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

by chan Cho-Yin. / "November 2005." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 128-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Text in English; abstracts in English and Chinese. / by Chan Cho-Yin.

Sandra Jane Chapman. / "October 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 163-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Yubing Li. / "September 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 173-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Este trabalho consistiu em aprimorar o entendimento da rota de migração do óleo no reservatório e verificar a possibilidade de variação da intensidade da biodegradação com as heterogeneidades existentes. Foram utilizadas como base para a dissertação sete amostras coletadas na bacia sedimentar do Paraná, no arenito asfáltico do Anhembi, afloramento da Fazenda Betumita. A Fazenda Betumita é considerada a ocorrência mais expressiva de óleo na região do alto estrutural do Anhembi, apresentando a maior acumulação de arenito asfáltico na borda leste da Bacia do Paraná. A ocorrência dos arenitos asfálticos na área de estudo é predominantemente por arenitos da Formação Pirambóia. Estes arenitos foram preenchidos por hidrocarbonetos relacionados ao sistema Irati-Pirambóia e são caracterizados como imaturo, devido ausência de n-alcanos e abundância de esteranos e terpanos. As amostras coletadas foram analisadas através da cromatografia líquida e gasosa e correlacionadas com a descrição das fácies do afloramento. A biodegradação do óleo apresentou a tendência de aumentar do topo para a base do afloramento, local caracterizado por fácies subaquosas, onde se encontra o contato óleo/água, propício para o crescimento dos microorganismos degradadoras de óleo. Na fácie de interduna, a biodegradação foi menor, pois este ambiente é caracterizado por partículas argilo-minerais e menores permo-porosidade, não propício para o crescimento de microorganismos capazes de degradar o óleo. Foi observada a presença de diasteranos e 25-norhopanos nas amostras coletadas, indicando que o óleo do afloramento está severamente biodegradado. Os esteranos apresentaram maior biodegradação na base do afloramento onde está o contato óleo/água e maior reposição de oxigênio pela infiltração de água meteórica, tornando-se ambiente propício para crescimento das bactérias aeróbicas tendendo a degradar preferencialmente os esteranos. Entretanto os hopanos apresentaram maior biodegradação no topo do afloramento, local com condições propícias para o crescimento das bactérias anaeróbicas, que tenderam a degradar preferencialmente os hopanos. As informações adquiridas nesta pesquisa são de grande relevância para o conhecimento na exploração do petróleo, pois geralmente esses conhecimentos não estão disponíveis nos dados de subsuperfície. Este trabalho servirá de parâmetro para o planejamento da produção e recuperação secundária e terciária de reservatórios com fácies sedimentares semelhantes da área estudada. / This report concerns to improve understanding of the migration route of the oil in the tank and check the possibility of varying the intensity of biodegradation with the existing heterogeneities. Were used as basis for the dissertation seven samples collected in the sedimentary Paraná Basin, in the Anhembi tar sandstone, outcrops of Betumita Farm. Betumita Farm is considered the more expressive occurrence of oil in the Anhembi High, showing the greatest accumulation of tar sandstone on the eastern margin of the Paraná Basin. The occurrence of tar sandstones in the study area is predominantly of sandstones of the Pirambóia Formation. These sandstones were filled by oil related Irati Pirambóia system and are characterized as immature due to the absence of n-alkanes and abundance of steranes and terpanes. The samples were analyzed by gas and liquid chromatography and related to the description of the facies outcrop. The biodegradation of the oil tended to increase from the top to the bottom of the outcrop, characterized by local subaqueous facies, where the contact oil/water, suitable for the growth of microorganisms to degrade oil. In the interdune facie, the biodegradation was lower, because this environment is characterized by clay-mineral particles and lower porosity and permeability, not conducive to the growth of microorganisms capable of degrading oil. Was observed the presence of 25-norhopanes and diasteranes in the samples, indicating that the oil outcrop was severely degraded. The steranes showed higher degradation at the bottom of the outcrop where the contact is oil/water and higher oxygen replenishment by infiltration of meteoric water, making it suitable environment for growth of aerobic bacteria tend to preferentially degrade steranes. However the hopanes showed higher biodegradation at the top of the outcrop, with local conditions conducive to the growth of anaerobic bacteria, which tended to degrade preferentially the hopanes. The information gained in this research are highly relevant to the knowledge on oil exploration, as these skills are not generally available in the subsurface data. This work will serve as a parameter for production planning and recovery secondary and tertiary of reservoirs with similar facies sedimentary of the studied area.

