Polyhydroxyalkanoate (PHA) is a cellular storage polymer with similar properties to some plastics when extracted from the cell and acts as a renewable and biodegradable plastic alternative. The objective of this research was to identify thermophilic PHA producing microorganisms and characterize their PHA accumulation, by which the process may become more competitive to current petroleum-based plastic production. This study characterizes growth and PHA production of Thermus thermophilus HB8 and three strains of T. filiformis (MOK, NGM and WRT), while simultaneously performing a genetic analysis for the presence of a phaC gene that encodes the PHA synthase. The results from the genetic analyses, together with attempts to produce PHA in T. thermophilus HB8 and T. filiformis strain (WRT, NGM and MOK) infer an absence of PHA accumulation. Contrary to our expectations, Thermus sp. is not a strong candidate for the thermophilic production of PHA.

Polyhydroxyalkanoate (PHA) is a cellular storage polymer with similar properties to some plastics when extracted from the cell and acts as a renewable and biodegradable plastic alternative. The objective of this research was to identify thermophilic PHA producing microorganisms and characterize their PHA accumulation, by which the process may become more competitive to current petroleum-based plastic production. This study characterizes growth and PHA production of Thermus thermophilus HB8 and three strains of T. filiformis (MOK, NGM and WRT), while simultaneously performing a genetic analysis for the presence of a phaC gene that encodes the PHA synthase. The results from the genetic analyses, together with attempts to produce PHA in T. thermophilus HB8 and T. filiformis strain (WRT, NGM and MOK) infer an absence of PHA accumulation. Contrary to our expectations, Thermus sp. is not a strong candidate for the thermophilic production of PHA.

A PHA producing bacterium (strain QN271) was selected from mangrove soil samples collected from Quang Ninh province by using the Nile red dying technique. PHA accumulation in the selected bacterium strain was confirmed by transmission electron microscope. With the exception of maltose or sucrose, the bacterium strain was found to be able to synthesize PHA from various carbon sources (glucose, xylose, fructose, glycerol, and glucose plus propionate). The strain accumulated poly(3-hydroxybutyrate) from glucose, fructose, xylose, and glycerol whereas poly(3-hydroxybutyrate-co-3-hydroxyvalarate) was produced when a combination of glucose and propionate was included in the culture medium. Fructose was found to be most suitable substrate for PHA synthesis by strain QN271. PHA content of 63.3% and CDW of 6 g/L were obtained after 32 hrs of cultivation in fructose medium. / Chủng vi khuẩn có khả năng sinh tổng hợp PHA đã được phân lập từ đất rừng ngập mặn tỉnh Quảng Ninh nhờ kỹ thuật nhuộm với Nile red. Ảnh quan sát dưới kính hiển vi điện tử dẫn truyền chứng tỏ rằng chủng vi khuẩn này có khả năng tích lũy lượng lớn PHA trong tế bào. Chủng vi khuẩn tuyển chọn có khả năng sinh tổng hợp PHA từ nhiều nguồn các bon khác nhau như glucose, xylose, fructose, glucerol, glucose và propionate nhưng không có khả năng tổng hợp PHA từ maltose hoặc saccharose. Chủng vi khuẩn tuyển chọn tổng hợp poly (3-hydroxybutyrate) từ các nguồn các-bon như glucose, xylose, fructose, hay glycerol, trong khi đó poly (3-hydroxybutyrateco- 3-hydroxyvalarate) sẽ được tổng hợp khi phối hợp sử dụng hai nguồn các-bon (glucose và propionate). Fructose là nguồn các-bon tốt nhất cho chủng QN271 sinh tổng hợp PHA, khi nuôi cấy trong môi trường có fructose chủng vi khuẩn này có thể tạo ra lượng sinh khối là 6 g/L trong đó có chứa 63.3% PHA sau 32 giờ.

