Orientador: Jonas Contiero / Coorientador: Francisco José dos Santos / Banca: Eliana Setsuko Kamimura / Banca: Miguel Jafelicci Junior / Banca: Flávio Faria de Moraes / Banca: Luis Henrique Souza Guimarães / Resumo: As ciclodextrinas (CDs) resultam da degradação do amido por ciclodextrina glicosiltransferase (CGTases) produzidas principalmente pelo gênero Bacillus. As CDs são oligossacarídeos cíclicos capazes de formar complexos de inclusão com uma grande variedade de moléculas modificando suas características indesejadas sendo assim muito utilizadas em vários setores industriais. A linhagem bacteriana Gram-positiva e formadora de esporos nomeada como CGII foi isolada de águas residuárias de uma fábrica de farinha de mandioca em Ribeirão Bonito, São Paulo, Brasil e submetidos a estudos filogenéticos e testes bioquímicos. O planejamento composto central foi então utilizado para optimizar a composição do meio e as condições de cultivo para a produção da enzima ciclodextrina glicosiltransferase (CGTase) em frascos agitados e em biorreatores. As características físico-químicas e bioquímicas da CGTase como pH e temperatura óptima; estabilidade em temperatura e pH; a influência de substâncias; cinética enzimática e produção de ciclodextrina foram avaliadas após purificação em cromatografia de afinidade usando a β-CD como ligante. Neste trabalho também foram investigadas novas aplicações da ciclodextrina produzida pela CGTase de B. lehensis: A síntese de fases sólidas de nanopartículas de óxido de ferro magnético (Fe4O3), óxido de cobre (CuO) e prata metálica (Ago) a partir de CDs por síntese hidrotérmica, usando CDs. As nanopartículas foram caracterizadas por difração de raios-x, espectroscopia de infravermelho e microscopia eletrônica de varredura. A enzima CGTase também foi imobilizada por ligação covalente no suporte magnetita síntetizada anteriormente, a qual foi silinalizado com 3-aminopropiltrimetilsilano e ativada com glutaraldeido. A sequência do gene 16S rRNA apresentou maior grau de similaridade com... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cyclodextrins (CDs) are obtained from starch degradation by enzyme cyclodextrin glycosyltransferase (CGTases) produced mainly by Bacillus genus. The CDs are cyclic oligosaccharides capable of forming inclusion complexes with a wide variety of molecules, modifying their unwanted characteristics and, therefore are widely used in various industrial sectors. The bacterial strain Gram-positive and spore-forming named CGII was isolated from wastewater from a cassava mill in São Paulo, Brazil and subjected to phylogenetic studies and biochemical tests. A central composite design was then used to optimize the medium composition and culture conditions for the production of the enzyme cyclodextrin glycosyltransferase (CGTase) in bioreactors. The physicochemical and biochemical proprieties of CGTase as pH and temperature optimum, stability to pH and temperature, the influence of substances, enzyme kinetics and production of cyclodextrin were evaluated after purification by affinity chromatography using β-CD as a ligand. In this work new applications of cyclodextrin produced by CGTase from B. lehensis were also investigated. The solid phase synthesis of nanoparticles magnetic iron oxide (Fe4O3), copper oxide (CuO) and metallic silver (Ag) by hydrothermal synthesis using CDs were characterized by x-ray, infrared, and scanning electron microscopy. The enzyme CGTase was also immobilized on the support by covalent bond to the synthesized magnetite, which has been silylazed with 3- aminopropil-trimetilsilano and activated with glutaraldehyde. The 16S rRNA gene sequence indicated the highest degree of similarity to strains of Bacillus genomic MLB2 lehensis (100%). The response surface methodology showed that the model for the optimization of CGTase of B. lehensis reached a good level of agreement with experimental... (Complete abstract click electronic access below) / Doutor

