Dissertations / Theses on the topic 'Halophilic bacterium'

In vitro protein synthesis in Vibrio costicola (polyuridilic acid (poly(U)) -directed incorporation of phenylalanine) was studied. As shown previously (Kamekura and Kushner, 1984), Cl$\sp-$ ions inhibited protein synthesis. The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. Washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. The main site of action of Cl$\sp-$ in this system is on the binding of ribosomes to the messenger RNA. I studied the protein synthesizing machinery of V. costicola using cell-free protein synthesis systems directed by natural mRNAs. One system, the endogenous system, was directed by a mixture of unidentified messengers present in V. costicola, the mRNAs endogenous to these cells. In another system, I have shown that the viral mRNA, that of the coliphage R17, directs the incorporation of ($\sp $C) valine with cellular extracts of V. costicola. The ionic requirements of both systems were similar; both were most active at NH$\sb4\sp+$ (as ammonium glutamate) and Mg$\sp{++}$ concentrations of 250-300 mM and 8 mM, respectively. As was the case with the poly(U) system, Cl$\sp-$ also inhibited protein synthesis in these systems. Added sodium or potassium glutamate inhibited the R17 RNA system but stimulated the endogenous system. It is believed that Cl$\sp-$ is more deleterious to the initiation than to the elongation of protein synthesis, and that glutamate stimulates elongation. Betaine stimulated the activity of both systems, but did not show the same protective effects against Cl$\sp-$ as those observed in the poly(U) system. I have successfully isolated a crude preparation of initiation factors of V. costicola. These factors were released from the ribosomes at a NH$\sb4$Cl concentration much higher than that required for the isolation of the initiation factors of E. coli (1.0 M). Cl$\sp-$ was more inhibitory to the retention of ($\sp $C) fmet-tRNA on nitrocellulose filters, than to the retention of ($\sp3$H) R17 RNA; the retention of these substances was presumably due to the action of IF$\sb2$ and IF$\sb3$, respectively. The enzymes choline dehydrogenase and betaine aldehyde dehydrogenase, which are involved in the synthesis of betaine from choline, were identified in V. costicola. Both enzymes are probably inducibly formed in the presence of choline and seem to be osmoregulated. V. costicola accumulates betaine in response to increasing external NaCl; the intracellular betaine concentration in cells grown in media containing 1.0 and 3.0 M NaCl is 0.25 and 1.2 M, respectively. This accumulation of betaine is also affected by the external concentration of choline. At the lowest choline concentration (0.002%), there is no difference in betaine concentration between cells grown in media containing 0.5, 1.0 and 2.0 M NaCl, while cells grown in the presence of 3.0 M NaCl have only slightly more (0.4 M). These results suggest that betaine may not be the only compatible solute in V. costicola and/or that it may be synthesized through an alternative pathway, and that these cells may also employ other means to maintain turgor pressure. (Abstract shortened by UMI.)

L’amenuisement des ressources pétrochimiques couplé à la pollution engendrée par l’exploitation des plastiques posent de nombreuses questions et conduisent à un besoin urgent de solutions alternatives. Les polyhydroxyalcanoates (PHA) sont des polymères qui ont su se démarquer et naturellement s’imposer comme matériaux de remplacement, de par leurs caractères à la fois biosourcé et biodégradable. Leur synthèse par un grand nombre d’organismes procaryotes et eucaryotes, à partir d’une large gamme de substrats carbonés, rend leur production quasi illimitée et conduit à l’obtention de polymères aux propriétés allant de celles d’un thermoplastique rigide à un élastomère. Malgré cela, les rendements et les coûts de production représentent toujours les principaux verrous à leur développement. Parmi les solutions envisagées, l’exploitation de ressources marines, telles que les bactéries halophiles, suscite un fort intérêt tant les capacités d’adaptation de ces souches sont étendues et avantageuses. Halomonas sp. SF2003 est une souche marine naturellement productrice de PHA possédant une grande capacité d’adaptation face à de nombreux substrats et conditions environnementales. L’étude et l’optimisation de sa production de PHA font l’objet de ce travail. Le séquençage de son génome, couplé à des tests phénotypiques, ont permis l’étude de différents gènes et voies métaboliques confirmant son caractère adaptatif. Dans le même temps, l’influence de différents paramètres sur la production de PHA a été étudiée. Ces travaux, faisant appel à des techniques de biologie moléculaire et des bioprocédés, contribueront au développement du potentiel biotechnologique de la souche Halomonas sp. SF2003
Depletion of oil ressources coupled to pollution caused by over-exploitation of plastics generate a plentiful of issues and lead to an urgent need for alternatives. PolyHydroxyAlkanoates (PHA) are biopolymers which have distinguished and naturally imposed themselves due to their biosourcing and biodegradability features. Their synthesis by a wide variety of eukaryotic and prokaryotic organisms, from various carbon substrates, makes their production almost unlimited and allows obtaining polymers exposing rigid thermoplastic to elastomeric properties. Despite that, the production yield and the cost are still the main locks to their development. Among possibilities studied, exploitation of marine resources, like halophilic bacteria, arouse a strong interest since adaptability of these strains is extensive and attractive. Halomonas sp. SF2003 is a PHA producing marine bacterium which naturally exposes a considerable versatility in front of carbon substrates and environmental conditions. Characterization and optimization of its PHA production are the main subjects of the presented study. Genome sequencing and annotation, in addition to phenotypic tests, allowed characterization of various genes and metabolic pathways attesting of the adaptative strain character. In the same time, impact of various parameters on Halomonas sp. SF2003 PHA production has been investigated. This work, employing molecular biology and bioprocess tools, will contribute to future development of biotechnological potential of Halomonas sp. SF2003

