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Doctoral Thesis
Full name
Cézar Vanzin
Knowledge Area
Date of Defense
São Paulo, 2008
Pradella, Jose Geraldo da Cruz (President)
Alterthum, Flavio
Ming, Chiu Chih
Valduga, Claudete Justina
Vicente, Elisabete Jose
Title in Portuguese
Estudo da biossíntese de poli-3-hidroxibutirato-co-hidroxialcanoatos de cadeia média (P3HB-co-3HAmcl) a apartir de ácidos graxos livres e óleo vegetal.
Keywords in Portuguese
Óleo de soja
Polímeros microbianos
Abstract in Portuguese
Os polihidroxialcanoatos constituem um grupo de poliésteres acumulados por inúmeras bactérias na forma de grânulos intracelulares, que podem representar até 80% da massa seca celular. No presente trabalho, foi avaliado o comportamento de diferentes linhagens quanto a capacidade de metabolizarem ácidos graxos livres e óleo de soja para a produção de biopolímeros e, posteriormente, em biorreator, algumas linhagens selvagens e recombinantes foram testadas na presença ou não de um inibidor da b-oxidação. Experimentos em frascos agitados, mostraram valores de teor de PHA em até cerca de 70% na biomassa total com cerca de 5% de monômeros de cadeia média permitindo selecionar as linhagens Cupriavidus necator e Burkholderia cepacia para os ensaios seguintes. Experimentos em biorreator mostraram que, as variáveis manipuláveis quanto ao fluxo de óleo de soja, de co-substratos e o inibidor de b-oxidação não influenciaram, significativamente, no rendimento de P3HB/3HAmcl formado quando utilizado Burkholderia cepacia IPT-048, entretanto, contribuíram na síntese de 3HV. Durante os ensaios, o aumento do número de cópias do gene phaB em Cupriavidus necator, aparentemente, não contribuíram para o aumento do rendimento de 3HHx, porém, outras variáveis manipuláveis deverão ser propostas para a confirmação.
Title in English
Biosynthesis study of poly-3hydroxybutirate-co-hydroxyalkanoates of medium chain length (P3HB-co-3HAmcl) from fatty acids and plant oil.
Keywords in English
Microbiol polyesters
Soybean oil
Abstract in English
The poly-3-hydroxyalkanoates (PHA) are a group of polyesters accumulated for several bacteria in the intracellular granule form, that can represent up to 80% of the dry mass cellular. The main advantage of the biopolymers on the synthetic materials is its degradation in the environment. Recycable raw materials can be used as carbon sources for the production biodegradable polymer. Some polymers have appeared in literature having mechanical characteristic considered appropriate in such a way for use in packings (flexible and covering films), how base for controlled release of asset to be applied in the pharmacological-medical and foods area. The P3HB-co-3HAmcl are 3-hydroxybutyrate copolymers and 3-hydroxyacyl of 6 or more carbon atoms which has aroused interest for to present intermediate properties between HB and HAmcl, having taken care of the requirements for diverse applications. Some strains are distinguished, however, assays in bottles agitated with the Cupriavidus sp. and Burkholderia sp. strains, using as substratum greasy free acid and soy oil, had led the promising results. In the present work, first, was evaluated the behavior of different strains (Cupriavidus necator DSM545 IPT-026, C. necator H16 IPT-027, Pseudomonas sp. IPT-066, Burkholderia cepacia IPT-048 and B. sacchari IPT-189) how much to metabolizer capacity of free fatty acids and soybean oil for the production of biopolymers and, later, in tank bioreactor some wild strains and recombinant had been tested together or not of an inhibitor of the boxidation (acid acrylic). Experiments in bottles with agitation, had shown values of PHA in about 70% of the total biomass with about 5% monomers chain average allowing to select the Burkholderia cepacia IPT-048 and Cupriavidus necator DSM545 strains for the following assays. Experiments in tank bioreactor had shown that, the interactions between the substratum, co-substratum and the inhibitor of boxidation had influenced in the amount of formed P3HB/3HAmcl when used the Burkholderia cepacia IPT-048 strains, contributing for the synthesis of 3HV and 3HHx. When used only the soybean oil (5g/L) was gotten an amount of HHx with about 6 mol% with Burkholderia cepacia strains. In this same condition was verified that after some determined period also proved monomer HV in an amount of 0.20 mol%. Adding the acid acrylic (0.18 g/L) were gotten 1.50 mol% of HHx, however, differently of the previous condition the amount of HV varied of 0.91 - 2.77 mol%. A conclusion for the condition soybean oil and acrylic acid is that, the strains is using the acrylic fatty acid as carbon source, or either, the formation of acrylil-CoA with consequent formation of chains with 5 carbons. Similar results had been gotten when analyzed extracted polymer, or either, after the stage of extraction with chloroform was verified that the amount varied of 1.60 - 2.14 mol%. When added to a cosubstratum in the phase of accumulation (caproic fatty acid) and absence of the acrylic acid, it did not have the increase of HHx in relation to the previous variable, as much that, the amount of monomer reached about 0.50 mol%. In this condition, the presence of the acrylic fatty acid also did not contribute for the HV synthesis, not having been observed monomers during the assays. In the following phase, in which we work with Cupriavidus necator and a recombinant (Cupriavidus necator::phaB) strains, was verified that the gotten amount of HHx had been similar, about 0.30 mol%. Our hypothesis, we believe that the increase of the number of copies of the gene phaB could contribute for the increase of the carbon flow in the direction to raise the amount of 3HB how much of other monomers. As in the assays in bottles with agitation substrat caproic and caprylic fatty acids they had been distinguished also when availabled 2g/L and the soybean oil of in a concentration of 5g/L, we analyze the strains recombinant with soybean oil and caprylic fatty acid with 5 g/L. The insistence in the use of the caprylic fatty acid was based on the fact of the possibility of strains to oxidate part of fatty acids for the production of cells and energy and, at the moment that had the nitrogen limitation, to begin the accumulation phase dividing two carbons for the production of acetyl-CoA and the remain of the chain was stored in the form of 3HHx. The caprylic fatty acid disponibility it did not have the monomer attainment, therefore, Cupriavidus necator and Cupriavidus necator::phaB had its replyed inhibited capacity by the substratum. In the added soybean oil bottles with and without the acrylic fatty acid the behavior it was the opposite to the observed with caprylic fatty acid. During the incubation period, for both the strains, the amount of PHA, and consequently, the percentage of 3HB in the dry mass cellular was of 22.95% (100 mol%) and 25.90% (100 mol%) in 28ª and 24ª hours, respectively. In this condition, in the recombinant strains, also was observed the presence of HHx between 32ª and 49ª hour with a maxim amount of 1.10 mol%. From the results it can be concluded that the Burkholderia cepacia is considered a promising strains for the attainment of HHx and HV allowing that soybean oil with and without the acrylic fatty acid, respectively. The increase in the number of copies of the gene phaB in Cupriavidus necator apparently did not contribute with the increase of 3HHx efficiency however, other handling variables will be proposed for confirmation.
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