Carbon dioxide is a viable resource, if used as a raw material for bioprocessing. It is abundant and can be collected as a byproduct from industrial processes. Globally, photosynthetic organisms utilize around 6’000 TW (terawatt) of solar energy to fix ca. 800 Gt (gigaton) of CO2 in the planets largest carbon-capture process. Photosynthesis combines light harvesting, charge separation, catalytic water splitting, generation of reduction equivalents (NADH), energy (ATP) production and CO2 fixation into one highly interconnected and regulated process. While this simplicity makes photosynthetic production of commodity interesting, yet photosynthesis suffers from low energy efficiency, which translates in an extensive footprint for solar biofuels production conditions that store < 2% of solar energy. Electron transfer processes form the core of photosynthesis. At moderate light intensity, the electron transport chains reach maximum transfer rates and only work when photons are at appropriate wavelengths, rendering the process susceptible to oxidative damage, which leads to photo-inhibition and loss of efficiency. Based on our fundamental analysis of the specialized tasks in photosynthesis, we aimed to optimize the efficiency of these processes separately, then combine them in an artificial photosynthesis (AP) process that surpasses the low efficiency of natural photosynthesis. Therefore, by combining photovoltaic light harvesting with electrolytic water splitting or CO2 reduction in combination with microbiological conversion of electrochemical products to higher valuable compounds, we developed an electro-microbial carbon capture and conversion setups that capture CO2 into the targeted bioplastic; polyhydroxybutyrate (PHB). Based on the type of the electrochemical products, and the microorganism that either (i) convert products formed by electrochemical reduction of CO2, e.g. formate (using inorganic cathodes), or (ii) use electrochemically produced H2 to reduce CO2 into higher compounds (autotrophy), three AP setups were designed: one-pot, two-pot, and three-pot setups. We evaluated the kinetic (microbial uptake and conversion, electrochemical reduction) and thermodynamics (efficiencies) of the separate processes, and the overall process efficiency of AP compared to photosynthesis. We address the influence of several parameters on efficiencies and time-space yields, e.g. salinity, pH, electrodes, media, partial pressures of H2 and CO2. These data provide a valuable basis to establish a highly efficient and continuous AP process in the future.

Polyhydroxyalkanoates (PHAs) are a type of biologically-produced plastic known for their biocompatibility and biodegradability. They have the potential to replace petroleum-based plastics as an environmentally-friendly alternative. This is beneficial because the release of plastics into environments such as the ocean and the buildup of plastics in landfills are major concerns facing society today. Currently, however, PHAs are significantly more expensive than their petroleum-based counterparts. This is largely due to the cost of carbon sources and of extracting the bioplastics from bacteria. The goal of these studies was to examine replacing traditional carbon sources used in PHA production like sugar and oils with sustainable carbon sources and to improve extraction procedures by inducing secretion of PHAs in bacteria. A few sustainable carbon sources were examined for use in PHA production. First, studies focused on the conversion of food waste into PHAs were reviewed. It was shown that utilizing food wastes as carbon sources may be a viable approach to producing PHAs. A second carbon source examined was methanol. A novel isolate of Methylobacterium that demonstrated the ability to produce PHAs from methanol was identified. A system of secreting PHAs that was constructed using synthetic biological engineering approach was introduced to this isolate. This secretion system was not shown to improve extraction of PHAs in Methylobacterium in its current form.

PHAs are a class of biopolymers consisting of (R)-3-hydroxy-fatty acids and are produced by the majority of eubacteria and some archaeal bacteria as carbon storage material. In general, PHA is synthesised when a carbon source is available in excess while another essential nutrient is limited. The key enzyme of PHA biosynthesis, the PHA synthase, catalyses the polymerisation of the substrate (R)-3-hydroxyacyl-CoA to PHA accompanied by the release of coenzyme A. PHA is stored intracellularly as inclusions, the so-called PHA granules. When the external carbon source becomes exhausted, bacteria can metabolise these carbon inclusions by degradation of the polymer. PHA granules are water-insoluble, spherical inclusions of approximately 50-500 nm in diameter which consist of a hydrophobic polyester core surrounded by a phospholipid layer with embedded and attached proteins. One could consider isolated PHA granules as bio-beads due to their structure and size. In this study we tested if the PHA synthase can be used as an anchor molecule in order to display proteins of interest at the PHA granule surface. Furthermore, these modified PHA granules were analysed for their potential applicability as bio-beads in biotechnological procedures. The concept of using the PHA synthase as granule-anchoring molecule for display of proteins of interest was established by the functional display of the ß- galactosidase at PHA granules. This “proof of concept” was followed by the display of biotechnologically more interesting proteins. The IgG binding domain of protein A as well as streptavidin, which is known for its biotin binding ability, were fused to the PHA synthase, respectively, and therefore localised at the PHA granule surfaces during PHA granule assembly, resulting in functional bio-protein A -beads and bio-streptavidin-beads. Moreover, their applicability in biotechnological assays was demonstrated. Recently, we fused the green fluorescent protein (GFP) to the PHA synthase and demonstrated that the PHA granule assembly does not start randomly distributed in the cytoplasm but occurred localised at or near the cell poles. To further investigate if the localisation of the PHA granule formation process is due to polar positional information inherent to the PHA synthase, different mutated versions of the PHA synthase of Cupriavidus necator were created and analysed for a potential alteration in localisation. Furthermore, the phasin protein PhaP1 of C. necator was fused to HcRed, a far-red fluorescent protein, and localisation studies were accomplished when the fusion protein was expressed under different conditions in Escherichia coli.