Mestrado em Microbiologia
Halophilic microorganisms and the metabolites involved in their adaptive strategies offer a wide variety of potential biotechnological applications and like other extreme environments, hypersaline habitats represent reservoirs of new bioactive compounds. The main objective of this work was to assess the biotechnological potential of halophilic bacteria from traditional salt pans of Ria de Aveiro in terms of biosurfactant production, plant growth promoting traits, quorum-quenching effect and extracellular enzymatic activity. Water from an active salt pan of Ria de Aveiro was collected in early autumn when the concentration of salt was at the saturation level. Using a culture-dependent approach, 14 bacterial strains were isolated and identified by sequencing of 16S rRNA gene fragments as species of Bacillus, Halobacillus, Idiomarina and Marinobacter. In order to assess the biotechnological potential of these halophiles, they were tested for biosurfactant production, plant growth promoting traits (motility, biocontrol effect and siderophore production), quorum-quenching effect and, activity of extracellular enzymes (phosphatase and esterase). None of the isolates demonstrated significant tensioactive or emulsification effects that could be interpreted as indicative of biosurfactant production. However, all isolates were motile even in presence of 10% NaCl and produced siderophores. All Idiomarina and Marinobacter strains caused growth inhibition of the phytopathogenic fungus Alternaria. Quorum sensing inhibition was also detected in isolates of the 4 genera although with differences between species and, in some cases, depending on the salinity of the culture medium. Esterase and phosphatase activity was detected in all isolates and maximum hydrolysis rates of the model substrates were the highest in Halobacillus species. Although biosurfactant production could not be demonstrated, the halophilic isolates displayed an interesting set of features in terms of plant-growth promoting traits with potential application on the emerging field of saline agriculture.
Os microrganismos halofílicos e os metabolitos envolvidos nas suas estratégias adaptativas apresentam uma ampla gama de potenciais aplicações biotecnológicas e, tal como outros ambientes extremos, os habitats hipersalinos são vistos como um reservatório de novos compostos bioativos. O objetivo deste trabalho foi avaliar o potencial biotecnológico de bactérias halofílicas de salinas da Ria de Aveiro em termos de produção de biossurfactantes, características de promoção do crescimento de plantas, efeito de quorum-quenching e atividade enzimática extracelular. No início do outono de 2016, quando a concentração de sal atinge os valores máximos colheu-se água do tanque de cristalização de uma salina ativa da Ria de Aveiro (Marinha de Santiago da Fonte). Utilizando uma abordagem dependente de cultivo, foram isoladas 14 estirpes bacterianas que foram posteriormente identificadas por sequenciação de fragmentos de genes de rRNA 16S como espécies de Bacillus, Halobacillus, Idiomarina e Marinobacter. Para avaliar o potencial biotecnológico destas halófilas, testou-se a produção de biossurfactantes, características de promoção do crescimento de plantas (mobilidade, efeito de biocontrolo e produção de sideróforos), efeito de quorum-quenching e atividade enzimática extracelular (fosfatase e esterase). Nenhum dos isolados demonstrou efeitos tensioativos ou de emulsificação significativos que pudessem ser interpretados como indicativos da produção de biossurfactantes. No entanto, todos os isolados exibiram mobilidade, mesmo na presença de 10% de NaCl e produziram sideróforos. Todas as estirpes de Idiomarina e Marinobacter causaram inibição do crescimento do fungo fitopatogénico Alternaria. A inibição de quorum sensing também foi detetada em isolados dos 4 géneros, embora com diferenças entre espécies e, em alguns casos, dependendo da salinidade do meio de cultura. A atividade de esterases e da fosfatases foi detetada em todos os isolados e as mais altas taxas de hidrólise dos substratos modelo foram observadas nas espécies de Halobacillus. Embora a produção de biossurfactantes não tenha sido demonstrada, os isolados halofílicos exibem um conjunto de características interessantes em termos de promoção do crescimento de plantas, com potencial aplicação no campo emergente da agricultura salina.