Orientador: Silvana Artioli Schellini / Banca: Erika Hoyama / Banca: Roberta Lilian Fernandes de Souza Meneghim / Banca: Patricia Mitiko Santello Araishi / Banca: Ana Estela Besteti Pires Ponce Sant'Anna / Resumo: A Nanoskin® é uma película de celulose bacteriana produzida pela bactéria Acetobacter xylinum,por meio de um processo biotecnológico. É composta por uma rede de nanofibrilas cuja estrutura cria uma extensa superfície, a qual permite a retenção de grande quantidade de água e importantes modificações em seu formato, sem perder suas características estruturais. Objetivo: visando acrescentar novas possibilidades para a reconstrução da pálpebra, em seus folhetos profundo ou superficial, o presente estudo foi desenvolvido com o objetivo de avaliar se a película de Nanoskin® poderia ser uma opção. Método: foram utilizadas 40 cobaias, do sexo masculino, que receberam fragmentos de Nanoskin® na região dorsal, em dois estudos, um direcionado para a utilização da Nanoskin nos tecidos profundos, quando o biomaterial foi colocado no subcutâneo e outro no qual a Nanoskin foi colocada na superfície da pele. Em ambos os estudos foram utilizados dois tipos de Nanoskin: grupo 1 (G1), no qual foi utilizada película de Nanoskin® (2X2 cm) sem recobrimento de gelatina no subcutâneo ou na superfície da pele e o grupo 2 (G2), que recebeu implante de Nanoskin® (2X2 cm) com revestimento de gelatina no subcutâneo e na superfície da pele. O grupo controle foi obtido com colocação de enxerto de pele de espessura total no tamanho de 2X2 cm, contíguo ao implante de Nanoskin®, em todos os animais de G1 e de G2 do experimento que estudou o biomaterial na superfície da pele. Cinco animais de cada grupo foram eutanasiados em quatro momentos experimentais: 7 dias (M1), 30 dias (M2), 90 dias (M3) e 180 dias (M4). Foram realizadas avaliações morfométricas do implante das lâminas histológicas, exame histológico e exame ultraestrutural. Resultados: a Nanoskin® quando implantada no subcutâneo não foi encontrada em um animal de M3 e em cinco animais de M4. Nos momentos M3, M4 e M5, houve separação entre as suas lamelas. Houve... / Abstract: The Nanoskin® is a bacterial cellulose film produced by the bacteria Acetobacter xylinum by means of a biotechnological process. It is composed by a network of nanofibrils whose structure creates a large surface area, which allows the retention of a large amount of water and significant changes in its shape without losing its structural characteristics. Purpose: aiming to add new possibilities for the reconstruction of the eyelid, in its superficial or deep lamellae, this study was developed in order to assess whether the Nanoskin® film could be an option. Method: 40 male guinea pig were used, that received Nanoskin® fragments in the dorsal region, in two studies, one directed to the use of Nanoskin® in deep tissue when the biomaterial was placed subcutaneously and another in which the Nanoskin® was placed on skin surface. In both studies we used two types of Nanoskin: group 1 (G1), which was used Nanoskin® film (2x2 cm) without gelatin coating on the subcutaneous or on the skin surface, and Group 2 (G2), which received Nanoskin® implant (2X2 cm) with gelatin coating on the subcutaneous and on the skin surface. The control group was obtained with full-thickness skin graft placement on the size of 2X2 cm Nanoskin® adjacent to the implant in all animals in G1 and G2 experiment that studied the biomaterial on the surface of the skin. Five animals from each group were euthanized at four experimental times: 7 days (M1), 30 days (M2), 90 days (M3) and 180 days (M4). Morphometric assessments of the implant and the histological slides were held, histological and ultrastructural examination. Results: the Nanoskin® when implanted subcutaneously was not found in one animal from M3 and in five animals from M4. In moments M3, M4 and M5, there was separation between their lamellae . There was a significant inflammation at the beginning of the experiment which reduced the following times, and formation of a pseudocapsule around the ... / Doutor

