Screening And Degradation Mechanism Analysis Of Effective Herbaceous Plant-microorganisms Of DEHP In Wetlands

Di（2-ethylhexyl）phthalate（DEHP）is the most typical phthalate esters（PAEs）.It is mainly used as plasticizer for plastic products and agricultural films.Based on the reproductive toxicity of DEHP and wide detection in the environment,it has been attracted much attention by humans.It is a typical endocrine disruptor and organic pollutant.Herbaceous plant is an important part of wetlands and play an important role in the removal of wetland pollutants.However,the research on DEHP pollut ion,potential risks and degradation of DEHP by herbaceous plant in plateau wetlands is still lim ited.The various wetlands in Qilu Lake watershed are typical wetlands polluted by plastic film for long time due to intensive agricultural plant ing activit ies.In this study,we investigated the level of DEHP pollution in surface sediments and water of different types of wetlands in Qilu Lake watershed.Nine typical herbaceous rhizosphere soils were collected from wetlands polluted by PAEs(PAEs content was about18.87～37.36 mg·kg^-1)in Fuxian Lake and Xingyun Lake watershed to breed DEHP-degrading microorganisms.A DEHP-degrading strain B6 was isolated and screened from rhizosphere soil of Cyperus alternif olius L.,and the main pathways and mechanisms of DEHP degradation by rhizosphere microorganisms of Cyperus alternif olius L.were analyzed by Cyperus alternif olius L.and Cyperus alternif olius L.rhizosphere microorganisms.The results are as follows:（1）The pollut ion level,heterogeneity and potential ecological risks of DEHP in different types of wetlands in Qilu Lake watershed were comprehensively analyzed.The results revealed that DEHP concentrations were significantly higher in rainy season than in dry season.During the rainy season,DEHP concentrations in surface water ranged from 0.035 to 2.32μg·L^-1,while they var ied from 0.05 to1.57μg·L^-1 in the dry season.Correspondingly,DEHP concentrations in surface sediments ranged from0.22 to 37.45μg·g^-1 in the rainy season,and 0.15 to 22.70μg·g^-1 in the dry season,respectively.Furthermore,the concentration of DEHP was considerably higher in r iver wetlands（RWs）than in other types of wetlands both in surface sediment and water.DEHP content in water ranging from 0.11 to2.32μg·L^-1 in RWs,0.1 to 1.67μg·L^-1 in CWs,and 0.035 to 1.32μg·L^-1 in LWs.DEHP was the dominant PAEs in the surface sediments and water of wetlands.Moreover,ecological r isk assessment suggested that DEHP posed a higher risk to the water of various wetlands in the Qilu Lake watershed,with calculated ecological risk coefficient（RQ）values of 4.5194 to 6.1328 for sensitive species of algae and 0.1022 to 0.6896 for crustaceans and fish.（2）The rhizosphere soils of 9 typical herbaceous plants were collected from the wetlands polluted by PAEs and screened a strain of DEHP efficient degrading bacteria B6.The nine herbaceous plants are Typha latif olia L.,Acoruscalamus L.,Canna indica L.,Phragmites australis T.,Tulbaghia violacea L.,Cyperus alternif olius L.,Nelumbo nucif era G.,Sagittaria sagittarif olia L.,and Pontederia cordata L.Strain B6 was isolated from the rhizosphere soil of Cyperus alternif olius L.in the Luju river wetland.The strain showed 100%similar ity with Bacillus simplex based on the experiment of physiological and biochemical characteristics,morphological ident if ication,and 16S r DNA sequence identif ication.The optimal degradation condit ions for strain B6 were p H of 7.0,temperature of 30℃,4%inoculum amount,and initial concentration of 100 mg·L^-1 DEHP.Under optimal condit ions,the growth curve of strain B6 showed a trend of first increasing and then decreasing with time,and the DEHP degradation rate gradually increased with time.The residual concentration of DEHP in the inorganic salt culture solution on the seventh day was 2.09 mg·L^-1,with a 97.91%degradation rate.（3）The degradation rate of DEHP by Cyperus alternif olius L.