Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

The diploma thesis is focused on the study of the mechanism of plasticization of polyhydroxyalkanoates (PHA) in vivo, focusing mainly on methods of isolation native PHA granules and physical and chemical methods of initiation their phase transitions. The literature search prepared in this work deals with this microbial polyester and further focuses on individual methods of isolating PHA from bacterial cells. The main task of the experimental work was to optimize the isolation process of native PHA granules, as well as to design and test processes that initiate the PHA crystallization in isolated granules, as well as analytical procedures to correctly detect this phase transition. Isolation of PHA granules from Cupriavidus necator was performed by enzymes, centrifugation several times and sonication of the cells. Fourier transform infrared spectroscopy (FTIR) and an enzymatic method in which amorphous PHA is selectively degraded by PHA-depolymerase with turbidimetric detection of its loss in dispersion was used to detect PHA crystallization. Techniques based on fluorescence staining of granules (fluorescence spectroscopy and flow cytometry) were used in a more detailed study of the mechanism of PHA plasticization in granules. The results of the analyzes confirmed, among other things, that the optimized procedure makes it possible to isolate granules in the amorphous state, which remain amorphous even after drying and are comparable to native granules in their basic physicochemical properties.

This diploma thesis deals with application of evolutionary engineering on PHA producing bacterial strains. Two bacterial strains, Cupriavidus necator H16 and Halomonas halophila, were chosen for the evolutionary experiments. Copper cations (Cu2+) and sodium chloride (NaCl) were chosen as the selective pressure for C. necator H16; acetic acid (AA) and levulinic acid (LA) for Halomonas halophila. The adapted strains were during long-time evolutionary experiments characterized by GC-FID and SEC-MALS. The growth of the adapted strains was studied by the mean of optical density measurement. The amount of viable cells was determined by spectral FC after their expositon to selected stress factors. Specific enzyme activities of enzymes involved in citrate and glyoxalate cycle, enzymes generating NADPH, LA metabolism enzyme and PHA biosynthesis enzymes were determined. The adapted strains were compared with the wild-type of strains. The successfull adaptation of C. necator H16 adapted to Cu2+ was detected. Biomass and PHA production of both wild and adapted H. halophila strains cultivated in lignocellulosis waste were determined. It was found out that H. halophila adapted to the LA is capable of producing more PHA than the wild strain of this bacteria.

In Cam Pha city (Quang Ninh province) there existes a lot of vegetation having a high degree of degradation. Findings of the research plots located in 4 years (2012-2015) have shown that, vegetation IC has the largest biomass (biomass fresh: 78.70 tons / ha; dry biomass: 36.65 tons / ha), followed by the grass vegetation (fresh biomass: 62.08 tons / ha; dry biomass: 25.67 tons / ha ). Vegetation IA has the lowest biomass (biomass fresh: 33.73 tons / ha; dry biomass: 15.18 tons / ha). The average amount of accumulated carbon in the biomass of vegetation IC is 18.33 tones / ha, in that vegetation IA was 7.60 tones/ ha, in the grass vegetation is 12.84 tones / ha. During the ecological succession, vegetation IC showed an increase in the ability to accumulate carbon (the amount of biomass carbon accumulation of vegetation IC in the 4th year was 21.97 tons / ha). In addition, the paper also proposed the suitable methods of using degraded vegetation in Cam Pha city, Quang Ninh province. / Ở thành phố Cẩm Phả, tỉnh Quảng Ninh tồn tại nhiều thảm thực vật có mức độ thoái hóa cao. Kết quả nghiên cứu trong các ô nghiên cứu định vị trong 4 năm (2012 - 2015) cho thấy, thảm cây bụi IC có sinh khối lớn nhất (sinh khối tươi: 78,70 tấn/ha; sinh khối khô: 36,65 tấn/ha), tiếp đến là thảm cỏ (sinh khối tươi: 62,08 tấn/ha; sinh khối khô: 25,67 tấn/ha). Thảm cây bụi IA có sinh khối thấp nhất (sinh khối tươi: 33,73 tấn/ha; sinh khối khô: 15,18 tấn/ha). Giữa các thảm thực vật này còn khác nhau về cấu trúc sinh khối và tỷ lệ sinh khối khô / sinh khối tươi. Lượng cacbon trung bình được tích lũy trong sinh khối của thảm thực vật cây bụi IC là 18,33 tấn/ha, ở thảm cây bụi IA là 7,60 tấn/ha, ở thảm cỏ là 12,84 tấn/ha. Trong quá trình diễn thế, thảm cây bụi IC có sự tăng lên về khả năng tích lũy cacbon (Lượng cacbon được tích lũy trong sinh khối của thảm cây bụi IC ở năm thứ 4 là 21,97 tấn/ha). Ngoài ra, bài báo còn đề xuất phương thức sử dụng hợp lý các thảm thực vật thoái hóa ở thành phố Cẩm Phả, tỉnh Quảng Ninh.