No presente trabalho foram estudados os metabólitos gerados no processo de biodegradação do corante Remazol brilliant blue R (RBBR) pelo fungo Tinctoporellus sp. em meio líquido. Foi investigada a descoloração do meio de fermentação causada por o processo de degradação, monitorando-se a absorbância da solução durante 17 dias por espectrofotometria UV-Vis. Na presença do fungo Tinctoporellus sp. observou-se uma perda de 90% da coloração do meio de crescimento contendo RBBR 90% em um período de 12 dias. Um experimento de degradação do RBBR em 6 L foi realizado e, após 12 dias, o micélio foi filtrado do meio líquido e os analitos foram extraídos do meio de cultura utilizando-se técnicas de extração em fase sólida. O extrato obtido neste processo foi submetido a separações cromatográficas em gel de Sephadex LH-20, sílica gel derivatizada com grupo C18, assim como purificações por HPLC. Estas separações permitiram o isolamento e identificação de quatro produtos da degradação do corante, derivados antraquinônicos, dos quais três ainda não conhecidos. Além disso, três sesquiterpenos irregulares tremulanos, também inéditos, foram isolados e identificados, a partir do meio de cultura de degradação do RBBR. / In the present study we have investigated metabolites generated during the biodegradation process of the dye Remazol Brilliant Blue R (RBBR), by the action of the marine-derived fungus Tinctoporellus sp. in liquid medium. The decolorization of the fermentation medium caused by the degradation process was monitored by measuring the medium absorbance by UV-Vis during 17 days. The growth medium of Tinctoporellus sp. decolorized up to 90% after 12 days. RBBR degradation experiment with Tinctoporellus sp. performed in large scale during 12 days afforded four dye degradation products, identified as anthraquinone derivatives, three of which not yet new reported in the literature. These anthraquinone derivatives have been isolated by chromatography techniques and identified by spectroscopic analysis. Additionally, three novel tremulane terpenes have also been isolated and identified.

Plastificantes podem ser definidos como aditivos de baixa volatilidade utilizados para aumentar a processabilidade, flexibilidade ou diminuir a dureza de materiais poliméricos. Os ftalatos e adipatos utilizados como plastificantes, por sua baixa solubilidade em água e pelo alto coeficiente de partição octanol/água, tendem a se acumular no solo e sedimentos. Estes compostos são considerados potencialmente carcinogênicos, teratogênicos e disruptores endócrinos. A presente pesquisa compreendeu a biorremediação ex-situ do solo contaminado com resíduos de uma unidade industrial de plastificantes, utilizando reatores aeróbios, com microrganismos indígenas e exógenos adaptados através da adição de inóculo retirado da Estação de Tratamento de Efluentes por Lodos Ativados desta indústria. Foram avaliados os plastificantes: DIBP (Di-isobutilftalato), DBP (Dibutilftalato), DOP (Dioctilftalato),DIDP (Di-isodecilftalato), DIAP (Di-isoamilftalato) e DOA (dioctiladipato). Após a caracterização geotécnica do solo da área de plastificantes em 10 diferentes pontos, foram retiradas as quantidades para a biorremediação em 8 diferentes pontos (100kg/ponto) com os teores totais de plastificantes compreendidos entre 17 mg/kg solo a 6222 mg/kg solo. Análises mineralógicas, físicas e químicas foram realizadas posteriormente e indicaram que possivelmente a capacidade de troca catiônica do solo era devida aos plastificantes. Na biorremediação, os teores iniciais de plastificantes no solo, variaram de 85 mg/kg a 1688 mg/kg e após 120 dias de biodegradação em reatores aeróbios, as eficiências de remoção foram de 75 a 97%. Conforme as análises de fingerprint da comunidade bacteriana, ao final do processo, as bactérias presentes no solo eram originárias do lodo e do solo inicial e as análises de CGMS identificaram os metabólitos MEHP e os sub-produtos finais da biodegradação. / Plasticizers are low volatility compounds that offer flexibility and processability to resins. The phthalates and adipates, used as plasticizers, have low water solubility e high partition octanol/water(Kow) and accumulate in soil and sediments. This compounds are considered teratogenics, carcinogenics and as endocrine disruptors. This study evaluated the bioremediation of tropical soil contaminated with plasticizers process wastes, in aerobic conditions, with and without introduction of acclimated bacteria. After geological analysis of soil, considering ten differents points on the factory area, it was selected the soil for biodegradation of eight points (100kg/point) representing 17mg total plasticizers/kg soil to 6222mg total plasticizers/kg soil. Mineralogical, physical and chemical analysis were done and the results showed that perhaps the cationic change capacity was due to plasticizers. The plasticizers contents in soil were 85-1688mg/kg and after 120 days of biodegradation in eight aerobic reactors, the removal efficiencies were 75-97%. The fingerprint analysis showed that the final bacteria present in reactors originated from soil and sludge and the CGMS analysis identified the metabolic MEHP, and showed the sub-products and final products of biodegradation.

