The endophytic fungus Aspergillus niger isolated from specie plant Hancornia speciosa, commonly known as mangabeira, was cultivated in different culture media and used for identification of six substances in their extracts, such as oxalic, citric, tartaric, p-acetylbenzoic, hexylitaconic acids and phyrofen. For quantification of pyrophen in different extracts, it was developed a method by High Performance Liquid Chromatography that was efficient for both extract analysis and fermented broth. The method allowed to evaluate the effect of different media ways and culture time on the pyrophen. The analyzed media for pyrophen production were mangaba juice, Czapeck and potato dextrose broth (PDB) and the highest yield was obtained using mangaba juice. The effect of culture time was evaluated by cultivating the fungus in the PDB medium, being the ideal period of fermentation for pyrophen production was 12 days. / O fungo endofítico Aspergilus niger isolado da espécie vegetal Hancornia speciosa, conhecida popularmente como mangabeira, foi cultivado nos meios suco de mangaba, Czapeck e Caldo de Batata Dextrose (CDB) e levou ao isolamento e identificação de seis substâncias em seus extratos, sendo elas os ácidos oxálico, cítrico, tartárico, p-acetil-benzóico e hexilitacônico e a substância pirofeno. Visando encontrar a melhor condição de cultivo para produção de pirofeno foi desenvolvido um método analítico por Cromatografia Líquida de Alta Eficiência para análise desta substância. O método desenvolvido foi eficiente tanto para análise dos extratos quanto para análise do caldo fermentado e permitiu avaliar o efeito dos diferentes meios e do tempo de cultivo na produção desta substância. A maior produção de pirofeno foi obtida utilizando suco de mangaba como meio de cultivo e o período ideal de fermentação para a produção de pirofeno foi de 12 dias, quando cultivado no meio líquido CDB.

Made available in DSpace on 2016-01-26T18:56:23Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 324266 bytes, checksum: 7cb8130236c8d36814fc6baebf56c4e4 (MD5) Previous issue date: 2011-03-17 / Plants are always exposed to aggression of biotic and abiotic agents. Their protection pathway is essential, and it would increase the food production. Navy beans (Phaseolus vulgaris L) are of high economic importance and of agronomic value. It is a susceptible crop to temperature and to water stress, what could lead to metabolic alterations as the synthesis of protein defences. The objective of this work was to study the effect of salicylic acid and the cold shock applied to germinating seeds over the water deficit tolerance, during the beginning of the germination process by means of physiological variables and soluble protein expression. Seeds were embedded in moistened paper substrate, half of the sample in water and half in the salicylic acid (0.01mM) in the first 24 hours. Part of both treatments was then subjected to cold shock 24 hours at 7°C. After that all seeds, from both treatments were subjected to different mannitol induced water potentials: 0;-0.3; -0.6 and -1.2 MPa. Water was considered the zero potential. Four combinations were considered: without shock and salicylic acid (SCHSAS), with shock and without salicylic acid (CCHSAS), without shock with salicylic acid (SCHCAS) and with shock and salicylic acid (CCHCAS). Treatments were evaluated by germination, vigour classification, shoot and root dry weight and length, as by the rate shoot/root. After germination evaluation, five normal seedlings were used to protein and another five to proline extraction. Proline was determined in the whole seedling and protein in shoot and in the root, being compared by SDS-PAGE. Results were analyzed as a completely random design in a factorial design (4x4 as a combination of salicylic acid cold shock with the water restriction) by the Tukey s test (P< 5%) for average comparisons and with polynomial regression for water deficit. Electrophoretic patterns were analyzed by absence presence of bands. Germination was not affected by salicylic acid or by the cold shock treatments, but by the increase in the water deficit. Vigour was altered by salicylic acid and by the shock, being the last one affecting positively and the cold shock negatively. Salicylic acid increased length and weight, especially in the intermediary levels of water deficit. Protein expression of the treatments CCHCAS and SCHCAS showed the same electrophorectical patterns for shoot and root, with an increase in the expression for the lower potentials. The pattern of CCHSAS and SCHSAS treatments exhibited some different bands. / As