Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2011 / Made available in DSpace on 2012-10-25T19:08:03Z (GMT). No. of bitstreams: 1 298275.pdf: 1289245 bytes, checksum: 547a991388b4d22250541d4f0e7b3fcf (MD5) / O alginato é um exopolissacarídeo constituído de quantidades variáveis de ácidos ?-D-manurônico e seu epímero C-5 ácido ?-L-gulorônico, unidos por ligações ?-1,4 glicosídicas. É extensamente utilizado na indústria de alimentos e biotecnológica. Atualmente a demanda do alginato para estas aplicações é suprida a partir da sua extração de algas marinhas, entretanto vários estudos reportam a produção de alginato por micro-organismos do gênero Pseudomonas e Azotobacter. A produção bacteriana de alginato apresenta-se como uma alternativa interessante e sua produção por micro-organismos, além de possibilitar a produção de biopolímeros de alta qualidade com características específicas e pré-determinadas, irá diminuir o impacto ambiental nas regiões em que as algas marinhas das quais é extraído são coletadas. Nos últimos anos, vários estudos relacionados à produção de alginato por micro-organismos foram realizados com o objetivo de avaliar sua produção e rota metabólica de biossíntese, para caracterizar o material produzido e para determinar as potencialidades de aplicação deste novo material. O conhecimento sobre a via metabólica de um organismo permite a compreensão da fisiologia celular e regulação de seu metabolismo, sendo a quantificação de fluxos metabólicos um importante objetivo, principalmente no que diz respeito à obtenção de produtos úteis comercialmente ou cientificamente. Neste estudo foi avaliado o efeito da relação C:N na produção de alginato. Foram realizados experimentos em frascos agitados a 240 rpm e 30°C, em meios contendo glicerol como fonte de carbono nas concentrações iniciais de 20 a 40 g.L-1 e (NH4)2SO4 como fonte de nitrogênio em concentrações de 0,5 a 2,0g.L-1. Fluxos extracelulares, medidos experimentalmente, foram utilizados para estimar fluxos intracelulares no metabolismo do glicerol em Pseudomonas mendocina na biossíntese de alginato. Foram utilizados modelos estequiométricos e técnicas de balanço de massa, conhecidas como Análise de Fluxo Metabólico (MFA). Para avaliação foi considerado o Estado Pseudo-estacionário (PSS). O objetivo foi avaliar o efeito das concentrações da fonte de carbono e da fonte de nitrogênio no fluxo de carbono. Os resultados indicaram um maior rendimento de bioconversão de substrato em alginato em concentrações menores de nitrogênio, sugerindo que nessas condições P. mendocina utiliza a fonte de carbono principalmente para produção de alginato. Assim, o aumento da relação C:N favorece a produção de alginato, diminuindo o fluxo de carbono na via Entner-Dourdoroff e o crescimento celular. / Alginate is an exopolysaccharide consisting of variable amounts of ?-D-mannuronic acid and its C5-epimer ?-L -guluronic acid linked via ?-1,4-glycosidic bonds. It is widely used in applications in the food and biotechnology industries. Currently the demand for these applications of alginate is supplied from the extraction of seaweed, however several studies report the production of alginate by micro-organisms of the genus Pseudomonas and Azotobacter. The bacterial production of alginate appears to be an interesting alternative and its production by micro-organisms, besides enabled the production of high quality polymers with specific characteristics and pre-determined, will reduce the environmental impact in areas where the seaweed is collected. In recent years, several studies relating the production of alginate by micro-organisms were carried out to evaluate their production and metabolic pathway of biosynthesis, to characterize the material produced and to determine the potential application of this new material. The knowledge about the metabolic pathway of an organism allows the understanding of cell physiology and regulation of their metabolism. The quantification of metabolic fluxes is an important goal, specially with regard to obtaining commercially or scientifically useful products. This study assessed the effect of C: N ratio in the production of alginate. Experiments were performed in shaken flasks at 240rpm and 30°C in medium containing glycerol as carbon source at initial concentrations of 20, 30 and 40 g.L-1 and (NH4)2SO4 as a nitrogen source at initial concentrations of 0,5; 1,0; 1,5 and 2,0 g.L-1. Extracellular fluxes, measured experimentally, were used to estimate intracellular fluxes of glycerol metabolism in Pseudomonas mendocina in the biosynthesis of alginate. It was used stoichiometric models and mass balance techniques, known as metabolic flux analysis (MFA). For evaluation was considered the pseudo-steady state (PSS). The objective was to evaluate the effect of concentrations of carbon and nitrogen sources in the flow of carbon. The results indicated a higher yield of bioconversion of substrate in alginate in lower concentrations of nitrogen, suggesting that in these conditions P. mendocina uses the carbon source mainly for alginate production. Thus, increased C:N ratio favors the production of alginate, decreasing the flow of carbon into the Entner-Dourdoroff pathway and cell growth.

