No description available.

Dissertation (Ph.D.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references (leaves 263-280).

Thesis (M.S.)--University of Akron, Dept. of Geology, 2005. / "August, 2005." Title from electronic thesis title page (viewed 11/29/2005) Advisor, Annabelle Foss; Committee members, Teresa Cutright, Ira Sasowsky; Department Chair, John Szabo; Dean of the College, Charles B. Monroe; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

No description available.

No description available.

This study examined the effect of in-situ biosparging on pentachlorophenol (PCP) degradation and bacterial communities in PCP contaminated groundwater. Bacteria were identified by sequencing the 16s rDNA fragment from DNA extracted from groundwater cultures and comparing those sequences to a database using a basic local alignment search tool, BLAST. The PCP-degraders Burkholderia cepacia and Flavobacterium (Sphingobium) chlorophenolicum were identified in multiple wells, as were the 4-chlorophenol degrader Herbaspirillum sp., and the common soil bacteria Pseudomonas sp., Aquaspirillum sp., and Rhodocista sp., among others. Numerous bacterial samples also appeared in the results as “uncultured”. Bacterial community changes were observed using terminal restriction fragment length polymorphism (TRFLP) analysis to identify operational taxonomic units of bacteria at various locations inside and outside the biosparging zone of treatment over time. Diversity measures including species richness, Simpson’s and Shannon’s indices, and species evenness were calculated from operational taxonomic unit results for each well at each sampling point in order to better understand changes in the bacterial community. Species richness tended to be higher at wells further away from the biosparging line, while diversity and evenness varied throughout the area. Correlations between PCP concentration, operational taxonomic units, and distance from biosparging wells were determined by Pearson’s product-moment correlation and Spearman’s rank correlation. Positive correlations were found between distance from biosparging wells and PCP concentration, species richness and distance, and to a smaller degree, diversity and distance. Biosparging remediation has a significant impact on the types of PCP-degrading bacteria within the groundwater matrix, and installations of this type of treatment should be applied to maximize the use of the native bacteria to assist in degradation of the contaminant.

Microbial populations from subsurface soil collected from a hydrocarbon contaminated site and a pristine site with no history of contamination had the ability to degrade pentachlorophenol (PCP) in anaerobic enrichment cultures. Increasing concentrations of PCP in nitrate, sulfate and yeast extract-mineral salts media were used to acclimate the cultures. Nitrate enrichments, previously incubated in an anaerobic phenol-mineral salts medium, showed 23% degradation in medium containing 40 μg ml⁻¹ PCP during a 32 d incubation period. Cultures not adapted to phenol degradation did not degrade PCP at concentrations over 20 μg ml⁻¹. Enrichment cultures grown in the anaerobic yeast extract-mineral salts medium did not degrade PCP at concentrations over 20 μg ml⁻¹ and phenol adaptation did not enhance PCP degradation. The sulfate reducing enrichment containing 1 μg ml⁻¹ PCP showed 71.3% degradation after 32 d incubation. No degradation occurred at or above 5 μg ml⁻¹ PCP. PCP intermediates, 2,4,6-trichlorophenol (TCP) and 3,4,5 TCP were found in the spent culture of the nitrate reducing enrichment. In the spent culture of the sulfate reducing enrichment, 3,4,5 TCP and 2,3,4,5-tetrachlorophenol were found. Attempts to obtain a pure culture of an anaerobic PCP degrading bacterium were unsuccessful. / Master of Science

