Orientador: Cláudio Cabello / Banca: Waldemar Gastoni Venturini Filho / Banca: Simone Damasceno / Resumo: Neste trabalho fez-se uma avaliação da utilização dos resíduos da agroindustrialização da mandioca como fonte de carbono para fermentação alcoólica, buscando um etanol, que possua qualidades adequadas para uso na indústria alimentícia. No sentido de promover uma alternativa viável, tanto tecnologicamente como economicamente, traçou-se um planejamento experimental que minimizasse os custos. Preocupou-se também, com a sustentabilidade ambiental do processo, pois a produção de álcool etílico pelo método aqui proposto, acena como uma alternativa para que o resíduo gerado, na indústria da mandioca, passe ao status de co-produto, gerando para a indústria não só receita como todos os benefícios mercadológicos de uma indústria limpa. O resíduo utilizado foi o amiláceo pré-hidrolizado que após o processo de fermentação foi destilado e teve seu produto separado e analisado. Desenvolveu-se uma coluna de destilação com recheio de vidro, para operação contínua a fim de estabelecer uma planta pré-piloto, onde ocorram os processos de hidrólise, fermentação e destilação. O substrato escolhido foi submetido a diversos ensaios e, o etanol obtido foi avaliado utilizando metodologias analíticas de derivatização. Para suprir as leveduras alcoólicas dos requisitos nutricionais necessários à sua manutenção durante o processo de fermentação, adicionou-se concentrações crescentes de mel residuário de sacarose aos substratos amiláceos previamente hidrolisados, nas proporções de 5, 10, 15 e 20% . Estas soluções foram padronizadas buscando manter a mesma concentração de carbono orgânico e na etapa subseqüente foram tratadas com enzimas amilolíticas para sacarificação completa. Após o inóculo com leveduras Saccharomyces cerevisiae, as fermentações foram...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this study an evaluation of the residue from the agricultural industrialization of cassava root is aimed. The main goal of this evaluation was towards the potential use of the root as a carbon source to the ethanol production via fermentation. The ethanol obtained must have characteristics good enough to be used in the food industry. Keeping in mind that, nowadays, industrial process must be sustainable and also economically feasible, an experimental scheme was designed. If the second goal may be proved, the first one appears immediatelly; due to promote an alternative use for a residue that otherwise may impact the environment. The experiment begins with the pre-hydrolysed amylaceous that is a by-product from the production of cassava flour. This starch was submited to fermentation under optimized conditions, the wine resultant went trough destilation, the final product was separated and analised. A destilation column, containing glass pearls, was designed in order to integrate a pilot project that may work in a process of continuos operation. This pilot plant was designed in order to achieve the process of hydrolysis, fermentation and destilation. The chosen substrate was then submited to various tests at diverse conditions and the ethanol obtained was evalueted trhough analytical methods with derivatization. In order to optimize the fermentation process by giving to the yeast better conditions of development, molasses, from the sucrose production, was added. The additions were of 5, 10, 15 and 20 %. These solutions were standardized in order to keep the same organic carbon content and after this were submited to a treatment with amylolitc enzymes. This treatment goal was to promote a complete saccharification...(Complete abstract, click electronic access below) / Mestre

Orientador: Gonçalo Amarante Guimarães Pereira, Juan Lucas Argueso Gomes de Almeida / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T13:31:37Z (GMT). No. of bitstreams: 1 Duarte_FabianadeMelo_M.pdf: 10932684 bytes, checksum: c31d50d399af1063941a0d931eee8101 (MD5) Previous issue date: 2010 / Resumo: O etanol de cana-de-açúcar brasileiro ocupa um lugar de destaque entre as alternativas energéticas disponíveis atualmente. No processo fermentativo de produção de etanol é utilizada a levedura Saccharomyces cerevisiae, com destaque para a linhagem industrial PE-2, utilizada por cerca de 30% das usinas brasileiras, o que representa 10% da produção mundial. Essa linhagem associa uma alta eficiência na produção de etanol com uma excelente capacidade de adaptação ao ambiente altamente hostil e competitivo das dornas de fermentação. O nosso grupo de pesquisa realizou previamente uma caracterização genética e molecular detalhada de uma linhagem diplóide derivada diretamente de PE-2 (JAY270), o que forneceu inúmeras possibilidades para a manipulação genética dessa levedura com o objetivo de desenvolver linhagens mais produtivas. No entanto, alguns estudos observaram a ocorrência de variação cariotípica nessa linhagem durante o processo fermentativo, o que pode representar uma barreira para a manipulação dessa levedura. Sendo assim, é extremamente importante estudar o comportamento do genoma dessa linhagem durante a produção de etanol. Este projeto de Mestrado teve como objetivo principal a determinação do mecanismo de recombinação genética responsável pela geração de rearranjos cromossômicos na linhagem JAY270 durante a produção de etanol. Foi realizado um experimento de fermentação em escala semi-industrial iniciado com um inóculo puro de JAY270, com duração de 50 dias. A partir de amostras coletadas ao final do processo foram obtidos isolados que foram analisados através de PFGE (Gel de Eletroforese em Campo Pulsado) para identificação de derivados de JAY270 portadores de variação cariotípica. Dos derivados analisados, 36% possuíam algum tipo de rearranjo cromossômico em relação à linhagem inicial, dos quais 11 foram selecionados (FDY1-FDY11) para análises detalhadas. As sequências genômicas de dois derivados haplóides de JAY270 possibilitaram o desenvolvimento de 9 marcadores moleculares para genotipar regiões heterozigotas de JAY270 com o objetivo de identificar eventos de recombinação genética. Os 11 isolados selecionados foram analisados com esses marcadores e, exceto por um deles, todos continuavam heterozigotos em todas as regiões genotipadas. Dois mecanismos de recombinação genética podem ser responsáveis pela geração dos rearranjos, recombinação mitótica, que ocorre em pontos isolados e recombinação meiótica, que envolve todo o genoma simultaneamente em um único ciclo celular. A possibilidade dos rearranjos terem sido gerados por recombinação meiótica foi excluída, visto que a probabilidade dos 10 isolados continuarem heterozigotos em todas as regiões analisadas após um evento de meiose seguido de cruzamento é extremamente baixa (entre 10-24 e 10-14). Sendo assim, concluiu-se que os rearranjos cromossômicos foram gerados durante o crescimento vegetativo, e que, portanto, o mecanismo de recombinação genética responsável pela geração dos mesmos é a recombinação mitótica. Os 11 isolados também foram analisados através de CGH-array, que possibilitou a comparação de sua dosagem gênica com a da linhagem JAY270, que lhes deu origem. Com essa metodologia foi possível detectar eventos de recombinação mitótica que resultaram em perda de heterozigosidade nas extremidades de alguns cromossomos. / Abstract: Brazilian sugar cane ethanol stands out among the energetic alternatives available