Os plásticos, polímeros geralmente derivados de petróleo com características bastante atraentes e valor baixo estão presentes em quase todos os objetos produzidos pelos homens, desde consumo doméstico até produtos de alta tecnologia. Em 2015, aproximadamente 320 milhões de toneladas de plásticos foram produzidas e a estimativa é que este consumo continue aumentando até 2020. No entanto, cerca de 60% do que é produzido, são plásticos não biodegradáveis, os quais são persistentes ambientalmente, afetando negativamente diversos seres vivos, inclusive os seres humanos. Diante deste cenário, e como alternativa ambientalmente correta, podemos fazer uso de biopolímeros, em especial o polihidroxibutirato (PHB). O PHB é um homopolímero biodegradável, biocompatível e possui características similares às do polipropileno, podendo servir como potencial substituto a esse polímero derivado de petróleo. No entanto, o PHB ainda não está difundido no nosso cotidiano, porque apresenta alto custo de produção e purificação, principalmente devido aos substratos utilizados, que na maioria das vezes são açúcares e chegam a representar 30% do preço final do produto. Visando diminuir o preço de produção, o objetivo deste trabalho é produzir o PHB utilizando bactérias metilotróficas, isoladas de ambiente estuarino e capazes de consumir metanol. Após isolamento seletivo, duas bactérias do gênero Methylobacterium sp. foram isoladas e identificadas por biologia molecular e espectrometria de massas (MALDI-TOF). A produção de PHB foi estudada em frascos agitados sem quaisquer ajustes de condições físico-químicas por um período de 168 horas sob 28 ºC e 180 RPM em agitador orbital de bancada e também em biorreator sob 28 °C, 180 RPM e pH 7,0 durante 96 horas. Os melhores resultados foram obtidos por M. extorquens sem ajuste de condições físico-químicas, tendo atingido produtividade do produto de 125 mg.L-1 e acúmulo de 30% de PHB em biomassa. M. rhodesianum obteve produtividade do produto de 38 mg.L-1 em seu melhor resultado, porém é uma produtora em potencial de polietileno glicol a partir de metanol. / Plastics, usually petroleum-derived polymers that have attractive characteristics and low price, are present in literally almost all products produced by humans. They are present since from home consuming to high technology products. In the year of 2015 approximately 320 million tons of plastics were produced and is estimate this consumption will continue to increase until 2020. However, about 60% of this production is non-biodegradable plastics, which makes them environmentally persistent, affecting negatively several living beings including humans. Considering this scenario, and as an environmentally correct alternative, we can make use of biopolymers, especially polyhydroxybutyrate (PHB). PHB is a biodegradable, biocompatible homopolymer and has characteristics similar to those of polypropylene, which may serve as a potential substitute for this petro derivative polymer. However, PHB is still not widespread in our daily lives, once has a high cost of production and purification, mainly due to the substrates used, which in most cases are sugars, and they can represent 30% of the final price of the product. In order to decrease the price of production, this work aims to produce PHB using methylotrophic bacteria, isolated from an estuarine environment and capable of consuming methanol. After a selective isolation, two bacteria of the genus Methylobacterium sp. were isolated and identified by molecular biology and mass spectrometry (MALDI-TOF). PHB production was studied in shake flasks without any physical-chemical adjustments for a 168 hour period under 28 ºC and 180 RPM in a rotary shaker and also in bioreactor under 28 °C, 180 RPM and pH 7.0 for 96 hours. The best results were obtained by M. extorquens without adjustment of physical-chemical conditions, reaching a product productivity of 125 mg.L-1 and accumulation of 30% of PHB in biomass. M. rhodesianum obtained product productivity of 38 mg.L-1 at its best result, but is a potential producer of polyethylene glycol from methanol.