Bench scale activated sludge reactors were acclimated to dextrose and then to low levels of pentachlorophenol. The metabolic activity of activated sludge was evaluated by its specific rate of dextrose uptake, Δs/Δt/X (measured by COD removal). Depression of the specific uptake rate, resulting from batch experiments in which the activated sludges were spiked with priority pollutants, indicated the relative inhibition caused by that toxin dosage. This study intended to determine if: (1) acclimation to low levels of PCP would provide any protection to the biomass against detrimental effects of higher shock loads of pentachlorophenol; (2) PCP-acclimation would decrease the inhibitory effect of related priority pollutants; (3) PCP would be consistently and efficiently degraded in the reactors. The practicality of this study was twofold. First, to determine the feasibility of introducing small concentrations of a toxin to the biological system of a treatment facility in order to gain protection against shock loads of that and related toxic chemicals. Secondly, to develop a rapid and easy method for evaluating the effects of a chemical load on activated sludge. The procedure was found to be applicable and it showed that acclimation of activated sludge to PCP provided protection against shock loads of pentachlorophenol as well as phenol, 4-nitrophenol, 2-chlorophenol, and 2,4,6-trichlorophenol. Gas chromatography analysis showed very little disappearance of PCP in the 1 mg PCP/L reactor; however, in the 15 mg PCP/L reactor, the penta concentration decreased to virtually zero for about a week and then it began to gradually increase. / Master of Science

by Wang Pui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 115-128). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / List of Figures --- p.vi / List of Tables --- p.viii / Abbreviations --- p.ix / Chapter 1. --- Introduction Pg no / Chapter 1.1 --- Ligninolytic enzyme systems --- p.1 / Chapter 1.2 --- Three main ligninolytic enzymes --- p.3 / Chapter 1.2.1 --- Lignin peroxidases (LiP) --- p.3 / Chapter 1.2.2 --- Gene structure and Amino acid sequence structure --- p.7 / Chapter 1.2.3 --- Regulation of expression --- p.8 / Chapter 1.3. --- MnP --- p.8 / Chapter 1.3.1 --- General properties --- p.8 / Chapter 1.3.2 --- Gene structure and Amino acid sequence --- p.9 / Chapter 1.3.3 --- Regulation of Expression --- p.12 / Chapter 1.4 --- Laccase --- p.12 / Chapter 1.4.1 --- General Properties --- p.12 / Chapter 1.4.2 --- Gene structure and Amino acid sequence --- p.14 / Chapter 1.5 --- Pentachlorophenol (PCP) --- p.16 / Chapter 1.5.1 --- Production --- p.16 / Chapter 1.5.2 --- Toxicity --- p.15 / Chapter 1.5.3 --- Persistence --- p.19 / Chapter 1.6 --- Oyster mushroom --- p.22 / Chapter 1.7 --- Application of ligninolytic enzymes in bioremediation --- p.23 / Chapter 1.7.1 --- Genetic modification --- p.23 / Chapter 1.7.2 --- Characterization of enzymes properties --- p.25 / Chapter 1.7.3 --- Ligninolytic enzymes Purification and extraction --- p.26 / Chapter 1.7.4 --- Immobilization of ligninolytic enzymes --- p.26 / Chapter 1.8 --- Fermentation --- p.29 / Chapter 1.8.1 --- Different types of fermentation --- p.29 / Chapter 1.8.1.1 --- Submerged fermentation (SF) --- p.29 / Chapter 1.8.1.2 --- Solid State Fermentation (SSF) --- p.30 / Chapter 1.9 --- Proposal and experimental plan of the project --- p.33 / Chapter 1.9.1 --- Objectives --- p.34 / Chapter 2. --- Methods --- p.36 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Culture maintenance --- p.36 / Chapter 2.1.2 --- Preparation of Pentachlorophenol (PCP) stock solution --- p.36 / Chapter 2.2 --- Optimization of production of ligninolytic enzymes by effective PCP concentration --- p.37 / Chapter 2.2.1 --- Preparation of mycelial homogenate --- p.37 / Chapter 2.2.2 --- Incubation --- p.37 / Chapter 2.2.3 --- Specific enzyme assays --- p.38 / Chapter 2.2.3.1 --- Laccase --- p.38 / Chapter 2.2.3.2 --- Manganese peroxidase (MnP) --- p.39 / Chapter 2.2.3.3 --- Lignin peroxidase (LiP) --- p.39 / Chapter 2.2.3.4 --- Protein --- p.39 / Chapter 2.3 --- Cloning of specific PCP-degradative laccase cDNA --- p.40 / Chapter 2.3.1 --- Isolation of total RNA --- p.41 / Chapter 2.3.2 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.41 / Chapter 2.3.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.4 --- Amplification of laccase cDNA --- p.43 / Chapter 2.3.4.1 --- Design of primers for PCR reaction --- p.43 / Chapter 2.3.4.2 --- Polymerase chain reaction --- p.44 / Chapter 2.3.5 --- Agarose gel electrophoresis of DNA --- p.44 / Chapter 2.3.6 --- Purification of PCR products --- p.45 / Chapter 2.3.7 --- TA cloning of PCR products --- p.46 / Chapter 2.3.8 --- Preparation of Escherichia coli competent cells --- p.46 / Chapter 2.3.9 --- Bacterial transformation by heat shock --- p.47 / Chapter 2.3.10 --- Colony screening --- p.48 / Chapter 2.3.11 --- Mini-preparation of plasmid DNA --- p.48 / Chapter 2.3.12 --- Sequencing --- p.49 / Chapter 2.3.13 --- Identification of sequence --- p.51 / Chapter 2.4 --- Study of regulation temporal expression of laccase genes by PCP --- p.51 / Chapter 2.4.1 --- Semi-quantitative PCR --- p.51 / Chapter 2.4.1.1 --- Design of gene-specific primers --- p.51 / Chapter 2.4.1.2 --- Determination of suitable PCR cycles --- p.54 / Chapter 2.4.1.3 --- Normalization of the amount of RNA of each sample --- p.54 / Chapter 2.5 --- Quantification of residual PCP concentration --- p.55 / Chapter 2.5.1 --- Extraction of PCP --- p.55 / Chapter 2.5.2 --- High performance liquid chromatography --- p.55 / Chapter 2.5.3 --- Assessment criteria --- p.56 / Chapter 2.6 --- Effect of other componds on laccase activity and laccase expression --- p.56 / Chapter 2.6.1 --- Study of different isoform of laccase --- p.57 / Chapter 2.6.2 --- SDS-PAGE analysis of proteins --- p.58 / Chapter 2.7 --- Study of laccase expression and laccase activity in fruiting process of oyster mushroom --- p.59 / Chapter 2.8 --- Statistical analysis --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Production of Ligninolytic Enzymes by oyster mushroom / Chapter 3.1.1 --- Optimization of laccase production --- p.62 / Chapter 3.1.2 --- Optimization of MnP production --- p.64 / Chapter 3.1.3 --- Change of Protein content at different PCP concentration and time --- p.64 / Chapter 3.1.4 --- Change of specific activity at different PCP concentration and time --- p.64 / Chapter 3.1.5 --- Toxicity of PCP towards mycelial growth --- p.67 / Chapter 3.1.6 --- Enzyme productivities of laccase and MnP --- p.67 / Chapter 3.1.7 --- Change of % of residual PCP concentrations during 14 days --- p.70 / Chapter 3.2. --- Cloning of PCP-degradative laccase genes --- p.70 / Chapter 3.3 --- Regulation of expression of the laccase genes by PCP --- p.74 / Chapter 3.3.1 --- Determination of suitable PCR cycles --- p.74 / Chapter 3.3.2 --- Normalization of total RNA amount of different samples --- p.74 / Chapter 3.3.3 --- Regulation of temporal expression of the laccase genes by PCP --- p.74 / Chapter 3.4 --- Effect of other compounds and physiological status on laccase activity and expression --- p.81 / Chapter 3.5 --- Study of different forms of laccase --- p.86 / Chapter 4. --- Discussion --- p.93 / Chapter 4.1 --- Production of Ligninolytic enzymes by Pleurotus pulmonarius / Chapter 4.1.1 --- Optimization of laccase and MnP production by PCP --- p.95 / Chapter 4.2 --- Cloning of laccase genes --- p.97 / Chapter 4.2.1 --- Cloning strategy --- p.97 / Chapter 4.2.2 --- Analysis of Nucleotide sequence of Lac1 - Lac3 --- p.99 / Chapter 4.2.3 --- Characterization and comparison of deduced amino acid sequences of Lacl-Lac3 --- p.99 / Chapter 4.3 --- Regulation of expression of the laccase genes by PCP --- p.100 / Chapter 4.3.1 --- Regulation of temporal expression by PCP --- p.100 / Chapter 4.4 --- Effect of the potential inducers on laccase activity and expression --- p.103 / Chapter 4.5 --- Effect of the physiological status on laccase activity and expression --- p.105 / Chapter 4.5.1 --- Production of PCP-degradative laccase by Solid-state fermentation --- p.107 / Chapter 4.5.2 --- Uses of molecular probe in bioremediation --- p.107 / Chapter 4.6 --- Different isoforms of laccase --- p.109 / Chapter 4.7 --- Conclusion --- p.112 / Chapter 4.8 --- Further studies / Chapter 4.8.1 --- Confirmation of PCP-degradation by gene product of Lac1 and Lac2 --- p.114 / Chapter 4.8.2 --- Optimization of PCP-degradative laccases production by solid-state fermentation --- p.114 / Chapter 5. --- References --- p.115

Yau Sze-nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 155-177). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.v / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Organopollutants and environment --- p.1 / Chapter 1.2 --- Pentachlorophenol --- p.3 / Chapter 1.2.1 --- Application of pentachlorophenol --- p.3 / Chapter 1.2.2 --- Characteristics of PCP --- p.4 / Chapter 1.2.3 --- Toxicity of PCP --- p.5 / Chapter 1.2.4 --- Environmental exposure of PCP --- p.6 / Chapter 1.3 --- Wastewater treatments of organopollutants --- p.9 / Chapter 1.3.1 --- Physical treatment --- p.10 / Chapter 1.3.2 --- Chemical treatment --- p.10 / Chapter 1.3.3 --- Bioremediation --- p.11 / Chapter 1.4 --- Biodegradation of PCP --- p.13 / Chapter 1.4.1 --- Biodegradation of PCP by bacteria --- p.13 / Chapter 1.4.2 --- Biodegradation of PCP by fungi --- p.14 / Chapter 1.5 --- Ligninolytic enzyme --- p.16 / Chapter 1.5.1 --- Lignin peroxidase --- p.16 / Chapter 