Pentachlorophenol (PCP) is a chloride-containing aromatic compound which is mostly used for preserving wood and leather, but still one can easily detect this compound present in the waste water generated by various industries such as petrifaction, oil-refining, and etc. PCP, due to its chemical property of being stable and highly toxic, would cause severe and irreparable environmental pollution once exposed to open air. This study is intended to explore the feasibility of dealing the problem of PCP with biodegradation. The examination results showed that, except for absorption, the suspension of contaminated soil (aerobic incubation), nonetheless, could effectively degrade PCP during a period of 90 days without the aid of any extra carbon source. (0.62 mg/L/day). The degradation rate was further greatly improved by adding sodium acetate, molasses, and sludge cake (sodium acetate added: 4.15 mg/L/day; molasses added: 1.05 mg/L/day; sludge cake added:0.83 mg /L/day). None of four experimental groups of aerobic sludge, anaerobic sludge, contaminated soil (anaerobic incubation), and Fe3+reaction could degrade PCP after 135 days, 174 days, 250 days, and 124 days, respectively, regardless of whether any sources of carbon were added or not. A bacterium which used PCP as the sole carbon source was isolated from the contaminated soil. After 16s rDNA sequence analysis, it had 98% degree of similarity to Pseudomonas mendocina and was designated as Pseudomonas mendocina NSYSU. The PCP (40 mg/L) degradation rate of Pseudomonas mendocina NSYSU was 9.33 mg/L/day, and the degradation rate would slow down as PCP concentration increased. At a PCP concentration of 320 mg/L, PCP degradation was completely inhibited, although an active population of Pseudomonas mendocina NSYSU was still present in these cultures. The study also indicated that the addition of various carbon sources such as sodium acetate and glucose did not facilitate the degradation of PCP with the degradation rate of 8.11 mg/L/day for sodium acetate, and that of 7.55 mg/L/day for glucose. Analysis from examining several environmental factors showed that the optimal condition for PCP degradation is that of 30¢J, pH6, and in the presence of oxygen. The end products of PCP degradation were detected by GC-MS. After 6 days of incubation, PCP was gradually disappeared and the metabolic intermediate product, acetic acid was detected. The chloride ion concentration also increased by 21.8 mg/L, which is approximately equal to the original total chloride content in PCP (66% of chloride content). In conclusion, PCP could be effectively and completely degraded by Pseudomonas mendocina NSYSU.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T07:55:46Z (GMT). No. of bitstreams: 1 284574.pdf: 1944066 bytes, checksum: ea84b90ef1fb7bb6f36e7a84a4a6ad60 (MD5) / O alginato é um polissacarídeo existente na parede celular de algas marinhas marrons (Phaeophyceae) e que também pode ser sintetizado por Azotobacter vinellandii e Pseudomonas spp. As aplicações do alginato microbiano podem ser as mesmas do alginato extraído de algas. Na indústria de alimentos, pode ser utilizado como gelificante, emulsificante, estabilizante e espessante. Ainda pode ser utilizado como agente de encapsulação e na formulação de curativos interativos, na indústria farmacêutica. Como ingrediente em pastas de impressão têxtil, detergentes e shampoos na indústria química. Na área médica, os alginatos têm sido empregados para imobilização de células vivas, no tratamento de doenças hormonais em cobaias e na manufatura de pele artificial com melhores propriedades cicatrizantes. O Alginato bacteriano pode ser produzido para fins específicos, com uma melhora nas suas propriedades qualitativas permitindo novas aplicações desse polímero no mercado. O objetivo deste trabalho foi avaliar a produção de alginato por Pseudomonas mendocina em diferentes substratos. Foram realizadas culturas em frascos agitados, utilizando glicose, sacarose ou glicerol, como fonte de carbono. Os experimentos foram delineados a um planejamento experimental para cada fonte de carbono, avaliando as diferentes concentrações de substrato e nitrogênio e limitando-se o tempo de cultivo em 48 h. A condição aximizada para a produção de alginato, a partir de glicose, dentro das condições estudadas foi 100 g.L-1 de glicose e 0,5 g.L-1 de sulfato de amônio. A produtividade obtida nessa condição foi 0,33 g.L-1h-1. Para meios utilizando glicerol como substrato, a condição maximizada foi 40 g.L-1 de glicerol e 0,7 g.L-1 de sulfato de amônio. A produtividade obtida nessa condição foi 0,23 g.L-1h-1. Utilizando sacarose como substrato, a condição maximizada para a produção de alginato dentro das condições estudadas foi 80 g.L-1 de sacarose e 0,7 de sulfato de amônio. Nas