Orientadores: Maria Regina Wolf Maciel, Reinaldo Gaspar Bastos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T08:48:28Z (GMT). No. of bitstreams: 1 Klein_BrunoColling_M.pdf: 2562214 bytes, checksum: f891de86d253786cf5d2101fec1f3eba (MD5) Previous issue date: 2013 / Resumo: A busca por uma maior sustentabilidade tem levado a uma mudança em direção à utilização de fontes renováveis para geração de energia em detrimento do uso de combustíveis fósseis, visando a uma modificação na matriz energética global. A utilização da biomassa de microalgas para produção de biocombustíveis vem sendo vista como uma alternativa promissora, uma vez que o seu cultivo proporciona produtividades em carboidratos e lipídios superiores às matérias-primas vegetais convencionalmente utilizadas na obtenção de etanol e biodiesel. Neste contexto, o objetivo da presente dissertação de mestrado foi avaliar a produção de biomassa da microalga clorofícea Chlorella vulgaris em fotobiorreator de placa plana em diferentes condições de fluxo luminoso, concentração de CO2 na alimentação gasosa e concentração de NaNO3 no meio de cultivo, visando o acúmulo de carboidratos para obtenção de bioetanol de terceira geração. As influências das variáveis nutricionais e de processo sobre a eficiência fotossintética das microalgas também foram estimadas para determinação do estado fisiológico das culturas. A produtividade média de biomassa e a concentração máxima final das microalgas foram significativamente afetadas pela incidência de radiação luminosa e pela suplementação de CO2 gasoso, obtendo-se maiores produtividades de carboidratos em cultivos com alto fluxo luminoso e concentrações de CO2 intermediárias (7,5%). Também foi observado o efeito positivo do aumento do fotoperíodo sobre o crescimento das microalgas. Através de hidrólise ácida foi possível atingir concentrações de até 2 g L-1 de açúcares fermentescíveis no hidrolisado a partir de biomassa de microalgas cultivadas em meio com baixo teor de nitrogênio. A fermentação etanólica foi então conduzida com a levedura Dekkerabruxellensis capaz de converter diferentes hexoses e pentoses em bioetanol, dada a presença de ambos os tipos de açúcares no hidrolisado / Abstract: The search for industrial processes with higher sustainability has led to a change towards the utilization of renewable sources for energy generation in substitution of fossil fuels, aiming the modification of the global energy matrix. The utilization of microalgal biomass for the production of biofuels is viewed as a promising alternative, since its cultivation yields carbohydrate and lipid productivities superior to those of conventional sources used in the obtention of bioethanol and biodiesel. In this context, the goal of this master thesis was to evaluate the biomass production of the chlorophycean microalga Chlorella vulgaris in a flat plate photobioreactor under different conditions of light flux, CO2 concentration in the gas feed and NaNO3 concentration in the culture medium, aiming carbohydrate accumulation for the production of third generation bioethanol. The influences of both process and nutritional variables on the photosynthetic efficiency of microalgae were estimated for the determination of the physiological condition of the cultures. The mean biomass productivity and the maximum final microalgae concentration were significantly affected by the incidence of light radiation and by the supplementation of gaseous CO2, the highest carbohydrate productivities being obtained in cultivations with high light flux and intermediate CO2 concentrations (7,5%). It was also observed the positive effect of increasing the photoperiod over microalgae growth. Through acid hydrolysis, it was possible to attain fermentable sugar concentration of up to 2 g L-1 from biomass of microalgae cultivated in low-nitrogen medium. The ethanolic fermentation was then carried out with the Dekkerabruxellensis yeast, capable of converting different hexoses and pentoses into ethanol, due to the presence of both sugar types in the hydrolysate / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química

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by Ng Wang Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Table of Contents --- p.xv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global picture --- p.3 / Chapter 2.1.2 --- Malaria mechanics --- p.4 / Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4 / Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5 / Chapter 2.2.1 --- Antimalarial drugs --- p.5 / Chapter 2.2.2 --- Drug resistance --- p.6 / Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7 / Chapter 2.3.1 --- Malarial vaccine developments --- p.7 / Chapter 2.3.2 --- Transmission blocking vaccines --- p.7 / Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9 / Chapter 2.3.4 --- Blood stage vaccines --- p.10 / Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11 / Chapter 2.5 --- Plants as bioreactors --- p.12 / Chapter 2.5.1 --- Products of transgenic plants --- p.13 / Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14 / Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15 / Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15 / Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16 / Chapter 2.7.1 --- Arabidopsis --- p.16 / Chapter 2.7.2 --- Tobacco --- p.17 / Chapter 2.8 --- Transformation methods --- p.17 / Chapter 2.8.1 --- Direct DNA uptake --- p.17 / Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17 / Chapter 2.8.1.2 --- Biolistic transformation --- p.18 / Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18 / Chapter 2.8.2.1 --- Leaf-disc technique --- p.18 / Chapter 2.8.2.2 --- In planta transformation --- p.19 / Chapter 2.9 --- Phaseolin --- p.20 / Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21 / Chapter 2.11 --- Aims of study and hypotheses --- p.22 / Chapter Chapter 3: --- Materials and Methods --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Chemicals --- p.24 / Chapter 3.3 --- Expression in tobacco system --- p.24 / Chapter 3.3.1 --- Plant materials --- p.24 / Chapter 3.3.2 --- Bacterial strains --- p.25 / Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25 / Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26 / Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30 / Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30 / Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33 / Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34 / Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35 / Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36 / Chapter 3.3.8 --- GUS assay --- p.37 / Chapter 3.3.9 --- Genomic DNA isolation --- p.37 / Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38 / Chapter 3.3.11 --- Southern blot analysis --- p.38 / Chapter 3.3.12 --- Total seeds RNA isolation --- p.39 / Chapter 3.3.13 --- RT-PCR --- p.39 / Chapter 3.3.14 --- Northern blot analysis --- p.40 / Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40 / Chapter 3.3.16 --- Western blot analysis --- p.41 / Chapter 3.4 --- Expression in Arabidopsis system --- p.42 / Chapter 3.4.1 --- Plant materials --- p.42 / Chapter 3.4.2 --- Bacterial strains --- p.42 / Chapter 3.4.3 --- Chimeric gene construction --- p.42 / Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43 / Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45 / Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45 / Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49 / Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49 / Chapter 3.4.7 --- Arabidopsis Transformation --- p.49 / Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50 / Chapter 3.4.9 --- Selection of successful transformants --- p.51 / Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51 / Chapter 3.4.11 --- GUS assay --- p.52 / Chapter 3.4.12 --- Genomic DNA isolation --- p.52 / Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52 / Chapter 3.4.14 --- Southern Blot analysis --- p.52 / Chapter 3.4.15 --- Total siliques RNA isolation --- p.53 / Chapter 3.4.16 --- RT-PCR --- p.53 / Chapter 3.4.17 --- Northern blot analysis --- p.53 / Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54 / Chapter 3.4.18 --- Western blot analysis --- p.54 / Chapter 3.5 --- In vitro transcription and translation --- p.54 / Chapter 3.5.1 --- In vitro transcription --- p.54 / Chapter 3.5.2 --- In vitro translation --- p.55 / Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56 / Chapter 3.6.1 --- Chimeric gene constructs --- p.56 / Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61 / Chapter Chapter 4: --- Results --- p.63 / Chapter 4.1 --- Tobacco system --- p.63 / Chapter 4.1.1 --- Chimeric gene constructs --- p.63 / Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65 / Chapter 4.1.3 --- GUS activity assay --- p.67 / Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68 / Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68 / Chapter 4.1.4.2 --- Southern blot analysis --- p.70 / Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72 / Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72 / Chapter 4.1.5.2 --- Northern blot analysis --- p.75 / Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76 / Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78 / Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78 / Chapter 4.1.7.2 --- Western blot analysis --- p.78 / Chapter 4.2 --- Arabidopsis system --- p.83 / Chapter 4.2.1 --- Chimeric gene constructs --- p.83 / Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85 / Chapter 4.2.3 --- Selection of transgenic plants --- p.87 / Chapter 4.2.4 --- Assay of GUS activity --- p.91 / Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92 / Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92 / Chapter 4.2.5.2 --- Southern blot analysis --- p.96 / Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99 / Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99 / Chapter 4.2.6.2 --- Northern blot analysis --- p.106 / Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107 / Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108 / Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108 / Chapter 4.2.8.2 --- Western blot analysis --- p.108 / Chapter 4.3 --- In vitro transcription and translation --- p.112 / Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115 / Chapter Chapter 5: --- Discussion and Future perspectives --- p.117 / Chapter 5.1 --- Failure in detecting transgene expression --- p.117 / Chapter 5.2 --- Poor transgene expression --- p.120 / Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120 / Chapter 5.2.2 --- AT-richness --- p.121 / Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123 / Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125 / Chapter 5.2.5 --- Codon usage --- p.126 / Chapter 5.3 --- Future perspectives --- p.127 / Chapter Chapter 6: --- Conclusion --- p.129 / References --- p.131

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2005. / The Up-flow Anaerobic Sludge Blanket Reactor (UASB) provides a state-of–the-art solution to effluent treatment by anaerobic digestion: sludge production is dramatically lower than in other digestion processes, and energy is gained from the system if the produced biogas is converted to electricity and/or heat. The UASB is a modified fluidised bed reactor, with the solid state ‘catalyst’ being granulated anaerobic sludge, and the liquid phase the effluent that needs to be treated. A gas cap is installed to serve as a carbon dioxide and methane collector. This biogas (carbon dioxide and methane) is produced by the stepwise decomposition of complex carbohydrates and proteins via a consortium of micro-organisms living in a symbiotic environment known as a granule. A typical UASB reactor has an organic removal rate of 89-93% Chemical Oxygen Demand (COD) and operates optimally at loadings of 9.8-11 kg COD/ m3 reactor volume/day. Unfortunately, one major problem hampers the efficiency of this reactor to such an extent that the unit is only economically viable in exceptional cases; if the reactor is inoculated with un-granulated anaerobic sludge, start-up times of up to 12 months can be expected. The lengthy start-up times motivated the search for an artificial way to cultivate USAB granules. Early research (done on lab-scale, 400ml vessel volumes) proved that, under a specified set of environmental conditions, granule growth can occur in an artificial environment. Yet these laboratory-scale vessels did not facilitate scale-up or the study thereof. This led to the main problem statement of this research project: namely to design, commission, and optimise benchscale bioreactors that will generate granulated anaerobic sludge in an incubation period of 20 days. These units should also facilitate in the determining of parameters that will assist in the design of a scale-up to a UASB granule producing reactor of economically viable size. Two bench-scale reactors were initially designed specifically to “mimic” the motion found in the laboratory-scale vessels. The results from these initial reactors proved that granulation cannot only be enhanced, but granules can actually be cultivated from dispersed anaerobic sludge in a larger artificial environment over an incubation period of only 20 days. The results were still far from satisfactory, as the granules produced were irregular in shape and the yield of usable granules (2.2 kg/m3 reactor volume) insufficient. A third test reactor was designed to “mimic” roller table movement and baffles were included. These results were much better and the yield was 4.4 kg/m3 reactor volume at a baffle tipspeed of 0.0055 m/s. The optimisation was extended further to include the inoculation sludge and the feed medium. A C:N:P ratio of 10:1:4 proved to yield the best results. Monovalent anions, hydrogen concentration and a pH-level outside the 6.5 to 7.2 range evidently had an inhibitory effect on the granulation rate. After the optimisation study the third test unit produced a usable granule yield of 15.2 kg/m3 reactor volume over the 20-day incubation period. The incubation period can be separated into 3 distinct phases, namely the acidification, stabilisation and growth phases. From the mass balance it was found that most of the COD and nutrients were used for ECP production in the acidification phase. During the stabilisation phase, the COD and nutrients were mostly used for nucleus formation, and finally in the growth phase the COD was used for granule growth. To study the effect the internal surface area of the reactor has on the granulation process, 3 scale-down versions of the third test unit were constructed. Within the studied range, a yield of usable granules of 40 kg/m2 reactor internal surface area was obtained.

