Isolation,Identification And Characteristics Of An Efficient Phenanthrene-Degrading Strain Of Acinetobacter Sp.

Polycyclic aromatic hydrocarbon(PAH)is a kind of organic compounds that contain two or more benzene rings,which have high adsorptivity,hydrophobicity,and low water solubility.They are typical environmental pollutants of carcinogenic,teratogenic,and mutagenic.The carcinogenicity enhanced with the increase in the number of benzene rings.The longer PAHs exist in environments,the higher their genetic toxicity is.At present,the methods for removing PAHs of the environment consist of physical methods,chemical methods,and bioremediation methods.Microbial degradation is one of the most important methods for effectively removing environmental PAHs.Phenanthrene is a model compound in the study of polycyclic aromatic hydrocarbon degradation.In this study,a highly efficient phenanthrene-degrading bacterium was screened from the oil-containing wastewater and identified.The growth characteristics,degradation characteristics of different hydrocarbon compounds and phenanthrene degradation kinetics were studied.The enzyme activity of degradation related genes were tested.In the laboratory condition,the strain F-1 was added to the artificial contaminated soil with phenanthrene to study the effect of the degrading strain on the phenanthrene-contaminated soil.The microbial diversity of the phenanthrene contaminated soil and the enhanced soil was analyzed.The results are as follows:A phenanthrene-degrading bacterium named F-1 was screened from oil contaminated water and identified as Acinetobacter johnsonii.Strain identification through the morphological,physiological and biochemical characteristics and 16 S r DNA sequence analysis.The growth characteristics of the strain F-1 were studied.This strain has stronger ability to adapt the environment.And the strain F-1 grew well at 25~ 40?,the initial p H range of 5.0~ 9.0,the Na Cl concentration of 0%~ 2%,and the phenanthrene concentration of 50~ 400 mg/L.The optimal phenanthrene growth conditions of the strain F-1 were as follows: temperature 30 ?,p H 7.0,and salinity 0.3%,phenanthrene concentration 250 mg/L.The degradation characteristics and the phenanthrene biodegradation kinetics of the strain F-1 were studied by the ultraviolet spectroscopy method and GC-MS analysis.The major phenanthrene-degrading related genes were detected by PCR amplification.The degradation rate was 43.57% with phenanthrene(100 mg/L)as the sole carbon source after 5 d and the degradation process complied with second order kinetics.The strain grew well in the LB broth with different organic substrates as sole carbon source including biphenyl,naphthalene,anthracene and pyrene.GC-MS analysis revealed that the bacterium could effectively degrade some C10-C28 straight-chain alkanes.The phenanthrene-degrading related genes such as catechol-1,2-dioxygenase,benzoate-1,2-dioxygenase,ferredoxin reductase,alcohol dehydrogenase,dihydroxy-acid dehydratase aldolase,ferredoxin genes were detected in the genome of the strain by PCR,which played an important role in the degradation process of the aromatic hydrocarbons.The activity of catechol-1,2-dioxygenase was detected in phenanthrene-based medium and liquid LB medium.The activity of catechol was significantly increased with the increase of phenanthrene in phenanthrene-based basal medium,while only trace amounts of enzyme activity were detected in liquid LB medium.Artificially contaminated soil has a strong ability to degrade and transform phenanthrene.The contents of phenanthrene in the contaminated soils gradually decreased during the bioremadation time.After 60 days,the content of phenanthrene in the contaminated soil decreases from 500 mg/kg to 0.405 mg/kg.The phenanthrene content of the soil containing the F-1 bacterial cells was 0.315 mg/kg,indicating that addition of the degrading bacterium in phenanthrene-contaminated soil contributed to the self-purification of phenanthrene-contaminated soil.The bacterial community diversity of phenanthrene-polluted soil and bacterial degrading remediation soil were studied by high-throughput sequencing analysis.Phenanthrene pollution can significantly reduce bacterial community diversity in soil.The natural soil contained an relative abundant microbial diversity before the pollution,including 22 phyla,57 classes,67 orders,111 families and 125 genera.After phenanthrene contamination for 60 days,the bacterial communities were reduced to 16 phyla,35 classes,48 orders,82 families and 105 genera respectively.The dominant bacteria phyla such as Acidobacteria,Gemmatimonadetes and dominant genera RB41,Sphingomonas were significantly reduced(p<0.05).Compared with the phenanthrene-polluted soil,the bacterial community of the polluted soil added with the strain F-1 increased with levels of 16 phyla,38 classes,51 orders,86 families and 116 genera respectively.It was showed that addition of the specificity degrading bacterial cells improved the bacterial community diversity in contaminated soils.