Lake Vanda is a meromictic, permanently ice-covered lake in the McMurdo Dry Valleys. The McMurdo Dry Valleys are a polar desert in Southern Victorialand Antarctica. This area experiences very little rainfall and very cold average temperatures, around –20°C. Lake Vanda has an unusual limnological profile, with a sharp physical and chemical gradient at about 60 m where the water transitions from cold, oxic, and fresh, to warm, hypersaline, and sulfidic; CaCl2 rather than NaCl is the dominant salt. Aerobic enrichment techniques were used to isolate what turned out to be several strains of a species of Chromohalobacter, a genus of the Gammaproteobacteria, from Lake Vanda deep waters, while targeted enrichments for anaerobic and spore-forming bacteria were negative. The isolates were characterized for their temperature and pH optima, carbon and nitrogen nutrition, and salt tolerance and requirements. The results showed the organisms to be obligately aerobic with a broad pH range (optima pH 7). The isolates used some sugars and organic acids but not alcohols or fatty acids for energy and cell carbon, and showed a moderate requirement for NaCl but no requirement for CaCl2, even though CaCl2 is the predominant salt in their environment.

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).

Alkaline saline lakes are unusual extreme environments formed in closed drainage basins. Qabar - oun and Um - Alma lakes are alkaline saline lakes in the Libyan Sahara. There were only a few reports (Ajali et al., 1984) on their microbial diversity before the current work was undertaken. Five Gram-negative bacterial strains, belonging to the family of Halomonadaceae, were isolated from the lakes by subjecting the isolates to high salinity medium, and identified using 16S rRNA gene sequencing as Halomonas pacifica, Halomonas sp, Halomonas salifodinae, Halomonas elongata and Halomonas campisalis. Two of the Halomonas species isolated (H. pacifica and H. campisalis) were chosen for further study on the basis of novelty (H. pacifica) and on dual stress tolerance (high pH and high salinity) shown by H. campisalis. Both species showed optimum growth at 0.5 M NaCl, but H. campisalis alone was able to grow in the absence of NaCl. H. pacifica grew better than H. campisalis at high salinities in excess of 1 M NaCl and was clearly a moderately halophile. H. pacifica showed optimum growth at pH 7 to 8, but in contrast H. campisalis could grow well at pH values up to 10. 13C - NMR spectroscopy was used to determine and identify the compatible solutes accumulated by H. pacifica and H. campisalis grown in rich and minimal media at different concentrations of NaCl. H. pacifica and H. campisalis accumulated betaine in rich (LB) medium with ectoine only appearing at the highest salinity tested (2.5 M NaCl). In contrast, in M9 minimal medium, no betaine was detected and ectoine and hydroxyectoine were accumulated at high salinities. H. campisalis was able to grow well with urea or nitrate as the sole source of nitrogen and was shown to be capable of efficiently removing nitrate from the medium under aerobic assimilatory conditions, where it is incorporated into biomass.

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.

Orientador: Lucia Regina Durrant
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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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

These en deux parties: d'une part etude d'une bacterie en milieu salin. L'addition de proline, choline, permet la croissance de la bacterie. Analyse de la composition proteique de la membrane bacterienne. D'autre part, etude de metalloproteinases et de certaines sequences impliquees dans le site actif de l'enzyme et dans l'activation du zymogene