O meio ambiente requer polímeros que possam ser degradados e desapareçam por completo pela atuação de vários fatores ambientais, incluindo microrganismos. No presente trabalho estudou-se a biodegradação de espumas de poliuretano obtidas a partir do óleo de mamona, um material renovável e de origem natural, se constituindo em uma alternativa viável para a substituição das tradicionais espumas de PU. O acompanhamento da biodegradação foi realizado para a espuma PU-vegetal e PU-petróleo, através das seguintes técnicas: zona de halo, microscopia eletrônica de varredura (MEV), análise termogravimétrica (TG) e espectrometria na região de absorção do infravermelho por transformada de Fourier, utilizando acessório de refletância total atenuada (FTIR ? ATR). Outras etapas do trabalho envolveram a realização de testes de biodegradação em espumas utilizadas na adsorção de um azocorante utilizado em curtumes (C.I. Acid Orange 61) e o estudo da biodegradação do material polimérico quando se realizam modificações em sua estrutura. As várias técnicas utilizadas indicaram mudança na estrutura química da espuma de origem vegetal, após o ataque de microrganismos, caracterizando um processo de biodegradação; o mesmo não ocorreu com a espuma derivada do petróleo, indicando a manutenção da estrutura da macromolécula. / The environment requires polymers that can be completely biodegraded through the action of several environmental factors, including microorganisms. The present work studied biodegradation of polyurethane foam obtained from castor oil, a renewable natural material, constituting a viable alternative to traditional PU foams. Biodegradation was compared for castor oil PU and conventional petroleum PU, using the following techniques: halo zone, scanning electron microscopy (SEM), thermogravimetric analysis (TG), and Fourier-transform infrared spectroscopy with accessory for attenuated total reflectance (FTIR ? ATR). Other steps in the work involved biodegradation testing for foams utilized in the adsorption of a tanning dye (C.I. Acid Orange 61) and the study of biodegradation in polymer material when submitted to structural modification. Utilization of these techniques indicated change in the chemical structure of the vegetable origin foam after undergoing attack by microorganisms, characterizing a biodegradable process. Petroleum derived foam did not show the same good results, indicating the retention of the macromolecular structure.

A capacidade de degradação de compostos xenobióticos por organismos vivos, principalmente bactérias, tem sido objeto intenso de pesquisa, principalmente por aqueles microrganismos isolados do solo. Dessa forma, a busca por novos nichos que resultem no isolamento de microrganismos altamente eficientes se torna cada vez mais necessária. Assim, dada a diversidade de interações inseto - bactérias, este estudo buscou explorar insetos resistentes a inseticidas como nicho de bactérias com capacidade de degradação de pesticidas para o desenvolvimento de estudos voltados à i) utilização dessas bactérias na biorremediação e ii) determinação de sua contribuição na metabolização de inseticidas pelo hospedeiro. Assim, esse trabalho teve por objetivos i) isolar, identificar e caracterizar microrganismos associados ao trato digestivo de linhagens de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) resistentes a lambda-cyhalothrin e deltamethrin (piretróide), chlorpyrifos ethyl (organofosforado), spinosad (naturalyte) e lufenuron (inibidor de síntese de quitina), ii) verificar o seu potencial de biodegradação desses compostos, iii) estudar sua associação a populações naturais de S. frugiperda e iv) determinar seu papel na metabolização de xenobióticos. Bactérias com potencial de biodegradação foram selecionadas via cultivo seletivo da flora associada ao trato intestinal de lagartas de quinto ínstar de S. frugiperda em meio mínimo M9 acrescido de 10 ?g/mL de um dos inseticidas em estudo como única fonte de carbono. As bactérias isoladas foram sujeitas à análise de PCR-RFLP do gene do 16S rDNA, com três enzimas de restrição (EcoRI, Rsa, DdeI), resultando na identificação de 16 isolados. Após sequenciamento, dez filotipos foram identificados, distribuídos em Firmicutes (Enterococcaceae e Staphylococcaceae), ?-Proteobacteria (Enterobacteriaceae e Pseudomonadaceae), ?