The ability of the ligninolytic fungus Pleurotus ostreatusto degrade 4 pharmaceutical drugs and 5 compounds which are either used during drug manufacturing or are created as by-products was studied. These compounds were detected in groundwater near a drug manufacturing plant. The maximum concentration levels of the selected compounds in tested groundwater samples variedfrom0.23 µg/lto 227.87 µg/l apart from 1 compound which was not detected in any sample. The degradation efficiency of P. ostreatus was examined with individual compounds as well as with the mixture of all 9 compounds. When degrading individual compounds P. ostreatus lowered the initial concentration (10 mg/l) of 5 compounds by 62-100% after 14-day cultivation in malt extract-glucose medium. When degrading the compound mixture P. ostreatus lowered the initial concentration (2 mg/l of each compound) of 5 compounds by 50-100% after 14-day cultivation in malt extract-glucose medium. Acute toxicity tests with Vibrio fischeri suggest the formation of metabolites which are more toxic than the original compounds. The EC50 value for individual compounds during toxicity tests with Vibrio fischeri was 5.45-131.98 mg/l. Keywords:biodegradation, pharmaceuticals, ligninolytic fungi, Pleurotus ostreatus, groundwater, toxicity, Vibrio fischeri

Orientador: Lin Chau Ming / Coorientador: Edson Luiz Furtado / Coorientador: Diego Cunha Zied / Banca: Kassandra Sussi Mustafé Oliveira / Banca: Meire Cristina Nogueira de Andrade / Resumo: Os cogumelos são uma alternativa muito viável na transformação de materiais lignocelulolíticos em produtos uteis, resultam na geração de alimento rico em proteínas, fibras, sais minerais, vitaminas, com baixo teor de lipídeos e carboidratos. Para a produção do cogumelo é necessário obter a Spawn, que pode ser feito a partir de um fragmento ou pseudotecido retirado do seu píleo. Para a fungicultura a fase laboratorial é tão importante quanto à fase no campo. O cultivo de cogumelos gera uma grande quantidade de composto pós-cultivo (SMC - "spent mushroom compost"). O presente trabalho buscou avaliar a influência da maturidade da Spawn na produtividade do cogumelo Pleurotus ostreatus utilizando tempos distintos de maturação. Após o cultivo do fungo foi testado o substrato exaurido na produção de mudas de alface, que foram levadas a campo para avaliar produtividade e análise bioquímica. Para a avaliação da Spawn foram utilizados sete sacos de 2 Kg de composto inoculado a cada três dias, onde a primeira inoculação ocorreu no 13º dia de maturação da Spawn e a ultima no 25º dia. Foram avaliados número de cachos, número de cogumelos, peso dos cachos, massa média de cachos e cogumelos e produtividade. Os maiores valores de produtividade foram obtidos pela 2ª e 3ª inoculação com 19,0 e 19,7 %, respectivamente, com 16 e 19 dias de maturação da Spawn, apresentando uma curva muito semelhante a de crescimento microbiano em geral, em fase Log. A primeira inoculação foi a menos produtiva, e ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mushrooms are a very viable alternative in the transformation of lignocellulolytic materials into useful products, resulting in the generation of food rich in proteins, fibers, minerals, vitamins, low in lipids and carbohydrates. For the production of the mushroom it is necessary to obtain the inoculum, which may be made from a fragment or pseudotecido taken from the colonus thereof. For fungicide the laboratory phase is as important as the phase in the field. Mushroom cultivation generates a large amount of spent mushroom compost (SMC). The present work aimed to evaluate the influence of the inoculum maturity on the productivity of the Pleurotus ostreatus mushroom using distinct maturation times. After the fungus was cultivated the exhausted substrate was tested in the production of lettuce seedlings, which were taken to field to evaluate productivity and biochemical analysis. For the evaluation of the Spawn, seven bags of 2 kg of inoculated substrate were used every three days, where the first inoculation occurred on the 13th day of mycelial run and the last on the 25th day. Number of bunches, number of mushrooms, weight of bunches, average