Isolation Of PAHs-degrading Bacteria And Characterization Of The Degradation Metabolism

Polycyclic aromatic hydrocarbons (PAHs) are typical persistent pollutants, which are highly toxic to the organisms, recalcitrant to biodegradation, and accumulated through the food chains. Remediation of PAHs-contaminated sites has been a continuous concern in environmental science. Since microbial remediation has the merits of lower cost and environmentally responsible, it is the potential and promising techniques in site remediation. In nature, PAH-degrading bacteria resources are abundant, but long time might be required for the natural evolution of high PAHs degradation capability due to the uneven distribution of the contaminants and lower mass transfer efficiency in contaminated site. The aim of the present work was to select the highly effective degrading bacteria from the active sludge of a chemical plant using high concentrations of phenanthrene and pyrene as environmental stresses. Two highly effective PAHs-degrading consortia which can use phenanthrene and pyrene as the sole carbon source were isolated. During the selecting process, the dynamic changes of flora composition and PAHs-degrading ability were monitored. Two highly efficient PAHs-degrading strains were chosen to be studied for their degradation mechanisms. The main research contents and results were as follows:1. Four gram per liter of phenanthrene or pyrene was used as the sole carbon source in the isolation media. After ten times passage acclimation, two highly efficient PAHs-degrading consortia named as Phe consortium and Pyr consortium were obtained from the active sludge with relatively stable composition and degradability. During domestification process, PCR-DGGE technique was used to monitor the community composition change. At the beginning, the original active sludge was shown to harbor a abundant and diverse bacteria species. However, under high concentrations of phenanthrene and pyrene, the flora species gradually reduced and the composition tended to stabilize as the domestification passage increased. The two consortia showed clear difference in their domestification difficulty, flora composition, and degradation ability. The first generation of Phe consortium was domesticated in5-7days whereas the first generation of Pyr consortium required almost one month to be domesticated. In the case of flora composition, Phe consortium was mainly composed of Acidovorax sp., Pandoraea sp., Pseudomonas sp., Enterobacter sp.. On the other hand, Pyr consortium was dominated by Achromobacter sp., Empedobacter sp., Pseudomonas sp. and some unknown ones. In the case of PAHs degrading ability, Pyr consortium exhibited higher efficiency in degrading high molecular weight (HMW) PAHs than Phe consortium. During7days, Pyr consortium degraded11kinds of16PAHs while Phe consortium was able to remove10P AHs. The Pyr consortium completely degraded NA, ACE, ACY, FLE, PHE, ANTH, FLA and PYR.36%of BAA,29%of CHR and13%of BaP were also degraded by Pyr consortium. On the other side, the Phe consortium degraded100%of NA, ACE, ACY, FLE and PHE,87%of ANTH,88%of FLA,66%of PYR,22%of BAA,11%of CHR. Moreover, both consortia showed high degrading ability of PAHs in the artificial contaminated soil.2. In order to study the degradation mechanisms of Phe consortium and Pyr consortium. Two highly effective degrading strains were isolated. Pseudomonas sp. JM2which could use PHE as the sole carbon source was isolated from Phe consorium, and Pseudomonas sp. Jpyr-1using PYR as the sole carbon was isolated from Pyr consortium. Results showed that the gene cluster encoding PHE degradation of strain JM2was located on the plasmid. By designing a series of degenerate primers, the gene encoding degradation pathway enzymes were amplified, sequenced, and analyzed. The gene sequences encoding the full-degrading enzymes from phenanthrene dioxygenase to2-carboxybenzaldehyde dehydogenase were analyzed. A comparison with14other PAHs degrading bacteria showed that phenanthrene dioxygenase of JM2, the key enzyme of its degradation pathway, had a homology of96%with that of Alcaligenes faecalis AFK2. Combined with the discovery of phthalate as a metabolite, it was proposed that strain JM2degraded PHE through the phthalate way which was similar to the degradation pathway of strain AFK2. On the other side, several metabolites, including phenanthrene-4-carboxylate,1-hydroxy-2-naphthoate, phthalate and phthalate3,4-dihydrodiol, were detected during pyrene degradation. According to previously reported PYR degradation pathways, it was proposed that strain Jpyr-1degraded PYR through o-phthalate pathway. According to known sequences of PAH dioxygenases, several degenerated primers were designed. The α and β subunits gene sequences of dioxygenases were obtained. Sequence analysis revealed that the α subunit of dioxygenase showed90%homology with a Gram-positive bacterial strain Mycobacterium sp.6PY1.3. The effects of different PAHs on the degradation capability of strains JM2and Jpyr-1were studied via designing systems consisting of two types of PAHs. Research results showed that the presence of FLE, ANTH and FLA inhibited the degradation of other PAHs by strain JM2whereas PHE promoted its degradation ability. In the first24hours, FLE, ANTH and FLA showed inhibition effects on PYR degradation while PHE enhanced the degradation in binary substrate systems. This observation was consistent with the performance of strain JM2. However,144hours later, the inhibition effect of FLA on PYR was reduced and ANTH promoted the degradation, while FLE continued to inhibit PYR degradation.In sumary, two highly effective PAH-degrading consortia were obtained under stresses of high concentrations of PAHs. The Pyr consortium showed high degradation ability to HMW PAHs. The degradation pathways of PHE and PYR were clarified by molecular work on PAH-degrading strains from the consortia. Based on these results, we investigated the influence of PAHs composition on degradation. Taken together, these results will provide valuable theoretical basis for the bioremediation of PAHs contaminated sites using high efficiency degrading bacteria.