Th.--Méd.--Paris, 1904-1905. / Paris, 1904-1905, tome 39, n ° 434.

With increasing energy demand, decreasing oil supply, and continuously accumulating waste in landfills, the interest in converting lignocellulosic biomass to liquid fuels has grown. The MixAlco™ process requires no exogenous enzymes, no sterility, can be adapted to any biodegradable feedstock, and converts lignocellulosic biomass into valuable chemicals and transportation fuels. This work focuses on the effects different feedstocks and inocula have on mixed-acid/hydrogen fermentations. When volatile solids (VS) are digested, mixed-acid fermentations produce hydrogen gas as a secondary byproduct. Hydrogen is only produced when there is an excess of NADH within the cell and when the energy selectivity (gamma) of the system has not been met. Continuous fermentations of paper produced 16.7 g carboxylic acid/L and 15.7 mL H2/g VS digested. Continuous fermentations of pretreated bagasse produced 17.1 g carboxylic acid/L and 41.1 mL H2/g VS digested. Both fermentations produced a fraction of the theoretical amount of hydrogen. The paper fermentation had a hydrogen percent yield of 6.9 percent, whereas the bagasse fermentation had a hydrogen percent yield of 22.6 percent. Hydrogen production was capped at this level because gamma had been met for these systems. The Bioscreening Project, a joint project between three departments, sought to improve the MixAlco™ process by finding natural cultures containing high biomass converters and high acid producers. A total of 505 inoculum samples were collected from 19 sites and screened using paper and yeast extract fermentations. The best converters were analyzed with Continuum Particle Distribution Modeling (CPDM). Nine inocula were run in paper and yeast extract countercurrent fermentations in which the overall performance varied less than 13 percent. Comparisons between six countercurrent train cultures showed an average culture similarity of 0.40 (Yue-Clayton similarity). With the dissimilar microbial cultures and the very similar fermentation performance, the performance of the MixAlco™ process depends on fermentation conditions, not on the microorganisms. Batch fermentations of office paper wastes, pineapple residue, Aloe vera rinds, wood molasses, sugar molasses, extracted algae, non-extracted algae, crude glycerol, obtained from the biodiesel process, and pretreated water hyacinths produced sufficient carboxylic acids and had sufficiently high conversions to be viable substrates for the MixAlco™ process.

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Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-23Bitstream added on 2014-06-13T18:31:29Z : No. of bitstreams: 1 avilaneto_pm_me_rcla.pdf: 2987014 bytes, checksum: 42f0c4d662fdc347535b329b4435956b (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Com a utilização crescente do biodiesel no mundo, houve uma grande disponibilidade de glicerol no mercado. No brasil, é esperado um mercado de 200 mil toneladas de glicerol de biodiesel com a utilização de 5% de biodiesel ao diesel. Apesar disso, a clarificação e purificação deste glicerol ainda é um processo muito oneroso, sendo que a utilização do glicerol na forma bruta, além de gerar produtos de alto valor agregado, pode contribuir para diminuir o valor do biodiesel no mercado, uma vez que os custos de armazenamento ou descarte do glicerol bruto está atrelado ao preço do biodiesel. O glicerol de biodiesel preferencialmente é utilizado para produção de 1,3 propanodiol por bactérias, porém pouco se tem estudado a respeito da sua fermentação por leveduras, que podem utilizar maiores quantidades de glicerol e maiores taxas de conversão. Foram realizadas fermentações com 30 cepas de leveduras capazes de assimilar o glicerol utilizando frascos tipo erlenmeyer e glicerol como única fonte de carbono. O caldo fermentado foi analisado em HPLC equipado com detector UV e IR na busca de etanol, propanol, butanol, lactato, propionato, citrato, succinato e acetato, 1,3 propanodiol, 1,2 propanodiol, e 2,3 butanodiol. Foi utilizando o método de planejamento experimental para otimização do crescimento em frascos e três fermentações em reator para avaliação do meio otimizado. Também foram realizados dois planejamentos experimentais para otimização do meio de produção em frascos e seis fermentações em reator variando a aeração e a agitação. Foi verificado