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Isolamento e caracterização de haloarqueas cultivadas em compostos aromaticos e construção de ferramentas moleculares para o estudo da secreção proteica no Dominio Archaea / Isolation and characterization of haloarqueas grown in aromatics and construction of molecular tools to study the protein secretion in the Domain ArchaeaCuadros Orellana, Sara 25 November 2003 (has links)
Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T18:02:39Z (GMT). No. of bitstreams: 1
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Previous issue date: 2003 / Resumo: O metabolismo de compostos aromáticos depende de fatores físico-químicos como temperatura, pH e salinidade, e é bem conhecido e caracterizado em microrganismos mesófilos e em condições ambientais moderadas. No entanto, pouco se conhece sobre o metabolismo desses compostos em ambientes hipersalinos. este trabalho, cinco ambientes hipersalinos foram analisados quanto à presença de arqueas halofílicas capazes de metabolizar compostos aromáticos: Salar de Uyuni (Bolívia), salinas de Cahuil (Chile), salinas de Cabo Rojo (Porto Rico), sabkhas da região do Golfo Pérsico (Arábia Saudita) e Mar Morto (Israel e Jordânia). A estratégia empregada para o enriquecimento e isolamento de arqueas halofílicas capazes de crescer em presença de compostos aromáticos foi bem sucedida. Foram isoladas 12 linhagens capazes de crescer em presença de 1 ,2--benzoantraceno (2 mM) e 44 linhagens capazes de crescer em presença de ácido p-hidroxibenzóico (10 mM) como única fonte de carbono e energia. O isolado MM 17, proveniente de uma amostra de água do Mar Morto, apresentou o melhor resultado de crescimento e foi capaz de degradar completamente os ácidos benzóico (10 mM) e p-hidroxibenzóico (10 mM) após 200 h de cultivo. As análises bioquímica e genética dos isolados, juntamente com a análise dos perfis de lipídeos polares, indicaram que as linhagens estudadas pertencem a pelo menos dois gêneros: Haloferax e Halobacterium. Foi determinada a secreção de uma proteína de alto peso molecular por Haloferax volcanii em resposta à presença de 2 mM 1 ,2-benzoantraceno no meio de cultivo. Com o objetivo de estudar o mecanismo de secreção protéica em haloarqueas, o qual não é completamente entendido_ foram realizadas a clonagem molecular dos genes secD e secY de Haloferax volcanii, a expressão heteróloga em Escherichia coli e a purificação dos produtos gênicos, além de uma tentativa preliminar de obtenção de anticorpos policlonais através da imunização de coelhos / Abstract: The metabolism of aromatic compounds depends on physical-chemical factors such as temperature, pH and salinity, and is well known and characterized in mesophilic microorganisms under mild environmental conditions. Little is known, however, about the metabolism of these compounds in highly saline environments. Here, five hypersaline sites were tested for the presence of halophilic archaea able to metabolize aromatic compounds: the Uyuni Salt Marsh (Bolivia), the crystallizer ponds in Cahuil (Chile), the crystallizer ponds in Cabo Rojo (Puerto Rico), the sabkhas in the Persian Gulf (Saudi Arabia) and the Dead Sea (Israel and Jordan). The strategy used for the enrichment and isolation of halophilic archaea able to grow in aromatic compounds was successful. Twelve strains able to grow in 1,2-benzoantracene (2 mM) and 44 strains able to grow in p-hydroxybenzoic acid (10 mM) as the sole carbon and energy source were isolated. Strain MM17, isolated from a Dead Sea water sample, showed the best growth and was able to degrade benzoic (10 mM) and p-hydroxybenzoic (10 mM) acids afier 200 h of cultivation. Biochemical and genetic analyses of the isolates, together with the analysis of polar lipid profiles, indicate that the strains belong to at least two different genera: Haloferax and Halobacterium. The secretion of a high molecular weight protein by Haloferax volcanii following cultivation in 2 mM 1,2-benzoantracene was observed. To study the mechanism of protein secretion in halophilic archaea, a process that is not completely understood, preliminary studies were conducted, which included cloning of secO and secY genes of Haloferax volcanii, their heterologous expression in Escherichia coli and the purification of the gene products. In addition, a preliminary attempt to obtain polyclonal antibodies through rabbit immunization was made / Doutorado / Doutor em Ciência de Alimentos
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Biological approach to improving the evaporation rates of mine wastewater desalination brine treated in evaporation pondsMoyo, Anesu Conrad January 2021 (has links)
Philosophiae Doctor - PhD / The disposal of brine effluent from inland wastewater desalination plants is a
growing global problem with adverse economic and environmental implications
because of the substantial cost associated with its disposal and the potential for
polluting groundwater resources. Currently, the best and most economical option
for brine disposal from inland desalination plants is the use of evaporation ponds,
which concentrate the liquid until getting a solid waste that can be valued or
directly managed by an authorized company. The effectiveness of these ponds is
therefore dependent on the evaporation rate, which has previously been improved
by the addition of dyes such as methylene blue. However, the addition of chemical
dyes to the evaporation ponds poses a threat to the environment, wildlife, and
humans.
