Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.

This thesis presents the results of three studies aimed at improving brine-quality at the Swartkops solar salt-works (Swartkops Sea Salt [Pty] Ltd) on the outskirts of Port Elizabeth, South Africa. This is a highly eutrophic salt-works, the management of which has become increasingly difficult in recent years. The fundamental problem is how best to operate the system at maximum capacity while limiting nutrient inputs from the nutrient-rich microtidal Swartkops Estuary. In the first study, brine-quality at several sites along the axis of the Swartkops Estuary, and the extent to which it is affected by a variety of factors, is compared. Sites were sampled on micro- and macrotidal time scales, and were selected by the management of the salt-works as possible locations for a new pump-house (for extracting brine from the estuary) for their salt-work operations at Swartkops and Missionvale. The study showed that there are incremental benefits to be had from moving the site of extraction downstream from its present position to a site closer to the mouth of the estuary, where the concentration of nutrients usually is lower and where salinity usually is higher. There is little to be gained from moving the site of extraction laterally, to the mouth of the Inlet from which brine currently is extracted, so that brine is extracted directly from the estuary itself. A set of models relating the concentrations of NH+ 4 , NO{u100000}3 , and PO34{u100000} to salinity is proposed. These take into account the influences of site and season and may be used to estimate the concentration of these nutrients from a measurement of salinity. The model for PO34{u100000} shows that it would be more damaging to the salt-works’ operations to pump “low”-salinity brine during the early months of summer than during autumn. Evidence is also presented to show that Wylde Bridge has no influence on nutrient concentrations in the estuary, with tidal flushing generally passing beyond the Wylde-Bridge break-point. The exceptionally heavy flooding of the estuary that occurred in September 2002 may, however, have biased this conclusion, because of its scouring effect. The second study concentrated on monitoring the effect of (1) decreasing pond depth and (2) increasing pond salinity—two readily available management tools—on brine quality at the salt-works. Pond depth throughout the salt-works was decreased by 40 cm, and the salinity of Pond 5, a pond in the middle of the system, was increased to 175 S. Both measures were kept in place for the duration of the study (Nov. 2002–Aug. 2004). The pond-depth experiment did not have the expected result, there being no evidence of the increase in microalgal growth in the water column that was predicted based on previous research. There was, however, a significant increase in benthic chlorophyll-a, and there was a general improvement in the condition of the sedimentary system of the salt-works. There was also a substantial decrease in particulate organic matter in the water column, with clear evidence that the remaining fraction was closely associated with living forms of particulate matter rather than with detritus. The pond-salinity experiment proves that there is a flourishing, and resilient, population of brine shrimp (Artemia salina L.) at the salt-works. Restocking the salina, or stocking it with a different strain of brine shrimp, is therefore not necessary. The results of this study show that the brine shrimp population at the salt-works needs salinities of greater than about 65–70 S to survive. As a living force they almost certainly need a protective salinity that is greater than about 120–140 S, perhaps even as great as 160 S. Brine shrimp thrived in the high salinity milieu of the experimental pond for the duration of the study, but dwindled from three other ponds of the system once their salinities fell to below 90 S, eventually to disappear from them, apparently completely, once salinity fell to below 65 S. The third and final study concentrated on establishing whether the products released by decomposing barley straw could be used in a solar salt-works to control macroalgal blooms without detrimentally affecting the benthic-mat. Previous research has shown that these products are effective inhibitors of macroalgal growth and that they remain effective under saline conditions. The results presented here show that the same products, or products released under similar conditions of decomposition, adversely effect both the structure and the function of the mat. Consequently, their use in a solar salt-works cannot be recommended.