-rhizosphere microorganisms was elucidat ed by indoor hydroponic experiment.Rhizosphere degrading bacteria and Cyperus alternif olius L.could promote the degradation of DEHP in water.Under DEHP stress,the degradation rates of Cyperus alternif olius L.to high DEHP concentration(10 mg·L^-1)and low concentration(1 mg·L^-1)treatment were 12.22%and 26.21%,the degradation rates of Cyperus alternif olius L.and rhizosphere degrading bacteria to high/low DEHP concentration treatment were 31.20%and 34.90%,and the degradation rates of Cyperus alternif olius L.and rhizosphere degrading bacteria to high/low DEHP concentration treatment were 44.34%and 45.15%.The degradation of Cyperus alternif olius L.without inoculat ion was not significant.There was no signif icant difference between DEHP degradat ion rate and init ial DEHP concentration in all treatments.（4）Analysis of main metabolites in rhizosphere of Cyperus alternif olius L.under DEHP stress.Metabonom ics studies showed that the main metabolites of rhizome under DEHP stress were lipids and lipid molecules,organoheterocyclic compounds,organic acids and derivatives,organic oxygen compounds,organic nitrogen compounds,phenylpropanoids and polyketides,nucleosides nucleotides,and analogues.Similar ly,lipids and lipid molecules,phenylpropanoids and polyket ides,organoheterocyclic compounds,organic oxygen compounds,benzenoids,organic acids,and derivatives were identified as the predominant metabolites in the root of Cyperus alternif olius L..Compared with the control,187 metabolit es were produced in root exudates by adding degrading bacteria,of which 170were up-regulated and 17 were down-regulated.There were 277 differential metabolites in the roots of Cyperus alternif olius L.,of which 103 were up-regulated and 174 were down-regulated.The most signif icant metabolic pathways for rhizosphere enrichment were glycerophospholipid metabolism（Glycerophospholipid metabolism）,pantothenate and cocoa biosynthesis（Pantothenate and Co A biosynthesis）,beta-alanine metabolism（Beta-Alanine metabolism）and tryptophan metabolism（Tryptophan metabolism）.The most significant enrichment pathways were phenylalanine metabolism（Phenylalanine metabolism）,nicotinate and nicotinamide metabolism（Nicotinate and nicotinamide metabolism）in the root of Cyperus alternif olius L.（5）Analysis of degradation pathway of DEHP in rhizosphere of Cyperus alternif olius L..Tryptophan metabolism and Phenylalanine metabolism were important metabolic pathways of DEHP degradation in rhizosphere of Cyperus alternif olius L..The potential degradat ion pathways for DEHP in the root exudates of Cyperus alternif olius L.were identified as follows:DEHP was degraded into MEHP and DBP init ially,with 2,3-butanol serving as an intermediate in the conversion of DBP.Subsequently,MEHP and DBP were converted to PA,w hich was then decarboxylated into benzoic acid derivat ives.The benzoic acid derivatives were primarily metabolized by the Tryptophan metabolism pathway,where they were converted to 2-amino-2-phenylacetic acid,2-keto-6-acetocaproic acid,and other acids then enter the tricarboxylic acid cycle.Similarly,the principal pathways of DEHP degradation in the roots of Cyperus alternif olius L.involved the init ial steps of DEHP degradation to MEHP and DBP by phthalic acid 2-ethylhexyl acyl hydrolase.MEHP and DBP were degraded into PA,which was then completely metabolized into benzaldehyde,p-hydroxybenzaldehyde,protocatechuic aldehyde,etc.,before conversion into benzoic acid derivatives that enter the benzoic acid metabolism.Phenylalanine metabolism pathway could be the main metabolic pathway of phenylacetaldehyde,which was further converted into nicotinic acid（C00253）,2,4,6-trimethoxyphenyl acetate,etc.This study analyzed the pollut ion level and potent ial ecological r isks of DEHP in different types of wetlands.Through screening and isolat ion of DEHP-degrading microorganisms in rhizosphere of 9typical wetland herbs,a DEHP-degrading strain B6 was screened in rhizosphere of Cyperus alternif olius L.,revealed the degradation characteristics and possible metabolic pathways of DEHP by rhizosphere microorganisms of Cyperus alternif olius L.Provide strain resources for the elim inat ion of DEHP in water environment,and theoretical basis or reference for the application of Cyperus alternif olius L.constructed wetland in the treatment of PAEs pollut ion in water environment.