mass of bunches and mushrooms were evaluated as a parameter for evaluation of productivity. The highest values of productivity were obtained by the 2nd and 3rd inoculation with 19.0 and 19.7%, with 16 and 19 days of seed maturation. It is concluded that the time of maturation of the inoculum influences the yield of the mushroom Pleurotus ostreatus, showing that the production of the inoculum is a primordial phase to reach high yields. Then, the residue of the Pleurotus ostreatus production was used for the cultivation of lettuce (Lactuca sativa L.) seedlings at doses of depleted compound (50%, 30% and 10%) mixed with the commercial substrate Carolina soil®. For comparison, a control with 100% commercial substrate was used. Seed productivity parameters were ... / Mestre

Orientador: Meire Cristina Nogueira de Andrade / Banca: Geisian Maria de Queiroz Fernandes / Banca: Tadeu Antonio Fernandes da Silva Júnior / Banca: Eustáquio Souza Dias / Banca: José Raimundo de Souza Passos / Resumo: Pleurotus ostreatus (shimeji) está entre os três cogumelos comestíveis mais consumidos no Brasil e no mundo. Dentre os fatores relacionados à sua produtividade elevada, a mais relevante é a produção do spawn. Spawn é a primeira etapa no cultivo de cogumelos e inicia-se com o crescimento micelial in vitro em meios de cultura, chamado de matriz primária, posteriormente transferida para substrato sólido nomeada como matriz secundária, e então utilizado como inóculo para produção de cogumelos. O objetivo desse estudo foi avaliar a influência dos diferentes processos de preparo do spawn sob efeito na eficiência biológica do fungo Pleurotus ostreatus. Na matriz primária avaliou-se número de repicagem, velocidade do crescimento micelial, concentração de nutrientes, concentração de dextrose e fontes diversas de nutrientes no meio de cultura em função da eficiência biológica do fungo. Na matriz secundária: tempo de armazenamento, atividade enzimática de lacase (LAC) e manganês peroxidase (MnP) do spawn em função da eficiência biológica de P. ostreatus. Para isto, a pesquisa foi subdividida em dois capítulos. No primeiro avaliou-se a velocidade do crescimento micelial em doze diferentes combinações de meio de cultura utilizando diferentes concentrações de batata, quirera de milho, composto à base de serragem e dextrose, sob efeitos na interferência da eficiência biológica de P. ostreatus. Posteriormente avaliou-se a atividade enzimática LAC e MnP nos spawns e foi correlacionadas à efic... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pleurotus ostreatus (shimeji) is among the three most consumed edible mushrooms in Brazil and worldwide. Among the factors related to its high productivity, the most relevant is spawn production. Spawn is the first step in the cultivation of mushrooms and begins with mycelial growth in vitro in culture media, called the primary matrix, later transferred to a solid substrate named as secondary matrix, and then used as an inoculum for the production of mushrooms. The objective of this study was to evaluate the influence of different spawn preparation processes under effect on the biological efficiency of the fungus Pleurotus ostreatus. In the primary matrix, the number of grains, mycelial growth velocity, nutrient concentration, dextrose concentration and various nutrient sources in the culture medium were evaluated according to the biological efficiency of the fungus. In the secondary matrix: storage time, enzymatic activity of laccase (LAC) and manganese peroxidase (MnP) of spawn as a function of the biological efficiency of P. ostreatus. For this, the research was subdivided into two chapters. In the first, the mycelial growth rate was evaluated in twelve different combinations of culture medium using different concentrations of potato, maize cherry, sawdust and dextrose based compounds, on effects on the biological efficiency of P. ostreatus. The LAC and MnP enzymatic activity in the spawns was then evaluated and correlated to the biological efficiency of the first and seco... (Complete abstract click electronic access below) / Doutor

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