condições ambientais desfavoráveis fazem com que os vegetais passem por adaptações e a forma como se protegem é essencial, podendo aumentar a produção e qualidade dos alimentos. O feijão (Phaseolus vulgaris L) é de grande valor econômico e de relevante interesse agronômico. Tratando-se de uma cultura de sensibilidade à temperatura e deficiência hídrica, podem ocorrer aclimatações e alterações no metabolismo celular, entre elas a síntese de proteínas de defesa. O objetivo deste trabalho foi estudar o efeito do ácido salicílico e do choque frio aplicados às sementes para a possível tolerância à restrição hídrica, durante o início do processo de germinação, por meio de variáveis fisiológicas e expressão de proteínas solúveis. As sementes foram embebidas em substrato papel a temperatura constante de 25°C em água ou solução aquosa correspondente a 2,5 vezes o peso do substrato seco, sendo parte das sementes em água e parte em solução de ácido salicílico à concentração de 0,01 Mm, pelas primeiras 24 horas. Metade das sementes embebidas em água como as embebidas em ácido salicílico foram transferidas para o choque frio, por 24 horas, à temperatura de 7°C. A seguir todas as sementes foram transferidas para um substrato simulando diferentes potenciais osmóticos: 0,0; -0,3; -0,6; -1,2 MPa induzidos por manitol, sendo considerado potencial zero o substrato umedecido com água, compondo quatro combinações sem choque sem ácido salicílico (SCHSAS), com choque sem ácido salicílico (CCHSAS), sem choque com ácido salicílico (SCHCAS) e com choque com ácido salicílico (CCHCAS). Os tratamentos foram avaliados por germinação, classificação de vigor, comprimento de parte aérea e raiz, massa seca da parte aérea e raiz e relação raiz/parte aérea. Após a avaliação da germinação, duas amostras de cinco plântulas normais, por repetição, foram utilizadas para determinação de prolina (plântula toda) e extração de proteínas totais e análise por eletroforese SDS PAGE de parte aérea e raiz. Os resultados foram analisados considerando o delineamento inteiramente casualizado em arranjo fatorial de 4x4 (combinação dos tratamentos com ácido salicílico e choque frio e níveis de deficiência de água no substrato), empregando-se Tukey (5% de probabilidade) para comparação de médias dos tratamentos qualitativos e análise de regressão polinomial para os níveis de deficiência. As análises de eletroforese foram avaliadas por imagem detectando-se presença e ausência de bandas. A germinação não foi influenciada pelos tratamentos com ácido salicílico e choque, mas diminuiu com a progressão da deficiência de água. O vigor foi alterado pelos tratamentos de ácido salicílico e choque, sendo que o tratamento com ácido salicílico influiu positivamente enquanto o choque negativamente. O tratamento com ácido salicílico proporcionou maiores comprimentos e massas secas de parte aérea e total das plântulas, principalmente nos níveis intermediários de deficiência de água. A expressão de proteínas dos tratamentos CCHCAS e SCHCAS revelou o mesmo padrão eletroforético tanto para parte aérea quanto para raiz com maior intensidade de expressão nos potenciais osmóticos menores. O perfil de bandas dos tratamentos CCHSAS e SCHSAS apresentou alteração revelando algumas bandas diferenciadoras.

Made available in DSpace on 2016-07-18T17:51:07Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 324266 bytes, checksum: 7cb8130236c8d36814fc6baebf56c4e4 (MD5) Previous issue date: 2011-03-17 / Plants are always exposed to aggression of biotic and abiotic agents. Their protection pathway is essential, and it would increase the food production. Navy beans (Phaseolus vulgaris L) are of high economic importance and of agronomic value. It is a susceptible crop to temperature and to water stress, what could lead to metabolic alterations as the synthesis of protein defences. The objective of this work was to study the effect of salicylic acid and the cold shock applied to germinating seeds over the water deficit tolerance, during the beginning of the germination process by means of physiological variables and soluble protein expression. Seeds were embedded in moistened paper substrate, half of the sample in water and half in the salicylic acid (0.01mM) in the first 24 hours. Part of both treatments was then subjected to cold shock 24 hours at 7°C. After that all seeds, from both treatments were subjected to different mannitol induced water potentials: 0;-0.3; -0.6 and -1.2 MPa. Water was considered the zero potential. Four combinations were considered: without shock and salicylic acid (SCHSAS), with shock and without salicylic acid (CCHSAS), without