Isolation,identification And Degradation Mechanism Of Efficient DEHP-Degrading Bacteria

Phthalate esters（PAEs）are one of the most widely used plasticizers in plastic manufacturing and the well-studied environmental pollutants with endocrine disrupting properties.The widespread use of plastic has led to the global occurrence of PAEs pollution.At present,PAEs can be effectively removed from polluted environments by microbe-mediated degradation.Di-（2-ethylhexyl）phthalate（DEHP）has the highest residual concentration in agricultural soil-contaminated areas compared to other PAEs in most of China.Due to the poor water solubility and complex structure of DEHP,it is difficult to degrade under natural conditions,and has strong biological toxicity.Therefore,it is urgent to remediate the DEHP-contaminated environment.In this study,the efficient DEHP-degrading bacterium DNHP-S2 was isolated from Mollisol in northeast China.Through metabolite detection,whole genome sequencing and transcriptional analysis,its intermediate metabolites were identified and the key DEHP degradation genes and gene clusters were deeply excavated.The degradation mechanism of DEHP by strain DNHP-S2was systematically and comprehensively revealed.The main results of this study are as follows:（1）An efficient DEHP-degrading bacterium,Rhodococcus pyridinovorans DNHP-S2,was isolated and identified from the soil covered with agricultural plastic film for a long time,and the degradation characteristics of DNHP-S2 were also investigated.First,the effects of different culture conditions on the growth and degradation characteristics of DEHP within 72 hours were investigated.Optimal growth conditions for strain DNHP-S2 were defined as p H 9.0,35°C,and800 mg·L^-1DEHP.Under optimal conditions(p H 7.0,35°C,500 mg·L^-1DEHP),>99.75%DEHP degradation was observed for this bacterial strain.The inoculum size(OD₆₀₀=1.0)was 1%（v/v）.DNHP-S2 retained its ability to efficiently degrade DEHP at a broad range of substrate concentration and p H levels.Importantly,strain DNHP-S2 was able to mediate 52.74%DEHP(500 mg·L^-1)degradation at 10°C,indicating that the bacterium is a promising germplasm for restoring PAEs-contaminated environment at low temperature.DNHP-S2 was also found to readily break down other PAEs including dimethyl phthalate（DMP）,di-n-butyl phthalate（DBP）,di-n-octyl phthalate（Dn OP）and n-butyl benzyl phthalate（BBP）,the first-order kinetic model describes the biodegradation properties of PAEs well.These results suggest that DNHP-S2 is ideally suited to deployment as a tool for remediating PAE-contaminated environments.（2）A new biological metabolic pathway for DEHP was identified.The metabolites of DEHP were detected by high performance liquid chromatography time-of-flight mass spectrometry（HPLC-TOF-MS/MS）,and four key compounds,2-ethylhexyl benzoate（EHBA）,DMP,phthalic acid（PA）and benzoic acid（BA）were identified,BA is an intermediate product of anaerobic metabolism.DMP was first identified as an intermediate metabolite of DEHP,which is different from the previously reported DEHP catabolic pathway.Strain DNHP-S2 can utilize protocatechuic acid（PCA）,BA,catechol（PC）and PA as the sole carbon source,and it grew very well in high concentrations of toxic PC(1000 mg·L^-1),suggesting that strain DNHP-S2 has a high survival and detoxification ability in a polluted environment.（3）The 0-gap genome of the strain DNHP-S2 was successfully sequenced,including a circular chromosome（4829321 bp,GC 67.99%）and two circular plasmids（plasmid 2,150504 bp,GC 66.30%;plasmid 4,67375 bp,GC 64.93%）.In the genome,there are 4935 protein coding sequences（CDSs）were predicted.According to the results of functional annotation,some key genes and gene clusters of DEHP degradation were explored,such as multiple esterase coding genes,pca BLIJCHG,xyl LZYXEGHI,mhp DEF and acetyl-Co A acyltransferase（fad A）.All esterases annotated by DNHP-S2 genome exhibit relatively limited amino acid similarity with the six esterases previously reported to degrade PAEs,suggesting that the amino acid sequence conservation of esterases was low.However,the amino acid sequence of catechol 1,2-dioxygenase and that of WP＿006553165.1（from Rhodococcus sp.）was 100%identical,and compared with many other strains,these identities were higher than 99%,suggesting that the amino acid sequence of catechol 1,2-dioxygenase is highly conservative.According to the subcellular localization results,the above key enzymes were all located in the cytoplasm,suggesting that the degradation of DEHP and its metabolic intermediates was intracellular degradation.（4）The genome sequencing data generated suggest that benzoate 1,2-dioxygenase,catechol2,3/1,2-dioxygenase may play integral roles in the process of DNHP-S2-mediated DEHP degradation.The real-time quantitative polymerase chain reaction（RT-q PCR）showed that when DNHP-S2 was grown on DEHP,benzoate 1,2-dioxygenase,catechol 2,3/1,2-dioxygenase m RNA levels were found to be 64.82-fold,26.93-fold,and 30.66-fold increased over those in glucose as the sole carbon source,respectively.With the increase of DEHP concentration,the expression of three target genes firstly increased and then decreased.These results suggested that DEHP or DEHP metabolic intermediates might induce the efficient expression of three target genes,indicating that there are two metabolic pathways for downstream degradation of DEHP.These DEHP degradation-related genes are still efficiently expressed at 10°C,indicating that strain DNHP-S2 holds great promise as a resource capable of fully degrading DEHP even at a lower temperature.