isolated from SD-1 consortium,designed as JM-1,J-1,J-11,KJ-1 and KJ-2.Their degradation efficiencies of DEHP were 92.51%,43.17%,40.32%,53.59% and 54.13%,respectively.Phthalic acid(PA)is the most common metabolic products of PAEs.The degradation experiment with PA as sole carbon and energy source indicated that KJ-2 and KJ-1 could not utilize PA and JM-1 could degrade PA with low degradation rate.The metabolic intermediates MEHP and PA of JM-1 degrading DEHP were detected and identified using GC-MS,and the degradation pathway was deduced as DEHP?MEHP?PA?….To obtain more efficient PA degraders,SD-1 was enriched using PA as solo carbon source,named as SD-P consortium.PA-1 and PA-3 capable of utilizing PA were isolated from SD-P.The microbial community of SD-P consortium was analyzed using MiSeq Illumina platforms,which demonstrates that Rhizobium spp.and Pigmentiphaga spp.were the dominant bacteria,and followed by Arthrobacter spp.and Bosea spp..PA-1 was identified as Bosea sp.and PA-3 was identified as Rhizobium sp.based on 16 Sr RNA gene sequence.PA-3 could completely degrade PA in 36 h,and PA-1 could remove 73.3% PA in 60 h.(3)Isolation and characterization of a DMP-degrading bacterial strainA bacterium,named as DB-7,capable of utilizing dimethyl phthalate(DMP)as sole carbon and energy sources for growth was isolated from soil with plastic film mulching by an enrichment culture technique.This bacterium was identified as Comamonas testosterone by 16 S rRNA gene sequence analysis and phospholipid fatty acid assay.DB-7 could degrade more than 99% of 450 mg/L DMP in 14 hours,and degraded DMP of different concentrations rapidly.The optimal degradation temperature and pH were 30-35 °C and pH 9.0,respectively.The degradation rate of DMP was positively related to inoculum volume of the bacterium.The result of HPLC and LC/MS analysis of metabolic products indicated that the major degrading intermediates were mono-methyl phthalate(MMP)and phthalate acid(PA)during the degradation of DMP by DB-7.Partial sequences of three genes involved in PA metabolism were detected in DB-7,and the expression of phthalate 4,5-dioxygenase was drastically induced in the present of DMP and PA.