-Proteobacteria (Comamonadaceae) e Actinobacteria (Micrococcaceae e Microbacteriaceae). Enterococcus, Pseudomonas e Microbacterium foram os únicos representados por dois filotipos, sendo ainda encontrados um filotipo de Delftia, Leclercia, Staphylococcus e Arthrobacter. Enterococcus casseliflavus e Enterococcus mundtii foram isolados de praticamente todas as linhagens resistentes de S. frugiperda. Análises de antibiograma e do metabolismo de carboidratos indicaram a similaridade entre os clones de E. casseliflavus, enquanto que os clones de E. mundtii diferiram de maneira expressiva. Apesar disso, os alelos analisados para tipagem via multilocus não resultaram em diferenças. Análises de PCR-diagnóstico do intestino de lagartas de S. frugiperda de linhagem suscetível de laboratório resultaram na detecção de quatro dos dez filotipos isolados do intestino de linhagens resistentes de S. frugiperda. Já em populações naturais foram encontrados cinco dos dez filotipos. O isolado com maior capacidade de crescimento em cada inseticida foi selecionado e todos apresentaram resposta dependente da concentração dos inseticidas, sendo encontrado efeito antimicrobiano em alguns inseticidas. A análise de cromatografia acoplada a espectrometria de massas comprovou a degradação dos inseticidas utilizados pelos diferentes isolados. A colonização do trato digestivo de linhagem suscetível de S. frugiperda com um dos filotipos (Microbacterium arborescens) isolado de lagartas resistentes a lufenuron resultou em CL50 cerca de 10 vezes superior àquela da linhagem apossimbionte. Assim, linhagens resistentes de S. frugiperda se mostraram um excelente reservatório de bactérias com capacidade de degradação dos inseticidas testados e que podem auxiliar na metabolização desses produtos pelo hospedeiro, assim como ser exploradas na descontaminação de áreas que apresentam resíduos desses xenobióticos. / The capacity of living organisms to degrade xenobiotics has been intensively studied, mainly for soil-associated bacteria. Thus, there is a growing need to search for new niches to lead to the isolation of highly efficient microrganisms. As insect-bacteria interactions are very diverse, we focused on exploiting insecticide resistant insects as a niche of pesticidedegrading bacteria to develop studies devoted to a) bacterial utilization in bioremediation and ii) determination of bacterial contribution to insecticide metabolization by the host insect. Our main objetives were to i) isolate, identify and characterize the microrganisms associated to the gut of resistant strains of Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) capable to degrade lambda-cyhalothrin,deltamethrin (pyrethroids), chlorpyrifos ethyl (organophosphate), spinosad (naturalyte) and lufenuron (chitin synthesis inhibitor), ii) verify their potential to degrade these insecticides, iii) to verify their association to natural populations of S. frugiperda, and iv) to determine their role in the metabolization of xenobiotics in the host. Bacteria with biodegrading capacity were selected from the gut flora of fifth instars of S. frugiperda on selective media based on the minimum media M9 added of 10 ?g/mL of one of the insecticides selected as the sole carbon source. The isolated bacteria were subjected to RFLP-PCR analysis of the 16S rDNA gene using three restriction enzymes (EcoRI, Rsa, DdeI), which led to the identification of 16 isolates. These isolates were grouped in ten phylotypes after sequencing, representing Firmicutes (Enterococcaceae and Staphylococcaceae), ?-Proteobacteria (Enterobacteriaceae and Pseudomonadaceae), ?- Proteobacteria (Comamonadaceae) and Actinobacteria (Micrococcaceae e Microbacteriaceae). Enterococcus, Pseudomonas and Microbacterium were the only represented by more than two phylotypes. Delftia, Leclercia, Staphylococcus and Arthrobacter were also represented. Enterococcus casseliflavus and Enterococcus mundtii were isolated from almost all resistant lines of S. frugiperda. Antibiogram and carbohydrate metabolism assays indicated the similarity among the isolates of E. casseliflavus, while those of E. mundtii displayed some differences. However, no molecular differences were observed by comparing different alleles in multilocus sequence analysis. Diagnostic-PCR analysis of a susceptible, lab-strain of S. frugiperda allowed the identification of four out of the ten phylotypes isolated from resistant strains. But no bacteria from the susceptible strain could be cultivated in the selective media. In natural populations of S. frugiperda, five out of the ten isolates were detected. The most active isolate to degrade each pesticide was selected for further tests and all displayed a dose-dependent response to the insecticides. Analyses by gas or liquid chromatography-coupled mass spectrometry demonstrated the capacity of each isolate to degrade the insecticides tested. Gut colonization of larvae from the susceptible strain with Microbacterium arborescens isolated from resistant larvae increased the LC50 to lufenuron in 10 fold when compared to the aposymbiont strain. Thus, resistant strains of S. frugiperda are an excellent reservoir of bacteria capable of degrading the tested insecticides, and have a potential to be exploited for the bioremediation of xenobiotic contaminated areas. Moreover, these bacteria can auxiliate the host insect to metabolize insecticides and may play a role in insect response to insecticides.