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It evaluated the quality of carne-de-sol in the period from June to September of 2010 sold in five cities of Rio Grande do Norte - Mossoró, Apodi, Areia Branca, Baraúna and Grossos. It was collect 80 samples of carne-de-sol, of which 44 were from small establishments and 36 purchased in supermarkets and stores. The samples were subjected to microbiological testing - Salmonella, determining the most probable number of fecal coliform, halophilic bacteria and Staphylococcus aureus, the samples were positive for this bacteria were analyzed for resistance to antibiotics, and physical-chemical properties (pH, water activity, minerals and humidity). There was an interaction between intrinsic pH value (μ = 5.702) and Aa (μ = 0.868), , indicating a favorable environment for their multiplication, resulting in values that stood out significantly in what is expected legislation counting S. aureus (μ = 4.81 log UFC at 78.75%), Salmonella (25%) and fecal coliform (μ = 2.36 LogNMP / g, 63.75%). A significant correlation (p <0.05) between the number of halophilic bacteria (μ = 4.87 log UFC) and S. aureus, such conditions may be used as an indicator of contamination in the sun-dried meat. To the antibiotics, 22.5% of strains were resistant to gentamicin, whereas for penicillin G, chloramphenicol, tetraciclina were 100%, 67.5% and 46.26% respectively. It was found that the meat produced and marketed in the sun studied region is improper (96.25%) for consumption in accordance with Brazilian law
Foram avaliadas a qualidade da carne-de-sol no período de junho a setembro de 2010 comercializada em cinco cidades do Rio Grande do Norte Mossoró, Apodi, Areia Branca, Baraúna e Grossos. Sendo coletadas 80 amostras de carne-de-sol, as quais 44 foram provenientes de estabelecimentos de pequeno porte e 36 adquiridas em supermercados e frigoríficos. As amostras foram submetidas à análises microbiológicas Salmonella, determinação do número mais provável de coliformes termotolerantes, bactérias halofílicas e Staphylococcus aureus, as amostras positivas para esta bactéria foram submetidas à análise de resistência à antibióticos; e físico-químicas (pH, atividade de água, cinzas e umidade). Verificou-se interação dos fatores intrínsecos, pH (µ=5,702) e Aa (µ=0,868), do produto com a contagem bacteriana, indicando ambiente favorável para a sua multiplicação, resultando assim em valores que sobressaíram de maneira significativa o que está previsto na legislação para contagem de S. aureus (µ=4,81LogUFC, em 78,75%), Salmonella (presença em 25%) e coliformes termotolerantes (µ=2,36LogNMP/g, em 63,75%). Houve correlação significativa (p<0,05) entre a contagem de bactérias halofílicas (µ=4,87LogUFC) e a de S. aureus, podendo aquelas serem utilizadas como indicador de contaminação na carne-de-sol. Quanto à sensibilidade das cepas S. aureus aos antibióticos, 22,5% das cepas apresentaram resistência à gentamicina, enquanto para penicilina G, tetraciclina e cloranfenicol foram 100%, 67,5% e 46,26% respectivamente. Ficou constatado que a carne de sol produzida e comercializada na região estudada apresenta-se imprópria (96,25%) para o consumo de acordo com a legislação brasileira

Les bacteries halophiles extremes vivent dans des milieux ultrasalins tels les marais salans et la mer morte. Les facteurs d'elongation tu, proteine qui interagit avec des aminocyl-trna, chez halobacterium marismortui a ete etudie, a ete purifie et ses proprietes halophiles demontrees, et la purification du gene codant pour cette monomolecule a ete realisee

碩士
元智大學
生物科技與工程研究所
106
Ectoine and Hydroxyectoine are compatible solutes produced by halophilic or halotolerant microorganisms in extremely saline environments to protect cellular activity. Ectoine and Hydroxyectoine have the functions of regulating osmotic pressure balance, protecting cell structure and maintaining enzyme activity. Between them, Hydroxyectoine has an extra -OH group in structure than Ectoine, which can replace water and partial water molecule loss in reaction. This stabilizes the natural structure of biopolymers, thereby enhancing the commercial value of Hydroxyectoine. Therefore, the main purpose of this study was to find a strain with superior performance in Hydroxyectoine production, and to achieve higher Hydroxyectoine production through regulation of culture conditions. Among the many strains that produce Ectoine and Hydroxyectoine, Corynebacterium glutamicum, which prefers to produce Hydroxyectoine, was used as a research object. In this study, we investigate the effect of Ectoine and Hydroxyectoine production by different environmental factors (i.e. rotation speed, temperature, pH and salt concentration) and medium compositions (i.e. carbon source, nitrogen source, iron ion and ketoglutaric acid). The results show that the best Ectoine and Hydroxyectoine production is 0.096 g/L and 2.579 g/L when C. glutamicum were incubated at 200 rpm, 30 °C, pH 9 and 0.5 M salt concentrations. In the screening results of the medium composition, it was found that when the carbon source was selected from yeast extract, the nitrogen source was Ammonium sulfate, and 0.5 mM iron ion and 75 mM ketoglutaric acid were added, the Ectoine yield was increased from 0.096 g/L to 0.1 g/L. Hydroxyectoine production increased from 2.579 g/L to 3.15 g/L. Taking into account the fact that the changes in culture conditions have no significant difference in the production of Hydroxyectoine by C. glutamicum, this study will follow up on different fermentation strategies and expect to significantly increase the production of Hydroxyectoine by C. glutamicum.