As a potential bioremediation system for contaminated soils, I evaluated the use of an arbuscular mycorrhizal (AM) fungus, Glomus intraradices on roots and shoots uptake of polycyclic aromatic hydrocarbons (PAHs), alkyl PAHs, and toxic metals in Echinacea purpurea, in using a controlled 20-week greenhouse study and a complimentary 2-year field study. E. purpurea seeds were either inoculated with the mycorrhizal fungus (AM) or not inoculated (non-AM) and grown in soil provided by the National Capital Commission (NCC) that have known contamination. In the greenhouse study, AM inoculation increased the uptake of alkyl PAHs in the roots of E. purpurea. The AM inoculation showed no effect on root uptake of PAHs and toxic metals over the 20-week study period. However, when I calculated the uptake rates (k1) for PAHs between both treatments, the AM treated roots ha 10-fold higher k1 values than non-AM treated roots. The soil concentrations of PAHs were found to increase over time with AM inoculation, suggesting, that AM fungi are causing a solvent depletion through root uptake of minerals and carbon, which concentrates the more hydrophobic PAHs in soils. Alkyl PAHs and metals showed no change over time amongst any of the treatments. Assessing the performance of AM fungi on the uptake of contaminants under field conditions, only PAHs showed increased bioaccumulation in the shoots of E. purpurea with AM inoculation. Alkyl PAHs and metals in plant material were unaffected by the AM inoculation, but increased significantly from year 1 to year 2. The uptake rates among treatments were similar, with non-AM roots having slightly greater uptake. Soil concentrations of PAHs and alkyl PAHs were unaffected over the course of the experiment. Our control soil, however, showed significant increases in concentration from year 1 to year 2 with alkyl PAHs. These results quantified the influence of AM hyphae-mediated uptake of organic and inorganic contaminant transfer from soil to plants and the bioaccumulation kinetics for contaminants by E. purpurea that will be useful for environmental models and phytoremediation strategies.

Les chloroéthènes sont les polluants majeurs des eaux souterraines et des nappes phréatiques. De par leur toxicité et leur effet cancérigène, ils représentent une préoccupation majeure pour les autorités publiques et sanitaires. La restauration des sites contaminés est possible par des techniques de dépollution biologique impliquant les microorganismes (bioremédiation microbienne). Cependant, la réussite des traitements dépend à la fois des conditions physicochimiques du site pollué et des capacités de dégradation de la microflore indigène. Ainsi, pour optimiser les processus de décontamination, l’identification et le suivi des différentes populations microbiennes sont indispensables avant et pendant le traitement. Les biopuces ADN fonctionnelles (FGA, Functional Gene Array), outils moléculaires haut débit, sont particulièrement bien adaptées pour des applications en bioremédiation. Leur élaboration nécessite de disposer de logiciels performants pour le design de sondes qui combinent à la fois une forte sensibilité, une très bonne spécificité et un caractère exploratoire, ce dernier étant indispensable pour la détection des séquences connues mais surtout de celles encore jamais décrites au sein d’échantillons environnementaux. Un nouveau logiciel, autorisant la sélection de sondes combinant tous ces critères, a été développé et nommé HiSpOD. Son utilisation pour la construction d’une FGA dédiée aux voies de biodégradation des chloroéthènes a permis d’évaluer l’effet de traitements de biostimulation sur la microflore indigène pour plusieurs sites industriels contaminés. Les données révèlent différentes associations entre microorganismes déhalorespirants qui sont fonction des paramètres environnementaux. / Chlorinated solvents are among the most frequent contaminants found in groundwater and subsurface ecosystems. Because of their high toxicity and carcinogenicity, they represent a serious risk for human health and the environment. Thus, such polluted sites need a rehabilitation treatment. Among remediation solutions, microbial bioremediation represents a less invasive and expensive alternative than physico-chemical treatments. However, the process efficiency greatly depends on the environmental conditions and the microbial populations’ biodegradation capacities. Therefore, bioremediation treatment optimization requires the identification and monitoring of such capacities before and during the treatment. Functional Gene Arrays (FGA), by profiling environmental communities in a flexible and easy-to-use manner, are well adapted for an application in bioremediation. But, constructing efficient microarrays dedicated to microbial ecology requires a probe design step allowing the selection of highly sensitive, specific and explorative oligonucleotides. After a detailed state of the art on probe design strategies suitable for microbial ecology studies, we present new software, called HiSpOD, generating efficient explorative probes for FGA dedicated to environmental applications. Finally, this bioinformatics tool was used to construct a FGA targeting most genes involved in chloroethenes biodegradation pathways which allowed the evaluation of biostimulation treatments conducted on indigenous bacterial populations for several industrial contaminated sites.