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Electricity generation and ethanol production using iron-reducing, haloalkaliphilic bacteriaPaul, Varun, January 2009 (has links) (PDF)
Thesis (M.S.)--Missouri University of Science and Technology, 2009. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed August 10, 2009) Includes bibliographical references (p. 58-64).
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NMR structure and relaxation studies of DHFR from Haloferax volcanii at high saltBinbuga, Bulent, January 2007 (has links)
Thesis (Ph.D.) Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.
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Improving Evaporation Rate of Mine WastewaterKhumalo, Londiwe Thandeka Precious January 2018 (has links)
Magister Scientiae - MSc (Biotechnology) / The treatment of mine water at the eMalahleni Water Reclamation Plant (EWRP) results in the
production of large volumes of brine. Different brine management methods have been applied
to dispose the brine but the evaporation pond method is regarded as the cheaper, most effective
and less laborious method for brine disposal. Brine wastewater is pumped into the pond where
it evaporates resulting in the mixture of salts. The rate at which evaporation occurs is influenced
by many factors such as temperature, salinity, humidity and wind. Due to high salinities in
brine the EWRP is currently experiencing a challenge with low evaporation rate. Here, a
comparative study was done to determine the efficiency of using a chemical and a biological
approach to enhance the evaporation rate of reject brine. The chemical approach involved the
addition of various concentrations of methylene blue dye (100 to 300 ppm with 50 ppm
increments) to 1L volumes of brine, and measuring the evaporation rate. On the other hand, the
biological approach involved the isolation of pigmented halophilic bacteria from eMalahleni
brine and Cerebos salt samples. Isolated bacterial strains were characterised based on their
morphology, biochemical and salt tolerance characteristics. Furthermore, the strains were
identified using 16S rRNA gene sequence analysis. Among the isolated halophilic bacterial
strains, EP-3, an Arthobacter agilis isolated from the eMalahleni brine produced a darker
pigment compared to the other strains. Therefore, EP-3 was evaluated for its effect on the
evaporation of brine using a culture inoculum or the addition pigment extracted from an EP-3
culture. The addition of MB above 100 ppm overcame the effect of salt precipitation and
resulted in higher evaporation (41%) rate. Addition of pigmented bacteria or bacterial extracted
pigment to the brine respectively resulted in 18% and 24% increase in the evaporation rate.