Made available in DSpace on 2015-04-22T22:02:04Z (GMT). No. of bitstreams: 1 Dissertacao Alcinei Pereira Lopes.pdf: 3280697 bytes, checksum: 535d2d7812f1764295a9b8c14b85a386 (MD5) Previous issue date: 2010-09-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The waters of the Rio Negro, in the border city of Manaus, receives large amounts of domestic and industrial waste from its main tributaries (Tarumã-Acu, Sao Raimundo, Pupils and Puraquequara) that may be compromising their natural features (water, plants, sediments and etc.). Due the ability to accumulate organic and inorganic compounds, the bottom sediments are used to assess the contamination levels of aquatic environments. As a result, we determined the concentrations of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH) and potentially toxic metals (MPT) in ten samples of bottom sediments collected in March 2009 in the confluence between the Rio Negro and major watersheds the city of Manaus. The aim of study was to classify the origin and level of contamination of these compounds. In the determination of hydrocarbons, sediments were freeze dried, extracted with hexane, fractionated by open column liquid chromatography. The determination of aliphatic hydrocarbons was performed by GC-FID and the PAH by GC-MS. After digestion with aqua regia the MPT (Al, Fe, Zn, Cu, Ni, Cr, Pb, Cd and Mn) in total fraction of sediments were determined by ICP-OES. The results showed that concentrations of total aliphatic hydrocarbons were 13.4 to 448 mg g-1, and the sum of 38 PAHs was 58.8 to 6832 ng g-1. These values are considered high for natural aquatic environments that have not suffered oil spill accidents. Among the metals determined Al (32 091 mg kg-1) and Fe (35 474 mg kg-1) were those with the highest concentrations.The sediments that received the largest anthropogenic contribution were those collected at the entrance of the basin of the streams São Raimundo and Educandos. The diagnosis index hydrocarbons the origin of in sediments showed a predominance of mixed sources and petrogenic sources in most seasons / As águas do rio Negro na orla urbana de Manaus recebem grande quantidade de resíduos domésticos e industriais de seus principais afluentes (Tarumã-Açu, São Raimundo, Educandos e Puraquequara) que podem estar comprometendo suas características naturais (água, plantas, sedimentos e etc). Por ter a capacidade de acumular compostos orgânicos e inorgânicos, os sedimentos de fundo são utilizados na avaliação dos níveis de contaminação de ambientes aquáticos. Em virtude disso, foram determinados as concentrações de hidrocarbonetos alifáticos, hidrocarbonetos policíclicos aromáticos (HPA) e metais em dez amostras de sedimentos de fundo, coletadas em março de 2009 nas confluências entre o rio Negro e as principais bacias de drenagem da cidade de Manaus. O estudo tem o objetivo de classificar a origem e o nível de contaminação destes compostos. Nas determinações de hidrocarbonetos, os sedimentos foram liofilizados, extraídos em soxhlet, fracionados por cromatografia líquida de coluna aberta. A determinação de hidrocarbonetos alifáticos foi realizada por CG-DIC e os HPA por CG-EM. Apos digestão com água régia, os metais (Al, Fe, Zn, Cu, Ni, Cr, Pb, V, Cd e Mn) presentes na fração total dos sedimentos foram determinados por ICP-OES. Os resultados mostraram que as concentrações dos hidrocarbonetos alifáticos totais foram de 13,4 a 448 μg g-1, e a somatória dos 38 HPA foi de 58,8 a 6832 ng g-1. Esses valores são considerados elevados para ambientes aquáticos naturais que não sofreram acidentes de derrame de óleo. Entre os metais determinados o Al (32091 mg kg-1) e o Fe (35474 mg kg-1) foram os que apresentaram as maiores concentrações. Os sedimentos que receberam a maior contribuição antrópica foram daqueles coletados na entrada da bacia dos igarapés São Raimundo e Educando. Os índices de diagnósticos da origem de ix hidrocarbonetos nos sedimentos mostraram predominância de fontes petrogênica e fontes mistas na maioria das estações.

