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Rhodococcus rhodochrous is an aerobic, non- pathogenic ,gram-positive bacterium that is often used in industries as a biocatalyst.R. rhodochrous DAP 96253 is capable of exhibiting contact-independent inhibition of selected fungal pathogens.The use of R. rhodochrous as a potential biocontrol agent against plant and animal fungi was examined.The fungi tested were Botrytis cinerea,Pseudogymnoascus destructans,Aspergillus flavus, Fusarium oxysporum’Cigar Tip’ , Rhizopus stolonifer’D1’ ,and other species isolated from berries.Each species was studied to establish the effect of dose (g/cells) and time of exposure to R. rhodochrous.Antifungal inhibition tests were done with the use of dosing,agar diffusion, frozen fermentation paste and exposed slides.Inhibition was observed with B.cinerea,P.destructans,A.flavus and D1,and reduced sporulation was observed with Cigar Tip. The results varied amongst the type of tests used on each target species.

Made available in DSpace on 2014-12-17T15:01:58Z (GMT). No. of bitstreams: 1 MarileideMA.pdf: 625410 bytes, checksum: 4571e21e5bf4561ee450806713d749af (MD5) Previous issue date: 2005-03-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In the last decade, biological purification of gaseous waste has become an important alternative to many conventional methods of exhaust air treatment. More recently, biofiltration has proved to be an effective and inexpensive method for the treatment of air contaminated with volatile organic compounds (VOCs). A biofilter consists in a reactor packed with a porous solid bed material, where the microorganisms are fixed. During the biofiltration process, polluted air is transported through the biofilter medium where the contaminant is degraded. Within the biofilm, the pollutants in the waste gases are energy and carbon sources for microbial metabolism and are transformed into CO2, water and biomass. The bed material should be characterized by satisfactory mechanical and physical properties as structure, void fraction, specific area and flow resistance. The aim of this research was the biofilter construction and study of the biological degradation of ethanol and toluene, as well as the modeling of the process. Luffa cylindrica is a brazilian fiber that was used as the filtering material of the present work. The parameters and conditions studied were: composition of nutrients solution; effect of microflorae strains, namely Pseudomanas putida and Rhodococcus rhodochrous; waste gas composition; air flow rate; and inlet load of VOCs. The biofilter operated in diffusion regime and the best results for remotion capacity were obtained when a microorganisms consortion of Pseudomanas putida and Rhodococcus rhodochrous,were used, with a gas flow rate of 1 m3.h-1 and molar ratio nitrogene/phosphore N/P=2 in the nutrients solution. The maximum remotion capacity for ethanol was around 90 g.m-3.h-1 and 50 g.m-3.h-1 to toluene. It was proved that toluene has inhibitory effect on the ethanol remotion When the two VOCs were present in the same waste gas, there was a decrease of 40% in ethanol remotion capacity. Luffa cylindrica does not present considerable pressure drop. Ottengraf and van Lith models were used to represent the results obtained for ethanol and toluene, respectively. The application of the transient model indicated a satisfactory approximation between the experimental results obtained for ethanol and toluene vapors biofiltration and the ones predicted it / Na ultima d?cada, a purifica??o biol?gica de rejeitos gasosos tem sido uma importante alternativa em detrimento dos m?todos convencionais de tratamento. Mais recentemente, a biofiltra??o tem se mostrado um m?todo efetivo e de baixo custo para tratamento de ar contaminado com compostos org?nicos vol?teis (COVs). O biofiltro ? um reator que possui um leito filtrante constitu?do por um material poroso, onde est?o fixados os microrganismos. Durante o processo de biofiltra??o, o ar polu?do ? transportado ao longo do leito onde o contaminante ? degradado. No biofilme, os poluentes presentes no efluente gasoso ser?o a fonte de carbono e energia para os microrganismos e ap?s serem metabolizados ser?o transformados em CO2, ?gua e biomassa. O leito filtrante deve ser caracterizado por propriedades f?sicas e mec?nicas como estrutura, fra??o de vazios, ?rea especifica e resist?ncia ao fluxo gasoso. O principal objetivo deste trabalho de pesquisa foi a constru??o de um biofiltro e o estudo da degrada??o do etanol e tolueno, bem como a modelagem do processo. Luffa cylindrica ? uma fibra bastante encontrada no nordeste do Brasil e foi utilizada no presente trabalho como leito filtrante. Os par?metros e condi??es estudos foram: composi??o da solu??o nutriente; efeito da esp?cie de microrganismos, denominadas Pseudomanas putida e Rhodococcus rhodochrous; composi??o do efluente gasoso; taxa de fluxo gasoso e carga de entrada do COV. O biofiltro operou em regime difusional, sendo os melhores resultados para a capacidade de remo??o foram obtidos quando utilizado o cons?rcio de microrganismos de Pseudomanas putida e de Rhodococcus rhodochrous, com uma vaz?o volum?trica de ar de 1 m3.h-1 e raz?o molar nitrog?nio/fosforo N/P=2 na solu??o nutriente. A m?xima capacidade de elimina??o foi de 90 g.m-3.h-1 para o etanol e 50 g.m-3.h-1 para o tolueno. Constatou-se que o tolueno tem efeito inibit?rio sobre a degrada??o do etanol, quando os dois VOCs est?o presentes no mesmo efluente, houve uma queda de 40% na remo??o do etanol. A Luffa cylindrica n?o apresentou grandes valores de perda de carga. Os modelos de Ottengraf e van Lith representaram bem os resultados obtidos para o etanol e tolueno, respectivamente. A aplica??o do modelo transiente evidenciou uma aproxima??o satisfat?ria entre os resultados experimentais obtidos para a biofiltra??o de vapores de etanol e tolueno e aqueles previstos pelo referido modelo

Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

Structural biology is one of the most quickly growing fields of research in life sciences. X-ray diffraction analysis is the technique that allows direct visualization of protein structure at the atomic or near-atomic level. Structure solution of proteins and protein complexes by X-ray crystallography provides important insights into their mode of action. The haloalkane dehalogenase proteins represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. In the present study, the structures of three haloalkane dehalogenase DhaA mutants DhaA04, DhaA14 and DhaA15 at atomic resolution are reported and compared to explore the effect of mutations on the enzymatic activity of modified proteins from a structural perspective. Besides that, in this work, the crystallization and initial X-ray diffraction characterization of DhaA wild type and its mutant variant DhaA13 in complex with environmental pollutant 1,2,3-trichloropropane and the crystallization of DhaA13 in complex with the fluorescence dye coumarin are described.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-07T13:57:20Z No. of bitstreams: 1 texto completo.pdf: 2824180 bytes, checksum: 35078d63ffeaef3e4935030bf53dc74e (MD5) / Made available in DSpace on 2017-04-07T13:57:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2824180 bytes, checksum: 35078d63ffeaef3e4935030bf53dc74e (MD5) Previous issue date: 2016-08-18 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Esse estudo é iniciado com uma revisão bibliográfica em que aborda a biorremediação de hidrocarbonetos pesados e hidrocarbonetos aromáticos policíclicos (PAHs). Na revisão, são explorados aspectos que incluem a obtenção de micro- organismos com capacidade de degradar hidrocarbonetos, vias metabólicas de biodegradação, as consequências ambientais da contaminação e por fim aborda novos estudos que utilizam estratégias e novas tecnologias de biorremediação possíveis em ambientes aquáticos e solos. A parte experimental de nosso estudo segue a mesma linha de pesquisa desenvolvida durante o mestrado. De maneira preventiva, busca-se avaliar e desenvolver metodologias para biorremediar as adjacências da Ilha da Trindade, ambiente pristino e de valor ecológico, caso ocorram acidentes envolvendo o derramamento de hidrocarbonetos. Inicialmente, objetivamos avaliar o genoma dos isolados bacterianos autóctones da comunidade microbiana existente nas águas marinhas que circundam a Ilha da Trindade, Rhodococcus rhodochrous TRN7 e Nocardia farcinica TRH1, isolados durante o trabalho de mestrado como sendo capazes de degradar uma grande variedade de hidrocarbonetos de petróleo. Nosso objetivo também foi avaliar a estrutura da comunidade microbiana e o catabolismo de fenantreno após contaminação de microcosmos contendo água coletada na mesma região e utilizar micro-organismos autóctones como estratégia de bioaumentação. Por fim, objetivou-se desenvolver um produto biotecnológico baseado em emulsões duplas W/O/W fertilizadas, capaz de promover a bioestimulação sem a perda instantânea de nutrientes em águas oceânicas oligotróficas contaminadas por compostos orgânicos hidrofóbicos. Os genomas de R. rhodochrous TRN7 e N. farcinica TRH1 possuem genes de degradação de compostos alifáticos e aromáticos, sendo que 17 genes envolvidos com a biossíntese de antibióticos foram anotados com o genoma de R. rhodochrous TRN7, enquanto que no genoma de N. farcinica TRH1 foram identificados genes relacionados à degradação de caprolactam, precurssor do nylon, e do pesticida atrazina, o que torna ambos os isolados candidatos a serem empregados tanto em estratégias de bioaumentação quanto em outras áreas biotecnológicas. Após a contaminação de microcosmos com hidrocarbonetos, a comunidade microbiana da água litorânea da Ilha da Trindade passa a ser dominada por Gammaproteobacteria, grupo que compõe mais de 72% das sequências analisadas, enquanto que no tratamento controle houve predomínio de Proteobacteria (95% das sequências). Destaca-se a abundância de representantes do gênero Alteromonas, presentes em todos os tratamentos contaminados, com maior valor de abundância (66% das sequências) no tratamento contendo apenas fenantreno como contaminante. Decorridos 30 dias da contaminação, os valores de diversidade foram reduzidos, havendo domínio de grupos bacterianos reconhecidos pela presença de membros com atividade hidrocarbonoclástica. A capacidade