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Aplicação de Bacillus cereus e Enterococcus faecalis, isolados de bacalhau seco salgado (Gadus sp), no tratamento de resíduos sólidos agroindustriais salinos por compostagem controlada / Aplication of Bacillus cereus and Enterococcus faecalis. strains isolated from salted dry cod (Gadus sp.) in the treatment of saline agrodindustrial solid waste by controlled microcompostingAraujo, Thayli Ramires 21 May 2018 (has links)
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Previous issue date: 2018-05-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / A atividade das agroindústrias tem causado impactos ao meio ambiente, dentre eles a contaminação de solo, águas subterrâneas e superficiais, atmosfera. Na indústria de alimentos o sal é utilizado em diversas etapas de processamento de alimentos, e também é considerado o ingrediente mais consumido pelos humanos. Na indústria pesqueira marítima o sal está presente desde a descarga de material e o efluente gerado quando tratado origina o chamado lodo de estação de efluentes de pescado salgado, esse resíduo lançado ao meio ambiente sem tratamento acarreta sérios problemas ambientais, tais como a inibição da microbiota natural do meio afetando o ciclo biogeoquímico. Uma das ferramentas para o tratamento desse resíduo salino é o processo de compostagem, que pode ser aplicada no tratamento de diversos resíduos sólidos agroindustriais. Uma questão a ser discutida é a presença de sal nesse resíduo. A pressão osmótica provoca a inibição do crescimento (atividade) da microbiota natural podendo levá-la a morte por extravasamento do conteúdo celular. Para solucionar esses problemas apresentados, a adição de cepas halofílicas e/ou halotolerantes seria uma alternativa para que ocorra a compostagem desse resíduo salino. Portanto, um dos objetivos desse trabalho foi isolar e identificar por PCR bactérias halofílicas de uma matriz alimentar salgada como o bacalhau seco salgado. Os resultados demostraram que as cepas Bacillus cereus, Enterococcus faecalis e Lactobacillus spp foram as mais promissoras quando submetidas a multiplicação bacteriana em caldo suplementado com 7,5% de NaCl. Outro objetivo deste trabalho constituiu em avaliar através de parâmetros físico-químicos a microcompostagem da mistura de lodo de pescado salino, casca-de-arroz e serragem com a adição de B. cereus, E. faecalis e consórcio delas nos dias de processo de compostagem. Os resultados demonstraram eficácia na queda da condutividade elétrica no período estudado pelas cepas utilizadas nos tratamentos. O pH apesar de ter apresentado queda durante a compostagem se manteve acima de 6, padrão estabelecido pela legislação brasileira para comercialização do composto. Durante todo o período de compostagem, o teor de nitrogênio encontrado permaneceu em porcentagens baixas, devido ao uso de bactérias halotolenrantes, apesar de serem utilizadas como probióticas causam a desnitrificação, assim elevando o percentual de nitrogênio no decorrer do experimento. Em relação ao carbono a porcentagem de carbono teve uma queda o insignificativa durante o processo, a porcentagem de carbono encontrada durante o experimento é respondido pela alta atividade microbiana obtido em porcentagens de carbono abaixo dos 45% ao final dos 26 dias de incubação. A umidade para os tratamentos com adição de probióticos estiveram dentro do padrão estipulado pelo MAPA, entre 40 e 60%, contrariamente dos tratamentos sem os inóculos que apresentaram valores acima. E a matéria mineral, apresentou valores maiores ao final da compostagem devido a ação microbiana e a redução da matéria orgânica e formação de minerais. O lodo de pescado salino de estação de tratamento de
efluentes obteve melhores resultados com adição de inóculo, como adição de outro resíduo rico em nitrogênio, modificação de volume/volume e material estruturante, e maior tempo de compostagem. Apesar dessas observações os resultados apresentados foram dentro dos padrões da legislação brasileira. / The activity of the agroindustries has caused impacts to the environment, among them the contamination of soil, groundwater and surface, atmosphere. In the food industry, salt is used in several stages of food processing, and is also considered the most consumed ingredient by humans. In the marine fishing industry salt is present since the discharge of material and the effluent generated when treated gives rise to so-called salty fish effluent seasoning, this waste released to the environment without treatment causes serious environmental problems such as the inhibition of the microbiota natural environment affecting the biogeochemical cycle. One of the tools for the treatment of this saline residue is the composting process, which can be applied in the treatment of various agroindustrial solid wastes. One issue to be discussed is the presence of salt in this residue. The osmotic pressure causes the inhibition of the growth (activity) of the natural microbiota and can lead to death by extravasation of the cellular content. To