nowadays. In the fermentation process to produce ethanol it is used the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is the industrial isolate PE-2, currently used by ~30% of Brazilian distilleries, generating ~10% of the world's ethanol supply. This strain combines a high performance in ethanol fermentation and an excellent adaptation to the hostile environment in the fermentation vats. Our research group previously carried out a thorough genetic characterization of an isolate of the PE-2 strain, known as JAY270. This analysis offers several opportunities for the improvement and modification of this strain. However, some studies observed the occurrence of karyotypic variation in this strain during the fermentation process, what can represent a barrier to the genetic manipulation of this yeast. Therefore, it is extremely important to study the genome behavior of this yeast during the ethanol production process. The main objective of this Master's Project was to determinate the genetic recombination mechanism responsible for generating chromosomal rearrangements in JAY270 during the ethanol production. A fermentation experiment in semi-industrial scale was carried out with a pure initial inoculum of JAY270. After 50 days, samples were collected and used to obtain single colony isolates. This isolates were analyzed through PFGE (Pulsed Field Gel Electrophoresis) to the identification of JAY270 derivatives carrying karyotypic variation. Of the analyzed derivatives, 36% had at least one chromosomal rearrangement. 11 of these derivatives were chosen to perform detailed analyses and were called FDY1-FDY11. The genome sequences of two haploid derivatives of JAY270 were used to develop 9 molecular markers to genotype heterozygous regions of JAY270 to identify genetic recombination events. The 11 isolates were analyzed with these markers and, except by one of them, all isolates were heterozygous in all genotyped regions. Two genetic recombination mechanisms can be responsible for generating the rearrangements, mitotic recombination, which occurs in isolate points of the genome, and meiotic recombination, that simultaneously affects all the genome in one single cellular cycle. The probability of the 10 isolates stay heterozygous in all genotyped regions after one event of meiosis followed by mating is extremely low (between 10-24 e 10-14). This result showed that these rearrangements couldn't be generated through meiotic recombination. Therefore, it was possible to conclude that the chromosomal rearrangements were generated during vegetative growth through mitotic recombination. The 11 isolates also were analyzed through CGH-array to compare their gene dosage with the strain JAY270. With this methodology it was possible to detect mitotic recombination events that resulted in loss of heterozygosity in peripheral regions of some chromosomes. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Dentre as leveduras selvagens mais comumente encontradas na fermentação alcoólica, destaca-se o gênero Saccharomyces apresentando colônias rugosas com células dispostas em cachos (pseudohifas). Este biótipo de levedura tem sempre sido associado com problemas na indústria, ocasionando queda no rendimento fermentativo. Sendo assim, o presente trabalho teve por objetivo realizar a caracterização genética, morfo-fisiológica e da resistência ao estresse de linhagens de Saccharomyces cerevisiae que apresentam colônias rugosas e mucosas, buscando alternativas que possam contribuir para o manejo dessas leveduras selvagens (rugosas) na fermentação alcoólica. Foram realizados testes de caracterização para crescimento invasivo, atividade killer, temperatura, pH, concentração de etanol e açúcar, presença de actidione, floculação e capacidade fermentativa, utilizando-se 22 linhagens de leveduras (11 rugosas e 11 mucosas) selecionadas previamente por seqüenciamento da região ITS do DNA ribossomal. O efeito do tratamento ácido em diferentes valores de pH sobre o crescimento de duas linhagens (52 rugosa e PE-02 mucosa) foi também avaliado, seguido do monitoramento do crescimento em meio de caldo de cana após tratamento ácido. Em seguida, testes fermentativos em meio de caldo de cana foram conduzidos em culturas puras e mista dessas linhagens. Os testes de caracterização morfofisiológica mostraram que a invasividade em meio YEPD Agar ocorreu em baixa freqüência dentre as 22 leveduras testadas; dez dentre onze leveduras rugosas apresentaram taxa de floculação expressiva, e entre as mucosas a floculação foi praticamente inexistente; nenhuma das linhagens apresentou produção de toxina killer; as linhagens com colônias rugosas apresentaram capacidade fermentativa significativamente inferior às linhagens com colônias mucosas, em sistema de batelada com ciclo único de 48 horas em meio de caldo de cana, demonstrando velocidade mais lenta de fermentação. Quanto à resistência ao estresse por temperatura, pH, etanol e concentração de açúcar, as linhagens mucosas tiveram maior resistência à acidez do meio, enquanto as linhagens rugosas foram mais resistentes às concentrações elevadas de etanol e açúcar. Nenhuma levedura foi resistente ao actidione. A análise genética, através dos loci microssatélites, revelou a presença de dois grupos principais relacionados geneticamente, sendo o primeiro ramo constituído principalmente de colônias rugosas (67%), contendo, no entanto, a linhagem PE-02, apresentando linhagens com alta taxa de floculação e tolerância às altas concentrações de açúcar e etanol; o outro grupo (com três subgrupos) compreendeu principalmente colônias mucosas, apresentando pouca resistência às situações estressantes aqui estudadas. A levedura com colônia rugosa (linhagem 52) foi bastante afetada pelo tratamento ácido em valores de pH 1,0 e 1,5, ao contrário da levedura com colônia mucosa (PE-02). A fermentação em sistema de batelada com reciclo celular e tratamento ácido conduzido em pH 1,5 teve efeito sobre o crescimento da levedura rugosa quando em cultura mista com a levedura mucosa, não resultando em prejuízo à eficiência fermentativa, quando comparada com a cultura pura da PE-02. Em cultura pura da levedura rugosa, constatou-se eficiência fermentativa por volta de 60%, confirmando o baixo desempenho destas leveduras. O conhecimento das respostas das leveduras rugosas 12 às situações estressantes pode ajudar a manejar a fermentação alcoólica para minimizar o efeito da levedura contaminante. / Among the wild yeasts more commonly found in the alcoholic fermentation, wrinkled colonies with pseudohyphal morphology belonging to Saccharomyces genus are highlighted. This yeast biotype has been associated with industrial problems, resulting in the decrease of the fermentative yield. In this context, this work aimed to perform the genetic, morphological/physiological characterization and stress tolerance of Saccharomyces cerevisiae strains which exhibited wrinkled and mucous colonies, searching for alternatives that could contribute to the management of these wild yeasts (wrinkled colonies) in the alcoholic fermentation. Characterization tests for invasiveness in Agar medium, killer activity, temperature, pH, ethanol and sugar concentration, actidione, flocculation and fermentative capacity were carried out with 22 strains (11 wrinkled and 11 mucous colonies), which were screened previously by the sequencing of the ITS region of the ribosomal DNA. The effect of the acid