Thesis (Master)--İzmir Institute of Technology, İzmir, 2004. / Includes bibliographical references (leaves. 65-74).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "May 2000." Typescript. Vita. Includes bibliographical references (leaves 112-121). Also available on the Internet.

Orientador: Telma Teixeira Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-25T04:11:32Z (GMT). No. of bitstreams: 1 Tagliari_CristianeVanessa_M.pdf: 4616067 bytes, checksum: 69983d195fdc3c5a4db642ac7b419d83 (MD5) Previous issue date: 1999 / Resumo: Para reduzir os problemas ambientais, a indústria de papel e celulose vem buscando alternativas para seus processos de branqueamento através da tecnologia enzimática. Desta forma, xilanases tem sido empregadas durante o processo de pré-branqueamento de polpas Kraft, com intuito de diminuir a carga de cloro utilizada nas etapas subseqüentes. Xilanases ativas a altas temperaturas (acima de 45 °C) e pH alcalino possuem elevado potencial de aplicação em branqueamento de polpas de papel, já que podem ser introduzidas livremente nos diferentes estágios do processo sem a necessidade de drásticas etapas de resfriamento ou ajustes de pH. Bactérias são aptas a produzir enzimas que atuam nestas condições, tornando atrativo o estudo da produção de xilanase por Bacillus pumilus. Para viabilizar a aplicação de xilanases em larga escala é necessário que esta seja obtida com alta produtividade e baixo custo. Neste trabalho foram investigadas as condições de produção de xilanases alcalinas por B. pumilus em fermentação submersa. O meio de cultivo foi estudado por planejamento estatístico, objetivando maximizar a produção de xilanases em frascos agitados. Após determinar as melhores condições de cultivo, um biorreator com volume útil de 2L foi utilizado para produzir a quantidade necessária de enzima para aplicação nos testes de branqueamento. A maior produção da enzima em frascos agitados (129 U/mL) foi verificada no ponto central do segundo planejamento experimental em 20 horas de fermentação, o qual apresentava 3% xilana, 0,6% peptona, 0,15% sulfato de amónio e pH 9,5. Foi verificado que a produtividade aumentou (170 U/mL em 10 horas) quando a enzima foi produzida em biorreator de 2 L, evidenciando o potencial do B. pumilus para a produção de xilanases alcalinas e termofílicas. Quanto a aplicação da enzima no pré-branqueamento de polpas Kraft, foi verificado uma deslignificação de 26% quando a polpa foi tratada com xilanases na dosagem de 50U/g polpa seca, resultado superior aos encontrados na literatura. / Abstract: Environmental concerns, have put a restriction on the usage of chlorine during bleaching process in the paper and pulp industry. Therefore, xylanase pretreatment has been used to lower bleaching chemical consumption. Enzymes which are active under alkaline conditions and higher temperatures, have great potential for industrial application, such as the bleaching process, without any need for cooling or changes in pH and with the advantage in lowering the release of polluting organic chlorine compounds. Bactérias are able to produce enzymes that act in these conditions, than being attractive xylanase production by Bacillus pumilus. For industrial large scale aplication, the enzyme should be obtained with high productivity and low cost. This work investigated the optimal conditions of alkaline xylanases produced by B. pumilus in submerged fermentation. The culture media was studied by statistical factorial design, aiming to increase the xilanases production. The best conditions were applied in 2L bioreactor to produce the enzyme necessary for application in bleaching process. The highest xylanase production in culture flasks (129 U/mL) was obtained at central point of the second factorial design in 20 hours of fermentation, which contained 3% xylan, 0.6% peptone, 0.15% ammonium sulfate and pH 9.5. It was observed that the productivity increased (170 U/mL in 10 hours) when the enzyme was produced in 2 L biorreator, evidencing the potential of the B. pumilus for the production of thermophilic and alkaline xylanases. The effect of xylanase treatment (50U/g pulp) on hardwood pulp was significant. A decrease of 2.5 units in kappa number was achieved (26% of delignification) suggesting that the xylanase from B. pumilus has a great potential in kraft pulp bleaching. / Mestrado / Mestre em Engenharia Química