condições otimizadas utilizadas neste trabalho, o meio com sacarose como fonte de carbono apresentou a maior produtividade, 0,42 g.L-1h-1, para a síntese de alginato por Pseudomonas mendocina. O biopolímero formado foi caracterizado através da técnica de espectroscopia de ressonância magnética (1HNMR) e pode-se verificar que o alginato sintetizado por Pseudomonas mendocina possui apenas blocos M. Foram realizadas medidas reológicas do meio de cultura ao final do cultivo e todos apresentaram comportamento pseudoplástico. Meios de cultivo com o alginato formado e utilizando sacarose como fonte de carbono, apresentaram uma viscosidade aparente muito superior em relação aos meios onde foi utilizado glicose ou glicerol. / Alginate is a polysaccharide that occurs as a structural components in marine brown algae (Phaeophyceae) as well as capsular polysaccharides in two bacterial genera, Pseudomonas and Azotobacter. Alginate produced by microorganisms can have the same use of alginate obtained from brown algae. In food industry, can be used as thickeners, emulsifiers, stablizers and gelling agents. Can be used as encapsulation agent and in formulation of dressings interactive, in pharmaceutical industry. As an ingredient in textil printing, detergent and shampoos in the chemical industry. In medical applications, to immobilizing living cells, as in treatment of hormonal diseases in experimental animal models and to manufacture wound dressing with improved healing properties. Bacterial alginate can be produced to specific applications with highquality, improved properties alowing new commercial aplications. This work was aimed to study the alginate production by Pseudomonas mendocina with different substrates as carbon source. Cultures were carried out on shake frasks with glucose, sucrose or glycerol as carbon source. The experiments were fitting by the Methodology of Experiment Planning for each one substrate to evaluate different substrate and nitrogen concentration in 48 h on process productivity. Optimal conditions for alginate production from glucose as carbon source, was 100 g.L-1 glucose and 0.5 g.L-1 ammonium sulfate, the maximum productivity was 0.33 g.L-1h-1. Using glycerol as carbon source, the optimal condition was 40 g.L-1 glycerol and 0.7 g.L-1 ammonium sulfate, the productivity found was 0.23 g.L-1h-1. Using sucrose as carbon source, the optimal condition for alginate production was 80 g.L-1 sucrose and 0.7 ammonium sulfate. Optimized conditions studied in this work, showed higher productivity (0.42 g.L-1h-1) in culture media containing sucrose as carbon source for alginate synthesis by Pseudomonas mendocina. The biopolymer was characterized by 1HNMR spectroscopy and could be verified that alginate synthesized by Pseudomonas mendocina only has homopolymerics M blocks. The culture media containing alginate was characterized by rheological measurements and exhibited the typical pseudoplastic behavior. Culture media containing sucrose as carbon source exhibited a higher apparent viscosity than culture media containing glucose or glycerol as carbon source.

The century poison ¡§dioxins¡¨ are hydrophobic compounds that can combine with many organic matters and persist in the environment as well as to accumulate in living organisms. Dioxins caused great risk to the health of living organisms and to the entire ecological environment. We had isolated previously one bacterial species, Pseudomonas mendocina NSYSU, which can use pentachlorophenol (PCP) as its sole carbon source and degrade dioxin compounds. In order to study the feasibility of using this bacterial strain to bioremediate an PCDD/Fs polluted site, four microcosm experiment groups were designed to test the degradation efficiency of this strain: sterile soil group, non-sterile soil group, soya lecithin group and non-sterile soil with soya lecithin group. In addition, we also analyzed the shift of community structure of each microcosm by PCR-DGGE. The results show that the soya lecithin group has the highest efficiency to degrade OCDD/OCDF. After fifty days of reaction, the degradation rates of OCDD/OCDF were 62% and 47% respectively. The microbial diversity analysis indicated that the soya lecithin group presented less abundant from the initial stage, but increasing gradually over time. This might related to the formation of micelles in water phase which contained higher concentration of PCDD/Fs dissolved from the soil particles. Therefore, soya lecithin not only can reduce the toxicity of PCDD/Fs, but also can enhance the bioavailability of the organic pollutants to the microorganisms. In conclusion, monitoring the transition of P. mendocina NSYSU as well as the microbial diversity can provide valuable information during the bioremediation process by applying soya lecithin.