treatment using different pH values over the growth of two strains (52 wrinked and PE-02 mucous) was also evaluated, following the growth monitoring in sugar cane juice after acid treatment. Fermentative tests in sugar cane juice were carried out with pure and mixed cultures of these strains. The morphological/physiological tests showed that the invasiveness in YEPD Agar medium occurred in low frequency among the 22 strains tested; ten out of eleven wrinked yeasts exhibited expressive flocculation, and among the mucous, the flocculation was near zero; none of the strains showed killer activity; the wrinkled colonies presented lower fermentative capacity comparing to mucous colonies, in a 48- hour cycle in batch system using sugar cane juice, with slower fermentation rate. Concerning the resistance to temperature, pH, ethanol and sugar concentration, the mucous colonies were more resistant to low pH, while the wrinkled colonies were more resistant to the elevated ethanol and sugar concentrations. None of the yeasts were resistant to actidione. The genetic analysis by microsatellite loci revealed the presence of two main genetic related groups, the first branch comprised mainly of wrinkled colonies (67%), containing however the PE-02 strain, showing strains with high flocculation rate and tolerance to high concentration of sugar and ethanol; the other group (with three subgroups) comprised mainly mucous colonies, showing lower resistance to the stressing conditions here studied. The yeast with wrinkled colony (strain 52) was severely affected by the acid treatment in pH values of 1.0 and 1.5, but the same did not occur with the mucous colony (PE-02). The batch fermentation with cell recycle and acid treatment in pH 1.5 had effect over the wrinkled yeast growth when in mixed culture with the mucous yeast, and did not impair the fermentative efficiency comparing to the pure culture of PE-02. In pure culture with the wrinkled colony, a fermentative efficiency as low as 60% was observed, confirming the low performance of these yeasts. The knowledge of the response to stressful conditions exhibited by the yeasts with wrinkled colonies can help to manage the alcoholic fermentation in order to minimize the effect of the contaminant yeast.

Corn is a valuable agricultural commodity in the United States and in the world. The causal agent of Stewart's wilt disease in corn, Pantoea stewartii subsp. stewartii, is a bacterial phytopathogen that is vectored into the plant by the corn flea beetle, Chaetocnema pulicaria. After entering the apoplast of the leaf, the bacteria cause water soaking symptoms before traveling to the plant xylem to form a dense biofilm, thereby blocking water transport and inducing necrosis and wilt. This results in reduced crop yield and may even lead to death of the corn plant. To better understand the in planta requirements of this pathogen, a whole transcriptome study was performed via RNA-Seq to determine genes differentially expressed in the bacteria while inside the corn. It was found that nutrient transporters and stress response genes were upregulated specifically when the bacteria are in their host plant, suggesting a response to nutrient availability and host defense in the xylem. Further elucidation of the genes required for the P. stewartii in planta lifestyle was performed via a reverse genetics approach where in-frame gene deletions and the corresponding complementation strains were constructed for genes that had shown a fitness defect in corn based on a previously published Tn-Seq study: genes encoding seven transcription factors, nsrR, iscR, lrp, nac, DSJ_00125, DSJ_03645, and DSJ_18135, as well as a hypothetical protein DSJ_21690. Investigation of the physiological role of these genes was performed using in planta virulence and competition assays for all strains. An in planta qRT-PCR analysis of bacterial gene transcription was also completed for the strains with deletions in nsrR and iscR. In vitro assays were performed on all strains to determine their capsule production and motility phenotypes. Taken together, it was seen that iscR is important for colonization capabilities in planta, both NsrR and IscR act as regulators, and lrp is important for full disease capabilities, perhaps due to reduced capsule and motility phenotypes. These findings lay the groundwork for finding potential disease intervention strategies not only against P. stewartii, but also other xylem-dwelling bacterial phytopathogens. In addition to exploring ways to enhance crop yield, an additional research area was on repurposing a byproduct of corn ethanol production, syrup. It was hypothesized that this corn-based syrup could be utilized as a carbon source to grown bacteria. In turn, the resulting bacterial biomass could then be added as a fish feed supplement in aquaculture. Syrup was tested as a growth medium for individual soil bacterial isolates as well as a full mixed bacterial community consortium to determine which bacteria could grow most efficiently, both in rate and yield. It was found that the highest growth rate and yield was from Bacillus species, some of which may have probiotic benefits to fish. Ultimately, the collective outcomes from these projects in basic research about a bacterial corn pathogen and applied research about beneficial microbes grown on a corn-based substrate are expected to improve scientific endeavors as well as agricultural practices. / Doctor of Philosophy / Corn is a top agricultural commodity in the United States, as a food for human consumption, a primary nutrient source used in animal feed, and a substrate consumed during biofuel production. These various corn-based industries are impacted by bacteria in multiple ways; in some cases, bacteria may cause disease that reduces crop yield, but other bacteria serve beneficial roles that enhance health. This dissertation research describes studies about the bacterium that causes Stewart's wilt disease in corn, Panteoa stewartii subsp. stewartii. In an initial experiment, the genes that P. stewartii expresses at the highest levels when it grows inside the corn plant were identified. These genes were deduced to be important for the ability of the bacterium to live successfully in this environment. This work was followed up with a more specific approach that examined the role of certain genes that were predicted to be master regulators of the expression of other genes in the ability of the P. stewartii to colonize the plant and/or cause disease. By identifying key bacterial genes, disease intervention strategies to combat Stewart's wilt and other similar bacterial plant pathogen diseases might become possible. Protecting corn yields is important for ethanol production. The final study of this dissertation examined the ability of bacteria to grow on a byproduct of ethanol production called syrup. The goal was to then use the biomass of these beneficial microbes as a food source for animals being produced in aquaculture facilities. Among the species tested, the highest growth rate and yield was from Bacillus subtilis, a safe-to-eat bacterium that has known beneficial health properties when consumed by fish. Overall, the research studies that were completed for this dissertation have the potential to improve agricultural practices by decreasing corn disease leading to increased corn yield and developing new downstream corn-based animal feed products.