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-01-30Bitstream added on 2014-06-13T18:29:32Z : No. of bitstreams: 1 bortolossi_jc_me_jabo_prot.pdf: 1992127 bytes, checksum: 4589472b07077a15576f670c58a3f88c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O cancro cítrico doença causada pela bactéria gram negativa Xanthomonas axonopodis pv. citri (Xac), representa muitos prejuízos para citricultura brasileira e mundial. Em 2002, a Xac teve todo seu genoma seqüenciado. Uma biblioteca de mütantes foi constituída e os mutantes foram analisados por inoculação in planta, deste modo tres mutantes foram selecionados por apresentarem diferenças nos sintomas, quando comparados com a estirpe selvagem Xac. Os genes nocauteados nos mutantes codificam três proteínas diferentes: DGTP pirofosfato tiamina sintase, envolvida na biossíntese de vitamina B 1, a subunidade E da enzima PHA sintase, que atua na síntese de polihidroxialcanoatos. que acumulados na forma de grânulos intracelulares constituem substâncias de reserva da célula, e podem formar biopolímeros e, ainda o terceiro gene nocauteado, que codifica uma proteína hipotética. As curvas de crescimento revelaram que o mutante da DGTP pirofosfohidrolase tiamina sintase não se desenvolve na planta hospedeira. O mutante da PHA sintase cresce menos que a Xac selvagem nos dois primeiros dias de avaliação, já o mutante da proteína hipotética sofre uma fase de declínio a partir do oitavo dia de avaliação experimental. A subunidade E da PHA sintase da Xac, gene codificador desta proteína foi donado em vetor de expressão e a proteína recombinante foi produzida em E.coli na forma solúvel. / The citric canker disease caused for the bacterium gram negative Xantlzomonas axonopodis pv. citri (Xac), presents many damages for Brazilian and world-wide citriculture. In 2002, aiming to know the mechanisms of attack of the disease, the Xac had alI its sequenced genome. A library of mutants was constituted and the mutants had been analyzed by inoculation in plant, three mutants had been isolated for not to show differences in the symptoms when compared with the wild lineage Xac. The mutants codify three different proteins: DGTP pyrophosphate tiamine synthase involved in biosynthesis B 1 vitamin; enzyme PHA synthase subunit E that is involved in the synthesis of polyhydroxyalkanoate, in the intracellular granule f01111, they constitute substances of reserve of the cell an can still to form biopolymer; and hypothetical proteins. The profile of growth of the wild Xac and the mutated Xacs in the three cases of the mutants had been evaluated in order to compare the profile of growth of the bacterial wild and mutant during the infection in citric host. The growth curves showed that the mutant of the DGTP pyrophosphate tiamine synthase does not develop the disease in the host plant. The PHA synthase grows less than the wild Xac in the first days of evaluaction, however the mutant of the hypotetical, shown a decline period for eighth day of the experimental evaluaction. The subunit E the PHA synthase of the Xac, the code gene of this protein was cloned in expression vector and the recombinant protein was produced in E.coli in the soluble form.

Resumo: O cancro cítrico doença causada pela bactéria gram negativa Xanthomonas axonopodis pv. citri (Xac), representa muitos prejuízos para citricultura brasileira e mundial. Em 2002, a Xac teve todo seu genoma seqüenciado. Uma biblioteca de mütantes foi constituída e os mutantes foram analisados por inoculação in planta, deste modo tres mutantes foram selecionados por apresentarem diferenças nos sintomas, quando comparados com a estirpe selvagem Xac. Os genes nocauteados nos mutantes codificam três proteínas diferentes: DGTP pirofosfato tiamina sintase, envolvida na biossíntese de vitamina B 1, a subunidade E da enzima PHA sintase, que atua na síntese de polihidroxialcanoatos. que acumulados na forma de grânulos intracelulares constituem substâncias de reserva da célula, e podem formar biopolímeros e, ainda o terceiro gene nocauteado, que codifica uma proteína hipotética. As curvas de crescimento revelaram que o mutante da DGTP pirofosfohidrolase tiamina sintase não se desenvolve na planta hospedeira. O mutante da PHA sintase cresce menos que a Xac selvagem nos dois primeiros dias de avaliação, já o mutante da proteína hipotética sofre uma fase de declínio a partir do oitavo dia de avaliação experimental. A subunidade E da PHA sintase da Xac, gene codificador desta proteína foi donado em vetor de expressão e a proteína recombinante foi produzida em E.coli na forma solúvel. / Abstract: The citric canker disease caused for the bacterium gram negative Xantlzomonas axonopodis pv. citri (Xac), presents many damages for Brazilian and world-wide citriculture. In 2002, aiming to know the mechanisms of attack of the disease, the Xac had alI its sequenced genome. A library of mutants was constituted and the mutants had been analyzed by inoculation in plant, three mutants had been isolated for not to show differences in the symptoms when compared with the wild lineage Xac. The mutants codify three different proteins: DGTP pyrophosphate tiamine synthase involved in biosynthesis B 1 vitamin; enzyme PHA synthase subunit E that is involved in the synthesis of polyhydroxyalkanoate, in the intracellular granule f01111, they constitute substances of reserve of the cell an can still to form biopolymer; and hypothetical proteins. The profile of growth of the wild Xac and the mutated Xacs in the three cases of the mutants had been evaluated in order to compare the profile of growth of the bacterial wild and mutant during the infection in citric host. The growth curves showed that the mutant of the DGTP pyrophosphate tiamine synthase does not develop the disease in the host plant. The PHA synthase grows less than the wild Xac in the first days of evaluaction, however the mutant of the hypotetical, shown a decline period for eighth day of the experimental evaluaction. The subunit E the PHA synthase of the Xac, the code gene of this protein was cloned in expression vector and the recombinant protein was produced in E.coli in the soluble form. / Orientador: Julio Cezar Franco de Oliveira / Coorientadora: Maria Ines Tiraboschi Ferro / Banca: João Martins Pizauro Junior / Banca: Roberto Willians Noda / Mestre