Orientador: Mauro Faber de Freitas Leitão / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-26T10:57:40Z (GMT). No. of bitstreams: 1 Zacarchenco_PatriciaBlumer_M.pdf: 17769563 bytes, checksum: 08109a10c7352f9e406cf56e29bd58d9 (MD5) Previous issue date: 2000 / Resumo: Nos últimos anos, em nível internacional, o microrganismo Bacillus sporothermodurans (BSP) vem sendo isolado a partir de leite processado pelo sistema UAT/UHT (Ultra Alta TemperaturalUltra High Temperature). Também no Brasil, mais recentemente, o microrganismo foi isolado a partir de amostras de leite UAT produzido em unidades industriais do Estado de São Paulo e Paraná. As amostras contaminadas apresentaram contagens em torno de 105UFC/ml sem evidências maiores de alterações organolépticas. No entanto, nestas condições, o leite UAT fica fora dos padrões exigidos pela legislação para o produto quanto a contagem de microrganismos aeróbios mesófilos, que é de 102UFC/ml(portaria SVSIMS n12451/97). Na primeira parte desta pesquisa, procurou-se avaliar e otimizar a metodologia para detecção de BSP, usando-se como referência a cepa DSMZ 10599, da Coleção Alemã de Microorganismos e Culturas Celulares (Deutsche Sammlug von Mikroorganismen und Zellkulturen). Em uma primeira etapa, avaliou-se qualitativa e quantitativamente o desempenho do ágar Infusão de Cérebro e Coração (BHI), ágar Padrão de Contagem (PCA) e o ágar Nutriente (NA) na contagem de BSP. Além disso, comparou-se as técnicas de inoculação em profundidade (pour p/ate) e espalhamento superficial (spread plate). Os melhores resultados foram obtidos pelo uso do ágar BHI (colônias mais diferenciadas e com melhor crescimento) e o uso da técnica de espalhamento superficial (maior uniformidade e crescimento das colônias). Numa segunda etapa formulou-se o meio diferencial (ágar BHI-E), com base na adição de 1g/I de esculina e 0,5gl1 de citrato férrico amoniacal, permitindo, assim, a melhor caracterização das colônias de BSP pelo seu enegrecimento e formação de halo escuro pela hidrólise da esculina. Numa terceira etapa, comprovou-se que a adição destes componentes não ocasionou quaisquer efeitos inibitórios comparativamente ao ágar BHI. Finalmente, numa última etapa, avaliou-se a eficiência da ágar BHI-E na diferenciação de BSP frente a outros Bacillus spp de ocorrência comum em produtos lácteos, caso de Bacillus cereus, Bacillus subtilis e Bacillus firmus. Na segunda parte da pesquisa, analisaram-se 100 amostras de leite UAT de 3 regiões do Brasil (Centro-Oeste, Sudeste e Sul) utilizando o ágar BHI-E desenvolvido na primeira fase. Verificou-se que 45% das amostras estavam fora dos padrões da Portaria SVS/MS no 451/97 e que 55% delas apresentavam esporos mesófilos termo-resistentes. Isolaram-se 300 colônias que hidrolisaram esculina e tinham aspecto semelhante às colônias do Bacillus sporothermodurans em ágar BHI-E. A maior parte dos isolados apresentou morfologia típica e produzia oxidase, sendo que poucos não fermentaram a glicose. Vinte e quatro cepas, da segunda etapa da pesquisa, tiveram o seu DNA cromossômico avaliado pelo método RAPD ("random amplified polymorphic DNA"). Os perfis de DNA obtidos permitiram dividí-Ias em 3 grupos que apresentaram perfiJde DNA muito semelhantes ao do BSP. / Abstrat: Bacillus sporothermodurans - BSP - has been isolated worldwide in milk processed by the ultra high temperature system (UHT-milk). More recently BSP has also been isolated in Brazil, partieularly in industrial samples processed in São Paulo and Paraná states. Ali the samples showed total counts around 105cfu/ml without any organoleptie ehanges. However based on Brazilian microbiological standards (Portaria SVS/MS nQ451/97) fixing a limit of 1& cfu/ml, the samples were eonsidered inadequate, being rejected. The objective of the first part of this research was the evaluation and improvement of the methodology for Bsp detection, employing as reference the strain DSMZ 10599 from Germany Colection of Microorganisms and Cells Cultures (Deutsche Sammlug von Mikroorganismen und Zellkulturen). In a first step, the performanee of Brain Heart Infusion agar (BHI agar), Total Plate Count Agar (PCA) e Nutrient agar (NA) were comparatively evaluated, considering tooa the inoculation methods (pour plate compared to spread