Thesis (MScEng)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Whereas fuel used for transport and electricity production are mainly fossil–derived, there has recently been an increased focus on bio-fuels due to the impact of fossil derived fuel on the environment as well as the increased energy demand worldwide, concomitant with the depletion of fossil fuel reserves. Paper sludge produced by paper mills are high in lignocellulose and represents a largely untapped feedstock for bio-energy production. The aim of this study was to determine the composition, fermentability and optimum paper sludge loading and enzyme dosage for producing ethanol from paper sludge. This information was used to develop a model of the process in Aspen Plus®. The mass and energy balances obtained from the Aspen Plus® model were used to develop equipment specifications which were used to source equipment cost data. A techno-economic model was developed from the equipment cost data to assess the economic viability of the simultaneous saccharification and fermentation (SSF) process utilising paper sludge as feedstock. Nine paper sludge samples obtained from Nampak Tissue (Pty) Ltd. were evaluated in terms of ethanol production and those samples yielding the highest and lowest ethanol titres were selected for optimisation. This allowed for the determination of a range of ethanol concentrations and yields, expressed as percentage of the theoretical maximum, which could be expected on an industrial scale. Response surface methodology was used to obtain quadratic mathematical models to determine the effects of solid loading and cellulase dosage on ethanol production and ethanol yield from paper sludge during anoxic fed-batch fermentations using Saccharomyces cerevisiae strain MH1000. This approach was augmented with a multi response optimisation approach incorporating a desirability function to determine the optimal solid loading and cellulase dosage in fed-batch SSF cultures. The multi response optimisation revealed that an optimum paper sludge loading of 21% (w/w) and a cellulase loading of 14.5 FPU g-1 be used regardless of the paper sludge sample. The fact that one optimal enzyme dosage and paper sludge loading is possible, regardless the paper sludge feed stock, is attractive since the SSF process can be controlled efficiently, while not requiring process alterations to optimize ethanol production when different batches of paper sludge are processed. At the optimum paper sludge loading and cellulase dosage a minimum ethanol concentration of 47.36 g l-1 (84.69% of theoretical maximum) can be expected regardless of the paper sludge used. An economic assessment was conducted to ascertain whether ethanol production from paper sludge using SSF is economically viable. Three scenarios were investigated. In the first scenario revenue was calculated from the ethanol sales linked to the basic fuel price, whereas in the second and third scenarios liquefied petroleum gas (LPG) consumption at the paper mill was replaced with anhydrous and 95% ethanol respectively. In all the cases, paper sludge feed rates of 15, 30 and 50 t d-1 were used. The production of ethanol from paper sludge for ethanol sales (scenario 1) resulted in higher IRR and NPV values, as well as shorter payback periods, compared to replacement of LPG at the paper mill (scenarios 2 and 3). At an assumed enzyme cost of $ 0.90 gal-1 (R 2.01 litre-1), IRR values of 11%, 22% and 30% were obtained at paper sludge feed rates of 15, 30 and 50 t d-1. A sensitivity analysis performed on the total capital investment and enzyme cost revealed that the SSF process is only economically viable at a paper sludge feed rate of 50 t d-1 irrespective of the variation in capital investment. For the SSF process to be economically viable the enzyme costs must be lower than $ 0.70 gal-1 (R 1.56 litre-1) and $ 1.20 gal-1 (R 2.68 litre-1) for paper sludge feed rates of 30 and 50 t d-1 respectively. The SSF process at a paper sludge feed rate of 15 t d-1 was not economically viable even assuming a zero enzyme cost. A Monte Carlo simulation revealed that the SSF process is economically viable at a paper sludge feed rate of 50 t d-1 as a mean IRR value of 32% were obtained with a probability of 26% to attain an IRR value lower than 25%. The SSF process at lower paper sludge loadings is not economically viable as probabilities of 70% and 95% were obtained to attain IRR values lower than 25% at paper sludge feed rates of 30 and 15 t d-1 respectively. From this study it can be concluded that paper sludge is an excellent feedstock for ethanol production for the sales of ethanol at a paper sludge feed rate in excess of 50 t d-1 with the added environmental benefit of reducing GHG emissions by 42.5%. / AFRIKAANSE OPSOMMING: Aangesien dat brandstof vir vervoer en energie meestal vanaf fossiel afgeleide bronne kom, is daar onlangs ʼn groter fokus op bio-brandstowwe as gevolg van die impak van fossiel afgeleide brandstowwe op die omgewing en 'n verhoogde aanvraag na energie wêreldwyd, gepaardgaande met die uitputting van fossielbrandstof-reserwes. Papier slyk geproduseer deur papier meule is hoog in lignosellulose en verteenwoordig 'n grootliks onontginde grondstof vir etanol produksie. Die doel van die studie was om vas te stel wat die samestelling, fermenteerbaarheid, optimale papier slyk en ensiem ladings is vir die vervaardiging van etanol uit papier slyk. Die inligting was gebruik om 'n model van die proses in Aspen Plus® te ontwikkel. Die massa-en energiebalanse wat verkry is van die Aspen Plus® model was gebruik om toerusting spesifikasies te ontwikkel wat gebruik was om toerusting kostes te bereken. ‘n Tegno-ekonomiese model is ontwikkel om die ekonomiese lewensvatbaarheid van die gelyktydige versuikering en fermentasie proses “SSF” wat gebruik maak van papier slyk as grondstof te assesseer. Nege papier slyk monsters verkry vanaf Nampak Tissue (Pty) Ltd. is geëvalueer in terme van etanol produksie. Die monsters wat die hoogste en laagste etanol konsentrasies opgelewer het, is geselekteer vir optimalisering omdat dit toegelaat het vir die vasstelling van etanol konsentrasies en opbrengste, uitgedruk as persentasie van die teoretiese maksimum, wat verwag kan word in industrie. Reaksie oppervlak metodologie “RSM” is gebruik om wiskundige modelle te ontwikkel om die impak van papier slyk lading en sellulase dosis op etanol produksie en etanol opbrengs te assesseer. Die RSM is aangevul met 'n multi effek optimiserings benadering wat 'n wenslikheid funksie inkorporeer om die optimale papier slyk lading en sellulase dosis in gevoerde-enkellading SSF kulture te bepaal. Die multi effek optimalisering het getoon dat 'n optimale papier slyk lading van 21% (w/w) en 'n sellulase dosis van 14.5 FPU g-1 gebruik moet word, ongeag van die papier slyk monster. Die feit dat die optimale ensiem dosis en papier slyk lading dieselfde is ongeag die papier slyk monster, is aantreklik aangesien die SSF proses meer doeltreffend beheer kan word omdat proses veranderinge nie nodig is om die proses te optimaliseer nie. By die optimale papier slyk lading en sellulase dosis kan 'n minimum etanol konsentrasie van 47.36 g l-1 (84,69% van die teoretiese maksimum) verwag word ongeag van die papier slyk wat gebruik word. 