Graduation date: 1993

Orientador: Marcelo Cristianini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T20:56:17Z (GMT). No. of bitstreams: 1 Senatore_MarcelaAndreaDuranHaun_M.pdf: 684290 bytes, checksum: 522970fe79261c7d2890c0a19a39b587 (MD5) Previous issue date: 2004 / Resumo: Um dos problemas que enfrenta a indústria de laticínios é a recontaminação do leite decorrente da formação de biofilmes em tanques de armazenamento e trocadores de calor. A tecnologia de esterilização de materiais por gás plasma tem sido utilizada com sucesso na esterilização de instrumentos cirúrgicos, oftálmicos e dentários. Os objetivos deste trabalho foram avaliar a eficiência de um sistema de esterilização a gás plasma sob células de Pseudomonas fluorescens aderidas em placas de aço inoxidável utilizando o leite como substrato. Foram avaliadas as variáveis tempo de pré plasma, tempo de exposição ao plasma e potência do mesmo na remoção do biofilme. As placas de aço inoxidável com a bactéria aderida foram submetidas a um tratamento com gás plasma, que foi formado a partir de um produto comercial composto por ácido peracético, peróxido de hidrogênio e ácido acético. Dois sistemas modelo foram utilizados para simular a formação de biofilmes de Pseudomonas fluorescens, um dinâmico e outro estático, simulando trocadores de calor e tanques de armazenamento, respectivamente. Através do modelo estático foi possível obter contagens acima de 105 UFC por placa de aço inoxidável, mesmo após três ciclos de lavagem das placas em água estéril sob agitação. Foi observado um efeito positivo na inativação de P. fluorescens em função do tempo de pré-plasma do sanificante. Exposições acima de 7 minutos foram capazes de produzir reduções superiores a dois ciclos logarítmicos na inativação do microrganismo. Através de um planejamento experimental fatorial 23 foi demosntrado que as variáveis tempo de pré-plasma, tempo de exposição ao plasma e potência do plasma apresentaram efeitos positivos na inativação de Pseudomonas fluorescens. O tempo de exposição (min.) apresentou o maior efeito na destruição da bactéria; mas sendo um pouco superior à potência do plasma (w) / Abstract: One of the problems that food industry is facing is the milk recontamination through biofims formation on storage tanks and heat exchanger. The technology of sterilization for materials through gas plasma has been used with succesfull for cirurgycal, ophathalmic and dentistry equipment. The goals of this work was to evaluate the efficiency of a gas plasma sterilization system on Pseudomonas fluorescens cells adhered on stainless steel plates using milk as substrate. Time of pre plasma, plasma exposition and potency (Watts) were evaluated as independent variables on cell destruction. The stainless steel plates were submitted to gas plasma treatment originated from a commercial product composed of peracetic acid, hydrogen peroxide and acetic acid. Two model systems were used to simulate a biofilm formation of Pseudomonas fluorescens, one dynamic and one static, in order to simulate heat exchangers and storage tanks, respectively. Through static model it was possible to get counts over 105 UFC/plate after washing three times using sterile water under stirring conditions. It was observed a positive effect on P. fluorescens inactivation by pre-plasma in a time dependent way. Expositions over seven minutes were capable to produce reductions higher than two logarithmic cycles on microorganism inactivation. A 23 factorial design indicated that the pre-plasma time, time exposition and potency showed positive effects on Pseudomonas fluorescens inactivation. Time exposition (min) was the most effective variable on bacteria destruction, being a little higher than plasma potency (w) / Mestrado / Mestre em Tecnologia de Alimentos

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is one of a group of bacteria known as cyanotrophs that exhibit the unique ability to grow on toxic cyanide as the sole nitrogen source. This ability has previously been genetically linked to a conserved cluster of seven genes (Nit1C), the signature gene (nitC) coding for a nitrilase enzyme. Nitrilases convert nitriles to ammonia and a carboxylic acid. Still, for the Pf11764 NitC enzyme (Nit11764), no in vivo substrate has been identified, and the basis of the enzyme's requirement for cyanide growth has remained unclear. Therefore, the gene was cloned and the enzyme was characterized with respect to its structure and function. These efforts resulted in the unique discovery that, aside from its nitrilase activity, Nit11764 exhibits nuclease activity towards both DNA and RNA. This ability is consistent with computer analysis of the protein providing evidence of a preponderance of amino acids with a high probability for RNA binding. A Nit11764 knock-out mutant was shown to exhibit a higher sensitivity to both cyanide (KCN) and mitomycin C, both known to induce chromosomal damage. Thus, the overall conclusion is that Nit11764, and likely the entire Nit1C gene cluster, functions as a possible repair mechanism for overcoming the damage inflicted on Pf11764 nucleic acids by toxic cyanide. Towards a further investigation of the Nit1C gene cluster in Pf11764, a second gene (nitH) annotated as a monooxygenase was also investigated. Interestingly, computer-based analysis shows that NitH also harbors a preponderance of RNA-binding amino acids. The nitH gene was cloned into an expression vector with the long-range goal of defining its role in CN utilization.

