Isolation And Characterization Of A DEHP Degrading Bacteria

As a major plasticizer,di(2-ethylhexyl)phthalate(DEHP)is widely used in agriculture,industrial,and daily life.Because its hardly photolyzed and hydrolyzed,it can accumulate in the environment,and cause persistent and serious pollution to various environments such as water,atmosphere,soil and sediments along with the transfer,absorption and evaporation.DEHP is considered to be an endocrine disruptor and it produces reproductive toxicity to the human body.Reports of developmental toxicity and teratogenic effects have been increasing.For the currently selected strains with low degradation efficiency and unsatisfactory results in practical applications,our research aims to screen out a strain capable of degrading DEHP completely and study its degradation characteristics,degradation mechanism and contaminated soil remediation.This study isolated a gram-negative aerobic strain Methylobacterium sp.F05 R capable of utilizing multiple phthalates and efficiently degrading di(2-ethylhexyl)phthalate(DEHP)from landfill topsoils.After optimizing the culture conditions including p H(8.0),temperature(35oC)and inoculum size(2.0%),sodium acetate with a 2:1 concentration ratio(sodium acetate/DEHP)was used as a co-metabolite to provide primary energy for strain F05 R so that it could degrade DEHP of 20 and 50 mg/L completely,and 200 mg/L 92.2% within 7 days.The growth kinetics of strain F05 R with Ks and Ki of 51.22 and 471.84 were described using the Haldane substrate inhibition model.With the increase of substrate concentration,the specific growth rate of strain F05 R first increased and then decreased.The maximum specific growth rate ?max could reach 0.02 h-1 at an initial concentration of about 200 mg/L,suggesting the existence of substrate inhibition,the higher substrate concentration,the greater the inhibitory effect on the strain,but the inhibition constant Ki is much higher than other DEHP degrading bacteria,indicating that the inhibition is relatively weak,that is,strain F05 R can be used for bioremediation of variable DEHP pollution environment.Substrate broad-spectrum experiment showed that strain F05 R could use DBP,BBP,and Dn OP of 200 mg/L as the sole carbon sources for growth.The degradation rates of these 3 PAEs were 85%,84%,and 74% within 6 days,respectively.Besides,strain F05 R has a greater ability to degrade short-chain DBP than long-chain Dn OPs and have potential to repairing environment contaminated with PAEs.DEHP degradation intermediates were detected by GC-MS,the analysis showed that DEHP was degraded to monoethylhexyl phthalate(MEHP)and phthalic acid(PA)before complete mineralization.Genome of F05 R was sequenced to obtain 5,832 coding genes with a size of 4,882,725 bp.The genome annotation indicated that strain F05 R had an esterase gene E1.1.1.60,dioxygenase genes similar to pht3,pht4 and lig A,and decarboxylase gene pht5 which encoded DEHP degradation.Combining with GC-MS mass spectrometry and the genomic annotation of F05 R,it is presumed that DEHP hydrolyzes the ester bond to PA via gene E1.1.1.60,then under the action of pht3 and pht4,PA hydroxylated to 4,5-Dihydroxyphthalate,then decarboxylation by pht5 to form protocatechuic acid.Protocatechuic acid is ligated with dioxygenase encoded by lig A to form 4-Carboxy-2-hydroxymuconate semialdehyde,subsequent passage via the action of lig C,lig I and lig K,eventually degrades into pyruvate and oxaloacetate into the TCA cycle,thereby achieving complete mineralization of DEHP.Soil experiments showed that strain F05 R had an effective degradation effect on DEHP-contaminated soil.When the water-soil ratio was 2:1 and the culture rotation speed was 180 r/min,it could be used individually to degrade the DEHP in contaminated soil from 50 to 11.6 mg/kg within 7 days,and the degradation rate reached 76.7%.Using the first-order kinetics equation to fit the degradation of DEHP in contaminated soil,it was found that strain F05 R,after bioenhancement of the DEHP-contaminated soil,formed a synergistic effect with the inherent microorganisms in the natural soil,greatly enhanced the removal rate of DEHP in the soil.And increased the DEHP degradation rate constant of nature soil from 0.0102 to 0.0146,which shortened the half-life of DEHP from 3.96 days to 1.98 days.It shows great potential in effectively restoring DEHP-contaminated soil.The above results of this study demonstrate strain F05 R is a promising biological resource,which has great potential for bioremediation of PAEs,not only has a greater degradation rate of DEHP in the aqueous phase,but also has a good advantage for degradation of DEHP in soil.