A biopolpação consiste no biotratamento de madeira por fungos degradadores de lignina como etapa prévia à produção de polpa celulósica. De uma forma geral, as madeiras biotratadas facilitam os processos posteriores de polpação. Entretanto, ensaios anteriores que avaliaram a biopolpação em escala piloto mostraram que é difícil estabelecer os cultivos de basidiomicetos de interesse livre de bolores contaminantes, já que não é fácil controlar perfeitamente a assepsia durante a inoculação. Para contribuir com o avanço do processo de biopolpação, este trabalho avaliou os cultivos mistos de Ceriporiopsis subvermispora e Phanerochaete chrysosporium sobre madeira de Eucalyptus grandis e Eucalyptus urograndis em regimes de incubação em temperaturas variáveis e em condições não assépticas. A estratégia de iniciar os cultivos em temperatura mais elevada (37°C) e depois manter as culturas em temperaturas variáveis de 27°C e 37°C se mostrou eficiente para inibir o crescimento de contaminantes indesejáveis tanto nos cultivos mistos como nos individuais de cada espécie avaliada. Para avaliar o efeito do biotratamento da madeira numa etapa subsequente de polpação quimiotermomecânica (CTMP) foi feito um estudo preliminar de ajuste de variáveis de polpação por refinamento mecânico de cavacos pré-digeridos com licor alcalino contendo sulfito de sódio. Os dados mostraram que é possível simular o rendimento e as características fisico-mecânicas de polpas industriais empregando uma etapa de pré-digestão dos cavacos com 6% de Na2SO3 e 3% de NaOH a 120°C por 2 h, seguida de desfibramento e refino em refinador de discos. As curvas de refino nesse processo CTMP mostraram que os cavacos biotratados deram origem a polpas com 300 mL de Freeness (CSF) consumindo menos energia do que o observado para o refino da madeira controle. As economias de energia chegaram a 60% em alguns casos, porém não foram maximizadas pelo efeito sinérgico do cultivo misto dos dois basidiomicetos em questão. Experimentos adicionais foram desenvolvidos empregando as espécies fúngicas Pycnoporus sanguineus e Trametes versicolor que foram eficientes para competir com contaminantes mesmo a 27oC. Neste caso, as economias de energia no processo CTMP subsequente somente foram obtidas com tempos mais longos de biotratamento (30 dias) e ainda assim os valores obtidos foram inferiores àqueles obtidos com as madeiras biotratadas por C. subvermispora e P. chrysosporium. Também a combinação das espécies C. subvermispora e Pleurotus ostreatus foi avaliada. Neste caso havia informação prévia da literatura indicando um efeito sinérgico da ação das duas espécies no que diz respeito a secreção da enzima manganês-peroxidase (MnP) e na degradação de lignina. Os resultados corroboraram que os cultivos mistos de C. subvermispora e P.ostreatus proporcionam maior produção de MnP do que os cultivos individuais de cada espécie. Por outro lado, P. ostreatus foi ineficiente para promover a redução do consumo de energia no processo CTMP subsequente. Os cultivos mistos também não proporcionaram efeito superior àquele observado com o cultivo individual de C. subvermispora. / Biopulping involves the wood biotreatment by selected white-rot fungi as a pretreatment step of conventional pulping processes. In general, the biotreated wood facilitates the subsequent pulping processes. However, previous studies on pilot scale showed that the process is susceptible to contamination by molds when the inoculation and biodegradation steps are carried out under non-aseptic conditions. To contribute with the biopulping progress this study evaluated the use of mixed cultures of Ceriporiopsis subvermispora and Phanerochaete chrysosporium acting on Eucalyptus grandis and Eucalyptus urograndis wood under non aseptic conditions where incubation was performed at varied temperatures. The simple strategy of initiating the incubation at 37°C for 3 days followed by a non-controlled step where the temperature could oscillate in the range of 27°C to 37°C proved to be efficient to inhibit the growth of contaminants. To evaluate the wood biotreatment effect in a subsequent step of chemithermomechanical pulping (CTMP), a preliminary study to adjust pulping variables was performed. Wood chips were predigested with alkaline-sulfite liquor, fibrillated and refined in a disk refiner. It was possible to simulate the yield and physico-mechanical properties of industrial CTMP pulps using a pre-digestion stage with 6% of Na2SO3 and 3% of NaOH at 120°C for 2 h. The refing curves in the CTMP process showed that the biotreated chips required less energy to reach 300 mL of Freeness (CSF) in the pulps than energy required to refine the control wood chips. Energy savings reached 60% in some cases, but were not maximized by the eventual synergic effect of the mixed cultures of the two basidiomycetes. Additional experiments were developed using the fungal species Trametes versicolor and Pycnoporus sanguineus that were efficient to compete with contaminants even at 27°C. In this case, the energy savings in the subsequent CTMP process were obtained only after long biotreatment times (30 days) and these values were lower than those obtained with the biotreated wood by C. subvermispora and P. chrysosporium. The combination C. subvermispora and Pleurotus ostreatus was also evaluated. In this case, previous literature information indicated some synergic effect of the two species regarding secretion of manganese peroxidase (MnP) and lignin degradation. The results corroborated that the mixed cultures of C. subvermispora and P.ostreatus provided an increased production of MnP compared to the individual cultivation of each species. On the other hand, P.ostreatus was not efficient to promote energy savings in the subsequent CTMP process. The mixed cultures did not provide increased energy savings as compared with the individual biotreatment with C. subvermispora.

