Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007. / Committee Chair: Sheldon W. May ; Committee Members: Donald F. Doyle, Leslie T. Gelbaum, Stanley H. Pollock, and James Powers. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 136-141). Also available via the Internet.

Production of poly(3-hydroxybutyrate) (PHB) was done in a cyclone bioreactor using various culture methods; including batch (lab and pilot scale) fed-batch and self-cycling fermentation with and without starvation periods. Alcaligenes eutrophus DSM 545 was used to accumulate about 87% (wt/wt) PHB to a total of 6.2 g L$ sp{-1}$ PHB at the end of a 48 hour batch. Similar pilot scale experiments contained a maximum of 96% (wt/wt) PHB with 4.9 g L$ sp{-1}$ accumulated. Fed-batch culture of A. eutrophus produced 96% (wt/wt) PHB with a final PHB concentration of 22.2 g L$ sp{-1}$ after 54 h. Self-cycling fermentation (SCF) production of PHB resulted in an average of 35% (wt/wt) PHB without starvation periods with production rates reaching 0.24 g L$ rm sp{-1} hr sp{-1}.$ With starvation periods of 4, 6 and 8 h extended on the cycle times, production of PHB decreased except in the 8 hour starvation period which was 59% (wt/wt). However, the rates of production all decreased to below 0.13 g $ rm L sp{-1} h sp{-1}$ as the lengths of the starvation periods were increased.

No description available.

The importance of Poly(3-hydroxybutyrate) (PHB) as a biodegradable material is well known. Due to ever increasing environmental awareness, significant efforts have been made to utilize PHB or its derivatives in producing disposable products. However, brittle mechanical properties of PHB hinder the direct application of this material in useful commodity items. In order to achieve toughened PHB, blending with other polymers which possess high relaxation behavior at room temperature seems attractive. Prior to such development, the fundamental characterization of the relaxation behavior of PHB itself is extremely important in order to assess the effect of any attempt to improve the situation in a quantitative manner. Dielectric thermal analysis was used in the study of the relaxation behavior of melt processed PHB. The approach was largely phenomenological, that was, based on the macroscopic theory of dielectric relaxation. The mean relaxation time of melt processed PHB was evaluated while PHB was undergoing crystallization at room temperature. The experimental conditions were kept as close as possible to actual shelf-life conditions. Dynamic temperature sweep experiments revealed multiple relaxation peaks at the glass transition region. Temperature plane curve resolution revealed, in the early stage of crystallization, two dynamically changing peaks whose behaviors, as the extent of crystallization progressed, were quite opposite in terms of the magnitude of the loss property. By analyzing the temperature dependence of loss property and mean relaxation time, it was concluded that the peak located at the lower temperature is related to pure amorphous chain movement, and the peak located at the higher temperature is related to the movement of amorphous chains which are confined in-between crystalline phases, such as lamellae and spherulites. For the evaluation of the mean relaxation time of binary blends or multiple relaxations arising from homopolymers and copolymers, an empirical model has been developed which is grounded in the theory of linear viscoelasticity with the aim of quantitatively assessing the effect of attempts to improve the toughness of PHB. In the course of data reduction and model development, the majority of empirical dielectric relaxation functions has been reviewed including the Havriliak-Negami model and the Kohlrausch-Williams-Watts stretched exponential function. It was found that the center of relaxation time in the Havriliak-Negami model was skewed toward short time scale of relaxation, while mean relaxation time reflected the relaxation behavior of PHB chains on average, including movement of chains which relax with difficulty as the extent of crystallization progresses. / Ph. D.

This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

This research followed acetate carbon (C) uptake, metabolism, and fate through a typical modern Kraft pulp and paper mill AS system. Freshly collected mill biomass (AS floc suspensions) was placed under conditions representing four key phases of AS biotreatment: (1) initial acetate uptake by aerated starved AS; (2) ongoing acetate uptake; (3) aerobic metabolism of acetate-loaded AS in acetate-stripped effluent; and (4) anaerobic, settled biomass metabolism. Conditions mimicked the mill system as closely as possible. Acetate carbon uptake kinetics and conversion to CO2, growth products, PHB, and secreted metabolites in each of the four phases were measured. The role of PHB synthesis in the initial stripping of acetate from mill effluent and the PHB production potential of this mill AS were also investigated. / Results showed that acetate was rapidly taken up by high-affinity systems in the AS. During the initial exposure of mill-starved AS, acetate greatly stimulated AS-O2 uptake, and was quickly converted to PHB and CO 2. Upon depletion of available effluent acetate, as occurs in the downstream sections of the aeration tank, O2-uptake rates decreased and the acetate-C stored in AS-PHB was slowly released as CO2, and partly used for growth. Under secondary clarifier-like anaerobic conditions, the AS released virtually no CO2. However, substantial amounts of PHB were used for growth under anaerobic conditions and a small proportion of the original acetate C exited the cells as organic acids.