shock with salicylic acid (SCHCAS) and with shock and salicylic acid (CCHCAS). Treatments were evaluated by germination, vigour classification, shoot and root dry weight and length, as by the rate shoot/root. After germination evaluation, five normal seedlings were used to protein and another five to proline extraction. Proline was determined in the whole seedling and protein in shoot and in the root, being compared by SDS-PAGE. Results were analyzed as a completely random design in a factorial design (4x4 as a combination of salicylic acid cold shock with the water restriction) by the Tukey s test (P< 5%) for average comparisons and with polynomial regression for water deficit. Electrophoretic patterns were analyzed by absence presence of bands. Germination was not affected by salicylic acid or by the cold shock treatments, but by the increase in the water deficit. Vigour was altered by salicylic acid and by the shock, being the last one affecting positively and the cold shock negatively. Salicylic acid increased length and weight, especially in the intermediary levels of water deficit. Protein expression of the treatments CCHCAS and SCHCAS showed the same electrophorectical patterns for shoot and root, with an increase in the expression for the lower potentials. The pattern of CCHSAS and SCHSAS treatments exhibited some different bands. / As condições ambientais desfavoráveis fazem com que os vegetais passem por adaptações e a forma como se protegem é essencial, podendo aumentar a produção e qualidade dos alimentos. O feijão (Phaseolus vulgaris L) é de grande valor econômico e de relevante interesse agronômico. Tratando-se de uma cultura de sensibilidade à temperatura e deficiência hídrica, podem ocorrer aclimatações e alterações no metabolismo celular, entre elas a síntese de proteínas de defesa. O objetivo deste trabalho foi estudar o efeito do ácido salicílico e do choque frio aplicados às sementes para a possível tolerância à restrição hídrica, durante o início do processo de germinação, por meio de variáveis fisiológicas e expressão de proteínas solúveis. As sementes foram embebidas em substrato papel a temperatura constante de 25°C em água ou solução aquosa correspondente a 2,5 vezes o peso do substrato seco, sendo parte das sementes em água e parte em solução de ácido salicílico à concentração de 0,01 Mm, pelas primeiras 24 horas. Metade das sementes embebidas em água como as embebidas em ácido salicílico foram transferidas para o choque frio, por 24 horas, à temperatura de 7°C. A seguir todas as sementes foram transferidas para um substrato simulando diferentes potenciais osmóticos: 0,0; -0,3; -0,6; -1,2 MPa induzidos por manitol, sendo considerado potencial zero o substrato umedecido com água, compondo quatro combinações sem choque sem ácido salicílico (SCHSAS), com choque sem ácido salicílico (CCHSAS), sem choque com ácido salicílico (SCHCAS) e com choque com ácido salicílico (CCHCAS). Os tratamentos foram avaliados por germinação, classificação de vigor, comprimento de parte aérea e raiz, massa seca da parte aérea e raiz e relação raiz/parte aérea. Após a avaliação da germinação, duas amostras de cinco plântulas normais, por repetição, foram utilizadas para determinação de prolina (plântula toda) e extração de proteínas totais e análise por eletroforese SDS PAGE de parte aérea e raiz. Os resultados foram analisados considerando o delineamento inteiramente casualizado em arranjo fatorial de 4x4 (combinação dos tratamentos com ácido salicílico e choque frio e níveis de deficiência de água no substrato), empregando-se Tukey (5% de probabilidade) para comparação de médias dos tratamentos qualitativos e análise de regressão polinomial para os níveis de deficiência. As análises de eletroforese foram avaliadas por imagem detectando-se presença e ausência de bandas. A germinação não foi influenciada pelos tratamentos com ácido salicílico e choque, mas diminuiu com a progressão da deficiência de água. O vigor foi alterado pelos tratamentos de ácido salicílico e choque, sendo que o tratamento com ácido salicílico influiu positivamente enquanto o choque negativamente. O tratamento com ácido salicílico proporcionou maiores comprimentos e massas secas de parte aérea e total das plântulas, principalmente nos níveis intermediários de deficiência de água. A expressão de proteínas dos tratamentos CCHCAS e SCHCAS revelou o mesmo padrão eletroforético tanto para parte aérea quanto para raiz com maior intensidade de expressão nos potenciais osmóticos menores. O perfil de bandas dos tratamentos CCHSAS e SCHSAS apresentou alteração revelando algumas bandas diferenciadoras.

