Polyhydroxyalkanoates (PHAs) are polyesters produced and degradable by prokaryotes, while biosurfactants/bioemulsifiers (BS/BE) are metabolites that reduces surface tension and synthesized by this group of organisms. Interest in the potential industrial applications for PHA and BS/BE has increased due to its eco-friendly appeal. In this study, 24 bacterial colonies were isolated from Atlantic forest soil and agro-industrial sludge (Coruripe-AL, Brazil) in minimum mineral medium, as indicative of the possible ability to synthesize PHA. All strains were submitted to biochemical characterization, while the phaC gene amplification showed that isolates BMA-05, BMA-10, BMA-13 and BDL-07 can express the key enzyme for the synthesis of PHA (PHA synthase). Sequencing of the partial 16S r-DNA region was able to identify these bacteria respectively as Pseudomonas fluorescens, Enterobacter aerogenes, Klebsiella oxytocaand Bacillus pumilus. The PHAs produced by each isolate were extracted with hot chloroform and the polymeric films were obtained. Experiments in shaken flasks (0 – 96 h) were conducted to compare the biomass, total reducing glycides, pH, and total protein in minimal medium containing different contents of glucose and peptone. Supplementation with peptone was able to induce the growth of these strains, and cell-free supernatant from the cultivation of Pseudomonas fluorescens BMA-05 showed no acidification in any of the conditions tested. To polyhydroxybutyrate production (evaluated after 24 h incubation), minimal medium was used with different nitrogen sources: ammonium acetate, ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, beef extract, yeast extract, glycine and peptone. The maximum accumulation of P (3HB), detected by UV-visible spectrophotometry, was obtained for P. fluorescens, E. aerogenes, K. oxytoca and B. pumilus grown in ammonium sulfate, ammonium chloride, meat extract and nitrate sodium, respectively. Observations in transmission electron microscope showed Pseudomonas fluorescens BMA-05 with eletronlucent granules when the strain was cultivated in the presence of ammonium sulfate or sodium nitrate. Although no granule was observed for the other strains in the presence of ammonium sulfate, the gas chromatography analysis confirmed the production of P (3HB). When exoenzymes expression tests were conducted, the results indicated that the strains were able to hydrolyse gelatine, starch and Tween 80, and B. pumilus caused hemolysis of sheep blood. Experiments for biosurfactant/bioemulsifier production indicate that E. aerogenes BMA-10 excreted a compound surfactant which collapses the hydrophobic surface and emulsifies the hydrocarbons kerosene, toluene, diesel oil and soybean oil. The emulsification index (E24) using the cell-free supernatant of this microorganism was thermally stable in toluene, but not in kerosene. Similar results were obtained when the pH of this microorganism cell-free supernatant was adjusted to 2, 7 and 10, as well as different concentrations of NaCl (2, 6, and 10 %) were added to the samples. These results reveal the potential of Enterobacter aerogenes BMA-10 as a P(3HB)-producing, and its growth medium free of cells an emulsifier for biotechnological purposes and also in bioremediation. / Polihidroxialcanoatos (PHAs) são poliésteres produzidos e degradados por procariotos, enquanto biosurfactantes/bioemulsificantes (BS/BE) são metabólitos que diminuem a tensão superficial e sintetizados por esse grupo de organismos. O interesse nas potenciais aplicações industriais para PHA e BS/BE tem aumentado devido ao apelo ecologicamente correto desses produtos. No presente estudo, isolaram-se 24 colônias bacterianas de solo de Mata Atlântica e lodo agroindustrial (Coruripe-AL, Brasil) em meio mineral mínimo, como indicativo da possível capacidade de sintetizar PHA. Todas as linhagens foram caracterizadas bioquimicamente, enquanto a amplificação do gene phaC evidenciou que os isolados BMA-05, BMA-10, BMA-13 e BDL-07 podem expressar a enzima-chave para a síntese de PHA (PHA sintase). O sequenciamento da região parcial 16S r-DNA dessas bactérias permitiu suas identificações respectivamente como Pseudomonas fluorescens, Enterobacter aerogenes, Klebsiella oxytoca e Bacillus pumilus. Os PHAs produzidos por cada isolado foram extraídos com clorofórmio quente e os filmes poliméricos foram obtidos. Experimentos em frascos agitados (0 – 96 h) foram conduzidos para comparar a biomassa, glicídios redutores totais, pH e proteínas totais em meio mínimo contendo