The aim of this work is to study microbial production of polyhydroxyalkanoates (PHA). Theoretical part is focused on production of PHA using microoganisms and transgenic plants. Bacterial strain Cupriavidus necator H16 was used for laboratory production of PHA. Various waste oils were used as sole carbon and energy source. Salt of propionic adic and 1-propanol were used as intermediate for 3-hydroxyvalerate monomer (3HV) unit. Incorporation of 3HV to polymer can improve material features of PHA. The major part of experimental work was focused to study influence of aeration (concentration of dissolved oxygen) to bacterial growth, selected metabolic pathways and formation of PHA. Furthermore, influence of aeration to monomer composition of polymer was evaluated. According to experimental conclusion of this work it was approved dependance between aeration and monomer composition of PHA. Moreover, it was approved that higher concentration of oxygen supports bacterial growth and influences PHA content in cells. In addition, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH inhibits TCA cycle and enhances accumulation of PHA in cells. For this reason, specific enzymatic aktivity of several selected intracelular enzymes were measured, including those enzymes which can generate NADPH.

A PHA producing bacterium (strain QN271) was selected from mangrove soil samples collected from Quang Ninh province by using the Nile red dying technique. PHA accumulation in the selected bacterium strain was confirmed by transmission electron microscope. With the exception of maltose or sucrose, the bacterium strain was found to be able to synthesize PHA from various carbon sources (glucose, xylose, fructose, glycerol, and glucose plus propionate). The strain accumulated poly(3-hydroxybutyrate) from glucose, fructose, xylose, and glycerol whereas poly(3-hydroxybutyrate-co-3-hydroxyvalarate) was produced when a combination of glucose and propionate was included in the culture medium. Fructose was found to be most suitable substrate for PHA synthesis by strain QN271. PHA content of 63.3% and CDW of 6 g/L were obtained after 32 hrs of cultivation in fructose medium. / Chủng vi khuẩn có khả năng sinh tổng hợp PHA đã được phân lập từ đất rừng ngập mặn tỉnh Quảng Ninh nhờ kỹ thuật nhuộm với Nile red. Ảnh quan sát dưới kính hiển vi điện tử dẫn truyền chứng tỏ rằng chủng vi khuẩn này có khả năng tích lũy lượng lớn PHA trong tế bào. Chủng vi khuẩn tuyển chọn có khả năng sinh tổng hợp PHA từ nhiều nguồn các bon khác nhau như glucose, xylose, fructose, glucerol, glucose và propionate nhưng không có khả năng tổng hợp PHA từ maltose hoặc saccharose. Chủng vi khuẩn tuyển chọn tổng hợp poly (3-hydroxybutyrate) từ các nguồn các-bon như glucose, xylose, fructose, hay glycerol, trong khi đó poly (3-hydroxybutyrateco- 3-hydroxyvalarate) sẽ được tổng hợp khi phối hợp sử dụng hai nguồn các-bon (glucose và propionate). Fructose là nguồn các-bon tốt nhất cho chủng QN271 sinh tổng hợp PHA, khi nuôi cấy trong môi trường có fructose chủng vi khuẩn này có thể tạo ra lượng sinh khối là 6 g/L trong đó có chứa 63.3% PHA sau 32 giờ.