O trabalho teve por objetivo avaliar fatores associados à ensilagem da cana-deaçúcar, com destaque para a aplicação de aditivos químicos e microbianos sobre a dinâmica fermentativa, composição bromatológica, atividade da álcool desidrogenase e desenvolvimento e diversidade da micloflora em silagens de cana-de-açúcar. No ensaio conduzido durante 110 dias o delineamento experimental adotado foi o inteiramente ao acaso, com 4 tratamentos, 2 repetições, e seis épocas de abertura (1, 3, 7, 15, 35, 110 dias). Os tratamentos foram: uréia 1% MV e os inoculantes microbianos Lactobacillus buchneri (3,65x105 ufc/g da MV) e a combinação de bactérias Pedioccocus pentosassus e Lactobacillus buchneri (1x106 ufc/g MV). As maiores variações na composição bromatológica e perdas de MS, das silagens controle ocorreram dos 7 aos 15 dias, estabilizando após esse período. As regressões ajustadas para perdas de MS e carboidratos solúveis foram bem similares e de forma contrária ao acúmulo de FDN. As perdas por gases alcançaram valores de 28,27%, de carboidratos solúveis em apenas 2,98% e FDN em torno de 67,77% da MS. Os aumentos nos teores de etanol e perda na digestibilidade nas silagens controle se extenderam até o 35º dia, com valores máximos de etanol de 12,23%. Foi possível relacionar etanol com a digestibilidade mostrando que cada 1% de aumento nos teores de etanol, 2 unidades de digestibilidade foram perdidas. Os aditivos uréia e o aditivo Lactobacillus buchneri mais Pediococcus foram eficazes em diminuir a produção de etanol (2,75 e 1,30 vs 8,27% no tratamento controle), em diminuir perdas de MS em 47 e 60%, e de carboidratos soluveis em 22 e 56% em relação à silagem controle, respectivamente. As silagens aditivadas com uréia obtiveram maiores valores de pH e maiores valores de ácido lático em relação às silagen controle. As silagens aditivadas com L. buchneri apenas foram as de maiores produções de etanol, acima da silagem controle (11.53 vs 8.27%), além de grandes perdas de matéria seca e baixa digestibilidade pelo acúmulo de FDN, comparáveis às silagens controle. A diferença entre aditivos na composição químico-bromatológica e perdas ocorreu após 7 dias de fermentação. Os dados apresentados pelos aditivos uréia e L. buchneri mais Pediococcus foram ajustados em curvas simples, através de modelos lineares, para descrever e predizer as variações durante a ensilagem. Os tratamentos controle e a aditivação com L. buchneri apenas, pelas altas taxas fermentativas, observaram melhor ajuste dos dados em polinômios de segundo e terceiro grau. Apesar dos altos teores de ácido acético em todas as silagens, principalmente nas silagens aditivadas com a combinação de bactérias, não foram verificadas efeitos deste sobre a população de leveduras. Os teores obtidos de ácido lático e ácido propiônico e a relação entre esses ácidos e o ácido acético, durante a fermentação, conseguiu explicar parte do sucesso dos tratamentos uréia e L. buchneri mais Pediococcus na redução da atividade da enzima álcool desidrogenase e na producão de etanol. A análise de grupamentos hieráquicos mostrou que os aditivos alteraram a diversidade bacteriana durante a ensilagem. / The present trial aimed to study the ensiling associated factors of sugarcane focusing on chemical and microbial additives on fermentation, chemical composition, enzymatic activity of alcohol dehydrogenase and the microflora development and diversity in sugarcane silages. A complete randomized design was set to a 110-d trial with 4 treatments, two replications within 6 opening dates (1, 3, 7, 15, 35, 110-d). Treatments were described as follows: urea 1% (wet basis), Lactobacillus buchneri (3.65x105 cfu/g of forage), a combination of Pediococus pentosassus and Lactobacillus buchneri (1x106). Major variation observed on the chemical composition and the DM losses in sugarcane silages without additives took place from the day 7 through the day 15. Losses of DM and soluble carbohydrates showed similar trend and in opposition to the NDF increase. Gases losses averaged 28.27%, while the soluble carbohydrates and NDF contents reached respectively, 2.98% and 67.77% when fermentation was stabilized. Conversely, ethanol and the digestibility were changed across the storage period up to the day 35, with ethanol content increasing to 12.23%. 1% of ethanol increase was associated with 2 percentage units of digestibility decrease. Both urea and the combination of microorganisms were effective in decrease the ethanol content (2.75, 1.30 vs 8.27% - without additives), decrease DM losses (47 and 60%) and reduce soluble carbohydrates losses (22 and 56%) when compared to the control treatment. The urea treated silages showed higher pH and lactic acid values. The L. buchneri treatment led to higher ethanol content (11.23 vs 8.27%) compared to the control, resulting in low DM recovery rate, higher losses and decreased digestibility as well as the silages without additives. The major changes on the chemical composition were noticed after the day 7 of fermentation. For the addition of urea and the combination of microorganisms L. buchneri and Pediococcus the variation was better described by linear equations whereas quadratic and cubic effects were more suitable for fitting the data from the control and the L. buchneri added silages. Even tough all silages has shown high acetic acid contents, mainly the combination of lactic bacteria, no significant effects were observed upon the yeast counts. However, the levels of lactic acid and propionic acid and the ratio of both over the acetic acid content were related to the decrease on the activity of the alcohol dehydrogenase enzyme and, furthermore, on the ethanol content of the silages. The cluster analysis based on molecular evaluation demonstrated a change promoted over the bacterial population mediated by the additives applied during the ensiling of sugarcane.

