| Dibutyl phthalate(DBP)is a kind of organic aromatic pollutants that has three induced toxicity,exists in most of the environment.And most of all,it is hard to be degraded.DBP as a plasticizer is used in a variety of industrial and agricultural production activities.At present,there is evidence shows that DBP can produce toxic effects on human body,animals,plants and microorganisms.While the DBP in the environment can also be enriched through the food web,and ultimately affect human health.Therefore,the research on the environmental remediation of DBP is urgent.The photolysis and hydrolysis of DBP under natural conditions are relatively slow,and the degradation of DBP is mainly done by microorganisms in the environment.However,in the complex environment,the degradation efficiency of microorganisms has been limited.Also,microorganisms in their way of DBP degradation process would produce toxic by-products,or some microorganisms can not completely degrade DBP,their degradation process stopped at the stadge of DBP degraded to phthalic acid,and phthalic acid is also a kind of environment pollutants.Therefore,it is of great value for environmental remediation study of the degradation process of DBP,and the study should analyze the intermediate product,and the degradation process.In this experiment,a Pseudomonas strains DNB-S1 with a high degradation ability of DBP were used as the research bio-material.The functional bacteria cultured in inorganic salt liquid medium contains two kinds of carbon source,DBP(500mg/L)and glucose(6g/L)were analyzed to obtain the changes of the metabolites,respectively.Metabolites were collected at 24 h and their metabolites were analyzed by LC-MS/MS technique.The comparison of metabolic DBP pathway under different conditions were conducted,and the information of DBP degradation was obtained by metabonomics analysis.Further study on the metabolic mechanism of DBP metabolism in DNB-S1 strain were investigated by measure the responses of DBP degrading bacteria to different concentrations of intermediate metabolites.The following results were obtained through experiments and data analysis:Through the HPLC-MS/MS detection,12689 ions were identified in the mass spectrometry data.And a total of 1329 differentially expressed ions were screened out by metabonomics analysis,of which there were 970 up-regulated metabolites,and 359 down regulated metabolites.Most of these metabolites are aromatic compounds and lipids,which are Benzenoids,Lipids and lipid-like molecules,Organoheterocyclic compounds,Organooxygen compounds and Phenylpropanoids and polyketides,specifically.Among these differential metabolites,only 3 of them are toxic intermediates.And the toxic intermediates including the phthalic acid which is known produced at the DBP degradation process.Compare these differential metabolites information to the KEGG database,the metabolites were found mostly participate in the metabolic pathways and Microbial metabolism in diverse environments pathways.Also,there were 12 of them participated in the amino sugar and nucleotide sugar metabolism,10 of them were in the starch and sucrose metabolism,4 in glycolysis,6 in carbon metabolic pathways,and these differential ions are mostly endogenous metabolite compounds.Through the comparison to the KEGG database,we found some important intermediates of DBP degradation pathway,such as phthalic acid,salicylic acid,4-hydroxy benzoic acid,acid,etc.It can be concluded that the DNB-S1 strain not only has the ability to degrade DBP efficiently,but also has the ability to completely mineralized DBP.The open loop cleavage pathway of DNB-S1 strain for DBP should be the pathway of protocatechuic acid and gentianic acid.DNB-S1 strain was cultured with 2mM phthalic acid,protocatechuic acid,gentianic acid and n-butanol as sole carbon source,respectively.The results showed that DNB-S1 strain could not directly use phthalic acid,protocatechuic acid,gentianic acid and n-butanol as the sole carbon source for the growth.The strains were cultured with different concentrations of gentianic acid,protocatechuic acid and n-butanol under the circumstances of DBP as the carbon source to determine the effects of three different concentrations of the intermediate products on growth and degradation rate of DBP in strain DNB-S1.The results showed that the concentration of 1 and 3mM gentianic acid could promote the growth rate of DNB-S1 strain,but with the concentration at 5mM,the strain's started after 48 h of shake flask test.The results of protocatechuic acid's effects showed a similar trend.However,n-butanol decreased the growth and degradation rate of the strain.DNB-S1 strain,as a highly efficient DBP degrading bacteria,can degrade DBP by the gentianic acid pathway and the protocatechuic acid pathway,and can completely mineralized DBP to carbon dioxide and water.The results showed that the DNB-S1 strain had great value in repairing the DBP polluted environment.Therefore,the study on the regulation of DBP metabolism in the functional strain is of great value. |
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