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Bacterial Consortium in the Biodegradation of Polycyclic Aromatic Hydrocarbons-Phenanthrene

John Farmer  
【摘要】: Many aromatic hydrocarbons are known to be toxic and carcinogenic to humans and their contamination of soils and water bodies is of great environmental concern. Hence microbial degradation of these hydrocarbons to less toxic compounds has become a vital tool in making the environment safe for humans as well as other life forms. In this study, industrial waste water samples were randomly collected from three different locations in Shuangyang local brewery. These samples were designated by the letters: HY, YY and WS. The waste water samples were used to isolate bacterium species using LB liquid medium. These bacterium cultures were transferred to a fresh media containing a concentration of Phenanthrene. The pH and Optical Density (OD) readings were measured and recorded on daily basis for a period of one week and this was the first transfer. This was done in other to ascertain the biodegradation capability of the bacterium strains in the Phenanthrene medium and the pH at which these strains degrade the pollutant- Phenanthrene. However, this process was repeated for another four consecutive weeks (five transfers). Meanwhile, from the fifth transfer the aliquot culture MSM-PHE medium was again transferred to a fresh liquid LB media and allowed to grow overnight. The bacterial cultures were again transferred to a solid MSM agar medium to obtain single colonies of bacterial strains. These cultures were transferred to a liquid MSM medium for 24 hrs and transferred again to the solid medium. This process was repeated for another three weeks to isolate pure clones. Pure clones were isolated with distinct colors and morphologies. The genetic DNA of these pure colonies was extracted, purified and two primers were used for the PCR analysis and the isolates were identified by blast analysis. These single isolates were later inoculated in a liquid MSM-PHE culture medium and their densities (biomass) were measured and recorded for a week. The isolates were also inoculated on solid MSM-PHE and Fluorine medium also MSM-Pyrene and Chrysene medium. One microliter of catechol was added into each of the small flasks containing culture medium of the strains and the color change was observed. However, these cultures were again inoculated on crude oil (diesel) and their emulsification index determined. These isolates were later inoculated on blood agar media to observe the clear zones formed by the isolates. From the Optical Density results it was clear that sample HY had many bacterium strains capable of degrading Phenanthrene followed by sample YY, but sample WS was the least degraded. This was also evidenced in the turbidity of the liquid medium. The bacteria were identified by their sequences through blast analysis. The names of genera of bacteria isolated were: HY1- Alcaligenes sp, HY32-Phyllobacteriaceae sp, HY52-Pseudochrobactrum sp, YY31-Pseudomonas sp, YY41-Alcaligenes sp, and WS11-Sphingobacterium sp. The strain WS11 had the highest bacterial density and degraded Phenanthrene effectively compared to the other strains when single isolates were inoculated in MSM-PHE medium. These isolates also degraded/grew on both Low (Phenanthrene, Fluorine) and High (Pyrene, Chrysene) molecular weight hydrocarbons with the exception of E.coli (control) which didn’t grow on these pollutants. When catechol was added to the bacterial cultures, there was a color change except E.coli (control) which had no color change. This indicated that the bacterial isolates formed catechol as an intermediate for the gene-encoding enzyme which degrades aromatic compounds. The strain YY31 formed a very clear zone or haloes when inoculated on blood agar media and formed surfactant which is vital for the bioremediation of hydrocarbons. The other isolates also formed haloes, but were not very clear. The strain YY31 formed a clear emulsion when added to crude oil while the other isolates formed foam, but HY32 formed the least. Therefore, strain YY31 was the best biosurfactant-producing bacterium and emulsified the crude oil effectively. This research is going on and will be completed by further studies.

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