M. Tech. (Chemistry Department, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Benzene, toluene and xylene (BTX) are water pollutants that appear very often in chemical and petrochemical wastewaters due to gasoline leakage from storage tanks and pipelines. These BTX compounds can cause adverse health effects on humans even at very low concentrations. Amongst the available pollutant removal methods from wastewater, adsorption has been used due to its ease of operation, simplicity and cost-effectiveness. Different adsorbents have been used for BTX removal, however the use of Magnetite-organic acid composites as an adsorbent seems to offer a much cheaper alternative. This work seeks to develop a one-step microwave synthesis and optimization of magnetite-oleic (MNP-OA) and magnetite-palmitic (MNP-PA) acid) composites. Response surface methodology was used to optimize the magnetite-organic acid composites. The optimum conditions estimated for MNP-OA acid composite were 78.3 % Fe content, 1561.9 S/cm conductivity, 82.2, 84.1, 85.3 mg/g for BTX adsorption capacity. The MNP-PA composite were 75.6 % Fe content, 1325.66 S/cm conductivity, 60.55, 64.47, 63.06 mg/g for BTX adsorption capacity. The materials were characterized, and the adsorption process was optimized for BTX removal from aqueous solution. X-ray analysis confirmed the formation of magnetite by the presence of both ferric and ferrous ion states on the surface. It was noted that after modification, the magnetite-organic acids characteristics peaks became broad and the height of the peaks decreased indicating that surface modification with organic acid controls the crystallinity of the material. The average cystalline size of MNP, MNP-OA, and MNP-PA composites were 19.7, 17.1 and 17.9 nm. FTIR analysis confirmed the target materials were produced and also to determine if the organic acids were imobilised on the surface of the magnetite. TEM images presented that the MNP, MNP-OA, and MNP-PA composites were spherical in shape with particle average sizes of 18.4 ± 0.5, 15.6 ± 0.5 and 16.5 ± 0.5 nm. The magnetite-organic acids show the particles with better isolated as compared to that of the MNP. The BET isotherms of the materials were described by a type IV characteristic related to uniform mesoporous materials. The magnetic saturation value for MNP, MNP-OA, and MNP-PA composites were 62.9, 59.0 and 51.0 emu/g. The decrease in magnetization was explained by the presence of the non-magnetic layer on magnetite surface. The pHpzc of MNP, MNP-OA, and MNP-PA composites were 6.9, 6.4 and 6.1. The decrease in pHpzc aftern modification was due to the charging acid-base interaction mechanism of metal oxide nanoparticles. The optimum pH for the adsorption of BTX onto MNP, MNP-OA, and MNP-PA composites was determined to be pH 7 for benzene, pH 8 for toluene and xylene. Among the three pollutants, xylene had the highest adsorption capacity followed by toluene and benzene. The optimum adsorbent dose for the adsorbents for the adsorption process was 0.1 g/dm3. The effect of time on the uptake of BTX onto MNP, MNP-OA, and MNP-PA composites show that initial adsorption of BTX occured between 0 and 3 min of contact time. The effect of initial concentration results shows the initial concentration of BTX increases from 100 to 350 mg/dm3 with an increase in adsorption capacity. The results suggest that the adsorption process is controlled by concentration driving force. The experimental data was fitted to the pseudo-first and pseudo-second-order kinetic models for all adsorbents and all pollutants. The pseudo-second-order models showed good correlation as compared to the first-pseudo model. Desorption studies for benzene, toluene and xylene using the pure magnetite, magnetite-palmitic and magnetite oleic acid composites indicate adsorption mrchanism can be explained in relation to acid–base chemistry. Electron donation from the phenyl ring of each benzene, toluene and xylene compound to surface iron atoms of magnetite has been suggested. The CH3OH and H2O desorbing agents were used and regeneration using five cycles show that the percentage desorption decreses from Benzene < Toluene < Xylene. The reduction in adsorption capacity after the cycles are attributed to decomposition of the adsorbents active sites and mass loss of the sample.

