Isolation And Characterization Of Phenanthrene-Degrading Bacteria And Their Preliminary Application In Plant-Microbe Combined Remediation Of Heavy Metals-Phenanthrene Co-Contaminated Soil

Two phenanthrene-degrading bacterial strains designated as PHE-1and PHE-2were isolated from the activated sludge sample of a wastewater-treating system of a chemical plant in Sheyang County of Jiangsu province. According to their morphological, physiological and biochemical characteristics and16S rRNA gene similarity analysis, strain PHE-1was identified as Sphingobium sp., strain PHE-2was identified as Ochrobactrum sp. Both of them grew well in pH6.0-9.0, their optimum growth pH and temperature were pH7.0and8.0and30℃. In mineral salts medium containing100mg/L phenanthrene as the sole carbon source, the degradation rates of phenanthrene by PHE-1and PHE-2within18h were99.12%and99.56%respectively. Cu2+above800mg/L or Cd2+above450mg/L would inhibit the degradation of phenanthrene by strain PHE-1. Cu2+above400mg/L or Cd2+above1000mg/L would show inhibitory effect on strain PHE-2. When the Cd2+was above300mg/L, the colony edge of PHE-2appeared transparent, the floc was produced in the internal. This phenomenon might be a resistance response of strain PHE-2to Cd2+toxicity.Polycyclic aromatic hydrocarbons dioxygenase is the first enzyme of involved in the phenanthrene degradation pathway, which determines the starting of degradation. With the primers designed according to the published sequence of a subunit of polycyclic aromatic hydrocarbons dioxygenase gene(ahdA1b), a gene fragment was obtained by PCR from the strain PHE-1, its length was1.49kb, its sequence showed99%similarity to ahdAlb gene of Sphingomonas sp. ZP1(EU082776) and97%similarity to that of Sphingomonas sp. P2(AB091693). Most of the strains with high ahdA1b similarity with strain PHE-1were from the Sphingomonadaceae, which showed the ahdA1b gene was highly conserved in strains from Sphingomonadaceae. A rapid and efficient chromosome walking method (SEFA-PCR) was employed to clone the upstream and downstream fragments of the ahdAlb gene. The obtaind sequences were spliced with the ahdA1b gene, resulting a fragment (9.2kb in length). The online Blast analysis showed that it showed96%identity to the gene cluster of Sphingomonas sp. P2(GenBank Accession No. AB091693), which contained six genes, they were xylA, xylM, ahdA2b, ahdA1b, ahdA2a and ahdA1a.Catechol dioxygenase is another key enzyme involved in the degradation process of phenanthrene, it catalyzes the ring cleavage of the metabolic intermediate. The xylE gene was cloned from PHE-1by PCR with the primers designed according to the reported sequence of catechol-2,3-dioxygenase gene (xylE), its length was1188bp. It showed99%identity to the sequence of xylE of Sphingomonas sp. B2-7(EU596533.2).The fluorescence quantitative PCR technique was used to investigate the effect of Cd2+, Cu2+and phenanthrene on the transcription of xylE gene and ahdA1b gene of strain PHE-1, the results showed that both ahdA1b gene and xylE gene were inducible gene. The copy number of ahdAlb gene was higher than that of xylE, when strain PHE-1was induced by phenanthrene. Under the stress of low concentration of Cd2+, Cu2+(100mg/L), the copy number of xylE and ahdA1b was higher than that of the control. Under the stress of500mg/L Cd2+, the copy number xylE and ahdA1b was lower than that of the control, while it was higher than that of the control under the stress of500mg/L Cu2+Ryegrass was combined with strains PHE-1and PHE-2respectively to establish two plant-microbial remediation systems, which was used to investigate the remediation of soils with different treatment(soil contaminated with500mg/kg phenanthrene); soil copper-phenanthrene combined contaminations(the concentration of phenanthrene and Cu2+were both500mg/kg; soil mixed with electronic rubbish). The inoculum size is1.5×10’cells/g soil. The results showed that the effect of plant-microbial remediation were significantly better than that of the treatment inoculated with microbes alone. In phenanthrene single pollution soil, the residue concentration of phenanthrene in soil inoculated PHE-1(plant and un-plant were101.9mg/kg,116.1mg/kg respectivity) and inoculated PHE-2(plant and un-plant were86.9mg/kg,90.6mg/kg respectivity) were lower than that in soil without the inoculation of functional bacteria(plant and un-plant were150.3mg/kg,204.1mg/kg respectivity). The average number of copies of the xylE gene in soil inoculated with strain PHE-1(plant and un-plant were5.18×108copies/g,3.23×108copies/g) and inoculated PHE-2(plant and un-plant were1.285×109copies/g,6.98×108copies/g) higher than that in without the inoculation of functional bacteria(plant and un-plant were1.64×108copies/g,1.31×108copies/g). In copper-phenanthrene combined contamination soil, the residue concentrations of phenanthrene in soil inoculated with PHE-1(plant and un-plant were101.2mg/kg,115.7mg/kg respectivity) and inoculated with PHE-2(plant and un-plant were140.8mg/kg,140.1mg/kg respectivity) were lower than that of soil without the inoculation of functional bacteria(plant and un-plant were150.3mg/kg,204.1mg/kg respectivity). The average number of copies of the xylE gene in soil inoculated with PHE-1(plant and un-plant were1.98×109copies/g,6.24×108copies/g respectivity) and inoculated PHE-2(plant and un-plant were7.27×108copies/g,4.01×108copies/g respectivity) higher than that in soil without the inoculation of functional bacteria(plant and un-plant were2.1×108copies/g,1.08×108copies/g respectivity). In soil mixed with electronic rubbish, no marked difference was found between the plant and un-plant treatment. Comparison of results of copper-phenanthrene combined soils revealed that the inoculation of strain PHE-1showed better effect than that of strain PHE-2.Two heavy metal-resistant strains capable of degrading phenanthrene were isolated and characterized, both of them were used in the plant-microbial remediation of heavy metal-phenanthrene contamianted soil. All theses results will provide theoretical base and technique support for the remediation of organic and heavy metal co-contaminated environments.