Orientador: Cassiana Maria Reganhan Coneglian / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-22T10:59:00Z (GMT). No. of bitstreams: 1 Rego_AnaPaulaJustiniano_M.pdf: 1374824 bytes, checksum: f4374ed0d3dafb4fb76f0bd4a5678bc9 (MD5) Previous issue date: 2013 / Resumo: A ametrina é um herbicida utilizado em culturas de cana-de-açúcar no período de pré e pós emergência das plantas daninhas. É um composto persistente no meio ambiente, podendo lixiviar para água subterrânea e escoar para águas superficiais, provocando impacto na comunidade aquática. No presente trabalho avaliou-se a atividade microbiana em solo com histórico de aplicação da ametrina em cultivo de cana de açúcar, acrescidos de concentrações de solução de ametrina e biofertilizante comercial Microgeo. Coletaram-se três amostras de solo em área de cultivo de cana de açúcar, sendo o primeiro solo caracterizado como argiloso, o segundo arenoso, o terceiro arenoso, mas esterilizado em estufa a 106ºC. As concentrações utilizadas da ametrina nos experimentos foram de 8 e 12 ?g/mL e, as de biofertilizante foram 1, 5 e 10%. Para avaliar a atividade microbiana do solo utilizou-se o método respirométrico de Bartha que quantifica a geração de CO2 em mg/L e após o processo de biodegradação o método enzimático de hidrólise de FDA para avaliar a estimativa da atividade microbiana. Quantificou-se a população de bactérias heterotróficas e fungos expressos em UFC/g de solo antes e após o experimento de respirometria. Realizou-se teste de toxicidade com o organismo-teste Daphnia similis exposta ao herbicida ametrina nas concentrações 0,01; 0,1; 1; 10 e 100 ?g/L, do biofertilizante nas concentrações 1, 10 e 100% e do solo antes e após o experimento de respirometria, utilizando a fração solúvel. Realizou-se teste de fitoxicidade com a semente Lactuca sativa antes e após os experimentos de respirometria, utilizando a fração solúvel do solo. De acordo com os resultados obtidos pode-se concluir que a produção de CO2 foi maior em solo arenoso e menor em solo argiloso e em solo estéril. A estimativa microbiana pelo método de FDA foi maior em solo argiloso e arenoso e menor em solo estéril. O herbicida ametrina apresentou toxicidade ao organismo Daphnia similis. Antes e após os experimentos de respirometria, o solo não apresentou efeitos tóxicos à Daphnia simillis, não sendo possível calcular o CE50. Os solos antes e após experimentos de respirometria não apresentaram inibição na germinação e alongamento das raízes de Lactuca sativa. Conclui-se que o herbicida ametrina como citado na literatura é lentamente biodegradado no solo e a adição de biofertilizante Microgeo não corroborou com a sua biodegradação, mas favoreceu a atividade microbiana do solo / Abstract: The ametryn is an herbicide used on crops of sugar cane in the pre and post emergence weed. It is a compound persistent in the environment and can leach into groundwater and surface water to drain, causing impact on the aquatic community. In this study we evaluated the microbial activity in soil with a history of application of ametryn in cultivation of sugar cane, plus concentrations of ametryn solution and commercial bio fertilizer Microgeo. Three samples were collected from soil in sugar cane cultivation area, the first being characterized as clayey soil, the second sandy, and the third gritty but sterilized in an oven at 106 °C. The concentrations used in the experiments of ametryn were 8 and 12 ?g / mL, and the bio fertilizers were 1, 5 and 10 %. To evaluate the microbial activity in the soil it was used the Bartha respirometric method that quantifies the CO2 generation in mg / L and after the biodegradation the process of the enzymatic hydrolysis of FDA to evaluate the estimation of microbial activity. We quantified the population of heterotrophic bacteria and fungi expressed in CFU / g of soil before and after the respirometry experiment. We held a toxicity test with the Daphnia similis organisms test exposed to herbicide ametryn at 0.01, 0.1, 1, 10 and 100 mg/L of bio fertilizer concentrations 1, 10 and 100% and soil before and after the respirometry experiment, using the soluble fraction. A test of fitoxicity was performed with Lactuca sativa seed before and after the respirometry experiments using the soluble fraction of the soil. According to the obtained results it can be concluded that the CO2 production was higher on sandy soil and smaller in clayey soil and sterile soil. A method for estimating microbial FDA was higher in clay soil and sandy soil and lower in sterile. The herbicide ametryn showed toxicity to the organism Daphnia similis. Before and after the respirometry experiments, the soil showed no toxic effects to the Daphnia simillis, it is not possible to calculate the EC50. The soils, before and after respirometry experiments showed no inhibition on seed germination and root elongation of Lactuca sativa. It is concluded that the herbicide ametryn as quoted in the literature is slowly biodegraded in soil and the addition of the bio fertilizer Microgeo has not substantiated its biodegradation, but favored the soil microbial activity / Mestrado / Tecnologia e Inovação / Mestre em Tecnologia