Call number: LD2668 .T4 1960 K55

Broccoli head rot is a destructive disease found in most broccoli production areas. The main pathogen is the bacterium Pseudomonas fluorescens. P. fluorescens 5064, which was first isolated from an infected broccoli head in SE Scotland, produces biosurfactants that are important for bacterial establishment on the plant surface prior to causing disease in broccoli. Preliminary experiments performed in this study showed that biosurfactant production in P. fluorescens 5064 was cell density dependent, which is a typical characteristic of the quorum sensing mechanism. Quorum sensing is a bacterial communication mechanism, which controls a number of key processes in growth, reproduction and virulence via signalling molecules (quorum sensing signal) in many gram-negative bacteria. One aim of this study was to determine if biosurfactant production in P. fluorescens 5064 is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of P. fluorescens 5064 were screened for their abilities to produce a quorum sensing signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum sensing signal production. In one mutant 6423, which contains a single Tn5 insertion, the production of the quorum sensing signal was almost eliminated. Addition of quorum sensing signal, either synthetic or extracted from wild type P. fluorescens 5064, was able to restore biosurfactant production in mutant 6423. This strongly suggests that quorum sensing regulates biosurfactant production in P. fluorescens 5064. Attempts were made to clone and sequence the Tn5 disrupted gene in mutant 6423, but the identity of the gene remains inconclusive. The quorum sensing signal in wild type P. fluorescens 5064 was identified in this study by High Pressure Liquid Chromatography and Mass Spectrometry as N-3-hydroxyoctanoyl-homoserine lactone, which has been shown by other researchers to be present in P. fluorescens strain 2-79, but not in the strains F113, 7-14 and NCIMB 10586. The discovery that biosurfactant production in P. fluorescens 5064 is regulated by quorum sensing opens up a possibility for novel control of broccoli head rot. Although only the control of biosurfactant production by quorum sensing was examined in this study, it is possible that other virulence factors, such as pectic enzyme production, are also controlled by quorum sensing as in other pathogenic bacteria. By blocking the quorum sensing system, the pathogenic P. fluorescens that use this mechanism to control virulence could potentially be rendered avirulent. In greenhouse pathogenicity tests, a quorum sensing signal-degrading bacterium, Bacillus sp. A24, was evaluated for biocontrol of head rot disease caused by P. fluorescens 5064 on broccoli. However, the Bacillus sp. A24 showed only limited control effects, despite its strong quorum sensing signal-degrading ability towards the pathogen in vitro. A subsequent test proved that Bacillus sp. A24 is a surfactant producer itself and this could explain its ineffectiveness in disease control. When screening the quorum sensing signals of the 35 biosurfactant mutants, mutant 6418 was found to produce a potent antibiotic-like compound. This was identified by thin-layer chromatography as pyrrolnitrin. Unlike wild-type P. fluorescens 5064, mutant 6418 has lost its ability to produce virulence factors and is thus non-pathogenic. It was therefore of interest to determine if mutant 6418 could be used as a biocontrol agent to control broccoli head rot disease. In greenhouse pathogenicity tests, mutant 6418 significantly reduced disease by 41 %. The practical application of this research to bacterial disease control – via the manipulation of quorum sensing to inhibit virulence gene expression – is discussed.

Biodegradation of poorly water soluble hydrocarbons, such as n-alkanes and polycyclic aromatic hydrocarbons (PAHs) is often limited by the low availability of the pollutant to microbes. Adhesion of microorganisms to the oil-water interface can influence this availability. Our approach was to study a range of compounds and mechanisms to promote the adhesion of a hydrophilic PAH degrading bacterium, Pseudomonas fluorescens LP6a, to an oil-water interface and examine the effect on biodegradation of phenanthrene by the bacteria. The cationic surfactants cetylpyridinium chloride (CPC), poly-L-lysine and chlorhexidine gluconate (CHX) and the long chain alcohols 1-dodecanol, 2-dodecanol and farnesol increased the adhesion of P. fluorescens LP6a to n-hexadecane from ca. 30% to ca. 90% of suspended cells adhering. The alcohols also caused a dramatic change in the oil-water contact angle of the cell surface, increasing it from 24° to 104°, whereas the cationic compounds had little effect. In contrast, cationic compounds changed the electrophoretic mobility of the bacteria, reducing the mean zeta potential from –23 to –7 mV in 0.01M potassium phosphate buffer, but the alcohols had no effect on zeta potential. This results illustrate that alcohols acted through altering the cell surface hydrophobicity, whereas cationic surfactants changed the surface charge density. Phenanthrene was dissolved in heptamethylnonane and introduced to the aqueous growth medium, hence forming a two phase system. Introducing 1-dodecanol at concentrations of 217, 820 or 4100 mg/L resulted in comparable increases in phenanthrene biodegradation of about 30% after 120 h incubation with non-induced cultures. After 100 h of incubation with LP6a cultures induced with 2-aminobenzoate, 4.5% of the phenanthrene was mineralized by cultures versus more than 10% by the cultures containing initial 1-dodecanol or 2-dodecanol concentrations of 120 or 160 mg/L. The production and accumulation of metabolites in the aqueous phase responded similarly to the addition of 1-dodecanol. Further experiments showed that the positive influence of the alcohols could not be attributed to the changes in surface and interfacial tension or increase in biomass concentration. The results suggest that enhanced adhesion of bacterial cells to the oil-water interface was the main factor responsible for the observed increase in phenanthrene biodegradation by P. fluorescens LP6a. / Chemical Engineering

Thesis (M.S.)--University of North Carolina at Wilmington, 2003. / Includes bibliographical references (leaves : 63-66).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves [207]-216).

No description available.

The adhesion of seven meat spoilage bacteria to model meat surfaces (thin fat and tendon slices) was studied in a specially designed flow chamber. In general, adhesion was not influenced by the physiological age of the cells and was not correlated with the cell surface characteristics (electrical charge, hydrophobicity) of the organisms. Also, adhesion data did not corroborate the predictions based on changes in the free energy of adhesion, calculated from contact angles and surface tension measurements. A more detailed study of the adhesion of Pseudomonas fluorescens to tendon was subsequently undertaken. Neither physiological activity nor the presence of flagella was found to be essential for adhesion. Selective chemical alterations of the cell surface pointed to no direct implication of carboxyl or amino groups in an adhesive bond with tendon. These groups may participate in adhesion, however, through electrostatic interactions as suggested from the variations of adhesion with ionic strength.