plate). The results showed the best performance of BHI agar with more uniform and better growth of Bsp, the same being valid eoncerning the use of spread plate compared to the pour plate method. In a seeond step, it was formulated a differential medium named BHI-E agar, based on addition of 19/1 of esculin and 0,5gl1 of ferrie ammonium eitrate; with this modification, BSP showed typical black colonies with a black halo due to esculin hydrolisis. In a third step, it was concluded that BHI-E agar did not show any inhibitory effect when compared to BHI agar. Finally, in a last step, BHI-E agar was evaluated in relation to the other Bacillus spp commonly found em milk products, particularly Bacillus cereus, Bacillus subtilis and Bacillus firmus. In the second part of the research work, a total of 100 comercial samples of UHT milk produced in 3 different Brazilian regions (Center-east, southern and southeast) were analysed using BHI-E agar previously developed and tested. The results showed that 45% of the examined samples did not attend the Brazilian standards (Portarja SVS/MS nQ451197)and 55% were contamined with mesophilic thermoresistant bacterial species. A total of 300 esculin positive cultures were isolated with morphological and cultural aspects similar to BSP besides being oxidase positive but generally showing a slow glucose fermenting activity. Twenty four strains isolated at the second part of this research were sub-tiped by molecular typing (RAPO). These 24 strains could be separated in 2 groups according to their ONA pattern. The profiles of 3 groups were very similar to that ONA pattern of Bacillus sporothermodurans (BSP). / Mestrado / Mestre em Tecnologia de Alimentos

Orientador: Valeria Maia de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:04:00Z (GMT). No. of bitstreams: 1 Crespim_Elaine_M.pdf: 1613061 bytes, checksum: 0510cf7cad319e1cd26f150b983f83bd (MD5) Previous issue date: 2008 / Resumo: A abordagem tradicional empregada em estudos de Microbiologia Ambiental, baseada em métodos de isolamento seletivo e cultivo de microrganismos em laboratório, embora seja útil para a determinação do potencial fisiológico dos organismos isolados, é inadequada para a realização de uma caracterização abrangente da comunidade microbiana destes ambientes ou para detectar microrganismos de difícil cultivo ou que vivem em consórcios. O emprego de técnicas moleculares em estudos de comunidades microbianas, as quais envolvem o isolamento direto de DNA a partir de amostras ambientais, a amplificação de genes conservados pela técnica de PCR e a análise de seqüências de rDNA 165, tem demonstrado resultados promissores em Ecologia Microbiana, uma vez que permite identificar organismos de ambientes naturais sem a necessidade de cultivo dos mesmos. A detecção de microrganismos com potencial para biodeterioração, biodegradação e biocorrosão encontrados em depósitos petrolíferos é de grande importância na medida em que estes organismos podem estar relacionados com a perda da qualidade do petróleo nos reservatórios e etapas subseqüentes de exploração (extração, . armazenamento e refino). O presente trabalho teve como objetivos desenvolver um método molecular para a detecção rápida de grupos específicos de microrganismos degradadores de hidrocarbonetos e avaliar a sua distribuição e abundância em diferentes amostras de óleo e água de formação provenientes de depósitos petrolíferos da bacia de Campos (RJ). Nossos resultados revelaram a presença dos grupos Bacíllus spp., Streptomyces spp., Achromobacter xy/osoxidans, Bacíllus pumilus, Micrococcus spp. e Dietzia spp. nas amostras de petróleo estudadas. Alguns destes microrganismos apresentaram ampla ocorrência, embora em baixa abundância, nas amostras dos cinco poços avaliados, independentemente do grau de biodegradação dos óleos e condições de pro{undidade e temperatura dos reservatórios. O desenvolvimento de um método molecular para a rápida detecção de grupos de microrganismos potencialmente biodegradadores em amostras ambientais seria extremamente útil como uma ferramenta de apoio para a avaliação da qualidade do óleo em