'n Ekonomiese evaluasie is gedoen om vas te stel of etanol produksie vanaf papier slyk met behulp van SSF ekonomies lewensvatbaar is. Drie moontlikhede is ondersoek. In die eerste moontlikheid is die inkomste bereken vanaf etanol verkope gekoppel aan die basiese brandstofprys, terwyl in die tweede en derde moontlikhede, LPG by die papier meul vervang is met anhidriese en 95% etanol onderskeidelik. In al die gevalle was daar gebruik gemaak van papier slyk voer tempo’s van 15, 30 en 50 t d-1. Die produksie van etanol uit papier slyk vir verkope (moontlikheid 1) het gelei tot hoër IRR en die NPV waardes, sowel as korter terugverdien tydperke, in vergelyking met die vervanging van LPG by die papier meul (moontlikhede 2 en 3). Met ʼn ensiem koste van $ 0.90 gal-1 (R 2.01 litre-1) is IRR-waardes van 11%, 22% en 30% verkry teen papier slyk voer tempo’s van 15, 30 en 50 t d-1 onderskeidelik. 'n Sensitiwiteitsanalise uitgevoer op die totale kapitale belegging en ensiem koste het aan die lig gebring dat 'n SSF proses slegs ekonomies lewensvatbaar is op 'n papier slyk voer tempo van 50 t d-1 ongeag van die variasie in die kapitale belegging. Vir die SSF proses om ekonomies lewensvatbaar te wees, moet die ensiem kostes laer wees as $ 0.70 gal-1 (R 1.56 liter-1) en $ 1.20 gal-1 (R 2.68 liter-1) vir papier slyk voer tempo’s van onderskeidelik 30 en 50 t d-1. Die SSF proses was op 'n papier slyk voer tempo van 15 t d-1 nie ekonomies lewensvatbaar nie, selfs teen 'n ensiem koste van nul. 'n Monte Carlo-simulasie het getoon dat die SSF proses ekonomies lewensvatbaar is met 'n papier slyk voer tempo van 50 t d-1 omdat 'n gemiddelde IRR-waarde van 32% verkry is met 'n waarskynlikheid van 26% om 'n IRR-waarde laer as 25% te verkry. Die SSF proses teen papier slyk voer tempo’s van 30 en 15 t d-1 is nie ekonomies lewensvatbaar nie omdat waarskynlikhede van 70% en 95% onderskeidelik verkry is om IRR-waardes laer as 25% te kry. Daar kan van die studie afgelei word dat papier slyk 'n uitstekende grondstof is vir die produksie van etanol mits 'n papier slyk voer tempo van meer as 50 t d-1 bereik kan word. Die produksie van etanol vanaf papier slyk het die bykomende voordeel dat kweekhuis gasse (GHG) met 42.5% verminder word.

Thesis (MscAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / Over the years, different techniques have been used to legally reduce the ethanol content of wines. Several physical processes are available for producing wines with less alcohol. Despite their efficacy, these treatments have a capital and operational cost influence. They can also affect the concentration of other wine components. On the other hand, vast amount of research has been conducted through genetic modification of wine yeast strains in order to reduce the ethanol yield of Saccharomyces cerevisiae by diverting sugar metabolism towards various byproducts. However, genetically modified yeasts are not currently accepted in most wine industries worldwide, including South Africa. Therefore, other approaches need to be envisaged. Commercial enzymes are commonly added during winemaking. Most enzymes essential for vinification naturally occur in grapes, but are inefficient under pH and sulphur levels associated with winemaking. Enzymes of fungal origin are resistant to such conditions. The most widely used commercial enzymes include pectinases, hemicellulases, glucanases and glycosidases. With the exception of glucanases, produced by Trichoderma harzianium, all the other enzymes are produced by Aspergillus niger. In this study, the possibility of using Gluzyme Mono® 10.000 BG (Gluzyme) (Novozymes, South Africa) to reduce the glucose content of synthetic grape must and grape must before fermentation in order to produce wine with a reduced alcohol content was investigated. Gluzyme is a glucose oxidase preparation from Aspergillus oryzae, currently being used in the baking industry. Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide in the presence of molecular oxygen. Gluzyme was initially used in synthetic grape must where different enzyme concentrations and factors influencing its activity were investigated for its use in winemaking. The results showed that up to 0.5% v/v less alcohol were obtained using an enzyme concentration of 20 kU compared to the control. This reduction in alcohol was increased to 1 and 1.3% v/v alcohol at pH 3.5 and pH 5.5 respectively in aerated synthetic grape must using 30 kU enzyme. Secondly, Gluzyme trials were carried out using Pinotage grape must. Gluzyme treated wines after fermentation contained 0.68% v/v less alcohol than the control samples at 30 kU enzyme. Colour and volatile flavour compounds of treated wine did not differ significantly from the untreated samples. Lower free anthocyanin and total phenol concentrations in treated than control samples were observed, possibly due to the hydrogen peroxide oxidation which could have led to polymerisation. The present study has clearly demonstrated that Gluzyme may be used in winemaking to produce reduced-alcohol wine without affecting its colour and aroma compounds. The enzyme in its current form is however, not ideal for winemaking; other forms such as liquid or powder form should be considered if the enzyme is to be used under winemaking conditions. Future work should focus on evaluating the potential new form of the enzyme and studying the effects of Gluzyme in various grape must in semi-industrial scale. A tasting panel should also evaluate its impact on the organoleptic properties and the overall quality of the resulting wines.

