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dc.contributor.advisorDu Preez, J. C.
dc.contributor.advisorKilian, S. G.
dc.contributor.authorKnox, Alison Margaret
dc.date.accessioned2018-04-03T07:57:55Z
dc.date.available2018-04-03T07:57:55Z
dc.date.issued2002-03
dc.identifier.urihttp://hdl.handle.net/11660/8074
dc.description.abstractEnglish: The direct fermentation of starch to ethanol using an amylase-producing yeast is of interest as an alternative to the conventional fermentation processes, which utilise commercial amylases. Starch is an abundant renewable biopolymer, comprising two major components, namely amylose (α-I,4-linked D-glucose residues) and amylopectin (α-I,4 and α-I,6-linked D-glucose residues), typically constituting 80 % of cereal starches. Efficient starch degradation, therefore, necessitates the use of α- and glucoamylases, together with an α-I,6 debranching activity. Naturally amylolytic yeasts are not suited to fermentations, whereas Saccharomyces cerevisiae, known for its strong fermentation capacity, lacks amylolytic activity (with the exception of S. cerevisiae var. diastaticus, which has weak glucoamylase activity). Consequently, the diploid "Sigma" strain of S. cerevisiae was transformed with different combinations of amylase genes from Lipomyces spencermartinsiae (with the PGK] promoter), and Saccharomycopsis fibuligera (with its natural promoters) using an integrating plasmid in a single copy form. These recombinant strains, provided by the University of Stellenbosch, were evaluated in respect of their ability to ferment starch to ethanol. Recombinant strains of S. cerevisiae containing the S. fibuligera amylase genes with non-integrating plasmids in a multi-copy and a single copy form, provided by the University of the Free State, were also evaluated. Notable differences in the hydrolysis zones on starch agar plates indicated that the type of starch medium used and the amylase produced exerted a significant effect. The dimensions of these hydrolysis zones were a poor indicator of the performance of the strains in liquid starch media. Anoxic cultivations in shake flasks and in 2-1 bioreactors containing a 2 % starch medium yielded less than 109 ethanol.l-1 over a 200 h incubation period. Aerobic growth yielded more biomass and, therefore, higher amylase values and higher rates of starch hydrolysis, but with no detectable amounts of ethanol. Initial evaluations indicated poor amylase activity, particularly with strains containing the S. fibuligera amylase genes. The limited amylase production by a strain containing the S. fibuligera amylase genes was not due to proteolytic activity or intracellular enzyme . accumulation. Strains containing the L. spencermartinsiae amylase genes gave the best overall results. However, a strain containing a combination of both a-amylase and . glucoamylase yielded disappointing results. The curious multi-phasic growth profile obtained with this strain, accompanied by a delay in amylase production, suggested regulation of the PGKl promoter by glucose. However, Northern blot analyses indicated very low levels of glucoamylase mRNA, with no clear indication of induction of the PGKl promoter. A recombinant strain (strain steIl7) with both glucoamylase genes (LKAll from L. spencermartinsiae and GLUI from S. fibuligera) proved to be the most prormsmg. Further evaluation in 15-1 bioreactors resulted in the production of ea. 21 g ethanol.l-1 from 55 g starch.l-1 by strain stell7. Despite a relatively high α.-amylase activity of 500 U.l-1 after 150 h, the slow rate of enzyme production remained the rate-limiting step. Although some of these recombinant strains were capable of complete starch hydrolysis, the slow rate of starch saccharification and the concomitant low ethanol productivity rendered these strains unattractive for commercial application.en_ZA