que Yarrowia lipolytica NRRL Y-1095 foi a cepa maior promissora na utilização de glicerol para produção de citrato. O meio de crescimento foi otimizado em glicerol 50 g.L-1, extrato de levedura 5 g.L-1, sulfato de amônia1 g.L-1, KH2PO4 7 g.L-1, Na2HPO4... / With the increasing use of biodiesel in the world, there is great availability of glycerol in the market. In Brazil, it is expected a availability of 200 thousand tons of biodiesel glycerol with the mixture of 5% biodiesel into mineral biodiesel. Nevertheless, the purification and clarification of glycerol is a very costly process, and the use of raw glycerol to generate products with high added value, may reduce the price of biodiesel in the market, since raw glycerol storing and disposing costs are put on the biodiesel price. Raw glycerol is preferably used to produce 1,3 propanediol by bacteria, but little has been studied about its fermentation by yeasts, which can use larger amounts of glycerol and have higher conversion rates. Fermentations were performed with 30 yeast strains able to use glycerol as the sole carbon source using Erlenmeyer flask. The fermented broth was analyzed on HPLC equipped with UV and RI detector searching ethanol, propanol, butanol, lactate, propionate, citrate, succinate and acetate, 1,3 propanediol, 1,2 propanediol, and 2,3 butanediol. The method of experimental design was used for growth optimization and three fermentation were performed in fermentor vase to evaluate the optimized medium. Two experimental designs were made for the evaluate the production medium and six fermentation in fermentor vase varying aeration and agitation. It was found that Yarrowia lipolytica NRRL Y-1095 strain was the most promising in the use of glycerol to produce citrate. The growth medium was optimized in glycerol 50.0 g.L-1, yeast extract 5.0 gL-1, ammonium sulfate 1.0 gL-1, KH2PO4 7.0 gL-1, Na2HPO4 2.5 gL-1, FeCl3 0.65 mg.L - 1, ZnSO4 1.2 mg.L-1, CuSO4.5H2O 0.31 mg.L-1 and MnSO4 0.27 mg.L-1, reaching 6.32 gL-1 of dry weight in bottles and 23gL-1 in fermentation reactor. It was found that glycerol was the only significant factor... (Complete abstract click electronic access below)

Orientador: Jonas Contiero / Banca: Fátima Aparecida de Almeida Costa / Banca: Edwil Aparecida de Lucca Gattas / Resumo: Com a utilização crescente do biodiesel no mundo, houve uma grande disponibilidade de glicerol no mercado. No brasil, é esperado um mercado de 200 mil toneladas de glicerol de biodiesel com a utilização de 5% de biodiesel ao diesel. Apesar disso, a clarificação e purificação deste glicerol ainda é um processo muito oneroso, sendo que a utilização do glicerol na forma bruta, além de gerar produtos de alto valor agregado, pode contribuir para diminuir o valor do biodiesel no mercado, uma vez que os custos de armazenamento ou descarte do glicerol bruto está atrelado ao preço do biodiesel. O glicerol de biodiesel preferencialmente é utilizado para produção de 1,3 propanodiol por bactérias, porém pouco se tem estudado a respeito da sua fermentação por leveduras, que podem utilizar maiores quantidades de glicerol e maiores taxas de conversão. Foram realizadas fermentações com 30 cepas de leveduras capazes de assimilar o glicerol utilizando frascos tipo erlenmeyer e glicerol como única fonte de carbono. O caldo fermentado foi analisado em HPLC equipado com detector UV e IR na busca de etanol, propanol, butanol, lactato, propionato, citrato, succinato e acetato, 1,3 propanodiol, 1,2 propanodiol, e 2,3 butanodiol. Foi utilizando o método de planejamento experimental para otimização do crescimento em frascos e três fermentações em reator para avaliação do meio otimizado. Também foram realizados dois planejamentos experimentais para otimização do meio de produção em frascos e seis fermentações em reator variando a aeração e a agitação. Foi verificado que Yarrowia lipolytica NRRL Y-1095 foi a cepa maior promissora na utilização de glicerol para produção de citrato. O meio de crescimento foi otimizado em glicerol 50 g.L-1, extrato de levedura 5 g.L-1, sulfato de amônia1 g.L-1, KH2PO4 7 g.L-1, Na2HPO4... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the increasing use of biodiesel in the world, there is great availability of glycerol in the market. In Brazil, it is expected a availability of 200 thousand tons of biodiesel glycerol with the mixture of 5% biodiesel into mineral biodiesel. Nevertheless, the purification and clarification of glycerol is a very costly process, and the use of raw glycerol to generate products with high added value, may reduce the price of biodiesel in the market, since raw glycerol storing and disposing costs are put on the biodiesel price. Raw glycerol is preferably used to produce 1,3 propanediol by bacteria, but little has been studied about its fermentation by yeasts, which can use larger amounts of glycerol and have higher conversion rates. Fermentations were performed with 30 yeast strains able to use glycerol as the sole carbon source using Erlenmeyer flask. The fermented broth was analyzed on HPLC equipped with UV and RI detector searching ethanol, propanol, butanol, lactate, propionate, citrate, succinate and acetate, 1,3 propanediol, 1,2 propanediol, and 2,3 butanediol. The method of experimental design was used for growth optimization and three fermentation were performed in fermentor vase to evaluate the optimized medium. Two experimental designs were made for the evaluate the production medium and six fermentation in fermentor vase varying aeration and agitation. It was found that Yarrowia lipolytica NRRL Y-1095 strain was the most promising in the use of glycerol to produce citrate. The growth medium was optimized in glycerol 50.0 g.L-1, yeast extract 5.0 gL-1, ammonium sulfate 1.0 gL-1, KH2PO4 7.0 gL-1, Na2HPO4 2.5 gL-1, FeCl3 0.65 mg.L - 1, ZnSO4 1.2 mg.L-1, CuSO4.5H2O 0.31 mg.L-1 and MnSO4 0.27 mg.L-1, reaching 6.32 gL-1 of dry weight in bottles and 23gL-1 in fermentation reactor. It was found that glycerol was the only significant factor... (Complete abstract click electronic access below) / Mestre

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Wine fermentation is a continuously changing biological process whereby the raw product, grape juice is transformed into a high value product wine. In an ideal situation the fermentation kinetics of batch fermentations should follow the same trend over time. This however is not the case in industrial wine fermentations where significant batch-to-batch variation is present. The time trajectories of fermentation processes are therefore often unpredictable in absolute terms. The monitoring of substrate (sugar) and product concentrations (ethanol) as well as other quality parameters during a wine fermentation, is therefore of extreme importance to ensure effective control and management of wine fermentation processes. Conventional methods for fermentation monitoring are however costly, time consuming and often unreliable. For these reasons the modern wine industry requires rapid, reliable, non-destructive monitoring techniques which would meet the criteria of providing critical real-time process information that is displayed in easily interpretable graphical format, in order to ensure the highest quality and continuous consistency throughout all the stages of a process. This research study in particular, addressed the current need for alternative fermentation monitoring strategies that meet these criteria, by evaluating the potential use of spectroscopy as an analytical technique for fermentation monitoring. The overall objective of this study was to use chemometric modelling of information obtained by Fourier transform mid-infrared (FT-MIR) and near-infrared (FT-NIR) spectroscopy, to quantitatively and qualitatively monitor both alcoholic (AF) and malolactic fermentation (MLF) processes. Towards this objective 11 batch fermentations elaborated with Oenococcus oeni and Lactobacillus plantarum strains in respectively a co-inoculation and sequential inoculation scenario, were sampled and analysed at regular time intervals with FT-MIR and FT-NIR spectroscopy and enzymatic reference methods during 2011. Samples were also analysed by gas chromatography flame ionisation detection (GC-FID) and mass spectrometry (GC-MS) at two critical stages during fermentation, namely 50% completion of MLF and 100% completion of MLF, in order to obtain a profile of the evolution of the aroma compounds associated with each inoculation scenario. Three clearly defined research objectives were set for this project. The first objective involved the expansion of the existing quantitative platform for fermentation monitoring. Towards the outcomes of this objective, partial least squares (PLS) calibration models for prediction of malic acid and lactic acid in fermenting must and wines