O ácido lático produzido por bactérias láticas constitui um sério problema nas fermentações industriais. Por este motivo, os efeitos deste ácido adicionado sobre a produção de etanol por levedura em meio sintético foi estudado no presente trabalho. Assim, foi possível evitar a interferências de um processo contendo uma cultura mista de bactérias láticas e levedura. Além disto, o meio sintético foi utilizado a fim de evitar as variações na composição da matéria-prima, no caso o melaço de cana-de-açúcar, durante a estocagem por um longo período de tempo. Inicialmente, dois planejamentos fatoriais foram realizados em batelada simples. Após a validação deste sistema, uma produção máxima de etanol (72,1 g.L-1) e uma boa retenção de viabilidade celular (86,4%) foram conseguidas utilizando-se inóculo bastante alto (40 g.L-1, massa seca) numa fermentação contendo 200 g.L-1 de sacarose inicial. Nestas condições, experimentos em batelada simples em mini-reatores foram realizados operando a 34 °C e em presença de 0 a 1,0 g.L-1 de ácido lático. Neste experimento, o pH do meio tornou-se bastante baixo (2,3 a 2,7). Por este motivo, outra fermentação em batelada simples foi realizada com o reajuste do pH inicial para 4,5 logo após adição de todos os componentes ao reator. O reajuste do pH inicial causou um aumento significativo tanto na produtividade e na velocidade específica máxima de etanol quanto na viabilidade final na batelada simples. Depois disto, comparações da batelada simples com a batelada alimentada continuamente e por pulsos também foram realizadas em presença de ácido lático. Observou-se que o processo de batelada com alimentação contínua consumiu açúcar mais lentamente, apresentou maior perda em viabilidade e maior produção de biomassa que a batelada simples enquanto que o processo de batelada alimentada por pulsos foi bem semelhante. A elevação da concentração do ácido lático (2,0 g.L-1 a 4,0 g.L-1) nos reatores permitiu a tamponagem do meio sintético logo após o reajuste inicial de pH para 4,5. Nestas condições, também houve semelhança entre os processos de batelada simples e batelada alimentada por pulsos. Em batelada alimentada por pulsos, verificou-se que a diminuição da quantidade de inóculo levou a perdas de viabilidade celular por causa do aumento do ácido externo em relação à quantidade de células no reator. De uma maneira geral, a presença de ácido lático causou aumentos no consumo de açúcar em todos os processos. Fermentações sucessivas foram realizadas, adicionado 3 pulsos de sacarose e 2,0 g.L-1 de ácido lático (em relação ao volume final do meio), a levedura 63M passou por um processo de adaptação até o terceiro ciclo. Além disto, houve uma redução na proteína intracelular final e um aumento na acidez total em presença ácido lático. No entanto, a produção de glicerol e a produção de trealose aumentaram e foram semelhantes tanto em presença quanto em ausência de pulsos de ácido lático. / Lactic acid produced by lactic bacteria is a problem serious in fermentation alcohol plants. For this reason, the effects of this added acid on ethanol by yeast in synthetic medium were studied in the present work. Thus, it was possible to avoiding interferences of a process containing a miscellaneous culture of lactic bacteria and yeast. Besides, synthetic medium was used to avoiding variations in raw material, sugar-cane molasses in this case, during the storage by a large period of time. Initially, two factorial planning were used in simple batch under agitation. After validation of these plannings, maximal ethanol production (72.1 g.L-1) with well retention of viability (86,4%) were obtained using high inoculum size (40 g.L-1, dry weight) in fermentation containing 200 g.L-1 initial sucrose. In these conditions, fermentations in simples batch in mini-reactors were carried out at 34 °C and in presence of 0 to 1,0 g.L-1 added lactic acid. In this experiment, the medium pH became very small (2.3 to 2.7). For this reason, another fermentation in simple batch was realized with initial pH readjusted for 4,5 after addition of all of medium compounds in the reactor. The readjusted of initial pH caused a significative increase as in productivity and maximal-specific rate of ethanol as in final viability in simple batch process. After this, comparisons among simple batch and continuous-fed batch and pulses-fed batch were made in the presence of lactic acid. In these experiments, continuous-fed batch process showed sugar uptake more slowly, showed more drops in viability and more biomass production than simple batch process, while the pulses-fed batch was similar than simple batch process. In addition, the elevation of lactic acid concentration for 2,0 g.L-1 to 4,0 g.L-1 in the reactors permitted the pH maintenance of synthetic medium after initial pH readjusted for 4,5. In these conditions, the simple batch and pulses-fed batch processes also were similar. In pulses-fed batch culture, the decrease of inoculum size caused drops in viability because of the increase of endogenous acid in relation to cell quantity in the reactor. Generally, the presence of lactic acid causes increases in sugar uptake in all of process. Fermentations with cell reuse were carried out in fed batch process with pulses of sucrose and 2,0 g.L-1 lactic acid (in relation to final medium volume), yeast 63M undertook to adaptation process until the third fermentation cycle. In adittion, a decrease of final intracellular protein and an increase of total acidity in presence of lactic acid. However, glycerol and trehalose productions increased with the fermentative cycles and were similar as in presence as in absence of added lactic acid.