de mineralização de fenantreno pela microbiota autóctone da Ilha da Trindade é reduzida quando outros PAHs e hidrocarbonetos de alto peso molecular estão presentes no ambiente. Em relação às emulsões produzidas, nossos resultados mostraram que elas podem ser utilizadas com o propósito de liberação gradual de nutrientes na fase aquosa dispersa. A emulsão é composta por gotículas que variam em um amplo espectro de tamanho que se desestabilizam temporalmente, resultando na liberação da inorgânicos. Quando utilizadas em hidrocarbonoclásticas, fase aquosa conjunto em microcosmos interna, ou contendo não, água contendo nutrientes com bactérias litorânea da Ilha da Trindade contaminada com petróleo, as emulsões W/O/W fertilizadas proporcionaram aumentos significativos da atividade microbiana. Como conclusões, nosso trabalho mostrou que a contaminação por hidrocarbonetos resulta na perda de diversidade microbiana da comunidade microbiana da água litorânea da Ilha da Trindade, e que um ambiente considerado pristino possui microbiota capaz de degradar hidrocarbonetos. Por fim, conclui-se que emulsões duplas W/O/W fertilizadas são uma alternativa para proporcionar aumento significativo na atividade catabólica de hidrocarbonetos em ambientes marinhos oligotróficos. / This study starts with a literature review approach in heavy hydrocarbon bioremediation and polycyclic aromatic hydrocarbons (PAHs). In the review are explored aspects from obtaining microorganisms capable of degrading hydrocarbons, metabolic biodegradation pathways, environmental consequences of contamination and finally discusses new studies using new strategies and bioremediation technologies in water and soil environments. The experimental part of our study follows the same research line developed during the Masters. Preventively, seeks to assess and develop bioremediation methodologies for Trindade Island shoreline, pristine environment with ecological value, should they occur involving the oil spill. Initially we aimed to evaluate the genome of indigenous bacterial isolates Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1, isolated during master’s work as being capable of degrading a wide range of petroleum hydrocarbons. Our aim was also to evaluate the microbial community structure and metabolism of PAHs after contamination in microcosms and use indigenous microorganisms as bioaugmentation strategy. Finally, it aimed to develop a biotechnolocal product based on double emulsion W/O/W fertilized able to promote biostimulation without the instant loss of nutrients in oligotrophic oceanic waters contaminated with hydrophobic organic compounds. The genomes of R. rhodochrous TRN7 and N. farcinica TRH1 presents aliphatic and aromatic compounds degradation genes. Moreover, 17 genes involved in the biosynthesis of antibiotics were recorded in the genome of R. rhodochrous TRN7 while in the genome of N. farcinica TRH1 were identified genes related to the degradation of caprolactam, a nylon precursor, and the pesticide atrazine, which makes the isolates candidates to be used in both strategies bioaugmentation as for other biotechnological activity. After contamination by hydrocarbons, in microcosms, the microbial community of the coastal water from Trindade Island becomes dominated by Gammaproteobacteria, a group that makes up more than 72% of the analyzed sequences, while in the control treatment predominated Proteobacteria (95% of the sequences). Noteworthy is the abundance of representatives of the genus Alteromonas, present in all the contaminated treatments with highest abundance (66% of the sequences) in the treatment containing phenanthrene as only contaminant. After 30 days of contamination, diversity values were reduced, with domain of bacterial groups recognized by the presence of members with hydrocarbonoclastic activity. The phenanthrene mineralization capacity by the indigenous microbiota of the Trindade Island is reduced when other PAHs and high molecular weight hydrocarbons are present in the environment, however, does not cease to occur. With respect to emulsions produced, our results showed that they can be used for the purpose of slow release nutrients in the dispersed aqueous phase. The emulsion comprises droplets ranging in a wide range of size that destabilize temporally, resulting in the release of the internal aqueous phase, containing inorganic nutrients. When used together or not, hydrocarbonoclastic bacteria in microcosms containing coastal water from Trindade Island contaminated with oil, the fertilized W/O/W emulsion provided significant increases in microbial activity. In conclusion, our work has shown that hydrocarbons contamination results in the loss of microbial diversity in marine environments, in addition to state that as environment considered pristine has microbiota capable of degrading hydrocarbons and therefore it becomes viable the use of strategies as biostimulation and bioaugmentation employing indigenous bacterial isolates previously selected. Finally, it is concluded that fertilized W/O/W emulsions are an alternative to provide significant increase in catabolic activity hydrocarbons oligotrophic marine environments.