A contaminação ambiental por pesticidas é uma preocupação cada vez mais presente, como indicam estudos que detectam frequentemente estes compostos em água e solo, e visto a importância destes, faz-se necessário a compreensão do seu impacto em organismos não alvo. Dessa forma, o estudo da resposta adaptativa bacteriana a herbicidas vem de encontro com a necessidade descrita, além de fornecer novos dados a cerca da regulação metabólica na possível biotransformação destes compostos. Neste estudo, o sistema antioxidante foi alvo de análises para a compreensão de sua participação neste processo, além de evidenciar a regulação diferencial de isoenzimas em bactéria exposta à herbicida. Para tanto, inicialmente um estudo de biodegradação apontou que o isolado bacteriano Enterobacter asburiae SD1 é capaz de degradar s-metolachlor em cerca de 30% com 13 h de cultivo em meio mineral, na presença de 1,25 mM do herbicida. Porém, a biodegradação também ocorreu em meio de cultura com fonte de carbono alternativa ao s-metolachlor, o que juntamente com o crescimento diferencial entre os meios, sugeriu uma resposta fisiológica adaptativa à diferentes condições nas quais o herbicida estava presente. Conhecido em estudos de resposta global a estresse, o sistema antioxidante foi avaliado na presença do herbicida e em diferentes consituições de meio de cultura, o que culminou em vias preferenciais ativadas conforme as características dos meios. Na presença de somente s-metolachlor como fonte de carbono (meio mineral), a enzima glutation S-transferase foi ativada, mostrando prioridade no reconhecimento e metabolização do s-metolachlor. Já na presença de outras fontes de carbono, além do herbicida, as vias de combate ao estresse oxidativo, representadas pelas enzimas catalase (CAT), superóxido dismutase (SOD) e glutationa redutase (GR), foram ativadas primeiramente. Além disso, foi possível também avaliar a ativação diferencial das isoenzimas de CAT e SOD frente ao s-metolachlor, sendo que a isoenzima Mn-SOD merece destaque pela importância na resposta adaptativa, visto que a deleção do gene sodA (Mn- SOD), causou danos a membrana e desequilíbrio no trabalho cooperativo enzimático no combate às ERO\'s (espécies reativas de oxigênio). Finalmente, a integração dos diferentes níveis de um metabolismo na resposta do sistema antioxidante bacteriano foi mostrado através do estudo com a via de controle traducional formada pela proteína EF-P (elongation factor P). Esta proteína atua na fidelidade do processo de síntese de proteína, e a deleção do gene efp que a codifica, bem como outras proteínas envolvidas na ativação desta via, resultou no desequilíbrio celular, gerando estresse oxidativo, potencializado pela presença do herbicida paraquat. Este trabalho reúne resultados bastante relevantes para a compreensão do papel do sistema antioxidante bacteriano, em condições de estresse gerado pela presença de herbicidas, e oferece dados importantes referentes a integração e regulação de vias bioquímicas na ativação do sistema antioxidante, bem como para uma eficiente resposta adaptativa deste sistema. / The environmental contamination by pesticides is an increasing concern, as frequently showed in studies with detection of these compounds in water and soil. In fact, the understanding of pesticide effect on non-target organisms is necessary given the importance of pesticide and its sustentable use. The study of bacterial adaptive response to pesticide can show how the metabolic regulation happens, as well as provide new data about biotransformation of these xenobiotics. For this propose, firstly the biodegradation analyse showed the Enterobacter asburiae SD1 strain has capacity to degrade the s-metolachlor herbicide in around 30% of your initial concentration (1,25 mM), in 13 h of cultivation in mineral medium. However, the degradation was also observed on the presence of alternative carbon source, what suggests, together the differential growth, the bacteria can show an adaptive response to different conditions represented by the media constitution. The antioxidant system, a global sensor to stress, was evaluated in E. asburiae SD1 cultivated in different media, to see what is the effect of s-metolachlor herbicide in the antioxidant response. It was possible to observe preferential pathways activated according to medium characteristics, such as the activation of glutathione Stransferase in the presence of only s-metolachlor as carbon source. This result was explained by the bacterial need to recognize and metabolize a new molecule. On the other hand, in the presence of alternative carbon source, in addition to herbicide, the pathways to combat oxidative stress, as those constituted by catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), were firstly activated. Besides this, the differential activation was observed among the isoenzymes of CAT and SOD, wich special attention to Mn-SOD, which one had an important role in the adptive responde to herbicide. This affirmation was justified when de delection of sodA gene (Mn-SOD) induced membrane damage and disruption in the cooperative work of antioxidant enzymes in bacteria exposed to s-metolachlor. Finally, the integration of different levels of metabolism in the antioxidant response was showed through study with a pathway envolved in the translational control which has the elongation factor-P (EFP) as one important protein. This enzyme acts in the fidelity during the protein synthesis, and its absence, caused by efp gene delection, as well others two protein important to activate the EFP, induced the oxidative stress, increased by the paraquat herbicide. The present work collects relevant results for understanding of the role of bacterial antioxidant system in the stress condition induced by herbicide presence, and also offers important data about integration and regulation of biochemical pathway in the antioxidant system activation for an efficient adaptative response.

O presente trabalho desenvolveu um estudo sobre a blenda polimérica Polipropileno/Poli(3-hidroxibutirato) (PP/PHB). Os principais objetivos desta pesquisa foram estudar a compatibilização da blenda PP/PHB, a influência da prévia fotodegradação sobre a biodegradação da blenda e o comportamento individual do PHB frente a fotodegradação. As blendas PP/PHB foram obtidas nas composições 90/10, 80/20, 70/30 e 60/40 (em peso) numa extrusora dupla-rosca. O estudo da compatibilização foi feito para a blenda PP/PHB 80/20 contendo ou não 10% dos seguintes compatibilizantes: polipropileno grafitizado com anidrido maleico (PP-g-MAn), poli(etileno-co-acrilato de metila) (P(E-co-MA)), poli(etileno-co-metacrilato de glicidila) (P(E-co-GMA)) e poli(etileno-co-acrilato de metila-co-metacrilato de glicidila) (P(E-co-MA-co-GMA)). A caracterização dos materiais foi realizada através de análises morfológicas, químicas e ensaios mecânicos (tração e impacto). Os resultados obtidos permitiram classificar a eficácia dos compatibilizantes na seguinte ordem: P(E-co-MA-co-GMA) >> P(E-co-MA) > P(E-co-GMA) PP-g-MAn. A fotodegradação do PHB foi investigada expondo-se este material numa câmara de envelhecimento artificial por 3, 6, 9 e 12 semanas. O efeito da radiação UV no PHB foi monitorado através de mudanças na sua massa molar, estruturas química e cristalina, bem como nas suas propriedades térmicas, morfológicas, óticas, mecânicas e de biodegradação. A radiação UV causou uma série de mudanças em todas as propriedades analisadas. Estes efeitos, entretanto, não se mostraram muito severos e um dos motivos apontados para isso foi a baixa transmitância da radiação UV apresentada pela amostra de PHB estudada, o que gerou um perfil de degradação muito pronunciado neste material. As blendas PP/PHB em todas as suas composições foram submetidas à radiação UV por 2 e 4 semanas e tiveram a sua biodegradabilidade avaliada por ensaios de perda de massa e de respirometria de Bartha (medida da produção de CO2). Os materiais antes e após as diferentes degradações foram caracterizados através de análises químicas, térmicas, morfológicas e de massa molar. Primeiramente, observou-se que, antes de qualquer degradação, a biodegradação da fase PHB foi suprimida dentro das blendas, o que foi atribuído ao PHB constituir a fase dispersa das misturas. A prévia fotodegradação retardou a biodegradação do PHB e acelerou a biodegradação do PP e de todas as blendas PP/PHB. A maior capacidade biodegradativa do PP e das blendas foi relacionada à cisão de cadeia e formação de grupos funcionais oxidados durante a exposição à radiação ultravioleta. / In this work Polypropylene/Poly(3-hydroxybutyrate) (PP/PHB) blend was studied. In particular the compatibilization and the influence of a previous photodegradation on the biodegradation of the blend were investigated. In order to understand the photodegradation of the blends it was also necessary to study the photodegradation of PHB. The compositions of the PP/PHB blends studied ranged from 90/10 to 60/40 (by weight). These blends were obtained using a twin screw extruder. The compatibilization was evaluated using a PP/PHB blend 80/20 containing or not 10% of the following compatibilizers: maleic anhydride grafted polypropylene (PP-g-MAH), poly(ethylene-co-methyl acrylate) (P(E-co-MA)), poly(ethylene-co-glycidyl methacrylate) (P(E-co-GMA)) and poly(ethylene-co-methyl acrylate-co-glycidyl methacrylate) (P(E-co-MA-co-GMA)). The blends obtained were