diferentes conteúdos de glicose e peptona. O suplemento com peptona demonstrou induzir o crescimento das linhagens, e o sobrenadante livre de células proveniente do cultivo de Pseudomonas fluorescens BMA-05 não apresentou acidificação em nenhuma das condições testadas. Na produção de polihidroxibutirato (avaliada após 24 h de incubação), utilizou-se o meio mínimo com diferentes fontes de nitrogênio: acetato de amônio, cloreto de amônio, nitrato de amônio, nitrato de sódio, sulfato de amônio, extrato de carne, extrato de levedura, glicina e peptona. O acúmulo máximo de P(3HB), detectado por espectrofotometria UV-visível, foi obtido quando P. fluorescens, E. aerogenes, K. oxytoca e B. pumilus foram cultivados no sulfato de amônio, cloreto de amônio, extrato de carne e nitrato de sódio, respectivamente. As observações em microscópio eletrônico de transmissão mostraram Pseudomonas fluorescens BMA-05 com grânulos eletronlucentes quando a linhagem foi cultivada na presença de sulfato de amônio ou nitrato de sódio. Embora nenhum grânulo tenha sido observado para as outras linhagens na presença do sulfato de amônio, as análises de cromatografia gasosa confirmaram a produção de P(3HB). Quando os testes de expressão de exoenzimas foram realizados, os resultados indicaram que as linhagens foram capazes de hidrolisar gelatina, amido e Tween 80, e B. pumilus causou hemólise em sangue de carneiro. Experimentos para a produção de biosurfactante/bioemulsificante indicam que E. aerogenes BMA-10 excretou um composto surfactante que colapsa na superfície hidrofóbica bem como emulsiona os hidrocarbonetos querosene, tolueno, óleo diesel e óleo de soja. O índice de emulsificação (E24) usando o sobrenadante livre de células desse microrganismo foi termalmente estável no tolueno, mas não em querosene. Resultados similares foram obtidos quando o pH do sobrenadante livre de células desse microrganismo foi ajustado para 2, 7 e 10, bem como quando concentrações diferentes de NaCl (2, 6 e 10 %) foram adicionadas às amostras. Tais resultados revelam o potencial de Enterobacter aerogenes BMA-10 como produtora de P(3HB), e o meio de cultura livre de suas células para finalidades emulsificantes em biotecnologia ambiental, bem como em biorremediação.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:03Z No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:33Z (GMT) No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) / Made available in DSpace on 2018-06-06T14:01:33Z (GMT). No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) Previous issue date: 2018-04-03 / Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study. / Os problemas ambientais gerados por pl??sticos ?? base de tereftalato de polietileno (PET) se devem ao extenso tempo de decomposi????o desses materiais no meio ambiente e a sua fonte de origem que ?? dependente de petr??leo. Diante disso, biopl??sticos t??m sido uma alternativa promissora devido ao fato de serem biologicamente degrad??veis, al??m de terem como origem mat??rias-primas renov??veis, o que os tornam sustent??veis. Um dos pol??meros mais relevantes desse cen??rio ?? o poli (??cido l??tico) (PLA) formado a partir de mon??meros de ??cido l??tico. As principais caracter??sticas do PLA s??o baixa toxicidade aos humanos devido ?? alta biocompatibilidade, como por exemplo em mat??rias biom??dicos, e biodegradabilidade, o que reduz seu tempo em aterros devido ao processo mais r??pido de decomposi????o. Essas propriedades proporcionam uma ampla aplicabilidade deste pol??mero em diversas ??reas como embalagens, ??reas t??xteis e materiais biom??dicos. Comumente, o processo qu??mico de polimeriza????o do PLA pode ser realizado por meio de duas formas, (1) abertura do anel para posterior polimeriza????o ou (2) por condensa????o dos ??cidos l??ticos. Nos dois casos, ?? necess??ria a presen??a de catalisadores met??licos como zinco, alum??nio e magn??sio. Estes, al??m de serem t??xicos atrapalham na utiliza????o do pol??mero, por exemplo, na ??rea biom??dica, por gerar res??duos met??licos. Uma alternativa a esses catalisadores ?? a utiliza????o de biocatalisadores, como a polihidroxialcanoato sintase (phaC), j?? foi previamente utilizada para polimeriza????o de ??cido l??tico produzido em cepas recombinantes de Escherichia coli. Assim, dentro da plataforma de produ????o de ??cido l??tico, em cepas de Komagataella phaffii recombinantes, o objetivo deste trabalho ?? referente ?? produ????o da enzima phaC com muta????es pontuais nos s??tios S325N e Q481I, pois essas altera????es