solve these problems, the addition of halophilic and / or halotolerant strains would be an alternative for the composting of this saline residue. Therefore, one of the objectives of this work was to isolate and identify, by PCR, halophilic bacteria from a salt food matrix such as salted dry cod. The results showed that the Bacillus cereus, Enterococcus faecalis and Lactobacillus spp strains were the most promising when submitted to bacterial multiplication in broth supplemented with 7.5% NaCl. Another objective of this work was to evaluate the microcompaction of the sludge mixture of saline fish, rice husk and sawdust with the addition of B. cereus, E. faecalis and their consortium in the days of the composting process. The results demonstrated efficacy in the reduction of the electrical conductivity in the period studied by the strains used in the treatments. The pH, even though it showed a drop during composting, remained above 6, a standard established by the Brazilian legislation for the commercialization of the compost. During the whole composting period, the nitrogen content remained low, due to the use of halotolenating bacteria, although they are used as probiotics, causing denitrification, thus raising the percentage of nitrogen during the experiment. In relation to carbon the percentage of carbon had an insignificant drop during the process, the percentage of carbon found during the experiment is answered by the high microbial activity obtained in carbon percentages below 45% at the end of the 26 days of incubation. The humidity for the treatments with addition of probiotics were within the standard stipulated by MAPA, between 40 and 60%, contrary to the treatments without the inocula that presented values above. And mineral matter presented higher values at the end of composting due to microbial action and reduction of organic matter and mineral formation. The saline fish sludge from the effluent treatment station obtained better results with addition of inoculum, as addition of another residue rich in nitrogen, modification of volume / volume and structuring material, and greater time of composting. Despite these observations, the results presented were within the brazilian legislation.
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Caracterização da comunidade microbiana em ambientes salinos e suas possíveis aplicações biotecnológicas / Caracterization of the microbiol community in saline environments and their possible biotechnological applicationsPiubeli, Francine Amaral 18 August 2018 (has links)
Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T08:43:54Z (GMT). No. of bitstreams: 1
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Previous issue date: 2011 / Resumo: A água residuária da extração de petróleo é altamente salina e contém uma mistura complexa de hidrocarbonetos, muitos dos quais são altamente tóxicos. Com o aumento da preocupação em preservar o meio ambiente, as tecnologias voltadas para a recuperação e despoluição de áreas degradadas tem ganhado a atenção. A biorremediação tem sido utilizada como método de biodegradação natural através da otimização de processos biológicos por ser econômica, versátil e a que mais se aproxima de uma despoluição ecologicamente aceitável. No entanto, a biorremediaçao depende de alguns fatores tais como: pH, temperatura, salinidade e pressão. Condições extremas desses fatores podem matar ou inibir espécies que não estejam adaptadas a ambientes extremos, por isso o desenvolvimento e otimização de processos de biorremediação de ambientes extremos contaminados por é de grande relevância. Desta forma, este trabalho teve como objetivos o isolamento e caracterização de bactérias halofílicas presentes na água residuária da extração de petróleo, além do estudo molecular dos genes envolvidos na degradação de compostos aromáticos para que posteriormente utilizássemos essas linhagens na biorremediação desse efluente, outra vertente desse trabalho foi o estudo da diversidade microbiana presente nesta água para auxiliar o direcionamento de esforços na minimização dos impactos causados com a exploração de petróleo. Foram isoladas sete linhagens de bactérias halofílicas da água de produção de petróleo, sendo todas pertencentes a família Halomoneaceae e com potencial para degradação de compostos aromáticos. A linhagem que apresentou maior taxa de crescimento nos compostos testados (ácido benzóico, fenol, ácido p-hidroxibénzoico) foi a denominada df2. Com relação à taxa de degradação dos compostos aromáticos testados, a dp3 degradou cerca 90% do fenol disponível, seguida pela df2 que degradou 80% desse mesmo composto. Quando a fonte de carbono foi o ácido benzóico, a df1 e DP3 degradaram 99% do composto e 70% do ácido p-hidroxibenzóico foi degradado em 12 dias de experimento pela linhagem df2. Para a degradação desses compostos a linhagem Halomonas organivorans seguiu a via de degradação do catecol na rota do ß- cetoadipato compreendendo a codificaçao de diferentes enzimas por catRBCA. A comparação das seqüências da biblioteca de bactérias com