Gram-negative bacteria cell envelope is a complex structure that is constantly exposed to its environment. It is composed of an Inner-membrane (IM), a thin peptidoglycan layer and an Outer-membrane (OM). The main component of the OM are Lipopolysaccharides (LPS), also termed as endotoxins. Those molecules are composed of three main parts: a polysaccharide named the O-antigen, a core oligosaccharide and a Lipid A. Endotoxins lacking the polysaccharide are termed Lipooligosaccharides (LOS). LPS are known to interact with mammal’s innate immunity through the Toll-like receptor 4 (TLR4) and Myeloid Differentiation factor 2 (MD-2) receptorial complex. Depending on their structure, and in particular on their Lipid A, LPS can either have an agonist or an antagonist activity. Discovering new LPS structure is hence necessary in order to develop new therapies, since agonist LPS can be used as vaccines adjuvant and antagonist as drugs against sepsis and septic shock. In this context, LPS structures from various bacterial sources are currently under study. This project present the characterization of LPS and LOS extracted from marine and halophilic bacteria. As those organisms live in a particular environment, they developed specific strategies to adapt themselves and were hence investigated, as their LPS structure can be shaped by the adaptation to their environment. In particular, the study of LPS from the following strains is here reported. Pseudoalteromonas sp1A1 is a sponge-pathogen bacterium isolated from Suberites domuncula. The full structure of its LPS was resolved using NMR spectroscopy and Matrix assisted laser desorption (MALDI) Mass spectrometry (MS). Its O-antigen is a branched polysaccharide that have two remarkable features: (i) it possesses a pyruvate linked at 4,6-position of a Glucosamine and (ii) it possesses a 9-carbon ulosonic acid that is the 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). The Lipid A from Pseudoalteromonas sp1A1 was studied by MALDI MS. It is constituted of a mixture of tri- to penta-acylated species, among the penta-acylated Lipid A species, at m/z 1474.6 and 1446.6 were bis-phosphorylated species composed respectively of four C12:0 (3-OH) and one C12:0 and two C12:0 (3-OH), one C10:0 (3-OH), one C11:0 (3-OH) and one C13:0. The structure of the core oligosaccharide from Pseudoalteromonas sp1A1 LPS was also resolved and turned out to be composed of a pentasaccharide containing one Kdo, one heptose, two galactoses and one glucose. Pseudoalteromonas sp1A1 biological activity was assessed using ELISA and Quanti-blue assays. It was found that its LPS does not possess any significant immunostimulant activity on human and murine cells. The Outer-Membrane properties of Pseudoalteromonas sp1A1 were also studied through Molecular Dynamic (MD) simulation, that showed how the asymmetric repartition of Pseudoalteromonas sp1A1 Lipid A influenced the properties of the phospholipid bilayer, increasing its flexibility. Finally, MD simulation was also performed with Pseudoalteromonas sp1A1 LOS in water and in 0.5 M of NaCl, that is closed to the natural marine environment of the bacterium. Results showed that the presence of salts influenced the conformational behavior of the Kdo-Lipid A region. The second bacterial strain studied was Spiribacter salinus M19-40T, a halophile isolated from an intermediate salinity pound of a marine saltern in Spain, the structure of the Lipid A was resolved using MALDI MS and MS2 experiment. It is a mono-phosphorylated and penta-acylated species bearing two C10:0 (3-OH), one C12:0, one C14:0 (3-OH) and one C14:0 (3-oxo). This structure possesses two interesting structural features: (i) the 2+3 symmetry that is unusual - as most penta-acylated Lipid A have a 3+3 symmetry and (ii) the occurrence of the C14:0 (3-oxo). Halopeptonella vilamensis is a halophilic bacterium that have been isolated from a saline lagoon in Argentina whose Lipid A was characterized using MALDI MS and MS2 experiments. Results showed that H. vilamensis has a highly heterogeneous mixture of Lipid A species, mono-phosphorylated and hexa-acylated, that differ for the length and saturation of their acyl chains. H. vilamensis main Lipid A species possesses two C10:0 (3-OH), two C12:0 (3-OH), one C12:0 and one C12:1. Another major species possesses only saturated C12:0. Immunological assays were performed on HEK293 hTLR4/CD14/MD2 murine cell line and on Monocytes-derived Macrophages (MoMs) and Bone marrow-derived macrophages (BMDMs) human cell lines with H. vilamensis LOS and demonstrated its slight immunopotency. Finally, the characterization of cell envelope components of Halomonas smyrnensis was also attempted. H. smyrnensis is an Exopolysaccharide (EPS) producing halophile isolated from a Turkish salt lake. It is known to be a high levan producer and two novel EPS were isolated. The first one is formed by α-(1→4)-Glc polymer and the second one by α-(1→3)-GlcNAc units.