reservatórios de produção / Abstract: The traditional approach used in Environmental Microbiology studies, based on selective isolation and cultivation methods, although very useful for determination of the physiological potential of the isolated organisms, is inadequate for a broad characterization of the microbial community in these environments, since it does not allow the recovery of fastidious microorganisms or the ones that live in consortia. The use of molecular techniques in microbial community studies, which involve the direct DNA isolation from environmental samples, amplification of conserved genes by PCR and sequence analysis, has shown promising results in Microbial Ecology, as it allows the identification of organisms trom natural environments without the need of cultivation. The detection of microorganisms with potential for biodeterioration, biodegradation and biocorrosion in petroleum deposits is of great importance, as these organisms may be related to a decrease in petroleum quality in the reservoirs and subsequent exploration steps (extraction, storage and refining). The present work aimed at developing a molecular method for the rapid detection of specific groups of hydrocarbondegrading microorganisms and evaluating their distribution and abundance in different oil and formation water samples originated trom petroleum reservoirs in the Campos basin (RJ). Our results revealed the presence of the target groups Bacillus spp., Streptomyces spp., Achromobacter xylosoxidans, Bacillus pumilus, Micrococcus spp. and Dietzia spp. in the environmental samples under study. Some of these microorganisms were of broad occurrence, although in low abundance, in ali the five samples trom the petroleum wells analyzed, in spite of the oil biodegradation levei and depth and temperature conditions. The development of a molecular method for the rapid detectión of specific groups of biodegrading microorganisms in environmental samples would be extremely useful as a supporting toei for the evaluation of oil. quality in the production reservoirs / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Orientador: Telma Teixeira Franco / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-21T04:20:39Z (GMT). No. of bitstreams: 1 Rodrigues_GiselledeArruda_D.pdf: 30208000 bytes, checksum: b1ed6a1c43dab6cee435816103c4f562 (MD5) Previous issue date: 2012 / Resumo: A produção de comppostos químicos a partir de resíduos lignocelulósicos tem atraído bastante a atenção da sociedade atual. A busca por alternativas produtivas não dependentes de combustíveis fósseis, o uso de fontes renováveis e a sustentabilidade são os principais motivos. Neste trabalho, a produção do ácido lático por fermentação dos açúcares obtidos dos bagaço da cana de açúcar, essencialmente glicose e xilose, foi estudada. Partindo de várias cepas de bactérias ácido láticas e utilizando-se metodologia qualitativa, 26 linhagens foram selecionadas para testes em frascos agitados e em biorreator. Os melhores resultados foram obtidos em fermentações utilizando-se B. coagulans 162, alcançando rendimento de 0,95 e produtividade volumétrica de 2,13 g L-1 h-1 em regime de batelada simples e meio de cultivo contendo glicose, xilose e arabinose. Em alimentação contínua, o meio de cultivo alcançou 105 g de ácido lático por litro de meio fermentado. Os efeitos dos tratamentos de hidrólise térmica e explosão a vapor do bagaço da cana de açúcar também foram estudados. Verificou-se a formação de ácido acético, furfural e hidroximetilfurfural (HMF) e liberação dos açúcares glicose, xilose e arabinose. As fibras do bagaço foram fotografadas utilizando-se microscopia eletrônica de varredura (MEV), antes e após tratamentos térmicos, para observação das modificações no arranjo da estrutura original. O bagaço in natura, contendo aproximadamente 43% de celulose, 28% de hemicelulose e 14% de lignina, foi submetido a tratamento hidrotérmico (220°C/5min) seguido de hidrólise pelas enzimas Celulase, Celobiase e Xilanase. O hidrolisado obtido pela degradação das frações celulósica e hemicelulósica continha basicamente glicose e xilose, e foi