Orientadores: Valéria Maia Merzel, Suzan Pantaroto de Vasconcellos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T20:16:20Z (GMT). No. of bitstreams: 1 Dellagnezze_BrunaMartins_D.pdf: 5318956 bytes, checksum: ea46ea243dab1c96e35480b724029b55 (MD5) Previous issue date: 2015 / Resumo: A poluição é um problema mundial amplamente discutido, incluindo os derramamentos de petróleo ocorridos através de acidentes ou por atividades humana, os quais acarretam grande impacto ambiental e econômico. O processo de biorremediação utiliza micro-organismos, associados ou não a outros compostos como biossurfactantes e até mesmo enzimas, com o objetivo de transformar compostos orgânicos em inorgânicos, levando à formação de compostos inertes ou não tóxicos. Deste modo, a biorremediação representa um modo efetivo e sustentável para se tratar áreas contaminadas. Neste trabalho foi possível avaliar o potencial de clones metagenômicos obtidos a partir da construção de uma biblioteca fosmidial e de linhagens de bactérias, todos provenientes de amostras de petróleo de reservatórios brasileiros em escala de microcosmos e mesocosmos, visando futura aplicação em processos de biorremediação. Em um primeiro ensaio os micro-organismos foram avaliados na forma livre, em 50 mL de água do mar artificial e petróleo bruto como única fonte de carbono, a cada sete dias durante 21 dias. Posteriormente, os micro-organismos com melhor potencial de biodegradação foram selecionados e aprisionados em esferas de quitosana e testados novamente em microcosmos, em diferentes escalas, durante 21 e 30 dias. Com base nos resultados observados nos ensaios de degradação em microcosmos, um último ensaio foi realizado empregando-se um consórcio contendo quatro clones metagenômicos e uma linhagem de Bacillus subtilis, o qual foi avaliado em ensaio de mesocosmos em 3000 litros de água do mar não-estéril. Nesta etapa, parâmetros como a contagem total dos micro-organismos (DAPI) e a demanda biológica de oxigênio (DBO) foram avaliados, e a cromatografia gasosa (CG) foi empregada para avaliar a degradação de hidrocarbonetos do petróleo. Os resultados demonstraram a capacidade desses micro-organismos em degradar compostos do petróleo bruto, tanto hidrocarbonetos alifáticos como aromáticos. Em microcosmos, na forma livre, as linhagens de Dietzia maris e Micrococcus sp. apresentaram o melhor desempenho, alcançando ao final de 21 dias 99% de degradação de hidrocarbonetos alifáticos e de 63-99% de degradação de aromáticos (fenantreno e metilfenantreno). Dentre os clones, o clone 2B apresentou o melhor desempenho para degradar tanto hidrocarbonetos alifáticos (47%) como aromáticos (94%). Na forma aprisionada, os micro-organismos também apresentaram capacidade para degradar petróleo bruto em mesocosmos, exibindo valores de degradação de 90 a 100 % para hidrocarbonetos saturados e 70 a 100% para aromáticos, ao final de 30 dias de avaliação. Os resultados indicam um resultado promissor e inédito, onde um consórcio combinado contendo clones metagenômicos e Bacillus subtillis pode ser futuramente utilizado em estratégias de bioaumento, em sistemas de contenção, como ferramenta para biorremediação de ambientes contaminados com hidrocarbonetos / Abstract: Pollution is a global environmental problem widely discussed, including oil spills that occur accidentally or due to human activities, which cause huge environmental and economic impacts. Bioremediation process uses biological agents, associated or not to other compounds like biosurfactants or even their enzymes, to mineralize or complex organic and inorganic pollutant compounds, transforming them into inert or non-toxic compounds. Thus, bioremediation represents an ecofriendly and effective way to treat impacted areas. In this work, the biodegradation potential of clones obtained from metagenomic libraries and bacterial isolates, all originated from Brazilian petroleum reservoirs, was evaluated in microcosm and mesocosm scale aiming at a future application in bioremediation process. In the first assay, microorganisms were evaluated as free cells, in 50 mL-volume of artificial seawater and using crude oil as sole carbon source. The experiment was monitored each seven days during 21 days. Further, the best performing microorganisms were selected, immobilized in chitosan beads and evaluated in microcosm assays, at different scales, during 21 and 30 days. Finally, in the last experiment, one consortium containing four metagenomic clones and a Bacillus subtilis strain was evaluated in mesocosmos assay in 3000 L-volume of non-sterile seawater. Parameters such as total counting of microorganisms by DAPI and biological oxygen demand (BOD) were evaluated, and petroleum degradation was monitored by chromatographic analysis. Results demonstrated the ability of the microorganisms