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Isolation and Identification of Three DBP-degrading Bacteria Strains and Related Research about Degradation Characteristics

Author: WangYangYang
Tutor: WuXueLing
School: Central South University
Course: Biological Engineering
Keywords: biodegradation PAEs metabolic pathway degradation gene real-time PCR
CLC: X172
Type: Master's thesis
Year: 2011
Downloads: 35
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Abstract

Phthalate acid esters (PAEs) are an important synthetic organic compounds in industry, it were widely used as additives in the production of plastic products such as plasticizers, packaging materials for food, infant toys, cosmetics and furniture industries. Due to the mass production and widespread use of PAEs, it has become a very serious environmental pollutant. PAEs in the environment can be eliminated by photolysis, hydrolysis and biodegradation; the biodegradation has become the main way to deal with PAEs pollution due to its speed and no secondary pollution. In this paper, three bacterial strains which can utilize the PAEs as the sole carbon source and energy were isolated from three samples collected from Guilin and Zhuhai, and were named as JDC-41, JDC-49 and JDC-36, respectively. According to molecular biological identification, JDC-41, JDC-49 and JDC-36 were Ochrobactrum sp., Agrobacterium sp. and Arthrobacter sp.. Their degradation conditions, degradation ability, degradation kinetics, degradation of PAEs mixtures, clone of degradation gene and differentially expression of the gene were investigated in this paper. The results obtained are as follows:(1) An Ochrobactrum sp. was isolated for the first time which can utilize DBP as the sole carbon source and energy and was named as JDC-41, the best temperature and pH of JDC-41 to degrade DBP are 30℃and 7.0. Substrate range tests result indicate that JDC-41 just can utilize the PAEs with relatively short side chain, but cannot utilize the PAEs with relatively long side chain. Kinetics of aerobic DBP degradation by Ochrobactrum sp. JDC-41 indicate that with the increase of the concentration of DBP, the half-life of degrade of DBP by JDC-41 will gradually increase and just about 90% of DBP (500mg/L) was degraded within 48 hours. Degradation of the mixed PAEs indicate that DMP can inhibit the degradation of DBP to certain extent, the degradation of DMP and DBP still cannot induce the degradation of DOP and JDC-41 still cannot degrade DOP within 48 hours. Substrate induction experiment showed that JDC-41 was induced by DBP can degrade DBP quickly; while JDC-41 has not been induced by DBP can observe a clear mind lag.(2) An Agrobacterium sp. was isolated for the first time which can utilize DBP as the sole carbon source and energy and was named as JDC-49. The best temperature and pH of JDC-49 to degrade DBP are 30℃and 8.0. Substrate range tests result indicate that JDC-49 can utilize PA as sole source of carbon and energy while cannot utilize the IPA and PTA, JDC-49 also can degrade both the PAEs with long and short side chain. Kinetics of aerobic DBP degradation by Agrobacterium sp. JDC-49 showed that when the concentration of DBP is not more than 100mg/L, the degradation of DBP by JDC-49 can fit well with the first order kinetics. Degradation of the mixed PAEs indicate that DMP can be quickly degraded, and its degradation have some inhibitory effect on the degradation of DBP at the beginning, but the overall effect of the degradation of DMP to the degradation of DBP is promotion. Finally, the degradation intermediates of DBP were identified as mono-butyl phthalate (MBP) and phthalate acid (PA). The pathway of DBP degradation was DBP to MBP and then to PA before mineralization.(3) A bacterial strain can utilize PAEs as sole source of carbon and energy was isolated from mangrove soil from Zhuhai, it was identified as Arthrobacter sp. and named as JDC-36. The diversity of the enrichments were analyzed by DGGE, it was indicated that with the increase of the number of the enrichment, the community diversity in the enrichment decreased significantly. The best temperature and pH of JDC-49 to degrade DBP are 30℃and 9.0. JDC-36 still cannot utilize IPA and PTA as sole source of carbon and energy which similar with the JDC-49, JDC-36 just can degrade some PAEs with short side chain and cannot degrade the PAEs with long side chain. Degradation of the mixed PAEs by JDC-36 indicates that it degrades DBP at first rather than the DMP with shorter side chain, and after the inducement of the DMP and DBP it still cannot utilize DOP. Finally, degradation gene was successfully cloned from JDC-36 and the real-time PCR was used to prove the correlation of this gene with the degradation of DBP.

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