characterized through their morphological, chemical and mechanical properties (tensile and impact tests). The results obtained enabled the classification of the compatibilizers efficiency in the following order: P(E-co-MA-co-GMA) >> P(E-co-MA) > P(E-co-GMA) PP-g-MAH. PHB photodegradability was investigated through its exposure to artificial UV radiation in a weathering chamber for 3, 6, 9 and 12 weeks. The photodegradation effect was followed by changes of molecular weight, of chemical and crystalline structures, of thermal, morphological, optical and mechanical properties, as well as of biodegradability. UV radiation caused several changes in all the properties evaluated, however, these effects were not very severe. These results could be explained in light of the low UV radiation transmittance of the PHB sample studied, which caused a strong degradation profile for this material. PP/PHB blends in all compositions were exposed to UV radiation for 2 and 4 weeks and had their biodegradability evaluated using the weight loss and the Bartha respirometer tests (CO2 production measurement). The materials before and after the different degradations were characterized by chemical, thermal, morphological and molar mass analysis. First, it was observed that, before any degradation, the biodegradation of the PHB phase was suppressed in the blends, most likely due to the fact that PHB was the dispersed phase within the mixtures studied. Previous photodegradation delayed PHB biodegradation and sped up the biodegradation of PP and all PP/PHB blends. The greater biodegradability of PP and blends was attributed to the chain scission and formation of oxidized functional groups taking place during ultraviolet radiation exposure.

O herbicida ametrina é relativamente persistente no meio ambiente e está entre os cinco mais usados junto à cultura da cana-de-açúcar no Brasil. Técnicas radiométricas e moleculares foram utilizadas neste estudo para avaliar o potencial de sorção/dessorção e a biodegradação da ametrina, além do impacto do tempo de residência deste herbicida sobre a estrutura e a diversidade da comunidade microbiana, respectivamente. Os solos foram coletados em áreas florestais, sem histórico de aplicação de ametrina, sendo que um deles apresentou textura mais argilosa (NVef) e o outro mais arenosa (RQo). A ametrina apresentou baixo a moderado potencial de sorção (Kd = 2,2 e 9,9 L kg-1 nos solos RQo e NVef, respectivamente). Paralelamente, a taxa de biodegradação (t1/2 = 16 e 36 dias nos solos NVef e RQo, respectivamente) e a formação de resíduos ligados de 14C-ametrina ao solo (59 e 38 % da quantidade aplicada nos solos NVef e RQo, respectivamente) foram muito maiores no solo NVef, mostrando sua maior taxa de dissipação. Houve também a formação de um metabólito de importância ambiental, o qual correspondeu a 19 e 26 % da quantidade aplicada após 70 dias da aplicação nos solos RQo e NVef, respectivamente. O DNA total foi extraído dos solos, com posterior uso da técnica de Reação em Cadeia da Polimerase associada à Eletroforese em Gel com Gradiente Desnaturante (PCR-DGGE). A aplicação da ametrina alterou a estrutura da comunidade microbiana em ambos os solos, sendo essa alteração mais evidente no solo NVef, principalmente nos períodos inicias da incubação. Os dados de seqüenciamento evidenciaram o predomínio dos seguintes filos: Proteobacteria, Acidobacteria, Actinobacteria e Bacteroidetes. Houve seleção de alguns filos no solo NVef mesmo com apenas 7 dias de incubação da ametrina; entretanto, não houve redução no número de filos no solo RQo. Isto permitiu concluir que a diversidade da população microbiana pode variar com a aplicação do herbicida ametrina, mas isto dependerá principalmente do tipo de solo / The herbicide ametryne is relatively persistent in the environment and is among the five most used in the sugarcane crop in Brazil. Radiometric and molecular techniques were applied in this study to evaluate sorption/desorption and biodegradation of ametryne and the impact caused its residence time on the structure and diversity of the soil microbial community, respectively. The soils were collected from forested areas, with no history of ametryne application. One of them showed a clayey (NVef) and the other one a sandy (RQo) texture. Ametryne presented low to moderate sorption potential (Kd = 2.2 e 9.9 L kg-1 in the RQo and NVef soils, respectively). In parallel, the biodegradation rate (t1/2 = 16 and 36 days in the NVef and RQo, respectively) and the formation of soil bound residues of 14C-ametryne in soil (59 and 38 % of applied amount in the NVef and RQo, respectively) were much greater in NVef soil, showing its higher dissipation rate. There was also formation of a