Inborn errors of metabolism (IEM) cause significant morbidity and mortality when left untreated. Urine organic acid (UOA) analysis is often a first-line investigation when an IEM is suspected. UOAs are usually qualitatively analyzed via the current gold standard, GC-EI-MS (Gas Chromatography-Electron Impact-Mass Spectroscopy). The Agilent 7890 GC in tandem with the Waters’ Xevo TQ-S MS contains an easily interchangeable LC-ESI (liquid chromatography-electrospray Ionization) and GC-APCI (Atmospheric Pressure Chemical Ionization) instrument set-up, while maintaining accuracy and sensitivity in both LC and GC applications. Utilizing this novel GC-APCI instrument, this project aims to develop and validate a new UOA method for clinical use. Furthermore, utilizing the machine’s MRM mode would increase sensitivities thus allowing for hopefully quantitative analysis. Chemical standards and patient urine samples were extracted via a liquid-liquid ether extraction and derivatized with BSTFA for proper GC elution. Results were compared on the current gold standard GC-EI-MS instrument and the new GC-APCI-MS instrument. Initial instrument suitability and method setup was then optimized. Source moisture levels were modified to explore the wet proton transfer and the dry charge transfer mechanism using [M+H]+ and [M+*]+ ion peak ratios, respectively. Elution times and APCI ion mass spectra profiles of UOA metabolites of interest were identified from full scan mode in preparation for MRM mode analysis. Exploration into the wet and dry mode settings of the APCI source determined that the former induced via methanol had greater peak areas and signal-to-noise ratios. Suitable MRMs were determined for clinically relevant organic acids from which a quantitative assay was developed for methyl malonic acid and several other compounds. The Waters’ Xevo TQ-S micro with Agilent 7890 GC demonstrated promising GC-APCI-MS detection of urine organic acids. With clear avenues for future work, the APCI technique hints at great benefits for biochemical genetic laboratories. / Thesis / Master of Science (MSc) / Inborn errors of metabolism (IEM) are a class of genetic diseases that when left untreated, cause reduced quality of life and sometimes death in newborns. Urine organic acid (UOA) analysis is used for detection using an instrument called GC-EI-MS (Gas Chromatography Electron Impact Mass Spectroscopy). This project explores how a new instrument, the Agilent 7890 GC and the Waters’ Xevo TQ-S MS, can detect these genetic diseases using a technique called APCI (Atmospheric Pressure Chemical Ionization) while still being accurate and sensitive. UOAs are isolated from urine and run through the new machine. When compared to the currently used technique, results were promising but further optimization is needed. Using the new machine, various UOA compounds that were elevated and/or decreased in newborns with genetics diseases were identified and quantified. With clear avenues for future work, the APCI technique can greatly improve newborn diagnosis of IEMs.