Thesis (PhD)--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production. / AFRIKAANSE OPSOMMING: Gewilde wynstyle word dikwels gemaak van volryp, goed ontwikkelde druiwe, gekarakteriseer deur intense aromas en smaakkomponente wat direk met spesifike kultivars geassosieer word. ’n Nadelige gevolg om druiwe te lank aan die wingerdstok te laat bly hang sodat meer intense geurkomponente kan ontwikkel, is die toename in suikerinhoud. Hierdie addisionele suiker lei tot wyne met hoër alkoholvlakke. Te hoë alkoholvlakke kan wyn ongebalanseerd laat voorkom en die smaak nadelig beïnvloed. Dit, tesame met gesondheidsredes en anti-sosiale gedrag wat gekoppel kan word aan die inname van te veel alkohol, strenger wetgewing aangaande dronkbestuur en die toename in belasting op wyne met ’n hoër alkoholinhoud, het aanleiding gegee tot ’n aanvraag vir wyn met ’n verlaagte alkoholinhoud, sonder dat aroma- en geurkomponente ingeboet word. Alhoewel daar sekere fisiese/gemeganiseerde prosesse beskikbaar is om die alkohol in wyn te verwyder of te verminder, is ’n nadeel dat hierdie prosesse baie duur en arbeidsintensief is, en dat dit deur sommige wynpuriste as te ingrypend in die ‘natuurlike’ proses van wynmaak beskou word. Sommige van hierdie alkoholverwyderingsprosesse kan ook die wyn se geur- en aromakomponente nadelig beïnvloed. As alternatief tot hierdie fisies-chemiese prosesse word wyngiste reg oor die wêreld deur verskillende navorsingsgroepe ontwikkel sodat van die druifsuikers nie na alkohol omgeskakel word nie, maar eerder ander metaboliete. Belowende navorsingsresultate in ’n voorafgaande studie het aanleiding gegee tot hierdie navorsingsprojek. In hierdie studie word daar klem gelê op die ontwikkeling, deur middel van genetiese manipulering, van ’n industriële wynras van Saccharomyces cerevisiae sodat dit in staat sal wees om glukose-oksidase (GOX; b-D-glukose:suurstof oksidoreduktase, EC 1.1.3.4) te produseer. GOX kan reeds b-D-glukose in die medium oksideer na glukoonsuur (GA), wat sodoende verhoed dat dit verder gemetaboliseer word via glukolise, en dit het tot gevolg dat ’n gedeelte van die beskikbare suiker nie omgeskakel word na alkohol nie. Die strukturele glukose-oksidase-geen (GOX) van die voedsel-gegradiëerde fungus, Aspergillus niger is gebruik tydens die konstruksie van drie kassette (GOX1, GOX2 en GOX2LOX). Binne hiedie geenkassette is A. niger se GOX-geen se transkripsieinisiëring en -terminering onafhanklik deur die fosfogliseraat-kinase-1-promotor (PGK1P) en termineerder (PGK1T ) bewerkstellig. Om uitskeiding van GOX uit die gis te bewerkstellig, is daar van die a-spesifieke gisferomoon-a-faktor (MFa1S) in GOX1 gebruik gemaak, en in GOX2, van A. niger se eie natuurlike sekresiesein. Hierdie geenkassette is binne-in die genoom van twee labaratoriumgisrasse van S. cerevisiae (BY4742 en S1278b) asook een industriële wyngisras (VIN13) geintegreer. ’n Addisionele integreringskasset (die sogenaamde GOX2LOX-kasset) is gemaak om die IME1-geen van S. cerevisiae te elimineer. Binne die GOX2LOXkasset is GOX2 aan ’n loxP-kasset gekoppel. Die nuwe wyngis VIN13-D1 is verkry deur ’n genomiese integrasie van GOX2LOX binne-in die IME1-lokus. Om die niesporulerende GOX-produserende wyngis VIN13-D2 te verkry, is VIN13-D1 gesporuleer, onderwerp aan mikromanipulasie en toegelaat om te herdiploidiseer. Ontledings het aangedui dat (i) GOX2 aanleiding gegee het tot hoër vlakke van ekstrasellulêre glukose-oksidase aktiwiteit in vergelyking met GOX1; en (ii) dat die