Microbial degradation and determination of polycyclic aromatic hydrocarbons
Creators: name: Li, Xing-Fang
Publisher: name: University of British Columbia
Year: 2009

description: Many polycycic aromatic hydrocarbons (PAHs) are carcinogenic, and it is important to understand their fate in the environment. In this thesis, the microbial degradation of PAHs has been studied, with emphasis on the degradation of phenanthrene and pyrene. Several cultures were enriched from sea water and sediment samples obtained from Kitimat Arm, B. C., an estuary environment that is contaminated by PAHs. It was demonstrated that microorganisms in the cultures are able to degrade phenanthrene very quickly and to utilize phenanthrene as the sole carbon and energy source. For example, complete degradation of 100 mg of phenanthrene in 10 ml of medium is achieved 23 hr after incubating phenanthrene with 0.5 ml of the culture and 10 ml of mineral salt medium at ambient temperature. A strain (LY) that is capable of degrading phenanthrene was isolated from a sea water culture and has been partially characterized. Another sixteen mixed cultures prepared from Kitimat samples readily degraded pyrene, in spite of the common perception that the microbial degradation of pyrene is very difficult. Microorganisms in the cultures grew on pyrene, without any additional carbon source. This is one of the few reports of microorganisms that can use pyrene as sole carbon source. The possibility of complete break-down of pyrene was studied by quantitatively measuring the amount of ¹⁴CO₂ produced from the mineralization of ¹⁴C- labeled pyrene. Up to an estimated 45% of the ¹⁴C-labeled pyrene that was initially added into the cultures was found to be mineralized to produce ¹⁴CO₂ and H₂O, after 12 days of incubation. The rest of the ¹⁴C activity is primarily accounted for metabolites. A typical metabolite, cis-4,5-dthydroxy-4,5-dihydropyrene, was identified in the cultures, by using various chromatographic and spectrometric techniques developed during this study. This metabolite was present in all the cultures catalyzing pyrene degradation and disappeared only after pyrene was completely degraded. Therefore, there was the possibility that cis 4,5-dihydroxy-4,5-dihydropyrene could be used as an indicator for the in situ microbial degradation of pyrene in the Kitimat Arm environment: cis-4,5-dihydroxy-4,5- dihydropyrene could be present because pyrene was continuously introduced to the Kitimat environment and is constantly available to the microorganisms. Analysis of environmental samples by using various analytical techniques showed that cis-4,5- dihydroxy-4,5-dihydropyrene is indeed present in some near shore sediment and pore water samples, in which a high concentration of pyrene is present. For the first time, these studies showed strong evidence for the in situ degradation of pyrene in the natural environment. Radioactive tracer studies using 14C-labeled pyrene also showed that untreated sediment samples collected from Kitimat Arm can mineralize up to an estimated 28% of pyrene. Two mixed cultures (Pysed-l and Pysed-2) were also tested for the ability to degrade benzo(a)pyrene and a standard mixture of sixteen PAHs. Some important factors influencing the degradation of these PAHs were studied, including temperature, the composition of culture medium, the concentration of the PAHs, and the population of the microorganisms. The degradation of pyrene at a temperature as low as 2 °C was observed. The addition of peptone and yeast extract was found to enhance the degradation of PAHs. Two methods were developed for the preconcentration of trace amounts of organics in water samples, one based on the use of C₁₈ solid phase cartridges the other on polytetrafluoroethylene tubing.