Hintergrund: Die Synthese von Chemikalien aus fossilen Rohstoffen wird wegen ihrer begrenzten Verfügbarkeit und ihren negativen Auswirkungen auf die Umwelt zunehmend kritisch bewertet. Eine Alternative bietet die „Weiße Biotechnologie“, insbesondere die Fermentation nachwachsender Rohstoffe mithilfe von Hefen. Die oleophilen Hefen Pseudozyma (P.) tsukubaensis und Yarrowia (Y.) lipolytica sind natürliche Säureproduzenten. Ihre Hauptprodukte sind Metabolite des Tricarbonsäurezyklus: Citrat (CA), α-Ketoglutarat und Malat. In kleineren Mengen werden auch andere Stoffe wie Isocitrat (ICA) oder Itaconat (ITA, nur von P. tsukubaensis) sekretiert. Das Interesse an den beiden Letztgenannten hat in den vergangenen Jahrzehnten stetig zugenommen. Bis heute gibt es allerdings keinen etablierten Wirtsorganismus für die ICA-Produktion. ITA hingegen wird mithilfe von Aspergillus terreus synthetisiert. Jedoch stößt die ITA-Produktivität dieses Hyphenpilzes auch mit großem wissenschaftlichem Aufwand an ihre Grenzen. Daher wird ein neuer Wirtsorganismus benötigt. Ergebnisse: In dieser Studie wurden ein vielversprechender P. tsukubaensis-Stamm für die Produktion von ITA und ein Y. lipolytica-Stamm für ICA konstruiert. Zunächst wurde das Genom von P. tsukubaensis sequenziert. Infolgedessen wurde ein Gencluster für die Synthese und den Export von ITA identifiziert, das homolog zu dem von Ustilago maydis ist. Die Überexpression von vier der fünf Clustergene erhöhte die ITA-Sekretion nicht deutlich. Das fünfte Gen kodiert den vermeintlichen Transkriptionsfaktor Ria1p, der vermutlich das Gencluster steuert. Die Überexpression des PtRIA1 Gens führte zu einer signifikant erhöhten ITA-Produktion von bis zu 31,4 g/l in Mikrotiterplatten. Durch die Optimierung der Wachstumsbedingungen wurden im Bioreaktor innerhalb von 7 d 113,6 g/l ITA ohne die Notwendigkeit eines Triggers produziert. Für die ICA-Produktion wurden zwei mutmaßliche mitochondriale Citrat-Transportproteine in Y. lipolytica identifiziert, welche von den Genen YlCTP1 sowie YlYHM2 kodiert werden. Die Funktionsweise der beiden Proteine scheint sich stark voneinander zu unterscheiden. Die Deletion von YlCTP1 führte zu einer leichten Verschiebung des ICA:CA-Verhältnisses, aber die Gesamtmenge beider Säuren nahm stark ab. Durch die Deletion von YlYHM2 stieg die ICA:CA-Produktrate von 12 % auf 95 % im Vergleich zum Wildtyp. Innerhalb von 5 d wurden bis zu 131,9 g/l ICA mit Sonnenblumenöl, bzw. 22,0 g/l ICA mit Glukose als einzige Kohlenstoffquelle in einem Bioreaktor unter kontrollierten Produktionsbedingungen erreicht. Durch die zusätzliche Hemmung des Isocitratlyase-Proteins mit ITA stieg das ICA:CA-Verhältnis bis 98 %. Fazit: Mittels Metabolic Engineering wurden im Rahmen dieser Arbeit die beiden Hefestämme P. tsukubaensis HR12 und Y. lipolytica ΔYHM2 erzeugt. Mit ihrer Hilfe ist es möglich, die hochwertigen Chemikalien ITA oder ICA in hohen Mengen (> 100 g/l) aus nachwachsenden Rohstoffen wie Glukose oder sogar Pflanzenölen herzustellen. / Background: The synthesis of chemicals from fossil fuels is being evaluated increasingly critically, mainly due to its expected exhaustion and negative impact on the environment. An alternative offers ‘white biotechnology’, especially the fermentation of renewable resources with the help of yeasts. The oleophilic yeast species Pseudozyma (P.) tsukubaensis and Yarrowia (Y.) lipolytica are both natural organic acid producers. Their main products are metabolites of the tricarboxylic acid cycle, namely citrate, α-ketoglutarate and malate. In smaller amounts, other compounds like isocitrate (ICA) or itaconate (ITA, solely with P. tsukubaensis) are also secreted. The interest for the latter two has been rising steadily during the last decades. However, to this date, there is no established host organism for the ICA production. ITA, on the other hand, is being synthesised with Aspergillus terreus. Even with great scientific effort, the ITA productivity of this hyphal fungus appears to reach its limits. Therefore, a different host organism is needed. Results: In this study, a promising P. tsukubaensis strain has been constructed for the production of ITA and a Y. lipolytica strain for ICA. First, the genome of the ITA producer P. tsukubaensis has been sequenced. As a result, a gene cluster for the synthesis and export of ITA, homologous to that of Ustilago maydis, has been identified. By overexpressing four of the five cluster genes, respectively, none to low increases in ITA secretion were observed. The fifth gene is encoding the putative transcription factor Ria1p which probably controls the gene cluster. The overexpression of the gene PtRIA1 led to a significantly increased ITA production of up to 31.4 g/l in micro-wells. By optimizing the growth conditions 113.6 g/l ITA could be produced within 7 d under controlled conditions in a bioreactor without the need of a trigger like phosphate limitation. For the production of ICA, two putative mitochondrial citric acid transporter proteins were identified in Y. lipolytica. One carrier protein is encoded by the novel gene YlYHM2, the other one by YlCTP1. The mode of function for the two deduced proteins appears to be very distinct from one another. The deletion of YlCTP1 led to a minor shift in the ICA:CA ratio but the total amount of acids decreased greatly. By deleting YlYHM2, the ICA:CA product ratio could be increased from 12 % to 95 % compared to the wild type strain. Within 5 d up to 131.9 g/l ICA with sunflower oil and 22.0 g/l with glucose as the sole carbon source could be achieved under controlled production conditions in a bioreactor. Further inhibition of the isocitrate lyase protein with ITA increased the ICA:CA ratio to 98 %. Conclusion: Within this work, the two yeast strains P. tsukubaensis (HR12) and Y. lipolytica (ΔYHM2) have been created via metabolic engineering. With their help, it is possible to produce the value-added chemicals ITA or ICA on a high scale (> 100 g/l) from renewable resources like glucose or even vegetable oils.