Chan Kam Che. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 199-219). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstracts --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of figures --- p.xiv / List of tables --- p.xix / Abbreviations --- p.xxii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Persistent organic pollutants --- p.1 / Chapter 1.2 --- DDT and DDE --- p.2 / Chapter 1.2.1 --- Background --- p.2 / Chapter 1.2.2 --- Health effects --- p.4 / Chapter 1.2.3 --- Environmental exposure of DDE --- p.4 / Chapter 1.2.4 --- Level of DDE in human --- p.9 / Chapter 1.2.5 --- Biodegradation of DDE --- p.10 / Chapter 1.3 --- Remediation methods --- p.11 / Chapter 1.3.1 --- Physical/ chemical treatment --- p.11 / Chapter 1.3.2 --- Bioremediation --- p.13 / Chapter 1.4 --- Fungal Bioremediation --- p.14 / Chapter 1.5 --- Ligninolytic enzymes --- p.15 / Chapter 1.5.1 --- Laccase --- p.15 / Chapter 1.5.2 --- Peroxidases --- p.20 / Chapter 1.5.2.1 --- Manganese Peroxidase (MnP) --- p.20 / Chapter 1.5.2.1 --- Lignin Peroxidase (LiP) --- p.24 / Chapter 1.6 --- Cultivation of Pleurotus pulmonarius --- p.27 / Chapter 1.7 --- Enzyme technology on environmental cleanup and its limitation --- p.28 / Chapter 1.8 --- Aims and objectives of this study --- p.29 / Chapter Chapter II --- Materials and Methods --- p.30 / Chapter 2.1 --- Organism and growth conditions --- p.30 / Chapter 2.2 --- Cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.30 / Chapter 2.3 --- Treatment of DDE by living P. pulmonarius --- p.31 / Chapter 2.3.1 --- Optimization of DDE removal in broth system --- p.31 / Chapter 2.3.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.32 / Chapter 2.3.1.2 --- Effects of inoculum size on the removal of DDE --- p.33 / Chapter 2.3.1.3 --- Effects of incubation time on the removal of DDE and transcriptional profiles of the ligninolytic enzyme-coding genes --- p.33 / Chapter 2.3.2 --- Optimization of DDE removal in soil system --- p.34 / Chapter 2.3.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.34 / Chapter 2.3.2.2 --- Effects of inoculum size on the removal of DDE --- p.35 / Chapter 2.3.2.3 --- Effects of incubation time on the removal of DDE --- p.35 / Chapter 2.3.2.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.35 / Chapter 2.4 --- Treatment of DDE by 1st SMC of p. pulmonarius grown on straw-based compost --- p.36 / Chapter 2.4.1 --- Optimization of DDE removal in soil system --- p.36 / Chapter 2.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.36 / Chapter 2.5.1 --- Optimization of DDE removal in broth system --- p.36 / Chapter 2.5.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.37 / Chapter 2.5.1.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.37 / Chapter 2.5.1.3 --- Effects of incubation time on the removal of DDE --- p.37 / Chapter 2.5.2 --- Optimization of DDE removal in soil system --- p.37 / Chapter 2.5.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.38 / Chapter 2.5.2.2 --- Effects of amount of crude enzyme preparations on the removal of DDE --- p.38 / Chapter 2.5.2.3 --- Effects of incubation time on the removal of DDE --- p.38 / Chapter 2.6 --- Soil characterization --- p.39 / Chapter 2.6.1 --- Identification of organic contaminants in soil sample from Gene Garden using Gas Chromatography/Mass Spectrometry (GC/MS) --- p.39 / Chapter 2.6.2 --- Determination of soil texture --- p.42 / Chapter 2.6.3 --- Fresh soil/air-dried sample moisture --- p.44 / Chapter 2.6.4 --- "Soil pH, electrical conductivity & salinity" --- p.44 / Chapter 2.6.5 --- Total organic carbon contents --- p.44 / Chapter 2.6.6 --- Total nitrogen and total phosphorus --- p.44 / Chapter 2.6.7 --- Available nitrogen --- p.45 / Chapter 2.6.8 --- Available phosphorus --- p.45 / Chapter 2.6.9 --- Potassium value --- p.46 / Chapter 2.7 --- Quantification of residual DDE level --- p.47 / Chapter 2.7.1 --- Preparation of DDE stock solution --- p.47 / Chapter 2.7.2 --- Extraction and quantification of DDE using Gas Chromatography with Electron Capture Detector (GC/μECD) --- p.47 / Chapter 2.7.3 --- Identification of DDE breakdown products by GC/MS --- p.50 / Chapter 2.8 --- Extraction of protein and ligninolytic enzymes --- p.53 / Chapter 