Os plásticos, polímeros geralmente derivados de petróleo com características bastante atraentes e valor baixo estão presentes em quase todos os objetos produzidos pelos homens, desde consumo doméstico até produtos de alta tecnologia. Em 2015, aproximadamente 320 milhões de toneladas de plásticos foram produzidas e a estimativa é que este consumo continue aumentando até 2020. No entanto, cerca de 60% do que é produzido, são plásticos não biodegradáveis, os quais são persistentes ambientalmente, afetando negativamente diversos seres vivos, inclusive os seres humanos. Diante deste cenário, e como alternativa ambientalmente correta, podemos fazer uso de biopolímeros, em especial o polihidroxibutirato (PHB). O PHB é um homopolímero biodegradável, biocompatível e possui características similares às do polipropileno, podendo servir como potencial substituto a esse polímero derivado de petróleo. No entanto, o PHB ainda não está difundido no nosso cotidiano, porque apresenta alto custo de produção e purificação, principalmente devido aos substratos utilizados, que na maioria das vezes são açúcares e chegam a representar 30% do preço final do produto. Visando diminuir o preço de produção, o objetivo deste trabalho é produzir o PHB utilizando bactérias metilotróficas, isoladas de ambiente estuarino e capazes de consumir metanol. Após isolamento seletivo, duas bactérias do gênero Methylobacterium sp. foram isoladas e identificadas por biologia molecular e espectrometria de massas (MALDI-TOF). A produção de PHB foi estudada em frascos agitados sem quaisquer ajustes de condições físico-químicas por um período de 168 horas sob 28 ºC e 180 RPM em agitador orbital de bancada e também em biorreator sob 28 °C, 180 RPM e pH 7,0 durante 96 horas. Os melhores resultados foram obtidos por M. extorquens sem ajuste de condições físico-químicas, tendo atingido produtividade do produto de 125 mg.L-1 e acúmulo de 30% de PHB em biomassa. M. rhodesianum obteve produtividade do produto de 38 mg.L-1 em seu melhor resultado, porém é uma produtora em potencial de polietileno glicol a partir de metanol. / Plastics, usually petroleum-derived polymers that have attractive characteristics and low price, are present in literally almost all products produced by humans. They are present since from home consuming to high technology products. In the year of 2015 approximately 320 million tons of plastics were produced and is estimate this consumption will continue to increase until 2020. However, about 60% of this production is non-biodegradable plastics, which makes them environmentally persistent, affecting negatively several living beings including humans. Considering this scenario, and as an environmentally correct alternative, we can make use of biopolymers, especially polyhydroxybutyrate (PHB). PHB is a biodegradable, biocompatible homopolymer and has characteristics similar to those of polypropylene, which may serve as a potential substitute for this petro derivative polymer. However, PHB is still not widespread in our daily lives, once has a high cost of production and purification, mainly due to the substrates used, which in most cases are sugars, and they can represent 30% of the final price of the product. In order to decrease the price of production, this work aims to produce PHB using methylotrophic bacteria, isolated from an estuarine environment and capable of consuming methanol. After a selective isolation, two bacteria of the genus Methylobacterium sp. were isolated and identified by molecular biology and mass spectrometry (MALDI-TOF). PHB production was studied in shake flasks without any physical-chemical adjustments for a 168 hour period under 28 ºC and 180 RPM in a rotary shaker and also in bioreactor under 28 °C, 180 RPM and pH 7.0 for 96 hours. The best results were obtained by M. extorquens without adjustment of physical-chemical conditions, reaching a product productivity of 125 mg.L-1 and accumulation of 30% of PHB in biomass. M. rhodesianum obtained product productivity of 38 mg.L-1 at its best result, but is a potential producer of polyethylene glycol from methanol.

No description available.

Upland soils are a significant sink of atmospheric methane, but the organisms responsible for methane consumption have yet to be identified. The ecology of methanotrophs was investigated in a beech forest soil which exhibited atmospheric-methane-uptake. Maximal methane-oxidation was observed in the upper mineral layer between the organic and inorganic horizons at a rate of 3.1 +/- 0.3 nmoles CH 4 g [fresh wt soil]-1 d-1. A clone bank of the methanotroph pmoA gene was constructed by PCR amplification from soil DNA extracts. The PCR primers used coamplify the related amoA gene of ammonia-oxidizers. The clones recovered grouped into three clusters: Nitrosospira-like sequences, a group somewhat related to alpha-Proteobacteria methanotrophs and previously referred to as the RA14 group, and a cluster which could not be characterized as either amoA or pmoA sequences. No pmoA genes closely related to genera of cultured methanotrophs were obtained. The 16S rDNA was also targeted using eubacterial and methanotroph-specific primers. 16S rDNA sequence analysis revealed the presence of organisms distantly related to known methanotrophs. Methanotroph enrichment cultures were established by inoculating mineral salts medium with soil and incubating under an atmosphere of 10% methane in air. Gene sequencing from the enrichments indicated the presence of organisms belonging to the genera Methylosinus and Methylocystis. The results suggest that the dominant methanotrophs in the forest soil are not related to known organisms and do not grow under conditions typically used to isolate methanotrophs.

Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

O gênero Methylobacterium é composto por bactérias de coloração rósea, metilotróficas e que podem colonizar endofiticamente a planta. Algumas espécies deste gênero são capazes de promover o crescimento vegetal e reduzir o ataque de fitopatógenos. A linhagem de M. mesophilicum SR1.6/6 foi isolada de ramos de citros e devido a sua interação com a planta hospedeira e com patógenos associados a planta, tem sido foco vários trabalhos. Os sistemas de secreção e as bombas de efluxo podem estar envolvidos na modulação das interações de bactérias endofíticas com seus ambientes. Assim, neste estudo foi analisada a composição dos exsudatos produzidos pelas plantas de milho e citros na interação com a SR1.6/6 por GC-MS, foi realizada a reanotação de genes relacionados ao sistema de secreção e bombas de efluxo e foi avaliada a expressão de alguns genes destes sistemas na interação com Zea mays e Citrus sinensis por qPCR. Foram encontrados sistemas de secreção tipo I, II e V, vias SEC e TAT e bombas de efluxo, principalmente super-expressos durante a interação com a planta hospedeira. / Methylobacterium genus is composed by pink-pigmented facultative methylotrophic bacteria. Some species of this genus are able to promote plant growth and reduce the incidence of pathogens. The SR1.6/6 strain of Methylobacterium mesophilicum is a bacterium isolated from citrus and due to its interaction with the plant has been the focus of several studies. Multidrug efflux pumps and secretion system can be involved modulating the interactions of bacteria with their environments. In this work was analised the root exsudates composition from two host plants citrus and corn interacting with SR1.6/6 by GC-MS technique, additionally was searched and reannotated genes related to secretion system and some multidrug efflux pumps and finally evaluated the gene expression of some of this genes during the interaction with Zea mays and Citrus sinensis by qPCR. Type I, II and V secretion system, SEC and TAT pathway and some multidrug efllux pumps were found in this strain according gene expression. This systems were up-regulated mainly during interaction with host plant.

Le chlorométhane est le plus abondant des composés organo-halogénés dans l’atmosphère et il est impliqué dans la destruction de l’ozone dans la stratosphère. Les sources et les puits de chlorométhane restent mal évalués. Bien que synthétisé et utilisé de manière industrielle, il est principalement produit naturellement, avec comme sources majeures les émissions provenant des végétaux et plus particulièrement de la phyllosphère, qui correspond aux parties aériennes des plantes. Certaines bactéries épiphytes de la phyllosphère sont des méthylotrophes capables d’utiliser des composés organiques sans liaison carbone-carbone comme le méthanol et le chlorométhane comme unique source de carbone et d’énergie pour leur croissance. La plupart des bactéries chlorométhane-dégradantes isolées jusqu’à présent utilisent une voie métabolique pour leur croissance sur chlorométhane appelée voie cmu (pour chloromethane utilisation), caractérisée par l’équipe. L’objectif principal de cette thèse a été de déterminer si des bactéries de la phyllosphère peuvent jouer le rôle de filtre pour l’émission de chlorométhane par les plantes. Dans ce but, un modèle de laboratoire a été mis en place, constitué de la plante Arabidopsis thaliana connue pour produire du chlorométhane par une réaction impliquant le gène HOL1, et la bactérie Methylobacterium extorquens CM4, souche de référence pour l’étude du métabolisme de dégradation du chlorométhane, qui possède la voie cmu et dont le génome complet a été séquencé et analysé. Des variants d’A. thaliana avec différents niveaux d’expression du gène HOL1 (le type sauvage, le mutant homozygote « knock-out » hol1 et un variant HOL1-OX avec surexpression) ont été sélectionnés par PCR et qPCR. Des souches bactériennes chlorométhane-dégradantes ont été isolées à partir de la phyllosphère d’A. thaliana, dont il a été montré qu’elles possèdent la voie cmu. Un bio-rapporteur bactérien pour le chlorométhane a été construit à l’aide d’un plasmide exploitant la région promotrice du gène conservé de la déshalogénase (cmuA) de la souche M. extorquens CM4. Il présente une réponse fluorescente rapide, sensible, et spécifique aux méthyl-halogénés de manière concentration-dépendante. L’application du bio-rapporteur aux trois variants d’A. thaliana étudiés suggère des niveaux d’émissions de chlorométhane différents. L’analyse, par qPCR et qRT-PCR, de l’ADN métagénomique extrait de la surface des feuilles a montré une corrélation entre la proportion relative de bactéries portant le gène cmuA et l’exprimant dans cet environnement, et l’expression du gène HOL1. Ces résultats indiquent qu’une production de chlorométhane, même très modeste par rapport aux fortes émissions de méthanol par A. thaliana, confère un avantage sélectif pour les bactéries épiphytes chlorométhane-dégradantes. Ces dernières pourraient ainsi bien jouer un rôle de filtre pour les émissions de chlorométhane de la phyllosphère vers l’atmosphère. En perspective, de nouvelles expériences complémentaires, basées sur l’analyse par génomique comparative des souches chlorométhane-dégradantes également effectuée dans le cadre du projet et sur une analyse par séquençage à haut-débit initiée dans ce travail, sont proposées pour améliorer la compréhension des mécanismes d’adaptation des bactéries chlorométhane-dégradantes dans la phyllosphère. / Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

