Isolation, Genetic Diversity And Degradation Characteristics Of PAEs-degrading Bacteria From Various Environmental Samples

Phthalic acid esters (PAEs), a class of refractory organic compounds which are widely used in the industries, have received extensive environmental concerns in recent years due to their release into the environment and their toxicity to human beings and other organisms. The metabolic breakdown of PAEs by microorganisms is considered to be one of the major routes of environmental degradation for this widespread pollutant since the rate of hydrolysis and photolysis is relatively low. The present study aims to investigate the biodegradation of PAEs by bacteria isolated from various environmental samples, including genetic diversity and activities of PAEs-degrading bacteria, mechanism of degradation of DOP, and the kinetics of biodegradation of DEP in the selected representative bacteria.32 strains of phthalic acid esters (PAEs)-degrading bacteria were isolated from 13 geographically diverse sites by enrichment using mixtures of PAEs as the sole source of carbon and energy. Sequence analyses of the 16S rRNA gene indicated that these isolates were from six genera(Arthrobacter, Gordonia, Rhodococcus, Acinetobacter, Pseudomonas, and Delftia). To evaluate the genetic diversity among them, the molecular typing method rep-PCR with primers based on ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR sequences was performed. Strain-specific and unique genotypic fingerprints were distinguished for most of these isolates. In addition, utilization of various PAEs and the central intermediate phthalic acid (PA) by representative isolates suggested between-isolate differences in substrate utilization and degradation pathways. Furthermore, HPLC analysis showed that the rate of dimethyl phthalate (DMP) degradation varied from 48.32% to 100% between strains. These results suggest a high level of genetic diversity among PAEs-degrading bacteria in the natural environment and their great potential to clean up PAEs-contaminated environments.Based on the previous study, Gordonia sp. JDC-2 and Arthrobacter sp. JDC-32 were chosen from the collection to investigate the mechanism of degradation of DOP by the co-culture of these two strains. Gordonia sp. JDC-2 rapidly degraded di-n-octyl phthalate (DOP) into phthalic acid (PA), which accumulated in the culture medium. Arthrobacter sp. JDC-32 degraded PA but not DOP. The co-culture of Gordonia sp. JDC-2 and Arthrobacter sp. JDC-32 degraded DOP completely by overcoming the degradative limitations of each species alone. The biochemical pathway of DOP degradation by Gordonia sp. JDC-2 was proposed based on the identified degradation intermediates. The results suggest that DOP is completely degraded by the biochemical cooperation of different microorganisms isolated from sludge.Furthermore, Acinetobacter sp. JDC-16 was further characterized for biodegradation of DEP. The degradation tests using diethyl phthalate (DEP) as the model compound show that the optimal pH and temperature for DEP degradation by Acinetobacter sp. JDC-16 is 8.0 and 35℃, respectively. Meanwhile, degradation kinetics under various initial concentration of DEP reveal that substrate depletion curves fit well with the modified Gompertz model with high correlation coefficient (R2>0.99). Furthermore, the substrate induction test indicates that DEP-induction can apparently shorten the lag phase and thus enhance the degradation rate. This study further highlights the potential of this isolate for bioremediation of DEP-contaminated environments.