2.8.1 --- Protein assay --- p.53 / Chapter 2.8.2 --- Laccase assay --- p.53 / Chapter 2.8.3 --- Manganese peroxidase assay --- p.54 / Chapter 2.8.4 --- Calculation of activity and specific activity of laccase and manganese peroxidase --- p.54 / Chapter 2.9 --- Estimation of fungal biomass --- p.55 / Chapter 2.9.1 --- Preparation of ergosterol standard solution --- p.56 / Chapter 2.9.2 --- Analysis of ergosterol content --- p.56 / Chapter 2.10 --- Expression of the ligninolytic enzyme-coding genes --- p.58 / Chapter 2.10.1 --- Preparation of ribonuclease free reagents and apparatus --- p.58 / Chapter 2.10.2 --- RNA isolation and purification --- p.58 / Chapter 2.10.3 --- cDNA synthesis --- p.59 / Chapter 2.10.4 --- Semi-quantification of ligninolytic enzyme-coding gene expression by RT-PCR --- p.59 / Chapter 2.11 --- Preparation of crude enzyme preparations from P. pulmonarius compost --- p.63 / Chapter 2.12 --- "Assessment criteria: removal efficiency, RE, and removal capacity, RC" --- p.63 / Chapter 2.13 --- Statistical analysis “ --- p.64 / Chapter Chapter III --- Results --- p.65 / Chapter 3.1 --- Soil characterization --- p.65 / Chapter 3.2 --- Cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.66 / Chapter 3.2.1 --- Mushroom yield --- p.66 / Chapter 3.2.2 --- Protein content --- p.66 / Chapter 3.2.3 --- Specific ligninolytic enzymes activities --- p.66 / Chapter 3.2.4 --- Ergosterol content --- p.69 / Chapter 3.2.5 --- Ligninolytic enzymes productivities --- p.69 / Chapter 3.2.6 --- Expression of the ligninolytic enzyme-coding genes during solid-state-fermentation --- p.72 / Chapter 3.3 --- Treatment of DDE by living P. pulmonaruis --- p.78 / Chapter 3.3.1 --- Optimization of DDE removal in broth system --- p.78 / Chapter 3.3.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.78 / Chapter 3.3.1.1.1 --- Effects of DDE on biomass development --- p.78 / Chapter 3.3.1.1.2 --- Protein content --- p.78 / Chapter 3.3.1.1.3 --- Specific ligninolytic enzyme activities --- p.78 / Chapter 3.3.1.1.4 --- Ligninolytic enzyme productivities --- p.79 / Chapter 3.3.1.1.5 --- DDE removal and removal capacity --- p.79 / Chapter 3.3.1.2 --- Effects of inoculum sizes on the removal of DDE --- p.84 / Chapter 3.3.1.2.1 --- Effects of DDE on biomass development --- p.84 / Chapter 3.3.1.2.2 --- Protein content --- p.84 / Chapter 3.3.1.2.3 --- Specific ligninolytic enzyme activities --- p.85 / Chapter 3.3.1.2.4 --- Ligninolytic enzyme productivities --- p.85 / Chapter 3.3.1.2.5 --- DDE removal and removal capacity --- p.85 / Chapter 3.3.1.3 --- Effects of incubation time on the removal of 4.0 mM DDE/g biomass --- p.89 / Chapter 3.3.1.3.1 --- Effects of DDE on biomass development --- p.89 / Chapter 3.3.1.3.2 --- Protein content --- p.89 / Chapter 3.3.1.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.89 / Chapter 3.3.1.3.4 --- DDE removal and removal capacity --- p.90 / Chapter 3.3.1.3.5 --- Putative degradation derivatives --- p.90 / Chapter 3.3.1.3.6 --- Expression of the ligninolytic enzyme-coding genes during the removal of 4.0 mM DDE/g biomass --- p.94 / Chapter 3.3.1.4 --- Effects of incubation time on the removal of 10.0 mM DDE/g biomass --- p.100 / Chapter 3.3.1.4.1 --- Effects of DDE on biomass development --- p.100 / Chapter 3.3.1.4.2 --- Protein content --- p.100 / Chapter 3.3.1.4.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.100 / Chapter 3.3.1.4.4 --- Expression of the ligninolytic enzyme-coding genes during the removal of 10.0 mM DDE/g biomass --- p.102 / Chapter 3.3.2 --- Optimization of DDE removal in soil system --- p.107 / Chapter 3.3.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.107 / Chapter 3.3.2.1.1 --- Ergosterol content --- p.107 / Chapter 3.3.2.1.2 --- Protein content --- p.107 / Chapter 3.3.2.1.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.107 / Chapter 3.3.2.1.4 --- DDE removal and removal capacity --- p.108 / Chapter 3.3.2.2 --- Effects of inoculum sizes on the removal of DDE --- p.111 / Chapter 3.3.2.2.1 --- Ergosterol content --- p.111 / Chapter 3.3.2.2.2 --- Protein content --- p.111 / Chapter 3.3.2.2.