Identifying hazards is fundamental for ensuring the safe design and operation of a system in process plants and other facilities. Several techniques are available to identify hazardous situations, all of which require their rigorous, thorough, and systematic application by a multi-disciplinary team of experts. Success rests upon first identifying and subsequently analyzing possible scenarios that can cause accidents with different degrees of severity. While hazard identification may be the most important stage for risk management, it depends on subjectivity issues (e.g., human observation, good judgment and intuition, creativity, expertise, knowledge) which introduce bias. Without a structured identification system, hazards can be overlooked, thus entailing incomplete risk-evaluations and potential loss. The present Thesis is focused on developing both managerial and technical aspects intended to standardize one of the most used techniques for hazard identification; viz. HAZard & Operability (HAZOP) study. These criteria have been carefully implemented not only to ensure that most of the hazardous scenarios will be identified, but also that US OSHA PSM Rule, EPA RMP, and Seveso Directive requirements will be accomplished. Chapter I pioneers the main research topic; from introducing the process safety concept up to the evidence of more detailed information is required from related regulations. A review of regulations (i.e., US, Europe legislation) focused on Hazard Identification has been conducted, highlighting, there is an absence of specific criteria for performing techniques intended to identify what can go wrong. Chapter II introduces the risk management system required to analyze the risk from chemical process facilities, and justifies that hazard identification stage is the Process Safety foundation. Hereafter, an overview of the key Process Hazard Analyzes (PHA) has been conducted, and the specific HAZOP weaknesses and strengths have been highlighted to establish the first steps to focus on. Chapter III establishes the scope, the purpose and the specific objectives that the research covers. It answers the following questions on the spot: why the present research is performed, which elements are included, and what has been considered for acquiring the final conclusions of the manuscript. Chapter IV gathers HAZOP-related literature from books, guidelines, standards, major journals, and conference proceedings with the purpose of classifying the research conducted over the years and finally define the HAZOP state-of-the-art. Additionally, and according to the information collected, the current HAZOP limitations have been emphasized, and thus, the research needs that should be considered for the HAZOP improvement and advance. Chapter V analyzes the data collected while preparing, organizing, executing and writing HAZOPs in five petroleum-refining processes. A statistical analysis has been performed to extract guidance and conclusions to support the established criteria to conduct effectively HAZOP studies. Chapter VI establishes the whole set of actions that have to be taken into account for ensuring a wellplanned and executed HAZOP study. Both technical and management issues are addressed, criteria supported after considering the previous chapters of the manuscript. Chapter VI itself is the result of the present research, and could be used as a guideline not only for team leaders, but also for any related party interested on performing HAZOPs in continuous chemical processes. Chapter VII states the final conclusions of the research. The interested parties should be released about the hazard identification related-gaps present in current process safety regulations; which are the key limitations of the HAZOP study, and finally, which are the criteria to cover the research needs that have been found Annex I proposes the key tools (tables, figures and checklists "ready-to use'') to be used for conducting HAZOPs in continuous chemical processes. The information layout is structured according to the proposed HAZOP Management System. This information is intended to provide concise and structured documentation to be used as a reference book when conducting HAZOPs. Annex II is intended to overview the most relevant petroleum refining processes by highlighting key factors to take into account in the point of view of process safety and hazard identification, i.e. HAZOP. In this sense, key health and safety information of specific petroleum refining units is provided as a valuable guidance during brainstorming sessions. Annex III illustrates the complete set of data