Orientadores: Wanderley Dias da Silveira, Eliana Gertrudes de Macedo Lemos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:23:49Z (GMT). No. of bitstreams: 1 Paganelli_FernandaLaroza_M.pdf: 1101767 bytes, checksum: e8882c8b6ec40ea617dd997584b239c3 (MD5) Previous issue date: 2009 / Resumo: Polihidroxialcanoatos (PHAs) são polímeros de hidroxialcarioatos produzidos, e acumulados, intracelularmente, como fonte de carbono e/ou outros materiais energéticos, em vários microorganismos. Freqüentemente, o acúmulo dos PHAs ocorrem em condições abaixo do limite nutricional de elementos como N, P, S, O ou Mg e excesso de carbono, podendo representar até 80% da massa seca total da célula. Mais de 300 diferentes microorganismos podem sintetizar e acumular PHAs. O polihidroxibutirato (PHB) é o mais conhecido dentre os polímeros bacterianos biodegradáveis denominados polihidroxialcanoatos. Por ter propriedades semelhantes ao polipropileno, o PHB pode ser usado na fabricação do plástico biodegradável. Além da busca por maior produção de tal polímero, pouco é conhecido sobre seu papel biológico, em especial nos rizóbios. Estudos revelaram que há variação na capacidade da produção e acúmulo de PHB nessas bactérias quando em simbiose, dependendo da espécie em questão e das condições de cultivo das mesmas, observando-se bactérias incapazes de acumular PHB quando bacterióides, como é o caso do Rhizobium meliloti, ou capazes de produzir e acumular PHB, nessas condições, como é o caso de Rhizobium etli, Bradyrhizobium japonicum e Bradyrhizobium elkanii. Desta forma, este trabalho teve por objetivo a identificação dos genes responsáveis pela síntese de PHB em Bradyrhizobium elkanii, clonagem e expressão dos mesmos em Escherichia coli, uma vez que esta bactéria é bem conhecida como ferramenta molecular e se multiplica rapidamente, podendo atingir uma alta produção do polímero esperado, em um curto período de tempo. Além disso, objetivou-se, também, aumentar a produção de PHB em B. elkanii através de mutações aleatórias (através da inserção do transposon TnphoA), já que esta é uma bactéria naturalmente produtora de PHB. Para isso, os genes phbA, phbB e phbC foram isolados através da técnica de PCR, amplificando-se os genes inteiros. Estes foram clonados em vetores de expressão tipo pET (NOVAGEN), sendo os genes phbA e phbB clonados em "operon " em um mesmo vetor e o gene phbC clonado separadamente. A expressão dos genes foi analisada, bem como sua capacidade de produzir PHB. Os mutantes de B. eíkanii obtidos através da inserção do transposon TnphoA foram analisados com o uso do corante Sudan Black, procurando-se selecionar linhagens maiores produtoras de PHB. A produção dos mutantes selecionados foi, posteriormente, analisada por cromatografia gasosa. Observou-se que a linhagem de E.coli com os três genes clonados teve a capacidade de produzir PHB, porém com baixa eficiência. Já os mutantes aleatórios de B. elkanii apresentação diferentes acúmulos em relação ao selvagem, com destaque para o MUT33 que teve 72% da sua massa seca acumulada na forma de PHB, enquanto o selvagem acumulou 51 % de PHB, nas mesmas condições. / Abstract: Polyhydroxyalkanoates (PHAs) are polymers of hydroxyalkanoate acids, produced and accumulated intracellularly as a source of carbon and energy storage material, in prokaryotic cells. Often, the PHAs accumulation occurs in conditions when the carbon source is in excess but one or several other nutrients are limited, and may represent up to 80% of the cell dry weight. More than 300 different microorganisms can synthesize and accumulate PHAs. The polyhydroxybutyrate (PHB) is the most studied polymer among the bacterial biodegradable polymers (PHAs). By having properties similar to polypropylene, PHB can be used in the manufacture of biodegradable plastic. On the search for greater production of such polymer, little is known about its biological role, especially in the genus Rhizobium. Studies have shown that there is a variation in the PHB capacity production and accumulation when these bacteria are in symbiosis. Depending on the species and cultivation conditions it has been observed either incapacity of PHB production and accumulation, when Bacteroides, as Rhizobium meliloti; or capacity and accumulation of PHB under bacteroides fase, as Rhizobium etli, Bradyrhizobium japonicum and Bradyrhizobium elkanii. Therefore, the present work aimed the identification, cloning and expression, in Escherichia coli, of responsible genes for PHB synthesis in Bradyrhizobium elkanii. Moreover, it was also expected to increase the production of PHB in B. elkanii, through random mutations (insertion of the TnphoA transposon), since B. elkanii is a good natural producer of PHB. For this, the entier phbA, phbB and phbC genes were isolated by PCR. Those genes were cloned in expression vectors such pET (NOVAGEN), where phbA and phbB genes were cloned in operon, in a single vector, whereas phbC gene was cloned separately, in another vector. The expression of those genes was analyzed, as well as its ability to produce PHB. Mutants B. elkanii, obtained by insertion of the TnphoA transposon, were analyzed using the dye Sudan Black, in order to select different strains that might produce higher quantities of PHB. The production of PHB by mutants was then analyzed by gas chromatography. It was observed that the E. coli with the three cloned genes had the ability to produce PHB, but with low efficiency. The B. elkanii random mutants show different accumulation compared to the wild, especially MUT33 that had 72% of its dry mass accumulated in the form of PHB, while the wild 51% of accumulated PHB under the same conditions. / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

No description available.