metabolite of environmental importance, which corresponded to 19 and 26 % of applied amount in the RQo and NVef soils, respectively. The genomic soil DNA was extracted with further application of the Polymerase Chain Reaction associated to the Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique. Ametryne application changed the structure of microbial community in both soils, but this change was more pronounced in the NVef soil, mainly in the initial periods of incubation. The sequencing data showed predominance of the following phyla: Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes. There was selection of certain phyla in the NVef, even only with 7 days of ametryne application; however, there was no reduction in the phyla number in the RQo. This led to the conclusion that microbial population diversity may vary with ametryne application, but it will depend on the soil type

O objetivo deste trabalho foi a seleção de bactérias capazes de biodegradar os pesticidas organoclorados dieldrin, DDD e PCP. Inicialmente, foram realizados os ensaios de tolerância visando à seleção das bactérias degradadoras; posteriormente foram realizados os ensaios de biodegradação em meio liquido utilizando a bactéria selecionada. Dentre as 14 linhagens bacterianas isoladas testadas, selecionou-se a linhagem Pseudomonas aeruginosa L2-1 por apresentar maior tolerância a todos os pesticidas. Os ensaios de biodegradação foram realizados em diferentes meios de cultura, variando-se a concentração de glicose, a fonte de nitrogênio e a presença de ramnolipídeo. Os ensaios de biodegradação foram realizados determinando-se a concentração de pesticida, a concentração de glicose, o número de células viáveis, e o pH. O meio de cultura que mais favoreceu a biodegradação dos três pesticidas foi o meio com nitrato de sódio e 0,5% de glicose, obtendo-se biodegradação de aproximadamente 50% para cada pesticida após três dias de incubação. Na ausência de glicose, o meio com nitrato de amônio e 0,1% de ramnolipídeo, favoreceu a biodegradação, obtendo-se após 14 dias de incubação 36,8% de biodegradação de dieldrin; 33,7% de DDD e 42,8% de PCP. De uma forma geral, as taxas de biodegradação pela P. aeruginosa L2-1 foram maiores em menores concentrações de glicose e na presença de ramnolipídeo. Ao alterar a fonte de nitrogênio foram observados resultados diversos sobre a taxa de biodegradação: na ausência de glicose, o nitrato de sódio favoreceu a biodegradação de PCP, enquanto o nitrato de amônio favoreceu a biodegradação de dieldrin, na presença de glicose observou-se o inverso. A taxa de biodegradação do DDD não foi significativamente alterada ao variar a fonte de nitrogênio. A bactéria selecionada P. aeruginosa L2-1 apresentou potencial para biodegradação de pesticidas organoclorados, sendo que as condições nutricionais do meio influenciaram diretamente a biodegradação. / The objective of this work was the selection of bacteria capable of biodegrading the organochloride pesticides dieldrin, DDD and PCP. Initially tolerance tests were conducted in order to select degrading bacteria subsequently, biodegradation tests were carried out in liquid medium using the selected bacteria. Among the 14 bacterial isolated strains, Pseudomonas aeruginosa L2-1 was selected due to its greater tolerance to all pesticides. The biodegradation tests were conducted on different culture media, varying the concentrations of glucose, nitrogen source and presence of rhamnolipid. Biodegradation studies were performed by measuring the concentration of pesticide, the concentration of glucose, the number of viable cell and pH during time. The best medium for the biodegradation of all three pesticides was composed of sodium nitrate and 0.5% glucose, giving approximately 50% yield after three days of incubation. In the absence of glucose, the medium containing ammonium nitrate and 0.1% rhamnolipid improved biodegradation yielding, after 14 days of incubation, 36.8% biodegradation of dieldrin; 33.7% DDD and 42.8% of PCP. In general, the biodegradation rates of pesticides by P. aeruginosa L2-1 were greater at lower concentrations of glucose and in the presence of rhamnolipid. Nitrogen source had different effects on the rate of biodegradation: in the absence of glucose, sodium nitrate favored the biodegradation of PCP, whereas ammonium nitrate favored the biodegradation of dieldrin; and in the presence of glucose, it was observed the opposite. The biodegradation rate of the DDD was not significantly altered by the nitrogen source tested. The selected bacteria, P. aeruginosa L2-1, showed potential for the biodegradation of organochloride pesticides demonstrating that nutritional conditions has a direct effect on degradation yields.