O gênero Methylobacterium é composto por bactérias de coloração rósea, metilotróficas e que podem colonizar endofiticamente a planta. Algumas espécies deste gênero são capazes de promover o crescimento vegetal e reduzir o ataque de fitopatógenos. A linhagem de M. mesophilicum SR1.6/6 foi isolada de ramos de citros e devido a sua interação com a planta hospedeira e com patógenos associados a planta, tem sido foco vários trabalhos. Os sistemas de secreção e as bombas de efluxo podem estar envolvidos na modulação das interações de bactérias endofíticas com seus ambientes. Assim, neste estudo foi analisada a composição dos exsudatos produzidos pelas plantas de milho e citros na interação com a SR1.6/6 por GC-MS, foi realizada a reanotação de genes relacionados ao sistema de secreção e bombas de efluxo e foi avaliada a expressão de alguns genes destes sistemas na interação com Zea mays e Citrus sinensis por qPCR. Foram encontrados sistemas de secreção tipo I, II e V, vias SEC e TAT e bombas de efluxo, principalmente super-expressos durante a interação com a planta hospedeira. / Methylobacterium genus is composed by pink-pigmented facultative methylotrophic bacteria. Some species of this genus are able to promote plant growth and reduce the incidence of pathogens. The SR1.6/6 strain of Methylobacterium mesophilicum is a bacterium isolated from citrus and due to its interaction with the plant has been the focus of several studies. Multidrug efflux pumps and secretion system can be involved modulating the interactions of bacteria with their environments. In this work was analised the root exsudates composition from two host plants citrus and corn interacting with SR1.6/6 by GC-MS technique, additionally was searched and reannotated genes related to secretion system and some multidrug efflux pumps and finally evaluated the gene expression of some of this genes during the interaction with Zea mays and Citrus sinensis by qPCR. Type I, II and V secretion system, SEC and TAT pathway and some multidrug efllux pumps were found in this strain according gene expression. This systems were up-regulated mainly during interaction with host plant.

Indiana University-Purdue University Indianapolis (IUPUI) / Long term no-till (NT) farming can improve the CH4 oxidation capacity of agricultural lands through creation of a favorable soil environment for methanotrophs and diffusive gas transport. However, limited data is available to evaluate the merit of that contention. Although the potential for biological CH4 oxidation may exist in NT soils, restricted diffusion could limit expression of that potential in fine-textured soils. A study was conducted to assess the CH4 oxidation potential and gaseous diffusivity of soils under plow till (PT) and NT for > 50 years. Intact cores and composite soils samples (0-10 and 10-20 cm) were collected from NT and PT plots located at a well-drained site (Wooster silt loam) and at a poorly-drained (Crosby silt loam) site in Ohio. Adjacent deciduous forest soils were also sampled to determine maximum rate expected in undisturbed soils in the region. Regardless of study sites and soil depth, CH4 oxidation rate (measured at near ambient CH4) and oxidation potential (Vmax, measured at elevated CH4) were 3-4 and 1.5 times higher in NT than in PT soils, respectively. Activity in the NT soils approached (66-80 %) that in the forest soils. Half saturation constants (Km) and threshold for CH4 oxidation (Th) were lower in NT (Km: 100.5 µL CH4 L-1; Th: 0.5 µL CH4 L-1) than in PT soils (Km: 134 µL CH4 L-1; Th: 2.8 µL CH4 L-1) suggesting a greater affinity of long-term NT soils for CH4, and a possible shift in methanotrophic community composition. CH4 oxidation rates were lower in intact soil cores compared to sieved soils, suggesting that CH4 oxidation was limited by diffusion, a factor that could lead to lower field-measured CH4 uptake than suggested by biological oxidation capacity measured in the laboratory. Regardless of soil drainage characteristic, long-term NT resulted in significantly higher (2-3 times) CH4 diffusivity (mean: 2.5 x 10-3 cm2 s-1) than PT (1.5 x 10-3 cm2 s-1), probably due to improved soil aggregation and greater macro-pores volume in NT soils. Overall, these results confirm the positive impact of NT on the restoration of the biological (Vmax, Km and Th) and physical (diffusivity) soil attributes essential for CH4 uptake in croplands. Long-term implementation of NT farming can therefore contribute to the mitigation of CH4 emission from agriculture.