vlakke van uitgeskeide biologies-aktiewe glukose-oksidase vir die wyngisrasse aansienlik hoër was. Dit het verdere kleinskaalse wynfermentasies geregverdig. Die getransformeerde wyngis VIN13-D1 is op eksperimentele skaal in die maak van rooi- en witwyn geëvalueer. Ontledings van hierdie eksperimentele wyne het daarop gedui dat glukose-oksidase deur die VIN13-D1-gisselle geproduseer en suksesvol uitgeskei tydens die wynmaakproses is, en dat VIN13-D1 die fermentasie gedomineer het en die alkoholiese gisting voltooi het. Resultate het egter ook aangedui dat die geproduseerde glukose-oksidase nie baie aktief was onder die wynmaaktoestande wat in hierdie eksperimentele wynmaakproses gegeld het nie, en gevolglik was daar nie ’n drastiese verlaging in die alkoholvlakke sigbaar toe VIN13-D1 se wyne met VIN13 se wyne vergelyk is nie. Wynmonsters is deur middel van Fourier-transformasie-mid-infrarooispektroskopie (FT-MIR) ontleed ten einde die chemiese samestelling te bepaal, en gaschromatografie-massaspektrometrie (GCMS) is aangewend om die wynaromakomponente te bepaal. Die vlakke van glukoonsuur is deur middel van ensiematiese reaksies bepaal. Multiveranderlike data-analise [hoofkomponentanalise (PCA) en parsiële kleinte kwadrate (PLS1) diskriminantanalise] is op die data van die verskeie analitiese tegnieke toegepas om onderliggende veskille tussen die wyne van VIN13 (wilde-tipe) en VIN13-D1 uit te wys. Chemometriese projeksies het aangetoon dat daar wel beduidende variasies sigbaar was tot en met drie hoofkomponente en/of PLS-komponente wat duidelik aandui dat glukoonsuur ’n sleutelfaktor was ten opsigte van die uitwerking wat GOX op VIN13-D1- fermentasies in vergelyking met VIN13-fermentasies. VIN13-D1 effek manifestasies is die beste waargeneem op grafieke wat PLS1-diskriminantanalise-data bevat. Verder het PLS1-diskriminantanalise ook aangetoon dat van die ‘groepe’ wat gebruik was tydens die analise, die FT-MIR-komponente en die GC-komponente beter resultate gelewer het. Die GC-komponente het hulle verder daartoe geleen om tussen die verskillende kultivars en VIN13/VIN13-D1-fermentasies te diskrimineer. Daar kan dus met sekerheid gesê word dat die grootste invloed in VIN13-D1 wyne sigbaar is in die aromakomponent, maar omdat daar wel ook variasies sigbaar was in die MIR-komponente, dat die smaakkomponente ook beïnvloed was. Die aktiwiteit van GOX in druiwesap is verder ondersoek deur gebruik te maak van kleinskaalse fermentasies in bioreaktors. Daar is bevestig dat die VIN13-D1- geproduseerde GOX biologies-aktief was tydens aerobiese kondisies, onaktief was tydens anaerobiese kondisies, en onmiddelik geaktiveer kon word wanneer ’n anaerobiese fermentasie aerobies gemaak word (gesimuleerde mikro-oksigenasie). Hierdie verskillende fermentasies dui daarop dat glukose-oksidase inderdaat aktief is in druiwesap, en dat suurstof ’n sleutelfaktor is tydens die aktivering van die ensiem. Met behulp van ’n vereenvoudigde model kon aangetoon word dat tydens ideale toestande dit wel moontlik is om die alkoholvlakke te verlaag na ’n voorafbepaalde konsentrasie vir die bereiding van lae-alkohol wyne. Hierdie studie verskaf verdere insig hoe om ’n GOX-produserende wyngis gedurende die wynmaakproses vir die verlaging van die alkoholvlakke te benut. Verder is dit duidelik dat suurstof van kardinale belang is vir die aktivering van die glukoseoksidase- ensiem en dat ’n tegniek soos mikro-oksigenasie ’n belangrike rol in hierdie verband tydens die wynmaakproses sou kon speel.