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.

A poluição do solo por uso de derivados de petróleo como o grupo de hidrocarbonetos denominado de BTEX (benzeno, tolueno, etil-benzeno e xilenos), apresenta grande risco às comunidades residentes próximas a esses locais. Esses compostos são tóxicos, comprovadamente cancerígenos e podem levar uma pessoa a morte, mesmo em concentrações extremamente baixas. O cidadão, muitas vezes não tem noção do que sejam estes contaminantes e o grande perigo que representam. Milhares de residências têm sido construídas sobre resíduos perigosos que continuam ativos, emitindo gases e/ou contaminando o lençol freático. Em particular, na região de Cubatão, o problema se agrava com o uso indevido de águas de rios e riachos, que por um processo de percolação, acabam sendo contaminados pelos poluentes presentes no solo, podendo ocorrer de o ponto de coleta de água ser próximo a um sítio contaminado. Este estudo relata o isolamento e caracterização de 4 cepas de bactérias isoladas de um solo contaminado com BTEX em Cubatão-SP, através da técnica de enriquecimento seletivo de culturas. A idéia é que estas cepas possam ser utilizadas em futuros processos de biorremediação (tratamento) destes solos. Além disso, o trabalho conta com o auxílio de programas sócio-ambientais existentes no Centro de Capacitação e Pesquisa em Meio Ambiente (CEPEMA-POLI-USP) os quais tem como um dos objetivos conscientizar a população carente cubatense, que faz uso indevido da água contaminada, aos perigos da exposição ao BTEX. / Contaminated soils by hydrocarbons known as BTEX (benzene, toluene, ethylbenzene and xylene), represent a risk to communities who live close to these places. These compounds are toxic, carcinogenic and can cause death to people, even in extremely small concentrations. Citizens often have no idea who and how hazardous are these contaminants. Millions of homes have been built on dangerous actives wastes, emitting gases and contaminating the groundwater. In particular, the problem is worst in Cubatão due inappropriate use of water from rivers and streams, which by a percolation process, contaminate soil. Most of times the water collected points are near from the contaminated site. This study reports the successful isolation and characterization of 4 bacteria strains isolated from an industrial soil in Cubatão-Brazil, using a culture enrichment technique. The idea is use these microorganisms in future soils bioremediation processes (treatment). This work have as partners, social and environmental programs from the Center for Training and Environmental Research (CEPEMA-POLI-USP), which has as goal educate poor people against dangers of BTEX.

Desde a revolução verde, na década de 1950, o processo tradicional de produção agrícola passou por mudanças com a inserção do uso intensivo de agrotóxicos como os piretróides, que são a terceira classe química de pesticidas mais comercializada no mundo. Estes compostos geralmente são ésteres que contêm um anel dimetilciclopropano com grupamentos variáveis e a presença de anéis aromáticos. Cada vez mais os cientistas vêm explorando a diversidade microbiana na biodegradação de pesticidas e neste contexto, o emprego de fungos de ambiente marinho possui grande potencialidade devido ao seu sistema enzimático único com a presença de compostos altamente oxigenados e halogenados, assim como o esfenvalerato empregado neste trabalho. Entretanto, estes micro-organismos não têm sido explorados na biotransformação de pesticidas piretróides. Neste estudo foi avaliada a eficiência de fungos de ambiente marinho [Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. Dr(A)6, Acremonium sp. Dr(F)1, Westerdykella sp. Dr(M2)4 e Cladosporium sp. Dr(M2)2] na degradação do pesticida piretróide esfenvarelato. Observou-se que o esfenvalerato e seus principais metabólitos de degradação causam efeitos inibitórios significativos no crescimento dos fungos, mas não o suficiente para inviabilizar o estudo da biodegradação por meio destes micro-organismos. Os resultados obtidos sugerem que diversas espécies fúngicas contribuem para a biodegradação do pesticida esfenvalerato, entretanto a eficiência da degradação