elaborated in our study, were established, based on the MIR and NIR spectra. The models showed excellent predictive abilities in independent test set validation. This outcome made a significant contribution to our existing PLS calibration capacity, particularly towards monitoring of MLF. Quantitative data obtained with the PLS models were also used to graphically project the rate of AF in the different batches, by non-linear fitted regression plots that easily visualised the overall patterns of sugar and ethanol metabolism in the different fermentations. The second research objective involved the qualitative monitoring of fermentations. This approach used FT-MIR and FT-NIR spectra together with chemometrics to identify trends between the different fermentation treatments. Principal component analysis (PCA) clearly projected the time trend from the onset of fermentation, through AF and MLF. No unique bacterial trend was however observed with spectroscopy. These results illustrate the potential of these techniques to be used for modelling of fermentations in industrial situations, through providing critical information about the evolution of the process. Furthermore, these techniques provide tools for identifying problematic and deviating fermentations. A spectral conformity test based on simple calculations of the standard deviation between the absorbance at each recorded wavenumber in the spectra, further confirmed identification of the critical fermentation stages. This technique by-passes the need for spectral interpretation and is a very useful addition, particularly from the industry perspective, to the portfolio of methods established in this study. The third research objective adressed the need to evaluate the possibility to discriminate between the different process stages and LAB treatments using univariate (ANOVA) and multivariate chemometric techniques such as PCA, PLS discriminant analysis (PLS-DA) and Soft Independent Modelling of Class Analogy (SIMCA) for possible future interpretative and classification purposes. The exploratory tool of PCA was used to investigate the similarities and differences between the chemical footprints of the different treatments. PCA showed clear differentiation between the two process stages using chemical quantified data. Differentiation between the LAB treatments was visible with PCA, showing a more prominent separation at 50% completion of MLF. Furthermore the ability of spectroscopy for potential classification was shown using PLS-DA and SIMCA. This profiling study can be seen as a preliminary study setting the ground work for further in-depth research into the profiling of different LAB treatments and inoculation strategies. / AFRIKAANSE OPSOMMING: Wyngisting is ‘n voortdurend veranderende biologiese proses waarvolgens die rou produk, druiwesap, in ‘n produk van hoë waarde, naamlik wyn, verander word. Onder ideale omstandighede sou die gistingskinetika van lotgistings dieselfde tendens oor tyd volg. Dit is egter nie die geval in industriële wyngistings nie, waar noemenswaardige wisseling van lot tot lot teenwoordig is. Die tydsbaan van gistingsprosesse is dus in baie gevalle in absolute terme onvoorspelbaar. Die monitering van substraat- (suiker) en produkkonsentrasies (etanol), sowel as ander kwaliteitsparameters tydens ‘n wyngisting, is van die uiterste belang om doeltreffende beheer en bestuur van wyngistingsprosesse te verseker. Konvensionele metodes vir die monitering van gisting is egter duur, tydrowend en ook soms onbetroubaar. Om hierdie redes vereis die moderne wynbedryf vinnige, betroubare en nie-destruktiewe moniteringstegnieke wat aan die kriteria sou voldoen vir die verskaffing van kritiese, intydse prosesinligting wat in ‘n maklik interpreteerbare grafiese formaat vertoon word om die hoogste kwaliteit en voortdurende konsekwentheid tydens al die stadia van ‘n proses te verseker. Hierdie navorsingstudie het in besonder die huidige behoefte aan alternatiewe gistingsmoniteringstrategieë wat aan hierdie kriteria voldoen, aangespreek deur die potensiële gebruik van spektroskopie as ‘n analitiese tegniek vir die monitering van gisting te evalueer. Die oorhoofse doelwit van hierdie studie was die chemometriese modellering van inligting wat met behulp van Fourier transform middel-infrarooi (FT-MIR) en naby-infrarooi (FT-NIR) spektroskopie verkry is om die prosesse van alkoholiese en appelmelksuurgisting (AMG) kwantitatief en kwalitatief te monitor. Ten einde hierdie doelwit te bereik, is 11 lotgistings met Oenococcus oeni en Lactobacillus plantarum rasse uitgevoer in scenario’s van ‘n gesamentlike inokulasie en opeenvolgende inokulasie onderskeidelik. Monsters van hierdie gistings is met gereelde tydintervalle in 2011 geneem en analises is met FT-MIR en FT-NIR spektroskopie en ensiematiese verwysingsmetodes gedoen. Monsters is ook met gaschromatografiese vlam ionisasie-opsporing (GC-FID) en massaspektrometrie (GC-MS) op twee kritiese stadia tydens gisting geanaliseer, naamlik toe AMG 50% en 100% voltooid was, om ‘n profiel te verkry van die evolusie van die aromaverbindings wat met elke inokulasie-scenario verband hou. Drie duidelik gedefinieerde navorsingsdoelwitte is vir hierdie studie bepaal. Die eerste doelwit het die uitbreiding van die bestaande kwantitatiewe platform vir gistingsmonitering behels. Hiervoor is gedeeltelike kleinstekwadraat [partial least squares (PLS)] kalibrasiemodelle vir die voorspelling van appelsuur en melksuur in die gistende mos en wyne in ons studie op die basis van MIR- en NIR-spektra bepaal. Die modelle het in onafhanklike geldigheidsbepaling van die toetsstel uitstekende voorspellingsvermoëns getoon. Hierdie uitkoms het ‘n noemenswaardige bydrae gemaak tot ons bestaande PLS kalibrasiekapasiteit, veral om AMG te monitor. Die kwantitatiewe data wat met die PLS-modelle verkry is, is ook gebruik om die tempo van alkoholiese gisting in die verskillende lotte grafies uit te beeld deur middel van kromlynige passing van regressiepersele, waarmee dit maklik was om die algehele patrone van suiker- en etanolmetabolisme in die verskillende gistings te visualiseer. Die tweede navorsingsdoelwit het die kwalitatiewe monitering van gistings behels. Hierdie benadering het FT-MIR en FT-NIR spektra tesame met chemometrie gebruik om tendense tussen die verskillende gistingsbehandelings te identifiseer. Hoofkomponentanalise [principal component analysis (PCA)] het duidelik die tydtendens vanaf die aanvang van gisting, deur alkoholiese gisting en AMG, geprojekteer. Geen unieke bakteriese tendens is egter met spektroskopie waargeneem nie. Hierdie uitslae illustreer die potensiaal van hierdie tegnieke om vir die modellering van gistings in industriële situasies gebruik te word deur kritiese inligting oor die evolusie van die proses te verskaf. Verder verskaf hierdie tegnieke gereedskap vir die identifikasie van problematiese en afwykende gistings. ‘n Spektrale gelykvormigheidstoets gebaseer op eenvoudige berekeninge van die standaardafwyking tussen die absorbansie by elke aangetekende golfgetal in die spektra het ook die identifikasie van die kritiese gistingstadia bevestig. Hierdie tegniek omloop die noodsaak vir spektrale interpretasie en is ‘n baie nuttige byvoeging tot die portefeulje van metodes wat in hierdie studie bepaal is, veral vanuit ‘n bedryfsperspektief. Die derde navorsingsdoelwit het die behoefte aangespreek om tussen die verskillende prosesstadia en melksuurbakterie-behandelings te onderskei deur gebruik te maak van eenvariant- (ANOVA) en meervariant- chemometriese tegnieke soos PCA, PLSdiskriminantanalise en sagte onafhanklike modellering van klasanalogie [soft independent modelling of class analogy (SIMCA)] vir moontlike toekomstige verklarende en klassifikasiedoeleindes. Die ondersoekende gereedskap van PCA is gebruik om ooreenkomste en verskille tussen die chemiese voetspore van die verskillende behandelings te ondersoek. PCA het duidelike differensiasie tussen die twee prosesstadia getoon op grond van chemies gekwantifiseerde data. Differensiasie tussen die melksuurbakterie-behandelings was met PCA sigbaar, met ‘n meer prominente skeiding teen 50% voltooide AMG. Verder is die vermoë van spektroskopie vir potensiële klassifikasie met PLS-diskriminantanalise en SIMCA getoon. Die profielsamestelling in hierdie studie kan beskou word as ‘n voorlopige studie vir verdere diepgaande navorsing oor die profielsamestelling van verskillende melksuurbakteriebehandelings en inokulasiestrategieë. / The National Research Foundation and Winetech