as sequências do gene RNA ribossomal 16S presente nos bancos de dados GenBank e RDP revelou a presença de micro-organismos pertencentes aos gêneros Marinobacter e Halomonas e na comparação da técnica clássica de isolamento com a técnica independente de cultivo (biblioteca do gene RNA ribossomal 16S), foi encontrada uma maior diversidade na última. Finalmente de todos os testes realizados na tentativa de biorremediar a água de produção de petróleo, a bioestimulação foi a mais efetiva, sendo conseguida uma diminuição de 77% da carga orgânica com a adição de solução de fosfato e alanina. Estes resultados demonstraram o grande potencial destas linhagens para a degradação de compostos aromáticos, bem como para a biorremediação da água de produção de petróleo, além de descrever a via metabólica utilizada por membros da família Halomoneaceas na degradação de compostos aromáticos, auxiliando assim nos esforços para biorremediação de ambientes salinos contaminados / Abstract: The wastewater from oil extraction (produced water) is highly saline and contains a complex mixture of hydrocarbons, many of which are highly toxic. With increasing concern about preserving the environment, the technologies for recovery and remediation of degraded areas has been gaining attention. Bioremediation has been used as a method of natural biodegradation by optimizing biological processes because it is economical, versatile and it is the closest to a ecologically acceptable decontamination. However, bioremediation depends on factors such as pH, temperature, salinity and pressure. Extremes of these factors can inhibit or kill species that are adapted to extreme environments, so the development and optimization of bioremediation processes for contaminated extreme environments is of great importance. Thus, this work aimed at the isolation and characterization of halophilic bacteria present in the wastewater from oil extraction, and molecular studies of the genes involved with the degradation of aromatic compounds so that later we could use these strains in the bioremediation of this wastewater. Another aspect of this work was to study the microbial diversity present in this wastewater to assist in directing the efforts to minimize the impact of oil drilling. Seven strains of halophilic bacteria were isolated from of the produced water of oil production, all belonging to the family Halomoneaceae having potential for degradation of aromatic compounds. The strain that showed the highest growth rate in the tested compounds (benzoic acid, phenol, p-hydroxybenzoic acid) was strain DF2. Regarding the rate of degradation of the aromatic compounds tested, dp3 degraded about 90% of available phenol, followed by DF2 that degraded 80% of this compound. When the carbon source was benzoic acid, DP3 and df1 and degraded 99% and strain DF2 degraded 70% when the compound was p-hydroxybenzoic acid. For the degradation of these compounds the strain Halomonas organivorans followed the catechol degradation pathway ending in the formation of ß-Ketoadipate and was shown to produce various enzyme activities encoded by catRBCA. Comparison of the 16S rRNA gene sequences of the isolates with the sequences present in the GenBank and RDP databases revealed the isolates belonged to the genera Marinobacter and Halomonas. Comparing these results with those obtained by classical taxonomic techniques demonstrated that the 16S ribosomal RNA gene analysis (culture independent method), found greater diversity. Finally, of all tests performed to bioremediate the produced water, biostimulation by addition of nutrients was the most effective, and achieved a reduction of 77% of the organic load with the addition of phosphate combined with alanine. These results demonstrate the great potential of these strains for the degradation of aromatic compounds, as well as for the bioremediation of produced water from crude oil production, and describe the metabolic pathway used by members of the family Halomoneaceas for the degradation of aromatic compounds, thus assisting in efforts to develop methods for the bioremediation of contaminated saline environments / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos
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Etude de la diversité des procaryotes halophiles du tube digestif par approche de culture / Study of the diversity of halophilic prokaryotes from gut by culturomics approachSeck, El Hadji 23 November 2017 (has links)