deste composto varia muito entre linhagens. Observou-se a degradação de 3 a 35% de 100 mg.L-1 de esfenvalerato presente na formulação comercial (SUMIDAN 150SC®) em 14 dias para diferentes fungos. Os metabólitos identificados [3-fenoxibenzaldeído, ácido 3-fenoxibenzoico, álcool 3-fenoxibenzílico e ácido 3-(hidroxifenoxi)benzoico] tornaram possível uma proposta de rota biodegradativa, onde se observou metabólitos cada vez mais polares, aumentando a possibilidade de carreamento para o meio aquoso. Constatou-se que em geral ocorre a formação de grandes quantidade do ácido 3-fenoxibenzoico e do ácido 2-(4-clorofenil)-3-metilbutanoico, compostos considerados tóxicos e que podem causar efeitos nocivos à saúde humana e ao meio ambiente. Para a linhagen Acremonium sp. Dr(F)1 que apresentou os melhores resultados de biodegradação, após 28 dias de cultivo com 100 mg.L-1 de esfenvalerato observou-se 20 mg.L-1 de acido 3-fenoxibenzoico, que posteriormente foi convertido ao ácido 3-(hidroxifenoxi)benzoico. Observou-se também uma biodegradação mais eficiente do princípio ativo esfenvalerato puro do que na formulação comercial, que pode ser causada por diversos fatores, como a toxicidade do xileno presente na formulação comercial ou a adsorção do esfenvarelato aos componentes da formulação emulsionável dificultando a ação enzimática. A partir destes resultados toma-se interessante o emprego destes micro-organismos visando a biorremediação deste pesticida. / Since the green revolution in the 1950s, the traditional agricultural production has undergone changes such as the intensive use of pesticides, including pyrethroids, which is the third most sold chemical class of pesticide. These compounds are generally esters containing a dimethylcyclopropane ring with different groups and aromatic rings. Scientists are exploring the microbial diversity for the biodegradation of pesticides. The use of marine fungi may show great potential to an efficient biodegradation, because of its unique enzymatic system and the presence of halogenated and oxygenated compounds, such as the pesticide esfenvalerate used in this work. However, these microorganisms have not been explored in the biotransformation of pyrethroid pesticides. In this study, marine-derived fungi [Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. Dr(A)6, Acremonium sp. Dr(F)1, Westerdykella sp. Dr(M2)4 and Cladosporium sp. Dr(M2)2] were applied in the biodegradation of the esfenvalerate. It was observed that the esfenvalerate and its degradation metabolites cause an inhibitory effect on the fungi growth, however not enough to make unfeasible the study of the biodegradation by these microorganisms. The results suggest that different fungal species contribute to the degradation of esfenvalerate, although the efficiency of degradation varies widely between strains. It was observed 3-35% of degradation at a concentration of 100 mg.l-1 of esfenvalerate present in the commercial formulation (SUMIDAN 15OSC®) in 14 days. The identified metabolites [3-phenoxybenzaldehyde, 3-phenoxybenzoic acid, 3- phenoxybenzyl alcohol and 3-(hydroxyphenoxy)benzoic acid ) enabled the proposal of a biodegradative pathway, in which was noted the increasingly polar character of metabolites, raising the possibility of entrainment for aqueous medium . It was observed, in general, the large formation of 3- phenoxybenzoic and 2-(4-chlorophenyl)-3-methylbutanoic acid, compounds that are considered toxic and may cause harmful effects to human health and the environment. For the strain Acremonium sp. Dr (F)1, which showed the best results of 100 mg.l-1 esfenvalerate biodegradation after 28 days, it was observed 20 mg.l-1 of 3-phenoxybenzoic acid, which was later converted to 3- (hydroxyphenoxy)benzoic acid. It was also observed a more efficient biodegradation of the pure active ingredient than the commercial formulation, what can be caused by various reasons, such as the toxicity of the xylene present in the commercial formulation or the esfenvarelate adsorption in the components of the emulsifiable formulation, difficulting the enzymatic activity. These results shows the potential use of these microorganisms in the bioremediation of esfenvalerate.