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.111 / Chapter 3.3.2.2.4 --- DDE removal and removal capacity --- p.112 / Chapter 3.3.2.3 --- Effects of incubation time on the removal of DDE --- p.115 / Chapter 3.3.2.3.1 --- Ergosterol content --- p.115 / Chapter 3.3.2.3.2 --- Protein content --- p.115 / Chapter 3.3.2.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.115 / Chapter 3.3.2.3.4 --- DDE removal and removal capacity --- p.116 / Chapter 3.3.2.3.5 --- Putative degradation derivatives --- p.116 / Chapter 3.3.2.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.121 / Chapter 3.4 --- Treatment of DDE by 1st SMC of p. pulmonarius grown on straw-based compost --- p.127 / Chapter 3.4.1 --- Optimization of DDE removal in soil system --- p.127 / Chapter 3.4.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.127 / Chapter 3.4.1.1.1 --- Ergosterol content --- p.127 / Chapter 3.4.1.1.2 --- Protein content --- p.127 / Chapter 3.4.1.1.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.127 / Chapter 3.4.1.1.4 --- DDE removal and removal capacity --- p.128 / Chapter 3.4.1.2 --- Effects of inoculum sizes on the removal of DDE --- p.132 / Chapter 3.4.1.2.1 --- Ergosterol content --- p.132 / Chapter 3.4.1.2.2 --- Protein content --- p.132 / Chapter 3.4.1.2.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.132 / Chapter 3.4.1.2.4 --- DDE removal and removal capacity --- p.133 / Chapter 3.4.1.3 --- Effects of incubation time on the removal of DDE --- p.136 / Chapter 3.4.1.3.1 --- Ergosterol content --- p.136 / Chapter 3.4.1.3.2 --- Protein content --- p.136 / Chapter 3.4.1.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.136 / Chapter 3.4.1.3.4 --- DDE removal and removal capacity --- p.137 / Chapter 3.4.1.3.5 --- Putative degradation derivatives --- p.137 / Chapter 3.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.142 / Chapter 3.5.1 --- The crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.142 / Chapter 3.5.2 --- Optimization of DDE removal in broth system --- p.143 / Chapter 3.5.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.143 / Chapter 3.5.2.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.145 / Chapter 3.5.2.3 --- Effects of incubation time on the removal of DDE --- p.147 / Chapter 3.5.2.4 --- Putative degradation derivatives --- p.147 / Chapter 3.5.3 --- Optimization of DDE removal in soil system --- p.151 / Chapter 3.5.3.1 --- Effects of initial DDE concentration on the removal of DDE --- p.151 / Chapter 3.5.3.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.151 / Chapter 3.5.3.3 --- Effects of incubation time on the removal of DDE --- p.154 / Chapter 3.5.3.4 --- Putative degradation derivatives --- p.154 / Chapter Chapter IV --- Discussions --- p.158 / Chapter 4.1 --- Quantification of the expression of the ligninolytic enzyme-coding genes --- p.158 / Chapter 4.2 --- Artificial cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.164 / Chapter 4.3 --- Treatment of DDE by living P. pulmonarius --- p.166 / Chapter 4.3.1 --- Optimization of DDE removal in broth system --- p.166 / Chapter 4.3.2 --- Optimization of DDE removal in soil system --- p.169 / Chapter 4.3.3 --- Phylogeny of the ligninolytic enzyme-coding genes --- p.170 / Chapter 4.3.3.1 --- Laccase coding genes --- p.170 / Chapter 4.3.3.2 --- MnP coding genes --- p.175 / Chapter 4.3.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.178 / Chapter 4.4 --- Treatment of DDE by 1st SMC of P. pulmonarius grown on straw-based compost --- p.183 / Chapter 4.4.1 --- Optimization of DDE removal in soil system --- p.183 / Chapter 4.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.184 / Chapter 4.6 --- Cost-effectiveness of the bioremediation method --- p.185 / Chapter 4.7 --- Further investigations --- p.194 / Chapter Chapter V --- Conclusions --- p.197 / References --- p.199

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

Thesis (M.S.)--Youngstown State University, 2009. / Includes bibliographical references (leaves 63-68). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2009. / Includes bibliographical references (leaves 38-42). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 57-64). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 33-36). Also available via the World Wide Web in PDF format.