Une consommation élevée de sel a été associée à beaucoup de maladies et à un risque accru de décès. Plusieurs mécanismes sous-jacents, y compris le stress oxydatif, ont été étudiés. Mais la salinité dans l'intestin et l'altération possiblement associée de son microbiote, récemment identifiées comme un symbiote critique de la santé et de la maladie, n'ont pas encore été explorées chez l'homme. En testant 1334 prélèvements de selles, nous avons montré qu'une salinité élevée était associée à une diminution de la diversité globale et à l'émergence de populations microbiennes halophiles dans l'intestin. La salinité fécale était associée au régime alimentaire salé et à l'obésité, conformément aux données épidémiologiques. Aucun procaryote halophile n’a été cultivé en dessous d'un seuil de salinité fécale de 1,5 %. Au-delà de ce seuil, nous avons découvert une diversité inattendue de microbiote halophile humain dont la richesse était corrélée avec les concentrations de sel; 64 espèces différentes ont été isolées, dont 21 nouvelles espèces et 43 espèces connues dans l'environnement mais non chez les humains. Trois procaryotes extrêmement halophiles ont été isolés, dont deux Archaea appartenant au genre Haloferax, avec une nouvelle espèce Haloferax massiliensis, et un nouveau genre bactérien, Halophilibacterium massiliense. D'autres études devraient spécifier les facteurs qui conduisent à la salinité intestinale et préciser si les altérations de microbiota intestinal associées à des niveaux élevés de sel peuvent être liées à des causes humaines / High salt intake has been linked with many diseases and an increased risk of death. Several underlying mechanisms, including oxidative stress, have been investigated, but salinity in human gut and the possible associated alteration of its microbiota recently identified as a critical symbiote of health and disease, have not yet been investigated in humans. Here, by testing 1,334 stools, we have shown that high salinity is associated with a decrease in overall diversity but the emergence of halophilic microbial populations in the intestine. Fecal salinity was associated with saline diet and obesity, according to epidemiological data. No halophilic prokaryote can be grown below a fecal salinity threshold of 1.5%. Beyond this threshold, we discovered an unexpected diversity of human cultured halophilic microbiota whose richness was correlated with salt concentrations; 64 different species were isolated, including 21 new unknown species and 43 known species in the environment but not in humans. Three extremely halophilic prokaryotes were isolated, including two Archaea belonging to the genus Haloferax, with a new species Haloferax massiliensis, and a new bacterial genus, Halophilibacteriums massiliense. Further studies should specify the factors driving gut salinity, and clarify if the gut microbiota alterations associated with high salt levels could be causally related to human diseas
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Carbon Dioxide Conversion to Value-Added Products using Microbial Electrosynthesis CellAlQahtani, Manal Faisal 11 1900 (has links)
Microbial electrosynthesis (MES) is an emerging biotechnology platform for the conversion of CO2 feedstocks into value-added chemical commodities. In MES, microbial catalysts use the cathode (electrons/ H2) as a sole source of energy for the reduction of CO2. Integrating MES technology with renewable energy sources, such as solar power, to convert CO2 to storable chemicals is an example of a perfect circular economy and a sustainable climate change mitigation strategy. However, many knowledge gaps need to be addressed to scale-up MES as an economically viable chemical production process.
Therefore, different in-depth approaches were tested in this dissertation by optimizing the cathode architecture and exploring the saline application to enhance MES
performance. A balance between various bio-physicochemical phenomena at the MES cathode, i.e., the three-phase interface between CO2 gas, cathodic-biofilm, and
electrolyte, is desirable for efficient microbial electrochemical CO2 capture and utilization.
To address this problem, this thesis investigated alternatives to the benchmark carbonbased plane cathode by applying a dual-functioning (cathode as well as a CO2 gas-transfer membrane) electrode architecture on MES performance. High Faradaic efficiencies for CO2 reduction were achieved with this novel cathode architecture. This hollow-fiber electrode architecture was also applied to MES operation in saline conditions (i.e., Saline-MES). Because seawater potentially acts as an endless source of saline electrolyte, and its high electrical conductivity useful to minimize the concentration overpotential losses occurs in MES. However, exploring robust halophilic microbial catalysts with high selectivity towards CO2 reduction to the desired end product(s) is necessary to develop the saline-MES process. Therefore, this thesis investigated natural saline habitats with hyper (Red sea brine pool) and moderate salinity (mangrove and salt marsh sediment) as a source of inoculum. Emphasis was placed on improving new knowledge in the direction of halophilic CO2 reducing communities enrichment using cathode selective pressure in the saline-MES. The fundamental insights demonstrated in this dissertation are useful for further development of MES technology, to bring MES one step closer to full-scale
applications, for overcoming the bottlenecks associated with reactor scaling-up related to cathode architecture, strategies for the enrichment of halophilic CO2 reducing
microbial communities, and saline-MES process optimization.