No description available.

Les Escherichia coli pathogènes de la volaille (APEC) font partie des E. coli extra-intestinaux pathogènes (ExPEC) et seraient un réservoir possible de gènes de virulence et de résistance aux antimicrobiens (RAM) des ExPEC chez l’humain. L’objectif de cette étude était d’évaluer l’effet d’un prébiotique et d’un mélange d’acide organique et d’huiles essentielles encapsulés sur la prévalence des gènes de virulence des ExPEC et de RAM, ainsi que les associations entre ces gènes chez E. coli de l’intestin du poulet sain. Des échantillons de contenus caecaux de poulets de 29 jours d’âge ayant reçu un de ces ingrédients alimentaires comparativement à des témoins ont été analysés pour la présence des gènes de virulence iucD, tsh, papC et des gènes de RAM blaTEM, blaSHV, tetA, tetC, blaCMY-2, aadA1, aac3 par PCR. La prévalence d’iucD était supérieure dans le groupe témoin comparativement aux groupes «prébiotique» et «acide organique» et la prévalence de papC était affectée dans le groupe «acide organique». La prévalence d’isolats d’E.coli positifs pour blaCMY-2 était supérieure dans le groupe témoin comparée aux groupes «prébiotique» et «acide organique», tel que démontré par la technique d’hybridation de l’ADN sur HGMF (Hydrophobic Grid Membrane Filter). De plus, la prévalence des isolats d’E. coli positifs pour tetA, blaTEM, aadA1 ou tsh était affectée par les ingrédients alimentaires. Dans l’ensemble, des associations entre la présence de tsh et iucD, blaTEM et aadA1, et iucD et blaCMY-2 ont été observées. .Cette étude démontre l’utilité de certains ingrédients alimentaires pour dimunier le risque d’exposition en santé publique. / Avian Pathogenic E. coli (APEC) belong to the extra-intestinal pathogenic E. coli (ExPEC) pathotype, and may be a virulence and antimicrobial resistance (AMR) gene reservoir for ExPEC in humans. The aim of this study was to evaluate the effect of addition to the feed of a prebiotic or an organic acid on the prevalence of ExPEC-associated virulence genes and antimicrobial resistance (AMR) genes and the association between these genes in E. coli of the intestinal microflora of healthy chickens. Caecal contents from 29-day-old chickens having received one of these feed ingredients in comparison to a control group were examined for the presence of virulence genes iucD, tsh, and papC and AMR genes blaTEM, blaSHV, tetA, tetC, blaCMY-2, aadA1, and aac3 by PCR. The prevalence of iucD was significantly higher in the control group than in the prebiotic and organic acid groups and prevalence of papC was affected by the use of the organic acid. The prevalence of blaCMY-2-positive E. coli isolates was higher in the control group than the prebiotic or organic acid groups, as demonstrated by Hydrophobic–grid membrane filter (HGMF) DNA probe colony hybridization. In addition, the prevalence of E. coli isolates positive for tetA, blaTEM, aadA1 or tsh was affected by the use of these feed ingredients. Overall, associations between the presence of iucD and tsh, blaTEM and aadA1, and iucD and blaCMY-2 were observed. This study demonstrates that the use of certain feed ingredients could reduce the risk of exposure in a public health perspective.

<p>This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy.</p><p>Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed.</p><p>Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. </p><p>A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (<1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (<1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.</p>