碩士
國立臺灣大學
海洋研究所
91
Abstract Halophilic Gram-negative bacteria are widely found in coastal zones and marine areas. They are also commonly present in the brackish water environments of estuary. In contrast, halophilic Gram-positive bacteria are rare in the microbial communities of estuary, coast and sea. Our laboratory had totally isolated 113 strains of halophilic or halotolerant bacteria, preliminarily identified as Gram-positive bacteria, from the sediment under mangroves at Tan-shui River estuary and Gau-Mei Taichung, and marine water at the coastal sites of Taiwan (Er-jen River estuary etc.) as well as Ba-dou-Zi Keelung and Shen-Ao. The propose of this study was to screen halophilic bacteria from the isolated strains and analyzed these bacteria for their diverse genera and species. There only 41 strains of 113 isolates were halophilic gram positive bacteria. According to Na+ requirement, endospore production, cell morphology, glucose fermentation and colony morphology, 41 strains were divided into 11 groups: the strains of group 1-8 required Na+ for growth, the strains of group 9-11 were not ; and only the strains of group 6 and 9 were facultatively anaerobes which were able to ferment glucose, the strains of other 9 groups were aerobe which were unable to ferment glucose. Group 1 contained two strains, which were the only group producing endospores. The cells of this bacterial group were regular rods and grew into orange circular colonies. Group 2 only included one strain, which could not ferment glucose and was characterized by its orange circular colonies and regular rod cells like group 1. However, the production of endospore was not observed in group 2, a distinction between group 1 and group 2. Group 3 only included one strain, which grew into pink circular colonies and utilized glucose as sole carbone source for growth, a distinction between group 3 and group 1-2. Group 4 contained 4 strains and group 5 contained 15 strains, which could not ferment glucose and the cells of this group were regular rod like group 1-3. However, the production of irregular gray colonies and white circular colonies separatly, a distinction between group 4-5 and group 1-3. Group 6 only included one strain, which were the only group grew into colorless circular colonies, ferment glucose and the cells of this group were irregular rods, a distinction between group 6 and group 1-5. Group 7 contained 2 strains, which the cells of this group were irregular rods, and the strain could not growthed in complex-medium whithout ultra Mg2+, a distinction between group 7 and other 10 groups. Group 8 only included one strain, which the cells of this group were irregular rods, and the only group grew into yellow colonies. Group 9 contained two strains, which the cells of this group were irregular rods, grew into white circular colonies and could not ferment glucose. Group 10 only included one strain, which were the only group grew into red circular colonies, and the cells of this group were regular rods. Group 11 contained 11 strains, which the cells of this group were regular rods,and grew into irregular gray colonies beside I strain grew into white circular colonies. The level of 16S rDNA sequence similarity revealed that the strains C1055 and J169 of Group 1 could be considered as Bacillus hwajinpoensis.The strain CS13 of Group 2 could be considered as Halobacillus trueperi. The strain B8 of Group 3, M336 of Group 7, A53 of Group 9 and A57 of Group 6 could be considered to represent a new species. The strains J149 and BS13 of Group 4, J607 and J717 of Group 5, and A2 of Group 11 Stappia aggregata the closest similary The strains A51 of Group 10 and Ba1a8 of Group 8 was identical to Rhodovulum sulfidophilum and Microbacterium ketoreductum, the closest similary. Based on the phylogenetic tree rebuilt by the 16S rDNA sequence analysis, it was revealed that the 11 groups contained at least 8 species in 8 genera. It is valuable to mention a strain in group 7, M336. Unlike other strains, it could grow neither in complex medium without added Mg2+, nor in 100％ marine water or 5% NaCl solution. In addition, it was only 94.6％ identical to 16S rDNA sequence of Devosia neptuniae, the closest similary. Therefore, there was no doubt M336 might be a new species or even a new genus. In this study, the selected strain in group 3, 4, 5, 6, 7, 9, 10 and 11 displaying Gram-positive was revealed that their closest species were all Gram-negative via 16S rDNA sequence analysis. A further study should be required to clarify why the outcome contradictory to common observation could occur.

