Degradation Of Dibutyl Phthalate By Glutamicibacter Sp. Strain 0426

Dibutyl phthalate(DBP)is a kind of phthalate acid esters(PAEs),which is a common used as plasticizer.It can cause functional changes in the central and peripheral nervous systems.And it also has teratogenic,carcinogenic,mutagenic effect[1].DBP is a priority pollutant by the US Environmental Protection Agency(EPA)and the European Union(EU)[2].In the process of environmental migration,hydrolysis,volatilization and photolysis are not important reaction processes for PAEs.In other words,they are difficult to be degraded in the environment and are refractory substances.The biological method is the main way to mineralize PAEs in the natural environment.Among various methods of PAEs removal,microbial degradation is considered to be most effective[3].The purpose of this thesis is to study biodegradationof DBP by bacterium.The studies were conducted as follows.(1)Isolation of DBP-degrading bacterial strains were carried out using oil samples collected from different polluted areas in eight provences.A DBP degrading strain which utilized DBP as the sole source of carbon and energy was isolated with a yellow colony.It was identified as Glutamicibacter sp.by 16S rRNA gene sequencing and named as Glutmicibacter sp.strain 0426.(2)The strain 0426 was inoculated into the carbon-free mineral medium supplemented with DBP to test its degradation ability,incubated in the dark at 30 ? and pH=7,shaking at 180 rpm.when the initial concentration of DBP was 300 mg/L,the degradation rate was 99.9%after 11 h,and acomplete degradation was achieved within 12 h.After biodegradation,it was observed that the medium turned light yellow and DBP oil-drop disappeared.Along with the degradation of DBP,strain 0426 gradually increased from 1.8 ×107 CFU/mL to 8 ×107 CFU/mL.As the initial concentration of DBP was 500 mg/L,99.6%of them could be degraded within 20 h.Additionally,DBP of 700 mg/L could be nearly completely degradaed(99.8%)after 36 h,while 99.5%of 1000 mg/L DBP could be degraded within 40 h.Biodegradation kinetics was analysed.And DBP degradation at different concentrations(300,500,700,1000 mg/L)could be described by exponential model,and followed the first-order kinetic equation,the degradation rate constant were 0.2351,0.1006,0.0719,0.0552,respectively,R2>0.91.The kinetic equation showed that with the increase of the initial concentration of DBP,the.degradation rate constant decreased and the degradation half-life prolonged,indicating that with the increase of the initial concentration of DBP,the degradation rate decreased.(3)The effect of temperature and pH on the degradation of 300 mg/L DBP was investigated,DBP could completely degraded within 12 h under the condition of 30,35,37 and 40 ?.And the degradation efficiency was higher at 35 ?.In contrast,the degradation efficiency of DBP was lower at 20 and 25 ?.The result indicated that DBP degradation efficiency decteased with the relatively low tempreture,while the optimal tempreture was 35?.On the other hand,DBP was nearly completely degraded after 12 h at pH 6,7 and 9,while the degradation rate was faster at pH 7.DBP could not be degraded at pH 5.Thus,DBP could not be degraded in acidic condition,while the optimal condition for degradation was at pH 7.(4)Genome DNA of strain 0426 was extracted and subjected for genome sequencing is available under GenBank accession number NZ_MPBI00000000.Strain 0426 has a genome size of 3,554,926 bp and aG G+C content of 63.05%.It contains 3304 CDSs,3 rRNA,64 tRNA,and 67 ncRNA.No plasmid was detected.The genes involved in degradation were predicted screened based on the genome annotation and their expression activity was analyzed.In the case of reverse transcription PCR analysis,all the tested genes were induced to express with DBP as the sole carbon source,demonstrating that the enzymes encoded by these genes are active and involved in degradation reactions.The transcription activity of these DBP metabolic genes was analyzed by real-time quantitative PCR,as strain 0426 was cultivated with DBP and fructose as the sole carbon source,respectively.The results showed that the transcriptional activity of the metabolic genes were up-regulated,indicating that they could be induced by DBP.(5)The metabolites of DBP were analyzed by GC-MS.Six degradation products were obtained,including phthalic acid,butyl methyl phthalate,ethyl methyl phthalate,dimethyl phthalate,methyl phthalate,and butanoic acid.Based on the identified metabolites and the putative enzymes involved in degradation,a complete DBP-degradation pathway pathway was deduced in strain 0426 was conducted.In the proposed pathway,DBP was converted to phthalic acid via hydrolysis.While the BMP,EMP,DMP,and methyl phthalate was formed via a transesterification reaction by phthalic acid.Phthalic acid was converted to protocatechuic acid via hydroxylation.Following a successive oxygenolytic ring-cleavage,rearomatization,decarboxylation and hydrolysis,protocatechuic acid was transformed into p-ketoadipate enol lectone,which finally transformed into succinyl-CoA and acetyl-CoA,and entered into TCA cycle.(6)Bioaugmentation of DBP-supplemented soil samples with strain 0426 was conducted.In the sterile soil sample bioaugmented with strain 0426,96.6%of DBP was removed after 3 d,and 98.1%was degraded after 6 d,in contrast to the non-bioaugmented sterile soil sample.While in the non-sterile soil sample,bioaugmentation with strain 0426 led to the complete removal of DBP after 6 d.The non-sterile soil sample without bioaugmentation also showed DBP deletion,however,only 45.6%of DBP was removed after 6 d and kept remaining afterward.The result showed that the indigenous flora in the soil could affect the degradation rate of DBP by strain 0426,however,it also had a certain promotion effect.