by Ho, Kai-Man. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 158-179). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstracts --- p.ii / Contents --- p.vii / List of figures --- p.xiv / List of tables --- p.xvii / Abbreviations --- p.xx / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Poly cyclic Aromatic Hydrocarbons (PAHs) / Chapter 1.1.1 --- Characteristics of PAHs --- p.1 / Chapter 1.1.2 --- Sources of PAHs --- p.3 / Chapter 1.1.3 --- Environmental fates of PAHs --- p.5 / Chapter 1.1.4 --- Effects of PAHs on living organisms --- p.7 / Chapter 1.1.5 --- Summary --- p.10 / Chapter 1.2 --- Target PAH: Benzo[a]pyrene (BaP) --- p.10 / Chapter 1.3 --- PAH contamination in Hong Kong --- p.14 / Chapter 1.4 --- Remediation for PAHs contaminated soils --- p.15 / Chapter 1.4.1 --- Chemical/ Physical methods --- p.15 / Chapter 1.4.2 --- Bioremediation --- p.16 / Chapter 1.5 --- Biodegradation of PAHs by bacteria and fungi --- p.18 / Chapter 1.5.1 --- Tolerance and degradation --- p.18 / Chapter 1.5.2 --- Biodegradation of PAHs by microorganisms --- p.20 / Chapter 1.5.2.1 --- Bacteria --- p.22 / Chapter 1.5.2.2 --- Fungi --- p.23 / Chapter 1.6 --- Environmental standards --- p.26 / Chapter 1.7 --- Strategies of soil sampling and microbial isolation --- p.26 / Chapter 1.7.1 --- Contaminated soil vs. uncontaminated soil --- p.26 / Chapter 1.7.2 --- Native species vs. foreign species --- p.29 / Chapter 1.7.3 --- Background of the sampling areas --- p.30 / Chapter 1.7.3.1 --- North Tsing Yi shipyard --- p.30 / Chapter 1.7.3.2 --- "Tsam Chuk Wan, Sai Kung" --- p.33 / Chapter 1.8 --- Objectives of this study --- p.33 / Chapter 2. --- Materials and Methods --- p.35 / Chapter 2.1 --- Soil Collection --- p.35 / Chapter 2.1.1 --- Abandoned shipyard soil and its sediment soil --- p.35 / Chapter 2.1.2 --- "Tsam Chuk Wan, Sai Kung" --- p.35 / Chapter 2.2 --- Characterization of soils --- p.35 / Chapter 2.2.1 --- Sample preparation --- p.36 / Chapter 2.2.2 --- Soil pH --- p.36 / Chapter 2.2.3 --- Electrical conductivity --- p.36 / Chapter 2.2.4 --- Salinity --- p.36 / Chapter 2.2.5 --- Total organic carbon contents --- p.38 / Chapter 2.2.6 --- Metal analys --- p.is / Chapter 2.2.7. --- Oil and grease content --- p.38 / Chapter 2.2.8 --- Soil texture --- p.39 / Chapter 2.2.9 --- Available ammoniacal nitrogen and oxidized nitrogen --- p.39 / Chapter 2.2.10 --- Available Phosporus --- p.40 / Chapter 2.2.11 --- Total Nitrogen and total Phosporus --- p.40 / Chapter 2.2.12 --- Moisture / Chapter 2.2.13 --- DTPA-extractable metals --- p.41 / Chapter 2.2.14 --- Extraction of PAHs and organic pollutants --- p.41 / Chapter 2.2.14.1 --- Extraction procedures --- p.41 / Chapter 2.2.14.2 --- GC-MSD conditions --- p.41 / Chapter 2.2.14.3 --- Extraction efficiency --- p.43 / Chapter 2.2.15 --- Soil colour --- p.43 / Chapter 2.3 --- Screening and selection of microorganismms --- p.43 / Chapter 2.3.1 --- Isolation of potential BaP-degrading microorganisms --- p.44 / Chapter 2.3.1.1 --- Isolation