碩士
元智大學
生物科技與工程研究所
104
A natural cyclic amino acid derivative, ectoine acts as a protective substrate in many bacterial cells as a compatible solute. Synthesized and accumulated by halophilic and halotolerant microorganisms. Preliminary study of our group as well as those stated in literatures indicated that ectoine is an innovative, multi-functional natural compound not only stabilize proteins and other macromolecules, but also a with an effect in protecting Langerhans cells and UV-induced degradation of DNA. Comparison of ectoine concentration among the three strains obtained from Bioresource Collection and Research Center (BCRC) in Taiwan, the best strain for ectoine synthesis is Halomonas salina BCRC17875. Experimental results indicated that 4.5% appears to be the optimal concentration of NaCl, producing a maximum ectoine production of 2968 mg/L by Halomonas salina BCRC17875. Moreover, ectoine production was optimal in batch cultures when the temperature, pH, agitation rate was controlled at 30 ℃, pH 7.5 and 150 rpm, respectively. While the maximum ectoine concentration was increased by 11-folds when the glucose and glutamate both were 4%, the maximum crude ectoine production reached 2968 mg/L. Furthermore, the chemical structure and molecular masses of the purified ectoine were confirmed by nuclear magnetic resonance (1H-NMR) spectrometer and fast atom bombardment mass (FAB-MS) spectrometer, respectively.

Soil salinization is a globally growing problem, and several approaches have been proposed to mitigate its effects on the productivity of plants of economic interest. The use of halophilic or halotolerant bacteria as plant growth promoters is one of the strategies recommended for the mitigation of salt stress. However, halotolerant bacteria isolated from the rhizosphere of halophyte plants are the most commonly used inoculum. The objective of this work was to evaluate the potential of halophilic bacteria, isolated from a salt pan, in the attenuation of saline stress Lactuca sativa, used as a model crop glycophyte. A collection of strains isolated from Santiago da Fonte saltpans (Aveiro) representing the genera Bacillus, Halobacillus, Idiomarina and Marinobacter, was analyzed for some characteristics considered as advantageous in the colonization and promotion of growth of host plants. The production of extracellular enzymes in presence of 0, 20 and 100 NaCl, as well as the ability to solubilize phosphate and produce 1-aminocyclopropane-1-carboxylate deaminase were tested. H. locisalis and I. seosinesis, considered as more interesting in terms of plant growth promoting traits, were tested separately and together as inoculum in lettuce seeds. A factorial experimental design was applied to test the effect of inoculation and salinity of the irrigation water on the efficiency of seed germination and plant growth. Germination efficiency was strongly affected by salinity and no significant effects of inoculation were observed. The germination efficiency was lower at 10 NaCl than at salinity 0 and the weight of the plants was significantly lower in the plants inoculated with the consortium of isolates than in the uninoculated plants. Plants inoculated with the consortium and grown at salinity 10 had lower water content. When used separately, H. locisalis or I. seosinesis caused an increase in leaf size in plants cultivated in salinity, in relation to the inoculated control. Inoculation did not cause a significant effect on chlorophyll fluorescence. Although no evidence of attenuation of saline stress by inoculation was detected, H. locisalis inoculum showed a positive effect on the growth of plants in non-saline conditions, indicating a potential as a growth promoting bacterium of plants of agricultural interest.
A salinização dos solos é um problema crescente a nível global e têm sido várias as abordagens propostas para atenuar os seus efeitos na produtividade de plantas de interesse económico. O uso de bactérias halófilas ou halotolerantes como promotoras do crescimento de plantas, é uma das estratégias preconizadas para a mitigação do stresse salino. No entanto, são normalmente usadas como inóculo bactérias halotolerantes isoladas da rizosfera de plantas halófitas. O objetivo deste trabalho foi avaliar o potencial de bactérias halófilas, isoladas de uma marinha de sal, na atenuação do stresse salino em Lactuca sativa, usada como modelo de glicófita de interesse agrícola. Uma coleção de estirpes isoladas da marinha de Santiago da Fonte (Aveiro) representando os géneros Bacillus, Halobacillus, Idiomarina e Marinobacter, foi analisada quanto a algumas caraterísticas consideradas como vantajosas na colonização e promoção do crescimento de plantas. Testou-se a produção de enzimas extracelulares em salinidades 0, 20 e 100 de NaCl bem como a capacidade para solubilizar fosfato e produzir ácido 1-aminociclopropano-1-carboxilato desaminase. H. locisalis e I. seosinesis, considerados como mais interessantes face às características promotoras do crescimento, foram testados separadamente e em conjunto, como inóculo em sementes de alface. Foi aplicado um desenho experimental fatorial para testar o efeito da inoculação e da salinidade da água de irrigação sobre a eficiência de germinação das sementes e crescimento das plantas. A eficiência de germinação foi fortemente afetada pela salinidade não tendo sido observados efeitos significativos de nenhum dos inóculos testados. Na condição de salinidade 10, a eficiência de germinação foi mais baixa do que com salinidade 0 e o peso das plantas foi significativamente menor nas plantas inoculadas com o consórcio de isolados do que nas plantas não inoculadas. As plantas inoculadas com o consórcio e cultivadas na salinidade 10 apresentaram menor teor de água. As plantas inoculadas separadamente com H. locisalis ou com I. seosinesis cultivadas em salinidade 0, revelaram um aumento do tamanho das folhas relativamente ao controle não inoculado. Embora não tenham sido encontradas evidências de atenuação do stresse salino, o inóculo H. locisalis apresentou um efeito positivo no crescimento das plantas em condições não-salinas, o que demonstra um potencial como bactéria promotora do crescimento de plantas de interesse agrícola.
Projeto nº 029736 - Programa Operacional Regional do Centro (02/SAICT/2017).
Mestrado em Ecologia Aplicada