The exploitation of lignocellulosic materials with the aim of producing high value-added products will potentially counteract concerns related to the depletion of fossil resources or exponential population growth. Bioethanol produced from lignocellulosic agriculture residue exhibits promising alternative to the petroleum-based fossil fuel which reduces net emission of greenhouse gases (GHG). But, due to certain technological barriers, the large scale production of lignocellulosic bioethanol has not been successfully commercialized. In this thesis, membrane filtration as an energy efficient separation process with low environmental impact was chosen with a possibility of improvement. Interconnected multi-staged microfiltration submerged membrane bioreactors (MBRs) set-up has been applied in order to separate suspended solids, obtain high concentration of yeast inside the bioreactor, and recover particle-free ethanol stream in a continuous high productivity process. The MBRs were effectively optimized comparing to different constant permeate fluxes of 21.9 LMH, 36.4 LMH, and 51 LMH. Moreover, membrane bioreactor performed effectively at low flux 21.9 LMH up to 262 h comparing to other applied fluxes. During continuous hydrolysis, membrane showed the capability of lignin recovery nearly 70% of medium SS content in all applied flux. Although the conversion rate of total sugars by concentrated cells were similar, yeast cells proved the capability of inhibitor tolerance, and to co-utilize 100% of glucose and up to 89% of xylose, resulted in bioethanol volumetric productivity of 0.78 g ethanol/l per hour 1.3 g ethanol/l per hour and 1.8 g ethanol/l per hour for 21.9 LMH, 36.4 LMH, and 51 LMH respectively. Moreover, the effect of different factors such as filtration flux, medium quality and backwashing on fouling and cake-layer formation in submerged MBRs during continuous filtration was thoroughly studied.

Este trabalho teve por objetivo verificar a influência da agitação no cultivo em meio sólido, FES, quanto a crescimento microbiano, homogeneização do meio e remoção de calor. As correlações obtidas contribuem na definição de critérios para ampliação de escala da FES. O modelo adotado foi o cultivo do fungo Monascus sp. em arroz. Os experimentos foram conduzidos em reator tubular horizontal de 40 l, com agitação interna intermitente, camisa de resfriamento e vazão de ar de 2 l.min-1.Kgms-1. Os cultivos foram realizados com base num planejamento fatorial rotacional: 12 a 60 rotações das pás em 24 horas; 2 a 12 horas de intervalo entre os eventos de agitação. A intensidade do crescimento celular foi considerada com base no consumo de O2, produção de CO2 , concentrações de proteína e ergosterol. O consumo de O2 apresenta correlação de 81% com os padrões de agitação sendo que tanto o número de rotações quanto o intervalo entre os eventos de agitação influenciam negativamente o crescimento celular assim estimado. Por outro lado, a máxima velocidade de consumo de oxigênio, OUR, obtida por volta de 24 horas, em cultivos com menores intervalos entre os eventos de agitação, indica efeito positivo da agitação sobre a velocidade do crescimento de fungos em superfície, enquanto não ocorre compactação do meio de cultivo. Conclui-se, portanto, que a natureza do substrato empregado, arroz, cuja reologia é sensivelmente alterada pela agitação, contribuiu de modo deletério à respiração celular e que a adoção de reatores com agitação na FES, requer substrato com baixo teor de amido e elevado teor de fibras. As medidas de ergosterol apresentaram correlação de 85% com os padrões de agitação mostrando que o intervalo entre os eventos de agitação é o fator com maior impacto nesta resposta e os ensaios com maiores intervalos entre os eventos de agitação e maior número de voltas apresentaram concentrações aproximadamente dez vezes maiores de ergosterol em relação aos outros ensaios. Os coeficientes de variação de umidade em cinco pontos do reator representam a homogeneidade, pois relacionam-se com os padrões de agitação com correlação de 95%. / This investigation aimed to verify the influence of mixing microbial growth, medium homogenization and heat removal within a solid state fermentation (SSF) bioreactor. The correlations obtained will help to establish the scale-up criteria. The model system involved the cultivation of the fungi Monascus sp. on rice. The assays were performed in a 40 l bioreactor under internal intermittent mixing with a cooling jacket and an air flux of 2 l.min-1 kgdm-1. The cultivations followed a rotational factorial plan: 12 to 60 paddle revolutions in 24 hours; with an interval of 2 to 12 hours between mixing events. Cellular growth rate was estimated