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Production, purification et caractérisation de peptides antimicrobiens d’archées halophiles isolées de la saline de Sfax en Tunisie / Production, purification and charactérization of antimicrobial peptides from halophilicarchaea isolated from Sfax solar saltern in TunisiaGhanmi, Fadoua 01 December 2016 (has links)
La saline de Sfax est un milieu hypersalin localisé dans la zone centrale de la côte est de Tunisie. Dans ce travail, nous avons isolé, identifié, et caractérisé des souches halophiles produisant des peptides antimicrobiens (halocines), afin de mieux comprendre leur rôle dans les interactions microbiennes au sein des milieux hypersalins. Deux étangs salins (TS18, 390 g.L-1 NaCl et M1, 200 g.L-1 NaCl) ont été choisis pour l’échantillonnage. Trente-cinq souches de procaryotes halophiles ont été isolées et caractérisées, dont 11 ont présenté une activité antimicrobienne. Parmi ces souches, 3 produisent des substances antimicrobiennes de nature protéique. A l’aide de PCR et RT-PCR nous avons montré que les souches Halobacterium salinarum ETD5 et ETD8 exprimaient le gène de l’halocine S8, un peptide de 3,6 kDa préalablement purifié d’une souche S8a non identifiée. Le peptide a été purifié à partir de cultures de la souche Hbt. salinarum ETD5. Après purification bioguidée, les fractions actives révèlent deux bandes de 8 et 14 kDa présentant une activité antimicrobienne. L’analyse par séquençage Nterminal et spectrométrie de masse a permis d’identifier ces deux halocines. La bande de 8 kDa correspond à une halocine S8 de 81 acides aminés qui subirait une maturation post-traductionnelle protéolytique différente de celle initialement décrite dans la littérature. Le clonage et le séquençage du gène codant le précurseur de l’halocine S8 démontrent que la séquence est identique chez les deux souches ETD5 et S8a. La bande de 14 kDa correspond à une nouvelle halocine, l’halocine S14. L’halocine S14 correspond à une forme tronquée en partie N-terminale de la Mn-superoxyde dismutase (SOD) d’Hbt. salinarum. Il pourrait s’agir d’évolution divergente d’un gène codant deux protéines distinctes, ou d’une maturation différente de la SOD. Ce travail permettra de mieux connaître les molécules intervenant dans les interactions microbiennes dans les milieux hypersalins, des milieux extrêmes susceptibles de révéler des structures et des modes d’action originaux. / The solar saltern of Sfax is a hypersaline located in the central area of the eastern coast of Tunisia. In this study, we isolated, identified, and characterized halophilic strains producing antimicrobial peptides (halocins), aiming to understand their role in microbial interactions in hypersaline environments. Two ponds (TS18, 390 g.L-1 NaCl and M1, 200 g.L-1 NaCl) were selected for sampling. Thirty-five halophilic strains have been isolated and characterized, among which 11 displayed antimicrobial activity. Three of them produced antimicrobial substances of proteinaceous nature. Using PCR and RT-PCR, we have demonstrated that Halobacterium salinarum ETD5 and ETD8 express the gene encoding halocin S8, a 3.6 kDa peptide previously isolated from a strain S8 unidentified. The peptide was purified from cultures of strain Hbt. salinarum ETD5. Following bioguided purification, the active fractions revealed two protein bands of 8 and 14 kDa exhibiting antimicrobial activity. N-terminal sequencing and mass spectrometry analyses allowed identification of these two halocins. The 8 kDa band corresponds to halocin S8, undergoing a different proteolytic post-translational processing from that originallydescribed. Cloning and sequencing of the gene encoding the precursor of halocin S8 showed that the sequence is identical for both strains ETD5 and S8a. The 14 kDa band is a new halocin termed halocin S14. Halocin S14 corresponds to an N-terminally truncated portion of the archaeal Mnsuperoxide dismutase (SOD). This could result from divergent evolution of a gene encoding two distinct proteins, or a different post-translational processing of SOD. Our study helps to better understand which molecules are involved in microbial interactions within hypersaline environments and and how they contribute to the competitions in such extreme environments, which are susceptible to give rise to original structures and modes of action.
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