utilizado como fonte de carbono no meio de cultivo. Na fermentação foi obtido rendimento de 0,96 e produtividade volumétrica igual a 4,11 g L-1. Hidrolisado obtido do bagaço explodido a vapor também foi testado. Constituído basicamente pela fração hemicelulósica, o hidrolisado continha essencialmente xilose. Nesta fermentação alcançou-se rendimento de 0,90 e produtividade volumétrica igual a 0,225 g L-1 h-1. Posteriores estudos mostraram que a adaptação da cepa a meios de cultivo contendo apenas pentoses (xilose e arabinose) reduzem a fase lag tendo como consequência o aumento da produtividade. Estudos de inibição em meio sintético permitiram avaliar o efeito que cada composto causou individualmente na multiplicação celular e na produção do ácido lático. O ácido acético foi o componente que mostrou maior efeito inibitório. Dentro da faixa estudada, furfural e HMF provocaram comportamentos semelhantes, tornando-se inibitórios em concentrações acima de 1,5 g L-1 na produção de biomassa, mas não na produção de ácido lático. Inibição por xilose também foi investigada. Rendimento máximo igual a 0,95 foi obtido quando 140 g L-1 de açúcar inicial foram utilizados. Concentrações acima de 150 g L-1 mostram-se inibitórias ao crescimento microbiano. Foi possível observar que o bagaço da cana de açúcar é um substrato promissor para produção biotecnológica de ácido lático, um produto químico com alto valor agregado e com versatilidade em aplicações. O microrganismo selecionado, a bactéria Bacillus coagulans 162, mostrou-se uma cepa robusta, com relativa tolerância a inibidores e capaz de converter homofermentativamente as pentoses presentes no meio de cultura, característica desejável do ponto de vista produtivo e econômico / Abstract: Production of chemical compounds from lignocellulose residues has attracted attention of actual world society. The search for productive alternatives fossil fuels non-dependent, the use of renewable resources and sustainability are the main reasons. In this work, lactic acid production by fermentation of sugars obtained from sugarcane bagasse, essentially glucose and xylose, was studied. From several strains of lactic acid bactéria and using a qualitative methodology, 26 microorganisms were selected for shaking flasks and bioreactor tests. The best results were obtained in fermentations using B. coagulans 162, reaching yield of 0,95 and volumetric productivity of 2,13 g L-1 h-1 in simple batch and medium containing glucose, xylose and arabinose. In continuous feeding, the cultivation medium reached 105 g of lactic acid per liter. The treatment effects of hydrotermal hydrolysis and steam explosion of sugarcane bagasse were also studied. It was verified acetic acid, furfural and hydroxymethylfurfural (HMF) formation and glucose, xylose and arabinose release. Bagasse fibers were photographed using Scanning Electron Microscopy (SEM), before and after termal treatment for visualization of modification in the arrangement of the original structure. Bagasse in natura, containing approximately 43% of cellulose, 28% of hemicellulose and 14% of lignina, was submitted to hydroythermal treatment (220°C/5min) followed by hydrolysis with the enzymes Cellulase, Cellobiase and Xylanase. The obtained hydrolysate of cellulose and hemicellulose fractions contained mainly glucose and xylose, and was used as Catbon source in the cultivation medium. In the fermentation was obtained yield of 0,96 and volumetric productivity equal to 4,11 g L-1. The hydrolysate obtained from steam exploded bagasse was tested. Constituted basically by hemicellulose fraction, the hydrolysate contained mainly xylose. It was reached yield of 0,90 and volumetric productivity equal to 0,225 g L-1 h-1. Later studies have showed that strains adaptation in cultivation medium containing only pentose (xylose and arabinose) reduces the lag phase resulting in increase of productivity. Inhibition studies on synthetic medium allowed to evaluate the effect that each compound caused individually on cell replication and on lactic acid production. Acetic acid was the compound that showed the higher inhibitory