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: There is a growing international consumer demand for the production of lower ethanol wines. This can be attributed to various qualitative, social, economic and health concerns that are associated with high ethanol wines (Kutyna et al., 2010; Varela et al., 2012). There is continuous development and research into methods and technologies to lower the ethanol concentration in wine. However, in addition to the added cost and complexity these technologies all have various shortcomings. The development of yeast strains with lower ethanol productivity, yet desirable organoleptic and fermentation capacity, therefore remains a highly sought after research and development target in the wine industry. Biologically based approaches aim to generate yeast strains with the capacity to divert carbon from ethanol production towards targeted metabolic endpoints (Kutyna et al., 2010). This should ultimately be achieved without the production of unwanted metabolites that can negatively affect wine characteristics. In the context of these challenges, this study aimed to investigate the use of fructans as carbon sinks during fermentation to divert fructose from glycolysis and ethanol production toward intracellular fructan production by generating levan producing strains. In addition, the impact of fructan production on metabolic carbon flux during fermentation by these strains was analyzed. This was the first attempt to analyze intracellular fructan production in Saccharomyces cerevisiae under fermentative conditions with fructans acting as carbon sinks. Fructans are fructose polymers that act as storage molecules in certain plants and function as part of the extracellular matrix in microbial biofilms, and are intensively studied due to their economic interest. Here we undertook the heterologous expression of a levansucrase (LS) M1FT from Leuconostoc mesenteroides, an enzyme producing β(2-6) levan-type fructans, in the S. cerevisiae BY4742Δsuc2 strains without invertase activity (encoded by SUC2). Levansucrases indeed utilize sucrose as both fructose donor and initial polymerization substrate, and the sucrose concentration is of import to maintain transfructosylation activity of enzyme. High intracellular sucrose accumulation was achieved by the heterologous expression of either a sucrose synthase (Susy; cloned from potato) or by growing strains expressing the spinach sucrose transporter (SUT) in sucrose containing media. Endogenous sucrose synthesis was of specific interest to the overall goal of the project, which was to reroute carbon flux away from glycolysis in grape must containing only hexoses as carbon source. In addition, this approach of combining intracellular sucrose production with intracellular levan production could be used in various applications to limit the need for sucrose in media as both carbon source and LS substrate. The extracellular LS M1FT was introduced into Susy and SUT strains as either the complete gene (M1FT) or 50bp truncation (M1FTΔsp) without the predicted signal peptide. The data show that intracellular levan accumulation occurred in aerobic, but not anaerobic conditions. The data also suggest that the production of levan did not impact negatively on general yeast physiology or metabolism in these conditions. However, no significant reduction in ethanol yields were observed, suggesting that further optimisation of the expression system is required. This is the first report of levan synthesis by S. cerevisiae, and contributes towards expanding the possibilities for further industrial applications of these compounds. / AFRIKAANSE OPSOMMING: Daar is toenemende aanvraag deur wynverbruikers na laër alkohol wyne. Hierdie neiging kan toegeskryf word aan verskeie kwalitatiewe, gesondheids en sosio-ekonomiese redes wat geassosieer word met die verbruik van hoër alkohol wyne. Daar is ’n deurlopende navorsing dryf toegespits op metodes en tegnologieë om die alkohol konsentrasie van wyne te verlaag. Hierdie tegnologieë het egter, bykomstig tot koste en kompliksiteits toename, verkeie tekortkominge. Die ontwikkeling van gisrasse met verlaagde alcohol produksie, maar steeds wenslike organoleptiese en fermentasie eienskappe, bly ‘n baie gesogte navorsings en ontwikkeling teiken in die internasionale wyn industrie. Biologiese benaderings streef om gisrasse te genereer met die vermoë om koolstof weg van etanol produksie te herlei na geteikende metabolise eindpunte. Hierdie doelwit moet ook uiteindelik bereik word sonder die produksie van ongewenste metaboliete wat die wyn negatief kan affekteer. In die konteks van hierdie uitdaging, het hierdie studie gestreef om die gebruik van