碩士
東吳大學
微生物學系
100
Purple non-sulfur bacteria are a group of extraordinary metabolic diverse bacteria, therefore may widely exist in the environment. The study the diversity of freshwater environment of the purple non-sulfur bacteria have been many reported, however, the diversity of the hemophilic purple non-sulfur bacteria are less studied. So this study target separation of pure strains of purple non-sulfur bacteria in different environments of Taiwan western coast, and the use of molecular biological methods for classification, differences strains caused by different environmental , isolated producing hydrogen and Polyhydroxybutyrate (PHB) capacity assessment. Experimental screening out 34 purple non-sulfur bacteria, of which three for halotolerant of purple non-sulfur bacteria, others were all the halophilic of purple non-sulfur bacteria. These bacteria using 16S rDNA sequences sequencing, they were all Rhodovulum sulfidphilum. Strain differences in colony size and bacteria liquid on the color rendering. Use 16S-23S ITS RFLP, 16S rDNA sequence similarity and relationship between phylogenetic tree, 16S-23S ITS (ribosomal intergenic spacer;ITS) sequence similarity and genetic relationship between trees and multi locus sequence typing (the Multiocus sequence type) trying to categorize strains do,but it can do no further grouping. Hydrogen parts, first detected using PCR method screening while nifD and nifH gene of strains, and gas production test, found under the strains of malic acid as carbon sources capable of producing gas. For gas analysis, recognizing the hydrogen gas generated, then selection two strains one was hydrogen yield the highest R. sulfidphilum SSa7, another was gas yield the higher then others R. sulfidphilum SSa3. Maximize hydrogen production efficiency of 6.31ml/h/L and 2.72/ml/h/L, respectively. PHB production parts, strain detected phaC gene using PCR method and then quantitative PHB. Isolated PHB yield were 6~15 (mg/g fresh wet) with a known PHB production ability of Alcaligenes latus 61.60 (mg/g fresh wet) comparative ability is poor. Wastewater section, since waste water usually family livelihood is a fresh water environment. Using halotolerant strains R. sulfidphilum SSa7 to test whether reduce the total organic carbon and to produce hydrogen and producing PHB. It could reduce total organic carbon and produced PHB without hydrogen in wastewater. The future can expect R. Sulfidphilum whole genome sequencing, we are convenient to classification this strain. PHB production and hydrogen-producing part trying to find the most suitable conditions to increase the yield. Strains are using in food or a leather factory, to test if it is capable of producing hydrogen and producing PHB.