effect. In the studied range, furfural and HMF showed similar behaviour becoming inhibitory in concentrations above 1,5 g L-1 on biomass production. The same effect was not observed on lactic acid production. Inhibition by xylose was also investigated. Maximum yield, equal to 0,95, was obtained when 140 g L-1 of initial sugar were used. Concentrations above 150 g L-1 showed to be inhibitory to the microbial growth. Sugarcane bagasse showed to be a promissor substrate to biotechnologic production of lactic acid, a chemical with high added value and versatile applications. The selected microorganism, the bacteria Bacillus coagulans 162, showed to be a robust strain, with relative tolerance against inhibitors and able to convert homofermentatively the pentose present in hemicellulose hydrolysate, desirable feature in productive and economic terms / Doutorado / Desenvolvimento de Processos Químicos / Doutora em Engenharia Quimica

Aisla y selecciona rizobacterias del género Azotobacter y Bacillus con potencial aplicación como bioinoculante en el cultivo de Mangifera indica L (mango). El aislamiento se realizó a partir de muestras de rizósfera de plantaciones de mango del departamento de Piura. Se logró aislar 23 cepas del género Bacillus, de las cuales, 8 (34,8%) presentaron antagonismo antifúngico frente a Lasiodiplodia theobromae, 7 (30,4%) frente a Colletotrichum gloeosporioides y 1 (4,3 %) frente a Phytophthora sp. Asimismo 6 (26,1 %) fueron antagonistas tanto a Fusarium sp. como a Alternaria sp. En el caso de Azotobacter de las 29 cepas aisladas, 5 (17,2 %) presentaron antagonismo antifúngico frente a Lasiodiplodia teobromae y Phytophthora sp., 6 (20,7 %) frente a Colletotrichum gloeosporioides, 12 (41,4 %) frente a Fusarium sp. y 11 (37,9 %) frente a Alternaria sp. Por otro lado 7 (30,4%) de las cepas de Bacillus sp y 18 (62,1 %) de las cepas de Azotobacter sp presentaron capacidad solubilizadora de fosfatos. El 39,1% (9) de Bacillus sp y el 65,5 % (19) de Azotobacter sp lograron producir acido indol acético (AIA). En ambos géneros el 100% presentó capacidad potencial de fijación de nitrógeno. Se realizaron pruebas a nivel de almácigo con 7 cepas del género Bacillus y 5 del género Azotobacter seleccionadas para uso como potenciales bioinoculantes debido a que presentaron los mejores resultados en las pruebas anteriores. Se observó que todas las cepas usadas tuvieron efecto positivo en cuanto a la 2 reducción del tiempo de germinación, incremento en la altura de tallo y número de hojas y ausencia de síntomas de micosis. Se demuestra de esta forma que la rizósfera del cultivo de mango presenta bacterias de los géneros Azotobacter y Bacillus con potencial aplicación como bioinoculante para la mejora de la producción de mango orgánico. / Tesis

Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. / Microsoft� Word 2010 / Adobe Acrobat 9.54 Paper Capture Plug-in

There are apprehensions that genetic modification of maize with Bacillus thuringiensis (Bt) may have negative effects on soil biodiversity, ecosystem processes and functions. This study aimed at determining the effect of Bt maize crop, Bt maize residues and its genetic modification on microbial biomass carbon (MBC), selected enzyme activities, vesicular arbuscular mycorrhizal (VAM) fungi and N and P release patterns. The study was conducted under field, glasshouse and laboratory conditions. In 2010/2011 season, four maize cultivars; DKC 61-25B (Bt), DKC 61-24 (non-Bt), PAN 6Q-321B (Bt) and PAN6777 (non-Bt) were planted. Determination of MBC, enzyme activities and fungal spore count was done at 42, 70, and 105 days after planting (DAP). A loam soil amended with Bt or non-Bt maize leaf residues from a study of 2009/2010 season was incubated to investigate effects of Bt maize residues on MBC and soil enzyme activities. Leaf residues of Bt and non-Bt maize cultivars (DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777) were used and soil without residues was used as a control. Samples were collected at 7, 28 and 56 days of incubation (DOI). An incubation study was also carried out in the laboratory to