fruktane as ’n koolstof poel tydens fermentasie, met die doel om fruktose te herlei vanaf glikolise en etanol produksie na intrasellulêre fruktane produksie. Om hierdie doelwit te bereik, is gisrasse ontwikkel wat levaan (’n spesifieke fruktaan) produseer. Die impak van fruktaan produksie op metaboliese koolstof vloei tydens fermentasie deur hierdie gisrasse is bykomsrig ontleed. Hierdie verslag beskryf die eerste poging om intraselullêre fruktaan produksie in Saccharomyces cerevisiae te bewerkstellig, met die doel om fruktaan as ’n koolstof poel te gebruik. Fruktane is fruktose polimere wat as bergings molekules optree in sekere plante en ook funksioneer as deel van die ekstrasellulêre matriks in mikrobiese biofilms. Hierdie polimere word tans internasionaal intensief bestudeer weens hul ekonomiese belang. Hierdie studie beskryf die uitdrukking van die levaansukrase (LS) M1FT van Leuconostoc mesenteroides, wat β(2-6) levaan-tipe fruktane produseer, in S. cerevisiae BY4742Δsuc2 rasse, sonder invertase (gekodeer deur SUC2). Levaansukrases gebruik inderdaad sukrose as beide ’n fruktose donor en ook as ’n aanvanklike polimeriserings substraat. Die fruktose konsentrasie is belangrik om transfruktosilerings aktiwiteit van die ensiem te handhaaf. Hoë intrasellulêre sukrose akkumulasie was bereik deur die heteroloë uitdrukking van ’n sukrose sintase (Susy; gekloneer van aartappel) of die spinasie sukrose transporter (SUT) in media bevattende sukrose. Endogene sukrose sintese was van spesifieke belang tot die algehele doelwit om koolstof te herlei, weg van glikolise tydens fermentase van druiwe sap. Die benadering om intraselullêre sukrose produksie met levaan produksie te koppel, kan ook gebruik word in verskeie toepassings om die afhanklikheid op sukrose in die media, as substraat vir LS, te verminder. Die ekstraselullêre LS, M1FT, was as vollengte geen (M1FT) of as ’n 50bp afkapping (M1FTΔsp), sonder seinpeptied, in die Susy en SUT gisrasse uitgedruk. Die data dui aan dat die produksie van levaan nie ’n negatiewe impak het op gis fisiologie of metabolisme in die toets kondisies nie. Daar was egter geen waarbeenbare afname in etanol opbrengs nie, wat aandui dat verdere optimisering van ekspressie sisiteem benodig word. Hierdie is die eerste verslag van levaan sintese in S. cerevisiae en dra by tot die uitbreiding van moontlikhede vir indutriële toepassings van die die verbindings. / IWBT and NRF

café é a segunda bebida mais consumida no mundo, ficando atrás apenas da água; o Brasil figura como o maior produtor e exportador desta commodity no cenário global. Apesar de possuir uma cadeia altamente consolidada em nível global, mudanças e refinamentos tecnológicos estão em constante evolução, visando melhoria ou elevação da qualidade final da bebida, para atender a mercados cada vez mais exigentes. As interações entre os fatores edafoclimáticos, as formas de processamento, secagem, armazenagem e torrefação, implicam nos resultados sensoriais, que são desempenhados pelos Q-Graders de café, consequentemente, afetam o preço final do produto. Pesquisas recentes vêm demonstrando, que mesmo por trás de toda complexidade, que envolve a descrição da qualidade do café, é possível modificar o perfil sensorial da bebida, com controles mais precisos no processamento da matéria após a colheita. Tais indicativos estão inerentes aos processos de fermentações que ocorrem nos frutos, sendo que as fermentações podem conferir notas especiais de aroma, fragrância e acidez, bem como prejudicar a qualidade, quando não bem controlada. Com objetivo de elevação, melhoria e maior controle de processo na fase via-úmida, esta tese aplicou quatro formas de processamento por via-úmida, em seis estratos de altitude distintos, visando potencializar a qualidade final da bebida e estimar melhores métodos para cada faixa de altitude, com base nos fatores edafoclimáticos e nos fatores de ordem de processamento (fermentações espontâneas e induzidas). A qualidade final dos cafés foi mensurada por meio de resultados sensoriais, físico-químicos e cromatográficos, para que fosse possível determinar os melhores métodos de processamento por via-úmida para zonas de produção montanhosas Além da relação do processamento, estudaram-se formas de se introduzir maior acurácia ao processo de análise sensorial, objetivando redução da subjetividade. Os resultados foram empreendidos mediante análise de variância