Propylene is the second most important starting chemical in the petrochemical industry after ethylene. Unlike ethylene, propylene readily undergoes substitution reactions including polymerisation, oxidation, halogenation, hydrohalogenation, alkylation, hydration, oligomerization and hydroformylation, which lead to a wide variety of important downstream products. One of the principal uses of propylene is to produce key chemicals from selective oxidation. In 2016, the world annual production of propylene is about 94 million tonnes, and the global proportion used to produce selective oxidation product is over 18%. They constitute a key part of the chemical industry and contribute towards substantial economic benefits. The application of Ag based heterogeneous catalysts to selective propylene oxidation is a key factor in the synthesis of nearly all downstream chemicals, however billions of pounds are lost every year due to unplanned reactor shutdown, safety control and environment unfriendly emission control as a results of inefficiency catalytic selectivity and activity. Despite, both theoretical and experimental research works have been intensively involved, the fundamental reason leading to these effects are not yet well understood. The work presented in this thesis explores a range of novel modification techniques that alter the activity of Ag nanocatalysts for selective propylene oxidation, especially in propylene epoxidation. Particular focus is placed on developing surface modified Ag catalysts through morphology control, surface architecture engineering with another sublayer metal. Using a combination of modelling, novel and traditional materials characterisation methods, it is found that these modification result in some significant electronic and/or geometric alterations to the Ag nanoparticles surface. The Ag-Ag bond distance can be dramatically enlarged by exposing a high-index Ag surface or a core-shell structure with monolayer Ag shell. When interacting with molecular oxygen, the molecular oxygen adsorption and dissociation behaviour is sensitive to the geometric changes in Ag surface. This leads to an enhanced selectivity toward propylene epoxidation than combustion resulting from preventing a C-H bond cleavage. Finally, be creating atomically dispersed Ag on zeolite, a completely different interaction between molecular oxygen and single atom Ag were discovered comparing to on a extensive silver surface. This leads to the observation of an excitingly new propylene oxidation reaction producing ethanol and CO₂ resulting from C=C bond cleavage. Overall, the research presented within this thesis demonstrated a number of methods for the intelligent design of novel heterogeneous Ag catalysts with remarkable activity and selectivity toward specific selective propylene oxidation. These modification methods are believed to be potentially applicable to a wide range of other catalytic reactions.

Currently there is great concern about the consumption of fossil fuels, as well as a possible depletion of traditional sources of production of raw materials mainly hydrocarbon derivatives. Parallel challenges to the preservation of the environment bring to discussions agenda every question involving biofuels and their means of obtaining. That said this issue still has major concerns about the use of raw materials that require less energy expenditure and prevent competition for land for food production, which end up entering the agenda of externalities related with this issue. In the case of the production of ethanol from starch sources, traditional processes still have high costs associated with the production of enzymes used and the great energy demand for starch gelatinisation at high temperatures. An alternative to the granular starch hydrolysis process is the use of enzymes capable of processing these starches at low temperatures which makes it unnecessary to gelatinization and reduces energy expenditure, also called simultaneous saccharification and fermentation (SSF), resulting in considerable economic advantage. Brazil is a traditional agricultural producer, and the rice production is quite significant and is as a by-product of rice bran that has low commercial value, being used for oil extraction, as an ingredient in animal feed and as fertilizer organic therefore be in perfect conditions to be used as raw starchy material to obtain ethanol. This work aims to investigate the use of this raw material and set the parameters for increased production scale-up with the use of commercial enzymes. The initial data to establish the working parameters were taken from the literature establishing this initial data for testing. In view of the large number of variables used to Plackett-Burman methodology to verify the effects of the main variables and select the ones that have most effect on the model.Thus for the job 1 in the shake flasks were selected following parameters: concentration of bran inoculum percentage, corn percentage of water macerated, soybean concentration, enzyme percentage yeast addition time and percentage of cellulase. This initial planning, among the most significant variables and, consequently, were selected for the planning application Outlining Central Composite Rotational (CCRD), are rice bran concentration, the percentage of inoculum and the percentage of cellulase. To work 2 were investigated variables agitation and temperature in ethanol production in bioreactor 3 liters. For analysis of the experiment of reducing sugar was used DNS method (Miller, 1959), and analysis of the ethanol present in the sample specimens were removed and subjected to Alcolyzer. For the tests performed in the first work the greatest amount of ethanol was obtained at 12 assay which was reached 172.70 grams of ethanol per kilogram of rice bran with 84% efficiency in the experiment. For experiments performed in the second study, with 3 liter reactor the best result is shown in Test 1 reaching 182.52 grams of ethanol per kilogram of rice bran with 89% efficiency. Thus, it was demonstrated that there is considerable potential for the production of ethanol using this raw material. Taking into account that the availability of this raw material is estimated at 1 million tonnes per year, the IRGA, we would have a much ethanol around 35 to 50 million liters per year. / Atualmente, existe grande preocupação em relação ao consumo de combustíveis fósseis, bem como um possível esgotamento das tradicionais fontes de produção de matéria-prima, principalmente os derivados de hidrocarbonetos. Paralelamente a isso, os desafios com a preservação do meio ambiente trazem para as discussões questões que envolvem os biocombustíveis e seus meios de obtenção. Isso posto, essa questão ambiental ainda traz grandes preocupações referentes à utilização de matérias primas que exijam menos gastos energéticos, bem como evitar a disputa por terras destinadas à produção de alimentos, o que acaba entrando na