to degrade aliphatic and aromatic hydrocarbons. In microcosms, using free cells, the strains of Dietzia maris and Micrococcus sp. showed the best performance, reaching 99% of aliphatic hydrocarbon degradation and 63-99% of aromatic compound degradation in 21 days. Among metagenomic clones, clone 2B presented the best performance to degrade aliphatic (47%) and aromatic hydrocarbons (94%). In chitosan beads, the microorganisms were also able to degrade crude petroleum, showing percentages between 90 and 100% for aliphatic hydrocarbons and 70 and 100% to aromatic. The results gathered in this work demonstrate that a microbial consortium containing metagenomic clones and one bacterial strain is able to achieve high extents of hydrocarbon degradation, offering a promising tool to be further used in bioaugmentation approaches for treating contaminated environments / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Bactérias aclimatadas a cobre foram isoladas a partir de amostras de solo e água coletadas na região da Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. foi escolhida, pois, apresentou a maior capacidade sortiva de Cu2+, Q = 40 mg/g. O cultivo em regime de batelada, meio mineral, com glicerol como fonte de carbono, resultou fator de conversão de glicerol a células, YX/S, de 0,49 g/g e velocidade específica máxima de crescimento, mmax, de 0,11 h-1. Alta concentração celular, 90 g/L, foi alcançada em cultivos em regime de batelada alimentada. Promoveu-se sorção de cobre pelas células, por meio de adição contínua ou em pulsos, de solução de CuSO4. A maior sorção específica de cobre, Q, de 30 (mg de Cu2+/g de células), foi verificada na adição por pulsos. Fotos de MET da bactéria na ausência e presença de Cu2+ mostram acúmulo de cobre na membrana e internamente, caracterizando biossorção e bioacumulação. / Bacteria acclimated to copper were isolated from soil and water samples collected in Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. was chosen as it had the highest sorptive capacity for Cu2+, Q = 40 mg/g. The fed-batch culture in mineral medium with glycerol as the carbon source resulted in a glycerol-to-cell conversion factor, YX/S of 0.49 g/g and maximum specific growth rate, mmax of 0.11 h-1. High cell concentration, 90 g/L, was achieved in cultures in fed-batch regimen. Cooper sorption by cells was promoted, by continuous or pulse addition of CuSO4 solution. The highest specific copper sorption, Q, 30 (mg Cu+2/g of cells) was seen with the addition by pulses. TEM photos of the bacteria in the absence and presence of Cu+2 show copper accumulation in the membrane and internally, featuring biosorption and bioaccumulation.

Orientador: Paulo Renato Matos Lopes / Resumo: O herbicida tebuthiuron é largamente utilizado na cultura da cana-de-açúcar e pode acarretar prejuízos ao ambiente devido seu elevado potencial tóxico e alta persistência no solo. Assim, esse trabalho teve como objetivo avaliar o potencial de espécies vegetais em diminuir a concentração de tebuthiuron no solo com aplicação de vinhaça. A eficiência do processo de biorremediação foi avaliada quanto: ao desenvolvimento vegetal, aos parâmetros físico-químicos do solo e à ecotoxicidade do meio. Os experimentos foram conduzidos em casa de vegetação utilizando vasos com solo sem histórico de aplicação do herbicida. As espécies potencialmente fitorremediadoras testadas foram: feijão-de-porco (Canavalia ensiformis), feijão-guandu (Cajanus cajan), milheto (Pennisetum glaucum), e mucuna-cinza (Mucuna pruriens (L.) DC.). Como espécie sentinela foi utilizada a Crotalaria juncea. Ao longo do experimento foram avaliados: o diâmetro do colo, a altura da planta e o número de folhas. Ao final, avaliações de massas fresca e seca foram realizadas para as cinco plantas testadas. A fitotoxicidade das amostras nos tratamentos foi determinada nos tempos inicial (zero) e final (50 dias), utilizando sementes de alface como organismos-teste. Os resultados revelaram que o feijão-de-porco e o feijão-guandu não resistiram a presença do herbicida. O milheto apresentou o menor índice de mortalidade e também o melhor desempenho em solos na presença do tebuhiuron associado ou não à vinhaça. Baseado em seu des... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The herbicide tebuthiuron is widely used in sugarcane cultivation and can cause damage to the environment due to its high toxic potential and high soil persistence. Thus, this work aimed to evaluate the potential of plant species in decreasing tebuthiuron concentration in the soil with vinasse application. The efficiency of the bioremediation process was evaluated for: plant development, soil physicochemical parameters and environment ecotoxicity. To evaluate these parameters, the experiments were conducted in a greenhouse using pots with soil with no history of herbicide application. Potential phytoremediation species tested were: jack bean (Canavalia ensiformis), pigeon pea (Cajanus cajan), millet (Pennisetum