Studies On Isolation, Identification And Degradation Characteristics Of PAEs-degrading Bacteria Strains

Phthalate acid esters (PAEs) are a group of synthetic chemicals, which are used mainly as additives in plastics to impart flexibility but also used in food wraps, furniture, toys, and cosmetics, for example. A large number of PAEs are not chemically bound to the products and may subsequently escape into the environment during the manufacturing process, during product use, and/or after product disposal. As a result of the large quantities produced and their widespread distribution, phthalates have become ubiquitous environmental pollutants. The available literatures suggest that the routs of environmental degradation of PAEs are hydrolyze, photodegradation and biodegradation, microorganisms are responsible for the complete destruction of PAEs in the environment.In this paper, six soil samples were obtained from six province ,china. And several PAEs-biodegrading strains were isolated from these samples. According to the conserved sequence of phthalate3, 4-dioxygenase from gram positive bacteria in the GenBank of NCBI, a set of degenerate primer were designed and successfully amplified the phthalate3,4-dioxygenase gene from the six strains which named JDC-1,JDC-3,JDC-8,JDC-9,JDC-11,JDC-12, respectively. Based on morphological, physio-biochemical characteristics and homology identification of 16S rDNA sequence,four strains (JDC-1,JDC-8,JDC-9,JDC-12) were identified as Arthrobacter sp. And JDC-3, JDC-11 were belonging to Delftia sp., Rhodococcus sp. respectively. In addition homology of phthalate3,4-dioxygenase between Arthrobacter sp.was compared; Experiments on the kinetics of DMP degradation by JDC-3 was conducted;Metabolism of DBP by strain JDC-11 was also analysised.The results are as below:(1) The gene encoding phthalate3,4-dioxygenase was cloned in Arthrobacter sp. for the first time in china .the results of compare the homology showed that they were highly homologous to each other . The optimal growth conditions and degradation rates of DBP were tested and the result indicated that the optimal growth conditions of the four bacteria strains were pH7.0～8.5 and 30～35℃. All the four bacteria strains performed efficient DBP degrading capabilities under optimal conditions. Results of degradation tests combined with the putative amino acid sequence of phthalate dioxygenase revealed that the bigger difference from the amino acid sequence of JDC-1 were, the lower degradation rate of the strain. Based on the different sites of amino acids, the key amino acids correlated to the degradation ability were preliminarily speculated.(2) A fragment of phthalate dioxygenase gene was successfully amplified from the genus of Delftia for the first time using a set of degenerate primers. Meanwhile, the degradation capability of JDC-3 was determined by HPLC using DMP as test substrate. The results showed that the optimal pH and temperature were at 7.0～8.0 and 30～35℃respectively. The degradation kinetics of JDC-3 was studied in different initial DMP concentration under optimal conditions. The results indicated that the degradation dynamic equation was In C = -0.06837 + A when DMP concentration was lower than 300 mg/L, with half life of 12.48 h. The degradation rate decreased and half life of JDC-3 prolonged as the initial concentration kept on increasing.(3) The optimum conditions for DBP degradation by strain JDC-11 are initial pH 8. 0, 30℃and agitation rate being 175 r/min. In 24 h , 1000 mg/L of DBP was degraded completely under the optimum conditions , It was demonstrated that JDC-11 is a high efficient strain in degrading DBP and will have valuable potential in practice. All concentration of glucose inhibited the degradation of DBP in the initial 12 h. Furthermore, When the glucose concentration was 200 mg/L, degradation of DBP also was inhibited in the latter 12 h. meanwhile, the concentration of glucose above 400 mg/L(including 400 mg/L) would increase the DBP degradation rate.(4) The biodegradation pathway of DBP by JDC-11 was evaluated by determining metabolites by GC/MS analysis,combined the result of the diversity of degradable substrates test and gene encoding the phthalate dioxygenase detected. a preliminary metabolic pathway of DBP was proposed: DBP was firstly hydrolyzed to MBP and subsequent PA, then was further metabolized to PCA and finally to produce CO₂ and H₂O.