by O2 consumption, CO2 production, and protein and ergosterol concentrations. The O2 consumption showed an 81% correlation with the revolutions pattern, and both the number of revolutions and interval between mixing events, influenced cell growth negatively. The maximal oxygen consumption rate (OUR) was reached after about 24 hours in cultivations submitted to shorter intervals between mixing events which indicates a positive effect of shaking on the fungal growth rate on the particle surface, as long as no medium compaction occurs. Thus it was concluded that the kind of used substrate (rice), whose reology was perceptively modified by the mixing process, acted harmfully on microbial respiration. If mixing is to be used in SSF bioreactors, the substrate used should have a low starch content and a high fiber content Ergosterol content showed an 85% positive correlation with the revolution pattern, indicating that the interval between mixing events is the most important factor. Assays performed with longer intervals between mixing events and greater numbers of turns achieved about 10 times higher ergosterol concentration than the others. The coefficient of variation of the moisture at five sites of the reactor represents the homogeneity, since they are related to the revolution patterns by 95%.

A velocidade específica de respiração (QO2) é um parâmetro fundamental para entender-se o metabolismo e o estado fisiológico celular, fornecendo informações úteis para o processo e controle em biorreatores. Neste trabalho, cultivou-se diferentes células de insetos em ambiente controlado medindo-se o QO2 e concentração crítica de oxigênio (Ccrít). Foram utilizadas nos ensaios células de insetos Spodoptera frugiperda (Sf9) não infectadas e células de Drosophila melanogaster (S2) selvagem e recombinantes, utilizadas na expressão de diferentes proteínas. Todas as experiências foram realizadas em biorreator Inceltech com volume de trabalho de 1L, mantido a temperatura de 28ºC, agitação de 100 rpm e oxigênio dissolvido (OD) a 40% da saturação de ar, com difusão por membrana de silicone com mistura gasosa (O2 e N2) e vazão gasosa constante. Foi utilizado meio de cultura Sf900II sem soro fetal bovino. O QO2 foi medido pelo método dinâmico e pelo balanço de oxigênio na fase líquida. Neste trabalho foi implementado um novo processo durante o método dinâmico para interromper completamente a transferência gasosa durante a execução deste método. Implementou-se também uma metodologia para medição de Ccrít. Chegou-se a concentrações máximas celulares (Xm), velocidades máximas específicas de respiração (QO2) na fase exponencial e Ccrít, conforme segue: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfectadas para expressão de GPV): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfectadas para expressão de EGFP): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfectadas para expressão de HBsAg): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. Conclui-se que as linhagens selvagens e transfectadas de S2 possuem entre si uma atividade respiratória diferente e também que as novas metodologias implantadas verificaram-se satisfatoriamente. / Specific respiration rate (QO2) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure QO2 and critical oxygen concentration (Ccrit). Wild Spodoptera frugiperda (Sf9) and wild and transfected Drosophila melanogaster S2 cells (able to produce different proteins) were used. All experiments were performed in 1-liter working volume Inceltech bioreactor, maintaining temperature controlled at 28ºC, agitation rate at 100 rpm, and dissolved oxygen (DO) at 40% of air saturation, through membrane diffusion of mixed gases (O2 and N2) at constant total flow rate. SF900II serum free medium was used. QO2 was measured through dynamic method and oxygen mass balance in the liquid phase. In this work a new process was implemented during the dynamic method to interrupt completely the oxygen transfer during the execution of this method. It was also implemented a methodology for measurement of Ccrít (determined when DO reduces its decay rate, without oxygen transfer). Maximum cell concentration (Xm), maximum specific respiration rate (QO2) in the exponential phase and Ccrít were reached, as follows: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfected for GPV expression): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfected for EGFP expression): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfected for HbsAg expression): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. From these results, it can be concluded that the studied cell lines have different respiration activity and the new developed methodologies behave satisfactorily.