collected during the field work of the present research, and also analyzed in Chapter V of the manuscript. Additionally, it depicts a statistical summary of the key variables treated during the analysis. Finally, the Nomenclature, References, and Abbreviations & Acronyms used and cited during the manuscript have been listed. Additionally, a Glossary of key terms related to the Process Safety field has been illustrated. / La present Tesis doctoral té com a objectiu estandarditzar l'aplicació d'una de les tècniques més utilitzades a la industria de procés per a la identificació de perills; l'anomenat HAZard & OPerability (HAZOP) study, específicament a processos complexes, com per exemple, unitat de refineria del petroli.El capítol I defineix el concepte de Seguretat de Processos, i progressivament analitza les diferents regulacions relacionades amb la temàtica, detallant específicament les mancances i buits d'informació que actualment hi ha presents a la primera etapa de la gestió del risc en industries de procés: la identificació de perills.El capítol II defineix el sistema de gestió del risc tecnològic que aplica a les industries de procés, i es justifica que l'etapa d'identificació de perills és el pilar de tot el sistema. Finalment, es mencionen algunes de les tècniques d'identificació més utilitzades, els anomenats Process Hazard Analysis (PHA), i es detallen les seves mancances i fortaleses, característiques que han acabat definint la temàtica específica de la Tesis. Concretament, es dóna èmfasis a la tècnica anomenada HAZard & OPerability (HAZOP) study, objecte principal de la recerca.El capítol III defineix l'abast, el propòsit i els objectius específics de la recerca. La intenció d'aquest capítol és donar resposta a les següents qüestions: el perquè de la recerca, quins elements han estat inclosos i què s'ha considerat per tal d'assolir les conclusions de la Tesis.El capítol IV descriu l'estat de l'art de la literatura relacionada amb el HAZOP. Aquesta revisió no només permet classificar les diferents línies de recerca relacionades amb el HAZOP, sinó que també permet assolir un coneixement profund de les diferents particularitats de la pròpia tècnica. El capítol finalitza amb un conjunt de mancances tant de gestió com tècniques, així com les necessitats de recerca que poden millorar l'organització i execució dels HAZOPs.El capítol V analitza la informació que ha estat recopilada durant la fase experimental de la tesis. Les dades procedeixen de la participació en cinc estudis HAZOP aplicats a la industria de refineria del petroli.En aquest sentit, el capítol V desenvolupa una anàlisi estadística d'aquestes dades per extreure'n conclusions quant a la preparació, organització i execució dels HAZOPs.El capítol VI estableix el conjunt d'accions que s'ha de tenir en compte per tal d'assegurar que un estudi HAZOP estigui ben organitzat i executat (la metodologia). Es defineix un Sistema de Gestió del HAZOP, i a partir de les seves fases, es desenvolupa una metodologia que pretén donar suport a tots aquells punts febles que han estat identificats en els capítols anteriors. Aquesta metodologia té la intenció de donar suport i guia no només als líders del HAZOP, sinó també a qualsevol part interessada en aquesta temàtica.El capítol VII descriu les conclusions de la recerca. En primera instància s'enumeren les mancances quant a la definició de criteris a seguir de diferents regulacions que apliquen a la Seguretat de Processos.Seguidament, es mencionen les limitacions de la pròpia tècnica HAZOP, i finalment, es descriuen quins són els criteris establerts per donar solució a totes aquestes febleses que han estat identificades.L'Annex I és una recopilació de diferents criteris que han estat desenvolupats al llarg de l'escrit en forma de taules i figures. Aquestes han estat ordenades cronològicament d'acord amb les diferents fases que defineixen el Sistema de Gestió HAZOP. L'annex I es pot utilitzar com a una referència concisa i pràctica, preparada i pensada per ésser utilitzada directament a camp, amb la intenció de donar suport a les parts interessades en liderar estudis HAZOP.L'annex II recopila informació relacionada amb aspectes clau de seguretat i medi ambient en diferents unitats de refineria. Aquest informació és un suport per tal de motivar el "brainstorming" dels diferents membres que conformen l'equip HAZOP.L'Annex III recopila les dades de les diferents variables que han estat considerades a la fase experimental de la recerca, juntament amb un conjunt de figures que mostren la seva estadística bàsica.

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