Orientador : Fumio Yokoya / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-16T22:24:44Z (GMT). No. of bitstreams: 1 RodriguezDeMassaguer_Pilar_M.pdf: 18942674 bytes, checksum: d2a1f007b2c7334faf9215e3e2c960c8 (MD5) Previous issue date: 1977 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Mestre em Ciência de Alimentos

Los explosivos son considerados contaminantes por sus características xenobióticas y constituyen un riesgo para la salud porque algunos son tóxicos y carcinogénicos. Entre los más energéticos y recalcitrantes está el pentaeritritol tetranitrato (PETN) por sus enlaces éster de difícil degradación. Dado que este explosivo es ampliamente usado en la explotación minera, petrolera y en otras actividades en el Perú, en el presente estudio se evaluó la capacidad degradativa de PETN por bacterias aisladas de ambientes mineros con la perspectiva de desarrollar técnicas de mitigación de la contaminación y riesgo por explosivos. Se identificaron las cepas J8A2 y M1B como Bacillus sp. y Enterobacter sp., respectivamente, las cuales demostraron la mayor eficiencia de degradación de PETN. En la cinética de degradación se obtuvo una velocidad específica de crecimiento de 0.057 h-1 para J8A2 y 0.042 h-1 para M1B, y una velocidad específica de degradación de 0.72 mmol de PETN/g de proteína.h y 1.16 mmol de PETN/g de proteína.h, respectivamente. Sin embargo, el rendimiento por sustrato de J8A2 fue de 39.7 ± 2.1 g de proteína/mol de PETN y de M1B 21.56 ± 1.5 g de proteína/mol de PETN. Además, el extracto crudo de la cepa J8A2 mostró una actividad específica (U) de 1.03 mmol de PETN/h y una actividad específica por proteína (U/g) de 138.6 mmol de PETN/g.h. En conclusión, estos resultados demuestran la presencia de bacterias degradadoras de PETN en ambientes mineros con potencial para ser empleadas en procesos de biorremediación. Palabras clave: Pentaeritritol tetranitrato, contaminación, minería, biodegradación, Bacillus sp., Enterobacter sp. / Explosives are considered pollutants for their xenobiotic characteristics. They constitute a health hazard because some of them are toxic and carcinogenic. Pentaerythritol tetranitrate (PETN) is included among the most energetic and xenobiotic explosive for its ester bonds that are difficult to degrade. As this explosive is widely used in mining, oil and other activities in Peru, in the present study we evaluated PETN degradation capacity of bacterial strains isolated from mining environments to develop techniques to mitigate pollution and explosives risk. J8A2 and M1B strains were identified as Bacillus sp. and Enterobacter sp. respectively, and demonstrated a great PETN degradation efficiency. Degradation kinetics results showed specific growth rate of 0.057 h-1 for J8A2 and 0.042 h-1 for M1B, and specific rate of PETN degradation of 0.72 mmol of PETN/g of protein.h and 1.16 mmol of PETN/g of protein.h, respectively. However, the growth yield of J8A2 was 39.7 ± 2.1 g of protein/mol of PETN and the growth yield of M1B was 21.56 ± 1.5 g of protein/mol of PETN. Furthermore, crude extract of J8A2 strain showed a specific activity (U) of 1.03 mmol of PETN/h and a specific activity per protein (U/g) of 138.6 mmol of PETN/g.h. In conclusion, these findings demonstrated the presence of PETN degrading bacteria in mining environments with potential to be applied in bioremediation processes. Keywords: Pentaerythritol tetranitrate, contamination, mining, biodegradation, Bacillus sp., Enterobacter sp. / Tesis