of bacteria --- p.44 / Chapter 2.3.1.2 --- Isolation of fungi --- p.44 / Chapter 2.3.2 --- Cultures preserving microorganisms --- p.46 / Chapter 2.3.3 --- Screening and selection of microbes --- p.46 / Chapter 2.3.3.1 --- Bacteria --- p.46 / Chapter 2.3.3.2 --- Fungi --- p.46 / Chapter 2.3.4 --- Survival test --- p.47 / Chapter 2.3.5 --- Removal efficiency (RE) towards BaP by the microorganisms --- p.47 / Chapter 2.3.5.1 --- Bacteria --- p.47 / Chapter 2.3.5.2 --- Fungi --- p.48 / Chapter 2.3.6 --- Removal efficiency (RE) --- p.48 / Chapter 2.3.7 --- Relationship of absorbance of bacterial culture and bacterial biomass --- p.49 / Chapter 2.4 --- Identification of selected microorganisms --- p.49 / Chapter 2.4.1 --- Identification of bacterium --- p.49 / Chapter 2.4.1.1 --- 16S rDNA sequencing --- p.49 / Chapter 2.4.1.1.1 --- Primers --- p.49 / Chapter 2.4.1.1.2 --- DNA extraction --- p.51 / Chapter 2.4.1.1.3 --- Specific PCR --- p.51 / Chapter 2.4.1.1.4 --- Gel electrophoresis --- p.51 / Chapter 2.4.1.1.5 --- Purification of PCR products --- p.52 / Chapter 2.4.1.1.6 --- DNA sequencing --- p.52 / Chapter 2.4.1.2 --- Midi Sherlock® Microbial Identification System (MIDI) --- p.53 / Chapter 2.4.1.3 --- Biolog MicroLog´ёØ system (Biolog) --- p.55 / Chapter 2.4.2 --- Identification of fungi --- p.56 / Chapter 2.4.2.1 --- ITS DNA sequencing --- p.56 / Chapter 2.4.2.2 --- Observation under electronic microscope --- p.58 / Chapter 2.5 --- Growth curve of the microorganism --- p.58 / Chapter 2.5.1 --- Bacterium --- p.58 / Chapter 2.5.2 --- Fungi --- p.58 / Chapter 2.6 --- Preparation of Benzo[a]pyrene (BaP) stock solution --- p.58 / Chapter 2.7 --- Comparison of isolated bacterium and fungi --- p.60 / Chapter 2.8 --- Optimization of BaP degradation by selected fungus --- p.60 / Chapter 2.8.1 --- Preparation of straw compost inoculated with selected fungus --- p.60 / Chapter 2.8.2 --- Effect of incubation time --- p.61 / Chapter 2.8.3 --- Effect of initial BaP concentration --- p.61 / Chapter 2.8.4 --- Effect of inoculum size / Chapter 2.8.5 --- Effect of temperature --- p.61 / Chapter 2.8.6 --- Effect of soil pH --- p.62 / Chapter 2.8.7 --- Study of BaP degradation pathway by the microorganisms using GC-MSD --- p.62 / Chapter 2.9 --- Chitin Assay --- p.62 / Chapter 2.10 --- Enzyme assay --- p.63 / Chapter 2.10.1 --- Laccase assay --- p.63 / Chapter 2.10.2 --- Manganese peroxidase assay --- p.63 / Chapter 2.10.3 --- Lignin peroxidase assay --- p.64 / Chapter 2.11 --- Toxicity of treated soil --- p.64 / Chapter 2.12 --- Statistical analysis --- p.65 / Chapter 3. --- Results --- p.66 / Chapter 3.1 --- Soil Collection --- p.66 / Chapter 3.1.1 --- North Tsing Yi shipyard --- p.66 / Chapter 3.1.2 --- "Tsam Chuk Wan, Sai Kung" --- p.66 / Chapter 3.2 --- Characterization of soil samples --- p.71 / Chapter 3.3 --- Extraction efficiency of Benzo[a]pyrene --- p.79 / Chapter 3.4 --- Screening and selection of microorganisms --- p.79 / Chapter 3.4.1 --- Isolation ofpotential BaP-degrading microorganisms --- p.79 / Chapter 3.4.2 --- Screening and selection of microbes --- p.87 / Chapter 3.4.2.1 --- Bacteria --- p.87 / Chapter 3.4.2.2 --- Fungi --- p.93 / Chapter 3.4.3 --- Growth curve of the microorganisms --- p.95 / Chapter 3.4.3.1 --- Bacterium --- p.95 / Chapter 3.4.3.2 --- Fungi --- p.99 / Chapter 3.5 --- Comparison of isolated bacterium and fungi --- p.99 / Chapter 3.6 --- Identification of selected microorganisms --- p.102 / Chapter 3.6.1 --- Identification of bacterium --- p.103 / Chapter 3.6.1.1 --- 16S rDNA sequencing --- p.103 / Chapter 3.6.1.2 --- Midi Sherlock® Microbial Identification System (MIDI) --- p.103 / Chapter 3.6.1.3 --- Biolog MicroLog´ёØ system (Biolog) --- p.107 / Chapter 3.6.2 --- Identification of fungi --- p.107 / Chapter 3.6.2.1 --- ITS DNA sequencing --- p.107 / Chapter 3.6.2.2 --- Observation under electronic microscope --- p.113 / Chapter 3.7 --- Optimization of BaP degradation by the selected fungus: Trichoderma asperellum --- p.107 / Chapter 3.7.1 --- Effect of incubation time --- p.107 / Chapter 3.7.2 --- Effect of inoculum size --- p.113 / Chapter 3.7.3 --- Effect of initial BaP concentration --- p.113 / Chapter 3.7.4 --- Effect of soil pH --- p.113 / Chapter 3.7.5 --- Effect of temperature --- p.117 / Chapter 3.8 --- Determination of breakdown products of BaP by BaP-degrading microorganisms --- p.117 / Chapter 3.9 --- Enzyme assay --- p.117 / Chapter 3.10 --- Evaluation of toxicity by using indigenous bacteria --- p.121 / Chapter 4. --- Discussion --- p.128 / Chapter 4.1 --- Physico-chemical properties of soil samples --- p.128 / Chapter 4.2 --- Determination of BaP and other organic compounds --- p.131 / Chapter 4.3 --- Identification of the microorganisms --- p.132 / Chapter 4.3.1 --- Bacteria --- p.132 / Chapter 4.3.2 --- Fungi --- p.134 / Chapter 4.4 --- Biodegradation by BaP-degrading microorganisms --- p.135 / Chapter 4.4.1 --- Isolation and screening of BaP-degrading microorganisms --- p.135 / Chapter 4.4.2 --- Biodegradation of BaP --- p.137 / Chapter 4.4.2.1 --- Bacteria --- p.137 / Chapter 4.4.2.2 --- Fungi --- p.138 / Chapter 4.4.3 --- BaP degradation pathway --- p.140 / Chapter 4.4.3.1 --- Bacteria --- p.140 / Chapter 4.4.3.2 --- Fungi --- p.140 / Chapter 4.5 --- Optimization of PAH degradation by T. asperellum --- p.143 / Chapter 4.5.1 --- Effect of incubation time --- p.143 / Chapter 4.5.2 --- Effect of initial BaP concentration --- p.144 / Chapter 4.5.3 --- Effect of inoculum size fungus --- p.144 / Chapter 4.5.4 --- Effect of soil pH --- p.145 / Chapter 4.5.5 --- Effect of temperature --- p.146 / Chapter 4.6 --- Comparison the selected bacterium and fungi --- p.146 / Chapter 4.7 --- Evaluation of toxicity by using in indigenous bacteria --- p.148 / Chapter 4.8 --- Post treatment by crude enzyme of Pleurotus pulmonarius --- p.149 / Chapter 4.9 --- Limiting factors for BaP degradation --- p.150 / Chapter 4.10 --- Further Investigations --- p.152 / Chapter 5. --- Conclusion --- p.155 / Chapter 6. --- References --- p.158