dc.description.abstractAfrikaans: Die gebruik van 'n amilase-produserende gis in die direkte fermentasie van stysel na etanol is van belang as 'n alternatief vir die konvensionele fermentasieproses wat van kommersiële amilase-ensieme gebruik maak. Stysel is 'n volop-beskikbare, hernieubare biopolimeer bestaande uit twee hoofkomponente, naamlik amilose (α-I, 4-gekoppelde D-glukose residue) en amilopektien (α-I,4 en α-I,6-gekoppelde D-glukose residue), wat tipies 80 % van graanstysels verteenwoordig. Effektiewe Stysel-afbraak noodsaak dus die gebruik van α en glukoamilases, tesame met 'n α-l,6 onttakkingsaktiwiteit. Amilolitiese giste is van nature nie geskik vir fermentasie nie, terwyl Saccharomyces cerevisiae, bekend vir 'n sterk fermentatiewe kapasiteit, nie amilolitiese aktiwiteit besit . nie (met uitsondering van S. cerevisiae var. diastaticus, wat 'n swak amilolitiese aktiwiteit besit). Gevolglik is die diploïde "Sigma" stam van S. cerevisiae met verskillende .kombinasies van die amilase-gene afkomstig van Lipomyces spencermartinsiae (met die PGKl promotor) en Saccharomycopsis fibuligera (met die natuurlike promotors) getransformeer, deur gebruik te maak van 'n integrerende plasmied in 'n enkelkopie formaat. Die rekombinante stamme, wat deur die Universiteit van Stellenbosch voorsien is, is met betrekking tot hul vermoë om stysel tot etanol te fermenteer, geëvalueer. Rekombinante stamme van S. cerevisiae, wat die S. fibuligera amilase-gene met die nie-integrerende plasmied in 'n veelvuldige kopie-formaat bevat het, is deur die Univérsiteit van die Vrystaat verskaf en is ook geëvalueer. Merkwaardige verskille in die hidrolitiese sones op die styselagarplate was 'n aanduiding dat die tipe styselmedium, sowel as die amilase wat geproduseer word, 'n betekenisvolle effek uitoefen. Die afinetings van hierdie hidrolitiese sones het 'n onbetroubare aanduiding van die werksverrigting van die stamme in vloeibare styselmedium gegee. Minder as 10 g etanol.l-1 is gedurende anoksiese kwekings in skudflesse en in 2-1 bioreaktors met 'n 2 % styselmedium na 'n inkubasietydperk van 200 h gelewer. Aerobiese kweking het meer biomassa, en dus hoër amilasewaardes en hoër styselhidrolise tempo's, gelewer, maar met geen waarneembare hoeveelhede etanol nie. Aanvanklike evaluasies het swak amilase-aktiwiteit aangedui, in besonder α-amilase aktiwiteit, vir die stamme met die amilase-gene vanaf S. fibuligera. Die beperkte amilase-produksie deur 'n stam met die S. fibuligera amilase-gene was nie as gevolg van proteolitiese aktiwiteit of intrasellulêre ensiem-akkumulasie nie. Stamme met die L. spencermartinsiae amilase-gene het oor die algemeen die beste resultate gelewer. 'n Stam wat 'n kombinasie van beide α-amilase- en glukoamilase-gene bevat, het egter teleurstellende resultate gelewer. Die merkwaardige multi-fase groeiprofiel wat met hierdie stam verkry is, tesame met 'n vertraging in amilase-produksie, het die regulering van die PGKl-promoter deur glukose geïmpliseer. 'Northern blot' analises het egter baie lae vlakke van glukoamilase-mRNA aangedui, met geen duidelike aanduiding van induksie van die PGKl promoter nie. 'n Rekombinante stam (stam stell7) met beide glukoamilase gene (LKAII vanaf L. spencermartinsiae en GLUI vanaf S. fibuligera) was die belowendste stam. Verdere evaluasie in 15-1bioreaktors het gelei tot die produksie van ca. 21 g etanol.l-1 deur stam stell7 vanaf 55 g stysel.l-1. Ondanks 'n relatief hoë α-amilase aktiwiteit van 500 U.l-1 na 150 h, het die stadige tempo van ensiemproduksie die snelheidsbeperkende stap gebly. Alhoewel sommige van hierdie rekombinante stamme tot volledige styselhidrolise in staat was, maak die stadige styselhidrolise en die gepaardgaande lae etanolproduksie hierdie stamme onaantreklik vir kommersiële toepassing.en_ZA
dc.description.sponsorshipFoundation for Research Development and the Industrial Development Corporationen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectRecombinanten_ZA
dc.subjectSaccharomyces cerevisiaeen_ZA
dc.subjectStarchen_ZA
dc.subjectSaccharomycesen_ZA
dc.subjectAmylasesen_ZA
dc.subjectDissertation (M.Sc. (Microbiology, Biochemical and Food Sciences))-- University of the Free State, 2002en_ZA
dc.titleThe growth kinetic characterisation of Saccharomyces cerevisiae strains transformed with amylase genesen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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