A casein agar diffusion method was developed to detect and quantify pertinent levels of proteinases produced in raw milk supplies by heat resistant Bacillus sporeformers. In order to optimize the required heat treatment conditions of raw milk samples, trials that involved a combination of different temperatures and times were evaluated. A heat treatment of 75°C for 20 min was the most effective for recovering the highest number of surviving spores. A sporulation broth containing five different minerals and supplemented with 0.2% nonfat dry milk was used to maximize spore production in all heat-treated samples. A β-casein based assay detected proteinase activity from raw milk samples that ranged from 0.093 to 4.034 units/mg which corresponded to zones of β-casein precipitation in the β-casein agar of 5.0 and 15.0 mm respectively, and was compared to Protease Type VIII (from B. licheniformis). This assay correlated well with the fluorescein isothiocyanate casein-labeled assay (FITC), R=0.995 (Protease Type VIII). Proteases of Bacillus origin such as Protease Type IX, X, XV and XXXI were also evaluated but were rejected in favor of a broader range of activity expressed by Protease Type VIII. For an initial set of 370 raw milk samples, no quality deterioration, such as coagulation or bitter taste was observed in heat-treated (75°C for 20 min) and incubated samples (7.2°C for 10 days). However, during the winter season, 18 of 75 incubated samples (7.2°C for 10 days) tasted slightly bitter and exhibited a slight degree of casein precipitation. One sample coagulated but exhibited no proteinase activity on the β-casein agar gel, hence it was considered a false negative. The positive results for proteinase activity from raw Grade A samples tested by the β-casein agar diffusion method did not correlate either with fresh spore counts (R=0.21) or post-heat treatment incubation counts (R=0.03) or with psychrotrophic sporeformer counts (R=0.06). The β-casein agar diffusion method is simple, rapid and sensitive to Bacillus spp. proteinases, but was unreliable in projecting results related to the psychrotrophic sporeformer count. Consequently, further research is required to establish optimum conditions (time and/or temperature) and inoculum volumes into sporulation broth for attainment of a more positive correlation between β-casein agar precipitation zones and psychrotrophic sporeformer populations of either raw or processed milk samples. / Graduation date: 1991

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a fermentable growth substrate (i.e. sucrose) from pH 7.5 to 10.0 with no significant change in specific growth rate, suggesting that this bacterium is a facultative alkaliphile. However, when strain TA2.A1 was grown on non-fermentable carbon sources like succinate, no growth was observed until the external pH was > 9.0, suggesting this bacterium is an obligate alkaliphile. Growth on succinate at pH 9.5 was sensitive to both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin revealing that both the proton and sodium motive force ([Delta][mu][H⁺] and [Delta][mu][Na⁺], respectively) were obligate requirements for growth at alkaline pH values. Transport of succinate was driven by a chemical gradient of Na⁺ ([Delta]pNa⁺) that was strictly coupled to [Delta][Psi]. A single transport system was detected for the uptake of succinate, with an apparent K[m] of 19 [mu]M and V[max] of 0.45 nmol succinate/min/mg protein. Succinate transport was pH-dependent, and showed optimal activity at pH values greater than 8.5. Other C₄-dicarboxylates (e.g. malate, fumarate) inhibited the uptake of succinate suggesting that the permease was general for other C₄-dicarboxylates. Cytochrome content, succinate dehydrogenase oxidoreductase, and F₁F₀-ATPase activities were lower in membranes from strain TA2.A1 cells grown at pH 7.5 compared to those cultured at 9.5. These data suggest that oxidative phosphorylation-linked processes are down-regulated at neutral pH values, an observation that mirrored oxygen consumption profiles of strain TA2.A1 in whole cells. To study this phenomenon at a molecular level, we measured ATP synthesis by the F₁F₀-ATP synthase from strain TA2.A1 as a function of pH. The strain TA2.A1 F₁F₀-ATP synthase had a pH optimum for ATP synthesis of 9.0-9.5, and significantly lower rates of ATP synthesis observed below pH 9.0. Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. strain TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. strain TA2.A1 numbering) within the a subunit of these F₁F₀-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F₁F₀) was developed in Escherichia coli DK8 ([Delta]atp). Amino acid substitutions were made in the a subunit of TA2F₁F₀ at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P[i]-loaded right-side out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F₁F₀ and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0 to 9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH�s 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue K180 is a specific adaptation within the a subunit of TA2F₁F₀ to facilitate proton capture at high pH. At pH values near the pK[a] of K180, the trapped protons readily dissociate to reach the subunit c binding sites but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H⁺ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited. The mechanisms where by alkaliphilic bacteria obtain iron remains unknown. Growth of strain TA2.A1 at pH 9.5 in the presence of the artificial iron chelators ethylenediamine O-hydroxyphenylacetic acid (EDDHA) and 2�2� dipyridal revealed that iron is an important requirement for aerobic growth at alkaline pH values. Furthermore, biochemical analysis showed that Bacillus alcalophilus and Bacillus pseudofirmus both synthesized orange catecholate siderophores, whilst Bacillus halodurans synthesized a hydroxamate siderophore. These tests showed that strain TA2.A1 synthesized both orange catecholate and hydroxamate siderophore/s. Attempts to purify the catecholate were unsuccessful. No homologues of previously identified non-ribosomal peptide synthase (NRPS) genes in Bacillus subtilis and B.halodurans were detected in the genome of strain TA2.A1 using both PCR and Southern hybridization using known non-ribosomal peptide synthase genes.