by Yuen Pui-yee, Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 213-221). / Acknowledgments --- p.i / Abstract --- p.ii / List of Tables --- p.ix / List of Figures --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problems of Pollution From Textile Industries --- p.1 / Chapter 1.2 --- Current Treatment Methods of Wastewater from Textile Industries --- p.5 / Chapter 1.3 --- Adverse Effects of Dyes on the Environment --- p.11 / Chapter 1.4 --- Classification of Dyes --- p.16 / Chapter 1.5 --- Azo Dyes --- p.17 / Chapter 1.6 --- Metabolisms of Azo Dyes in Microbial and Animal Systems --- p.21 / Chapter 1.7 --- "Toxicity, Mutagenicity and Carcinogenicity of Azo Dyes" --- p.31 / Chapter 1.8 --- Removal of Azo Dyes --- p.35 / Chapter 1.8.1 --- Biological Methods --- p.35 / Chapter 1.8.2 --- Physico-chemical Methods --- p.49 / Chapter 1.9 --- Purposes of Study --- p.50 / Chapter 2. --- Objectives --- p.53 / Chapter 3. --- Materials and Methods --- p.54 / Chapter 3.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-Dimethyl-p-phenylene diamine-degrading Microbial Isolates" --- p.54 / Chapter 3.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil" --- p.54 / Chapter 3.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.56 / Chapter 3.1.3 --- "Enrichment of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing wastewater, Activated Sludge and Soil" --- p.59 / Chapter 3.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.60 / Chapter 3.1.5 --- Selection of N，N-Dimethyl-p-phenylene diamine-degrading Bacteria --- p.60 / Chapter 3.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.61 / Chapter 3.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.63 / Chapter 3.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.2 --- Change of UV-Vis Spectra of Methyl Red and N，N-Dimethyl-p- phenylene diamine at Different pH and Matrixes --- p.64 / Chapter 3.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.66 / Chapter 3.2.4 --- "HPLC separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.67 / Chapter 3.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.68 / Chapter 3.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved, Degradation Value of N,N-Dimethyl-p-phenylene diamine and o- Aminobenzoic acid, and Growth of Selected Methyl Red- degrading Bacteria by Spectrophotometric Analysis " --- p.68 / Chapter 3.3.2 --- Study of Degrading Products of Methyl Red by Selected Methyl Red-degrading Isolates --- p.71 / Chapter 3.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.73 / Chapter 4. --- Results --- p.74 / Chapter 4.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.74 / Chapter 4.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil " --- p.74 / Chapter 4.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.79 / Chapter 4.1.3 --- "Enrichment of N,N-dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing Wastewater, Activated Sludge and Soil " --- p.85 / Chapter 4.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.85 / Chapter 4.1.5 --- "Selection of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.94 / Chapter 4.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.108 / Chapter 4.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.123 / Chapter 4.2.4 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.129 / Chapter 4.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.138 / Chapter 4.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved and Degradation Value of N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid and Growth of Selected Methyl Red- degrading Bacterial Isolates by Spectrophotometric Analysis " --- p.138 / Chapter 4.3.2 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC --- p.175 / Chapter 4.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.175 / Chapter 5. --- Discussion --- p.181 / Chapter 5.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.181 / Chapter 5.1.1 --- "Isolation and Selection of Methyl Red-degrading Microbes from Dye-containing Wastewater, Activated Sludge and Soil " --- p.181 / Chapter 5.1.2 --- "Isolation and Selection of N,N-Dimethyl-p-phenylene diamine- degrading Microbial Isolates from Dye-containing Wastewater, Activated Sludge and Soil " --- p.183 / Chapter 5.1.3 --- Identification of the Selected Methyl Red-degrading and N，N- Dimethyl-p-phenylene diamine-degrading Bacteria --- p.185 / Chapter 5.1.4 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.185 / Chapter 5.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.186 / Chapter 5.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid in 0.05 M phosphate buffer and 0.2MHC1 " --- p.186 / Chapter 5.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.187 / Chapter 5.2.3 --- "Change of UV-Vis Spectra of N,N-Dimethyl-p-phenylene diamine in Different Matrixes at Different pH " --- p.187 / Chapter 5.2.4 --- "UV-Vis Spectra and Standard Curve of Methyl Red, N,N- dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.188 / Chapter 5.2.5 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.189 / Chapter 5.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.190 / Chapter 5.3.1 --- Effect of Glucose --- p.194 / Chapter 5.3.2 --- Effect of Ethanol --- p.196 / Chapter 5.3.3 --- Effect of Ammonium Sulphate --- p.198 / Chapter 5.3.4 --- Effect of Yeast Extract --- p.199 / Chapter 5.3.5 --- Effect of Phosphate Buffer (pH 7) --- p.200 / Chapter 5.3.6 --- Effect of pH --- p.201 / Chapter 5.3.7 --- Effect of Temperature at Static and Shaking Conditions --- p.203 / Chapter 5.3.8 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC Analysis --- p.206 / Chapter 5.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.207 / Chapter 6. --- Conclusion --- p.209 / Chapter 7. --- References --- p.213 / Chapter 8. --- Appendix 1: Composition of Media --- p.222 / Appendix 2: Composition of Buffers --- p.225 / Appendix 3 --- p.228

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