pauta das externalidades envolvendo o tema. No caso da produção de etanol de fontes amiláceas, os processos tradicionais ainda apresentam elevados custos de produção associados às enzimas empregadas e à grande demanda energética para a gelatinização do amido com altas temperaturas. Uma alternativa ao processo de hidrólise do amido granular é a utilização de enzimas capazes de processar esses amidos a baixas temperaturas, o que torna desnecessária a gelatinização e reduz o gasto energético, também denominado sacarificação e fermentação simultânea (SSF), apresentando considerável vantagem econômica. O Brasil é um tradicional produtor agrícola, sendo que a produção de arroz é bem expressiva e tem como um dos subprodutos o farelo de arroz. Este subproduto possui baixa valor comercial e é utilizado para extração de óleo, além de funcionar como ingrediente de ração animal e como fertilizante orgânico, estando, portanto, em perfeitas condições para utilização como matéria-prima amilácea para obtenção de etanol. A partir disso, este trabalho visa investigar a utilização e a viabilidade técnica do farelo de arroz, com a utilização de enzimas comerciais. Os dados iniciais para estabelecer os parâmetros de trabalho foram retirados da literatura. Tendo em vista o grande número de variáveis, utilizou-se a metodologia Plackett-Burman, visando verificar os efeitos das principais variáveis e selecionar as que tiveram maior efeito sobre o modelo. Dessa forma para o trabalho 1 em frascos agitados foram selecionadas as seguintes variáveis: concentração de farelo, percentual de inoculo, percentual de água de milho macerada, concentração de farelo de soja, percentual de enzima, tempo de adição de levedura e percentual de celulase. Desse planejamento inicial, entre as variáveis mais significativas e que, consequentemente, foram selecionadas para a aplicação do planejamento Delineamento Composto Central Rotacional (DCCR), estão a concentração de farelo de arroz, o percentual de inoculo e o percentual de celulase. Para o trabalho 2 em biorreator de 3 litros, foram investigadas as variáveis agitação e temperatura na produção de etanol. Para análise do açúcar redutor dos experimento foi utilizado a metodologia de DNS (Miller, 1959), e para análise do etanol presente nos ensaios foram retirados amostras e submetidas ao Alcolyzer. Para os ensaios realizados no primeiro trabalho a maior quantidade de etanol foi de 172,70 gramas de etanol por quilograma de farelo de arroz, com eficiência de 84%. Para os experimentos realizados no segundo trabalho, com reator de 3 L o melhor resultado foi 182,52 gramas de etanol por quilograma de farelo de arroz com eficiência de 89 %.Dessa forma, foi demonstrado que existe um potencial considerável para produção de etanol utilizando esta matériaprima, tendo em vista que há a estimativa de 1 milhão de toneladas por ano, com a possibilidade de produção de 35 a 50 milhões de litros adicionais de etanol por ano.

Made available in DSpace on 2014-06-11T19:35:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-07Bitstream added on 2014-06-13T20:26:28Z : No. of bitstreams: 1 dalchiavon_fc_dr_ilha.pdf: 1313078 bytes, checksum: 6554536e2e3f1a0493c8f1868557714c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cultura da cana-de-açúcar desempenha, frente à realidade brasileira, sobretudo no aspecto relacionado às fontes energéticas alternativas, importantíssimo papel. Assim, num talhão de cana-de-açúcar (variedade RB855035, em seu terceiro corte) da Usina Vale do Paraná S/A Álcool e Açúcar, localizada no município de Rubinéia (SP), Brasil, 20°17’53’’ de Latitude Sul e 51°02’23’’ de Longitude Oeste, manejado para duas formas de colheita da cana-crua (com manutenção e com remoção da palhada), foram estudadas as correlações (lineares de Pearson e as geoestatísticas) entre a produtividade de colmos e atributos físico- químicos do solo, com os objetivos de verificar, entre as duas modalidades de colheita da cana-crua: a) o atributo do solo que possui a melhor correlação de Pearson com a produtividade da cana-de-açúcar; b) o atributo do solo que possui a melhor cokrigagem (correlação geoestatística) com a produtividade da cana-de-açúcar; e c) analisar se há diferença entre os dois métodos de colheita de cana-crua. Para tanto, foram instaladas duas malhas geoestatísticas regulares, contendo cada uma 1,30 ha com 121 pontos amostrais georreferenciados, em um Argissolo Vermelho eutrófico (declive homogêneo de 0,065 m m- 1 ), no ano de 2011. Os atributos avaliados das plantas foram: a) produtividade de colmos de cana-de-açúcar, b) plantas por metro, c) quantidade de palhada sobre o solo, d) estoque de carbono na palhada, e e) açúcares totais recuperáveis. Os do solo foram: a) resistência mecânica à penetração, b) umidade gravimétrica, c) densidade, d) estoque de carbono, e e) pH (CaCl2), nas profundidades de 0-0,20 e 0,20-0,40 m, além da altitude do terreno. A análise estatística dos dados ficou compreendida de análise descritiva, teste F, análise das correlações lineares e análise geoestatística... / The sugarcane crop play a very important role on the Brasilian reality, mainly in the aspect related to the alternative sources. So, in a sugarcane area (variety RB855035, third cut) at Vale do Paraná S/A Alcohol and sugar destillary, lacated in Rubinéia-SP, Brazil, 20°17'53''S and 51°02'23''W, managed for two forms of harvest of the raw cane (with maintenance and with removal of the straw) they were studied the correlations (linear of Pearson and the geostatistical) between the productivity of stems and physiochemical attributes of the soil, with the objectives of verifying, between the two modalities of crop of the raw cane: a) the attribute of the soil that provides the best correlation of Pearson with the productivity of the sugarcane; b) the attribute of the soil that provides the best cokriging (correlation geoestatistical) with the productivity of the sugarcane; and c) to analyze if there is difference between the two methods of crop of raw cane. For this were installed two regular geoestatistical meshes containing each one 1.30 ha with 121 sampling from a Typic Haplustult (homogeneous slope of 0.065 m m-1), in 2011. The appraised attributes of the plants were: a) productivity of sugarcane stems, b) plants for meter, c) amount of straw on the soil, d) stock of carbon in the straw, and e) recoverable total sugar content in sugarcane. The attributes of the soil were: a) mechanical resistance to the penetration, b) gravimetric moisture, c) bulk density, d) stock of carbon, and e) pH (CaCl2), at depths of 0-0.20 and 0.20- 0.40 m, besides the altitude of the land. The statistical analysis of the data was performed by descriptive analysis, test F, analysis of the linear correlation and geostatistical analysis. For the crop of the raw cane with maintenance of the straw, the bulk density, at depth of 0-0.20 m, was the attribute of... (Complete abstract click electronic access below)