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a fermentable growth substrate (i.e. sucrose) from pH 7.5 to 10.0 with no significant change in specific growth rate, suggesting that this bacterium is a facultative alkaliphile. However, when strain TA2.A1 was grown on non-fermentable carbon sources like succinate, no growth was observed until the external pH was > 9.0, suggesting this bacterium is an obligate alkaliphile. Growth on succinate at pH 9.5 was sensitive to both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin revealing that both the proton and sodium motive force ([Delta][mu][H⁺] and [Delta][mu][Na⁺], respectively) were obligate requirements for growth at alkaline pH values. Transport of succinate was driven by a chemical gradient of Na⁺ ([Delta]pNa⁺) that was strictly coupled to [Delta][Psi]. A single transport system was detected for the uptake of succinate, with an apparent K[m] of 19 [mu]M and V[max] of 0.45 nmol succinate/min/mg protein. Succinate transport was pH-dependent, and showed optimal activity at pH values greater than 8.5. Other C₄-dicarboxylates (e.g. malate, fumarate) inhibited the uptake of succinate suggesting that the permease was general for other C₄-dicarboxylates. Cytochrome content, succinate dehydrogenase oxidoreductase, and F₁F₀-ATPase activities were lower in membranes from strain TA2.A1 cells grown at pH 7.5 compared to those cultured at 9.5. These data suggest that oxidative phosphorylation-linked processes are down-regulated at neutral pH values, an observation that mirrored oxygen consumption profiles of strain TA2.A1 in whole cells. To study this phenomenon at a molecular level, we measured ATP synthesis by the F₁F₀-ATP synthase from strain TA2.A1 as a function of pH. The strain TA2.A1 F₁F₀-ATP synthase had a pH optimum for ATP synthesis of 9.0-9.5, and significantly lower rates of ATP synthesis observed below pH 9.0. Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. strain TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. strain TA2.A1 numbering) within the a subunit of these F₁F₀-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F₁F₀) was developed in Escherichia coli DK8 ([Delta]atp). Amino acid substitutions were made in the a subunit of TA2F₁F₀ at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P[i]-loaded right-side out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F₁F₀ and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0 to 9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH�s 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue K180 is a specific adaptation within the a subunit of TA2F₁F₀ to facilitate proton capture at high pH. At pH values near the pK[a] of K180, the trapped protons readily dissociate to reach the subunit c binding sites but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H⁺ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited. The mechanisms where by alkaliphilic bacteria obtain iron remains unknown. Growth of strain TA2.A1 at pH 9.5 in the presence of the artificial iron chelators ethylenediamine O-hydroxyphenylacetic acid (EDDHA) and 2�2� dipyridal revealed that iron is an important requirement for aerobic growth at alkaline pH values. Furthermore, biochemical analysis showed that Bacillus alcalophilus and Bacillus pseudofirmus both synthesized orange catecholate siderophores, whilst Bacillus halodurans synthesized a hydroxamate siderophore. These tests showed that strain TA2.A1 synthesized both orange catecholate and hydroxamate siderophore/s. Attempts to purify the catecholate were unsuccessful. No homologues of previously identified non-ribosomal peptide synthase (NRPS) genes in Bacillus subtilis and B.halodurans were detected in the genome of strain TA2.A1 using both PCR and Southern hybridization using known non-ribosomal peptide synthase genes.

Thesis (M.Sc.) -- University of Adelaide, Dept. of Biochemistry, 1981.