proporcionam uma maior especificidade do complexo enzima-substrato, para que atue como biocatalisador na polimeriza????o de ??cido l??tico em Komagataella phaffi. Neste estudo, foram realizadas tr??s estrat??gias de clonagem entre o inserto phaCPs e vetor pGAPZ??B. At?? o presente, n??o houve transformantes em nenhuma das estrat??gias. Entretanto, a Estrat??gia C ainda n??o foi executada completamente, o que resulta ainda na possibilidade de clonagem entre inserto phaCPs e vetor de express??o pGAPZ??B com a sequ??ncia correta. A expectativa deste estudo ?? a conclus??o da clonagem, verifica????o da sequ??ncia correta do DNA recombinante atrav??s do resultado do sequenciamento e inser????o do plasm??deo ao genoma da levedura Komagataella phaffii.

Hydrogen, which is a clean energy source, is one of the alternatives for fossil fuels. Biological hydrogen production is one of the hydrogen production methods. Rhodobacter capsulatus is a photosynthetic bacterium that produces hydrogen via photofermentation. R. capsulatus can also synthesize some valuable by-products such as Poly-beta- hydroxy butyric acid (PHB), which is a biodegradable bioplastic. In a two stage biohydrogen production system, which is combination of dark fermentation and photofermentation, dark fermentor effluents are used for photofermentation by R.capsulatus. Dark fermentor effluents usually contain high amount of acetate. High amount of acetate may decrease the efficiency of hydrogen production by causing high amount of PHB production. Therefore, it is significant to determine optimum acetate concentration for photofermentation. In this study, the effects of acetate concentration on hydrogen and PHB production by R.capsulatus were investigated by growing bacteria at various acetate concentrations (10 mM-65 mM). In addition, gene expression analysis was performed to investigate the effects of acetate at transcriptional level. For this purpose, expression levels of the genes that encode nitrogenase which is the enzyme that catalyzes hydrogen production and PHB synthase, which is the key enzyme of the PHB synthesis pathway, are examined. Optimum acetate concentration for photofermentation with high hydrogen yield and low PHB amount was determined to be in the range 25 mM-50 mM. nifD expression was found to be high at optimum acetate concentrations and phaC expression was found to be the highest at 65 mM.

The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.

Objective: The future of public health in Canada depends on the competence of today’s public health students. The Public Health Agency of Canada (PHAC) core competence categories are designed to guide public health practice and the training of public health students. The objectives of this study were to understand public health graduate students’ perception of the PHAC core competencies and report the usability of a practicum placement software in the facilitation of competence-based learning. Methods: Twelve students in the first year of the graduate program in public health participated in two focus group sessions. Participants were asked to select their top and least desired PHAC competencies and then discuss the reasons for their selection. Factors that may have influenced the category selection and their opinion on improving the competence categories were discussed. The system usability scale (SUS) was administered to the student participants and two staff members to help understand the usability of the practicum placement software in the facilitation of competence-based learning. Results: Partnership, collaboration, and advocacy emerged as the top-desired, with public health sciences being the second top-desired. The assessment and analysis category was the least desired, followed by the Leadership competence category. Prior educational background, future career goals with respect to job prospects were among the key factors that influenced the students’ competence selection. Conflict resolution, outreach, and community engagement were some of the suggestions of categories that could be included in the core competence categories. The system usability score for InPlace platform was 61.8 (95% 56.7- 66.9). Conclusions: Overall, students believe that the PHAC core competencies are comprehensive. They suggested seeing certain terminologies become a prominent part of the competence categories. The use of InPlace platform in the facilitation of competence-based learning may require more time for adequate user experience. / Thesis / Master of Public Health (MPH)