glaucum), and velvet bean (Mucuna pruriens (L.) DC.). As bioindicator species was used Crotalaria juncea. Throughout the experiment were evaluated: the neck diameter, the plant height and the number of leaves. At the end, fresh and dry mass evaluations were performed for the five plants tested. The phytotoxicity of the samples in the treatments was determined at the initial (zero) and final (50 days) times, using lettuce seeds as test organisms. Results revealed that pigeon bean and jack bean did not resist the herbicide presence. Millet plants showed the lowest mortality rate among all plant species and also presented the best performance in tebuthiuron presence associated or not with vinasse. However, despite of these results for millet, velvet bean was the most suit... (Complete abstract click electronic access below) / Mestre

Polaromonas sp. strain JS666 is the only isolated bacterium capable of aerobic growth using the groundwater pollutant cis-1,2-dichloroethene (cDCE) as a sole carbon source. Its genome has a wealth of evidence of recent gene acquisition through horizontal gene transfer, and contains gene clusters predicted to encode enzymes allowing the metabolism of a wide variety of xenobiotic compounds. Culture growth using each of these hypothesized substrates was tested experimentally, and many were confirmed as sole carbon sources for strain JS666. In addition to pollutant degradation, many of these metabolic pathways have applicability in the field of biocatalysis, as does the genome-assisted pathway prediction approach to biocatalyst discovery. During (or immediately following) growth on cDCE, cultures of Polaromonas sp. strain JS666 oxidize ethene to epoxyethane at an increased rate, and also cometabolically oxidize several other chlorinated ethenes. Given the involvement of a monooxygenase in other species' 1-chloroethene (vinyl chloride) oxidation, it was hypothesized that alkene oxidation in strain JS666 was due to the activity of a monooxygenase that also was responsible for the first step in cDCE oxidation. The alkene oxidation activity of strain JS666 was investigated using gene expression analysis, proteomics, and whole-cell kinetic assays. Results of these experiments pointed to the upregulation of a cyclohexanone monooxygenase (CHMO) during growth on cDCE and during oxidation of ethene. To determine the activity of this cyclohexanone monooxygenase, its gene was cloned and heterologously expressed in an E. coli host. Our CHMO expression system exhibited activity on cyclohexanone, but not cDCE or ethene, disproving our hypothesis about its involvement in alkene oxidation. The heterologously expressed monooxygenase was also investigated for enantioselective oxidation of racemic cyclic ketones to chiral lactones, and was discovered to have very high enantioselectivity with the tested compounds. Chiral lactones and other single-enantiomer oxidation products are valuable for fine chemical synthesis, and their biocatalytic production is more environmentally sustainable and often less expensive than traditional techniques. The research described in the following chapters illustrates the many opportunities that arise when the fields of bioremediation and biocatalysis converge. The shared research goals and methods of these two areas lend themselves to interdisciplinary research, and increased communication and crossover between them should provide benefits for both environmental remediation and sustainable chemical synthesis.

Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene, chlorinated solvents often used in plastics and dry cleaning solvent manufacturing. The treatment of VC-contaminated sites by bacteria that can biodegrade VC has been demonstrated to be a practical and potentially cost-effective alternative to traditional "pump and treat" site cleanup options. However, little is known about the biochemical pathways involved in VC-assimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. The results of these studies demonstrated the successful extraction and identification of proteins involved in VC-assimilation from ethene-enriched groundwater samples. The presence and diversity of VC-assimilating bacteria in several ethene-enriched groundwater samples were examined using tandem mass spectrometry analysis to identify the protein biomarkers EtnC and EtnE. VC-assimilating organisms can evolve in vitro from bacteria that grow on ethene but very little is known about the molecular changes involved. Proteomic investigations comparing three strains of Mycobacterium strain JS623, a wild type and two VC-adapted strains, validated previous studies indicating that protein expression changes are involved in VC-adaptation. Tandem mass spectrometry and spectral counting were used to identify proteins and semi-quantitatively estimate protein expression levels in the three ethene-grown JS623 variants. The results of this study suggest that multiple VC-adaptation mechanisms are involved in the two VC-adapted strains