1.5.2 --- Manganese peroxidase --- p.19 / Chapter 1.5.3 --- Laccase --- p.21 / Chapter 1.5.4 --- Biodegradation of PCP and other organopollutants by ligninolytic enzymes --- p.25 / Chapter 1.6 --- Structure and gene regulation --- p.27 / Chapter 1.6.1 --- MnP gene and structure --- p.27 / Chapter 1.6.1.1 --- Structure of MnP --- p.27 / Chapter 1.6.1.2 --- MnP gene regulation --- p.30 / Chapter 1.6.2 --- Laccase gene and structure --- p.31 / Chapter 1.6.2.1 --- Structure of laccase --- p.31 / Chapter 1.6.2.2 --- Laccase gene regulation --- p.32 / Chapter 1.7 --- Pleurotus pulmonarius --- p.36 / Chapter 1.8 --- Aims of study --- p.37 / Chapter 2 --- MATERIALS & METHOD --- p.39 / Chapter 2.1 --- Optimization of PCP induction in broth system --- p.39 / Chapter 2.1.1 --- Specific enzyme assays --- p.41 / Chapter 2.1.1.1 --- Assay for laccase activity --- p.41 / Chapter 2.1.1.2 --- Assay for manganese peroxidase (MnP) activity --- p.41 / Chapter 2.1.1.3 --- Assay for protein assay --- p.41 / Chapter 2.1.2 --- PCP effect on biomass gain --- p.42 / Chapter 2.1.3 --- Extraction of PCP --- p.42 / Chapter 2.1.3.1 --- Preparation of PCP stock solution --- p.43 / Chapter 2.1.3.2 --- Extraction efficiency of PCP --- p.43 / Chapter 2.1.3.3 --- Quantification of PCP by HPLC --- p.43 / Chapter 2.1.3.4 --- Study of PCP degradation pathway using GC-MS --- p.44 / Chapter 2.2 --- Isolation of laccase and manganese peroxidase coding genes --- p.46 / Chapter 2.2.1 --- Preparation of ribonuclease free reagents and apparatus --- p.46 / Chapter 2.2.2 --- Isolation of RNA --- p.46 / Chapter 2.2.3 --- Quantification of total RNA --- p.47 / Chapter 2.2.4 --- First strand cDNA synthesis --- p.47 / Chapter 2.2.5 --- Polymerase Chain Reaction (PCR) --- p.48 / Chapter 2.2.6 --- Gel electrophoresis --- p.50 / Chapter 2.2.7 --- Purification of PCR products --- p.50 / Chapter 2.2.8 --- Preparation of Escherichia coli competent cells --- p.51 / Chapter 2.2.9 --- Ligation and E. coli transformation --- p.51 / Chapter 2.2.10 --- PCR screening of E. coli transformation --- p.52 / Chapter 2.2.11 --- Isolation of recombinant plasmid --- p.52 / Chapter 2.2.12 --- Sequence analysis --- p.53 / Chapter 2.2.13 --- Construction of dendrogram for Pleurotus sp. laccase and manganese peroxidase dendrogram --- p.54 / Chapter 2.2.13.1 --- Dendrogram of laccase genes --- p.55 / Chapter 2.2.13.2 --- Dendrogram of manganese genes --- p.55 / Chapter 2.3 --- Differential regulation profiles of laccase and manganese peroxidase genes --- p.57 / Chapter 2.3.1 --- Time course of the effects of PCP on levels of laccase and manganese peroxidase mRNAs --- p.57 / Chapter 2.3.1.1 --- Isolation of RNA --- p.57 / Chapter 2.3.1.2 --- RT-PCR --- p.57 / Chapter 2.3.2 --- The effect of different stresses --- p.65 / Chapter 2.3.2.1 --- Pollutant removal analysis --- p.66 / Chapter 2.3.2.2 --- Differential gene expression under different stresses --- p.69 / Chapter 2.4 --- Construction of full-length cDNA --- p.69 / Chapter 2.4.1 --- Primer design --- p.69 / Chapter 2.4.2 --- First-strand cDNA synthesis --- p.71 / Chapter 2.4.3 --- RACE PCR reactions --- p.71 / Chapter 2.5 --- Statistical analysis --- p.73 / Chapter 3 --- RESULT --- p.74 / Chapter 3.1 --- Optimization of PCP induction in broth system --- p.74 / Chapter 3.1.1 --- Enzyme Assay --- p.74 / Chapter 3.1.1.1 --- Protein content --- p.74 / Chapter 3.1.1.2 --- Specific laccase activity --- p.74 / Chapter 3.1.1.3 --- Specific MnP activity --- p.76 / Chapter 3.1.1.4 --- Laccase productivity --- p.78 / Chapter 3.1.1.5 --- MnP productivity --- p.78 / Chapter 3.1.2 --- PCP effect on biomass development --- p.80 / Chapter 3.1.3 --- PCP removal --- p.80 / Chapter 3.2 --- isolation of laccase and manganese peroxidase coding genes --- p.83 / Chapter 3.2.1 --- Dendrogram construction for heterologous MnP and laccase coding genes --- p.83 / Chapter 3.2.2 --- Phylogeny of ligninolytic enzyme coding genes of P. pulmonarius --- p.85 / Chapter 3.2.2.1 --- Phylogeny of MnP coding genes --- p.88 / Chapter 3.2.2.2 --- Phylogeny of laccase coding genes --- p.88 / Chapter 3.3 --- differential regulation profiles of laccase and MnP genes --- p.91 / Chapter 3.3.1 --- Time course of the effects of PCP on levels of MnP and laccase mRNAs --- p.91 / Chapter 3.3.1.1 --- Time course of the effects of PCP on levels of MnP mRNAs --- p.91 / Chapter 3.3.1.2 --- Time course of the effects of PCP on levels of laccase mRNAs --- p.97 / Chapter 3.3.2 --- The effects of different stresses and two lignocellulosic substrates --- p.99 / Chapter 3.3.2.1 --- The effect on laccase and MnP enzyme activities --- p.99 / Chapter 3.3.2.1.1 --- Protein content --- p.99 / Chapter 3.3.2.1.2 --- Specific laccase activity --- p.100 / Chapter 3.3.2.1.3 --- Specific MnP activity --- p.102 / Chapter 3.3.2.1.4 --- Dry weight of P. pulmonarius --- p.102 / Chapter 3.3.2.1.5 --- Laccase productivity --- p.105 / Chapter 3.3.2.1.6 --- MnP productivity --- p.105 / Chapter 3.3.2.2 --- Organopollutant removal --- p.107 / Chapter 3.3.2.3 --- Differential gene expression under different stresses --- p.107 / Chapter 3.3.2.3.1 --- The effect on MnP mRNAs --- p.107 / Chapter 3.3.2.3.2 --- The effect on laccase mRNAs --- p.115 / Chapter 3.4 --- Construction of full-length cDNA --- p.116 / Chapter 3.4.1 --- PPMnP5 --- p.117 / Chapter 3.4.2 --- PPlac2 --- p.120 / Chapter 3.4.3 --- PPlac6 --- p.120 / Chapter 4 --- DISCUSSION --- p.123 / Chapter 4.1 --- Optimization of PCP induction in broth system --- p.123 / Chapter 4.2 --- Isolation of MnP and laccase coding genes --- p.126 / Chapter 4.3 --- Differential regulation profiles of MnP and laccase genes --- p.128 / Chapter 4.3.1 --- The effects incubation time and PCP on levels of MnP and laccase mRNAs --- p.128 / Chapter 4.3.1.1 --- MnP --- p.129 / Chapter 4.3.1.2 --- Laccase --- p.129 / Chapter 4.3.2 --- Regulation of MnP and laccase by different substrates --- p.130 / Chapter 4.3.2.1 --- Regulation of MnP and laccase activities --- p.131 / Chapter 4.3.2.2 --- Organopollutant removal --- p.132 / Chapter 4.3.2.3 --- Regulation of MnP coding genes --- p.136 / Chapter 4.3.2.4 --- Regulation of laccase coding genes --- p.137 / Chapter 4.4 --- "Characterization of full length cDNAs of PPMnP5, PPlac2 and PPLAC6" --- p.140 / Chapter 4.4.1 --- PPMnP5 --- p.140 / Chapter 4.4.2 --- PPlac2 and PPlac6 --- p.144 / Chapter 4.4.3 --- Real-time PCR --- p.146 / Chapter 4.4.3.1 --- Methodology for SYBR-Green real-time PCR --- p.146 / Chapter 4.4.3.2 --- Comparison of conventional PCR and real-time PCR --- p.148 / Chapter 4.5 --- APPLICATION AND FURTHER INVESTIGATION --- p.150 / Chapter 5 --- CONCLUSION --- p.152 / Chapter 6 --- REFERENCES --- p.155