Degradation Mechanism Of Typical PAEs In Soil Applied With Acinetobacter Baumannii DP-2

Plastic film,which was the fourth largest agricultural production material after seeds,fertilizers and pesticides,and was widely used in various agricultural production practices.However,a large amount of plastic film has been discarded in soil due to the incomplete recycling system in the past decades.This can not only bring microplastic pollutions to the soil environment,but also cause phthalic acid esters（PAEs）pollutions in soil.Due to the"three toxicity" characteristics of PAEs,their accumulation in soil can seriously endanger the soil ecological security.Therefore,it is particularly important to carry out in-depth research on the PAEs pollution remediation technology to maintain soil health.Microbial remediation technology,with the characteristics such as environmental friendliness and no secondary pollution,had become a potential effective technology for soil PAEs pollution remediation.However,there were still some scientific problems,such as the lack of excellent broad spectrum PAEs degrading strains,the incomplete microbial degradation mechanism of PAEs,need to be investigated.Therefore,this study investigated the degradation characteristics and mechanisms of typical PAEs in soil using stain Acinetobacter baumannii DP-2（hereinafter referred to as DP-2）,which was previously screened by our research group.Firstly,a total of fifteen PAEs pollutants was selected to investigate the PAEs degradation broad spectrum of DP-2.Secondly,the environmental conditions of DP-2 were optimized by the combination of single factor and response surface methodology tests in which,dibutyl phthalate（DBP）,bis（2-ethylhexyl）phthalate（DEHP）and dicyclohexyl phthalate（DCHP）were selected as the representatives of typical straight-chain,side-chain and cyclic PAEs.Then,the PAEs degradation performance of DP-2 in soil was verified in a soil culture experiment.Finally,the degradation mechanism of DP-2 on typical soil PAEs was clarified with a combination of metabolite analysis,whole genome sequencing and transcriptomics analysis,The main results are as follows:（1）This study confirmed the good broad-spectrum PAEs degradation performance of strain DP-2.For the eight tested straight-chain PAEs,the PAEs degradation rate of strain DP-2 increased gradually with the increase of the number of alkyl carbon atoms.The DBP treatment exhibited the highest degradation performance with a value of 94.52%,while the DMP treatment showed the lowest degradation rate（42.30%）.Among the four tested side-chain substituted PAEs,strain DP-2 had the highest degradation rate（92.33%）of diisobutyl phthalate（DIBP）and the lowest degradation rate（27.75%）of dimethoxyethyl phthalate（DMEP）.For the three tested cyclic substituted PAEs including butyl benzyl phthalate（BBP）,diphenyl phthalate（DPHP）and dicyclohexyl phthalate（DCHP）,the corresponding degradation rates were 89.87%,80.56%and 62.91%,respectively.（2）The cultivation conditions of DP-2 for the degradation of DBP,DEHP and DCHP were optimized by single factor and response surface experiments.The optimal cultivation conditions of strain DP-2 degrading typical straight-chain PAEs（DBP）were as follows:NaCl concentration was 5g·L-1,inoculation amount was 15.14%,and the initial DBP concentration was 10.81 mg·L-1.The theoretical and measured DBP degradation rates were 86.45%and 85.86%,respectively.The optimal cultivation conditions of DP-2 for typical side chain PAEs were:NaCl concentration of 20.00 g·L-1,inoculation amount of 19.10%,and initial concentration of DEHP of 10.66 mg·L-1.the theoretical and measured DEHP degradation rates were 82.09%and 81.75%,respectively.The optimal cultivation conditions for DP-2 degrading typical cyclic PAEs（DCHP）were:NaCl concentration of 19.18 g·L-1,inoculation amount of 16.85%,initial DCHP concentration of 7.18 mg·L-1.The theoretical and measured DCHP degradation rates were 78.90%and 78.86%,respectively.（3）This study verified the remediation performance of strain DP-2 in DBP,DBP/DEHP,DBP/DEHP/DCHP contaminated soil,and analyzed the changes of soil microbial diversities after DP-2 was inoculated in to soil for PAEs remediation.The results showed that the strain DP-2 could maintain a relatively stable PAEs degradation rate（64.81%-69.42%）in both single PAEs contaminated soil and multiple PAEs contaminated soil,moreover,the PAEs degradation amount of DP-2 increased to 97.50 mg·kg-1 in multiple PAEs contaminated soil while it was only 34.71 mg·kg-1 in single PAEs contaminated soil,which proved DP-2 had a higher PAEs degradation ability in multiple PAEs contaminated soil.Strain DP-2 had a significant on the soil bacterial community composition and structure when it was inoculated into three types of PAEs contaminated soil.The relative abundance of Firmicutes and Proteobacteria in soil was significantly increased,and the relative abundance of Bacillus niacini in Firmicutes was significantly increased.In addition,the application of strain DP-2 had no significant effect on fungal community composition and structure in soil.（4）This study investigated the main degradation pathways of PAEs by DP-2 by metabolite analysis,and verified the functional genes of the key biochemical processes of PAEs degradation based on whole genome sequencing.Taking DBP as the typical PAEs,it was first degraded through β-oxidation,demethylation and hydrolysis into dimethyl phthalate（DMP）and monobutyl phthalate（MBP）.The above substances were further converted into phthalic acid（PA）and benzoic acid（BA）,which were subsequently converted into adipic acid,and then enter into the tricarboxylic acid cycle（TCA）,finally,converted into CO2 and H2O.The biodegradation pathways of DBP by DP-2 were verified according to whole gene sequencing analysis.The homology comparison analysis annotated one monooxygenase（pobA）and three hydrolases（EstDP1,EstDP2,EstDP3）,which were related to the biodegradation process relating the conversion from PAEs to PA.In the benzoic acid metabolic pathway of xenobiotics biodegradation and metabolism pathways,we screened out a total of nine functional genes,namely mhpA,pcaG,pcaH.pcaB,pcaC,pcaI,pcaD,pcaJ and fadA,which were mainly related to the biological transformation process of PA.（5）The differential degradation mechanisms of DBP,DEHP and DCHP by strain DP-2 were preliminarily analyzed by transcriptome sequencing.KEGG Pathway enrichment analysis showed that the expressions of encoding genes fahA,alkB1₂,hprT and catB,which were related to aromatic compounds degradation such as benzoic acid and styrene,and drug metabolism were upregulated during PAEs degradation process of DP-2.Compared with CK,the expression of encoding gene fahA increased 2.1 times in DP-2 under DBP treatment,while the expressions of alkB1₂ and hprT in DEHP treatment increased about 12.75 and 2.01 times,respectively,and the encoding genes of catB,alkB1₂ and hprT were upregulated 2.04,3.75,and 2.04 times under DCHP treatment.In summary,this study confirmed the good PAEs degradation broad-spectrum characteristics of strain DP-2,and clarified the degradation pathway of PAEs by strain DP-2,moreover,the biodegradation mechanisms of strain DP-2 which can efficiently degrade typical PAEs in soil were revealed using whole genome and transcriptome sequencing technology.The research results can enrich the PAEs degrading microbial strain resources in China and provide theoretical basis and technical support for microbial remediation of PAEs pollution in soil.