conjunta de experimentos, para os resultados sensoriais e físico-químicos (compostos fenólicos, acidez titulável total e pH), sendo as médias comparadas pelo teste de Tukey, considerando o nível de significância de 5%, e os modelos de regressão foram testados pelo teste F e os parâmetros pelo teste t. Além das análises de variância, adotou-se o método de agrupamento hierárquico com ligação completa, para as análises multivariadas. Os resultados indicam que o uso de cultura de arranque na fase de fermentação por via-úmida constitui em uma alternativa relevante para zonas menos elevadas, onde naturalmente os cafés tendem a notas sensoriais mais baixas, quando aplicados métodos tradicionais, e que as fermentações espontâneas possuem maior potencial de entrega de resultados sensoriais em faixas acima dos 900 metros de altitude. Os resultados demonstram que o processamento adotado após a colheita pode ser determinante para a formação do perfil sensorial, físico-químico e cromatográfico que o café terá, indicando que o controle de processo constitui em uma fase indispensável no pós-colheita. Tais evidências indicam que a qualidade pode ser modificada, para atender determinados nichos de mercado, visando à produção de um produto mais consistente e que dependa menos dos fatores climáticos ou do acaso. / Coffee is the second most consumed drink in the world, behind only water, Brazil figures as the largest producer and exporter of this commodity in the global scenario. Despite having a highly consolidated chain globally, technological and refinements changes are constantly evolving, aimed at improving or raising the final quality of the drink, to meet increasingly demanding markets. The interactions between the edaphoclimatic factors, the forms of processing, drying, storage and roasting, imply in the sensorial results, which are performed by the coffee tasters, consequently affect the final price of the product. Recent researches have shown that even behind all complexity, which involves the description of the quality of the coffee, it is possible to modify the sensorial profile of the beverage, with more precise controls in the processing of the matter, after the harvest. Such indications are inherent to the fermentation processes that occur in the fruits, and the fermentations can confer special notes of aroma, fragrance and acidity, as well as impair quality, when not well controlled. With objective of elevation, improvement and greater process control in the wet phase. This thesis applied four forms of wet processing in six distinct altitude strata, aiming at enhancing the final quality of the beverage and estimating better methods for each altitude range, based on edaphoclimatic factors, and processing order factors (spontaneous and induced fermentations). The final quality of the coffees was measured by sensorial, physico-chemical and chromatographic results, to determine the best wet processing methods for mountainous production areas In addition to the processing relationship, we studied ways to introduce greater accuracy to the process of sensory analysis, aiming to reduce subjectivity. The results were carried out through the analysis of joint variance of experiments for the sensory and physicochemical results (phenolic compounds, total titratable acidity and pH). The averages were compared by Tukey's test, considering a significance level of 5% and the regression models were tested by the F test and the parameters by the t test. In addition to the variance analysis, the hierarchical grouping method with complete linkage was used for the multivariate analysis. The results indicate that the use of starter culture in the wet fermentation phase constitutes a relevant alternative for less elevated areas, where naturally the coffees tend to lower sensorial scores when traditional methods are applied and that spontaneous fermentations have greater potential for delivery of sensory results in bands above 900 meters altitude. The results show that the post-harvest processing can be determinant for the formation of the sensorial, physico-chemical and chromatographic profile that the coffee will have, indicating that the process control is an indispensable post-harvest phase. Such evidence indicates that quality can be modified to meet certain market niches, aiming at producing a product that is more consistent and less dependent on climatic or chance factors.