determine the effect of Bt maize residues (i.e. leaf, stem and root) and its genetic modification on N and P release patterns. Residues of DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777and soil without residues as a control were incubated in the laboratory. After destructive sampling at 0, 7, 14, 28, and 56 DOI, N in the form of NH4-N and NO3-N and P mineralisation were determined. Amendment of soil with residues enhanced MBC (p < 0.05) at all the sampling dates. For example MBC increased from 95 in the control to 146.3 mg/kg in the DKC 61-25B treatment at the end of the glasshouse trial. In the field DKC 61-25B had 9.1 mg/kg greater MBC than DKC 61-24, while PAN 6Q-321B had 23.9 mg/kg more MBC than PAN6777 at the end of the trial. However, no differences (p < 0.05) were observed in enzyme activities under field and glasshouse conditions except for dehydrogenase that had greater activity where DKC 61-25B and PAN 6777 were grown. There were no differences between the type of residues (Bt and non-Bt) on enzyme activities tested. However, differences were observed among the sampling dates. No effects of Bt maize crop on fungal spore count were observed. Similarly no differences were observed in leaf, stem and root tissues composition between Bt and non-Bt maize cultivars. Net N and P mineralisation from Bt maize cultivars did not differ from that of non-Bt maize cultivars. However, differences were observed among the cultivars. The results of this study suggested that Bt maize with Bt MON810 event can be grown in the central region of the Eastern Cape (EC), South Africa without affecting MBC, soil enzyme activities, VAM, and release of N and P nutrients from its residues.

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No description available.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system. / AFRIKAANSE OPSOMMING: In hierdie studie is die chromosomale arseen weerstandbiedendheidsgene (ars gene), teenwoordig in alle Acidithiobacillus caldus isolate, gekloon en die DNA volgorde daarvan vanaf At. caldus #6 bepaal. Tien oopleesrame (ORFs) is geïdentifiseer op ‘n kloon wat arseen weerstandbiedend is, met drie homoloog aan ars gene, nl. arsC (arsenaat reduktase), arsR (reguleerder) en arsB (membraan-geleë pomp wat arseniet uitpomp). Die ars operon is gerangskik met die arsRC en arsB gene wat in teenoorgestelde rigtings getranskribeer word. Analise van die afgeleide aminosuurvolgorde van dié ars gene het getoon hulle is naverwant aan die ars gene van Acidithiobacillus ferrooxidans. Die ars gene was funksioneel na transformasie na ‘n E. coli ars mutant (ACSH50Iq), en het ‘n hoër vlak van weerstand teen arsenaat en arseniet gebied. ArsC was nodig vir weerstand teen arsenaat, maar nie vir weerstand teen arseniet nie. Geen van die ander ORFs het arseen weerstandbiedendheid in E. coli bevorder nie. Voorheen is ‘n ars operon, geleë op ‘n transposon (TnAtcArs), in ‘n hoogs arseen-weerstandbiedende stam van die middelmatige termofiliese, swawel-oksiderende, bio-ontgunning (“biomining”) bakterie Acidithiobacillus caldus #6 beskryf. In laasgenoemde studie is gevind dat TnAtcArs hoër vlakke van weerstand bied teen arsenaat en arseniet as die chromosomale operon. TnAtcArs is na ‘n lae arseen-weerstandbiedende At. caldus stam (C-SH12) gekonjugeer. Die resultaat was ‘n groot verhoging in arseen weerstandbiedendheid. RT-PCR analise het onthul dat arsR en arsC saam getranskribeer word. Benewens die feit dat ORF1 (kadmium induseerbare protein) en ORF5 (afgeleide integrase vir profaag CP-933R) nie betrokke is in weerstand teen arseniet and arsenaat nie, is ORF1 saam met arsRC getranskribeer en ORF5 saam met arsB. Deur gebruik te maak van die fusie-gene arsR-lacZ en arsB-lacZ is bewys dat die chromosomale ArsR reguleerder van At. caldus as ‘n inhibeerder van die arsR en arsB promoter uitdrukking funksioneer. Indusering van geen uitdrukking vind plaas wanneer arseniet of arsenaat bygevoeg word. Die chromosomaal-geleë ArsR is ook in staat om TnAtcArs te inhibeer, terwyl die transposon geleë ArsR nie daartoe in staat is om die chromosomale ars sisteem te reguleer nie.