Show simple item record

dc.contributor.advisorSmit, M. S.
dc.contributor.advisorDu Preez, J. C.
dc.contributor.advisorSetati, M. E.
dc.contributor.authorMokgoro, Masego Marjorie
dc.date.accessioned2017-05-10T06:45:20Z
dc.date.available2017-05-10T06:45:20Z
dc.date.issued2003-05
dc.identifier.urihttp://hdl.handle.net/11660/6206
dc.description.abstractEnglish:Dicarboxylic acids are value added products, which can be prepared by diterminal oxidation of n-alkanes by bacteria or yeasts with a block in β-oxidation. Yarrowia lipolytica is one of the alkane-utilizing yeasts for which genetic tools have been developed. Y lipolytica has a complex set of five acyl Coenzyme A (CoA) oxidase isozymes (encoded by POXI through POX5 genes) with different substrate specificities. Abundant information is available on dioic acid accumulation from n-alkanes by C. maltosa and C. tropicalis strains deficient in β-oxidation. In many cases β-oxidation had been blocked at the level of the acyl CoA oxidases. In comparison very little information was available .on dioic acid accumulation by Yarrowia lipolytica. We had through our collaboration with Dr J-M Nicaud of the INRA-CNRS in France access to the above mentioned series of Y lipolytica strains with the acyl CoA oxidase encoding genes disrupted. It thus became the purpose of my studies to investigate dioic acid .accumulation by Y lipolytica strains with impaired β-oxidation. In order to study the growth of different Yarrowia lipolytica strains on or in the presence of a range of liquid and solid n-alkanes or alkane derivatives we required a rapid method using small samples to estimate biomass production. Insoluble hydrophobic substrates iriterfere with turbidimetric measurements. This problem is more severe if the hydrophobic substrate is a solid. It is not possible to efficiently separate Y lipolytica cells from a hydrophobic substrate by centrifugation in the presence of an organic solvent, because Y lipolytica cells are hydrophic and a large percentage of cells cling to the water/solvent interface. We established that pre-treatment of samples for turbidimetric analysis with 5 M NaOH, thus increasing the pH of samples to 14, abolished to a large extent the hydrophobicity of Y lipolytica cells. We also established that washing of the cells with cyclohexane and NaOH when cells were harvested by filtration, did not result in any significant loss in biomass. Based on these observations we developed a simple, cost effective sample preparation procedure for turbidimetric analysis, which gave accurate, repeatable turbidity measurements with no Interference from the hydrophobic substrates. This method involved the pre-treatment of small samples (500 µl)with 5 M NaOH plus cyclohexane, prior to harvesting the biomass by centrifugation. Sample preparation was carried out in microcentrifuge tubes and turbidimetric measurements were done with a microtitre plate reader. The newly developed procedure was used to investigate the toxicity of dodecane and hexadecane as well as their terminal and diterminal oxidation products to Y lipolytica wild type strain H222. The alkanes and dicarboxylic acids were never toxic to Y lipolytica. Dodecanol severely inhibited growth of Y lipolytica strain H222 in YP media with glucose whereas in a semi-synthetic YNB medium without additional carbon source dodecanal and dodecanoic acid were the most toxic. Hexadecanol and hexadecanoic acid did not inhibit growth of Y lipolytica in yP medium with glucose, but were toxic to Y. lipolytica in a semi-synthetic YNB medium without glucose. The results obtained in the first round of experiments indicated to us the possibility of preparing dodecanol-tolerant strains. Two dodecanol-tolerant strains were subsequently prepared. The first H222A was prepared by step-wise increasing dodecanol concentrations in YP broth supplemented with glucose to 7.5% (v/v). The second strain MTL Y35A was prepared on YP agar plates without glucose by step-wise increasing dodecanol concentrations to 8.5% (v/v). Dodecanedioic acid was not accumulated by the dodecanol-tolerant strains H222A or by the triple POX-deleted strain MTL Y35A, when grown on glucose in the presence of dodecanol. Two dodecanol concentration were tested 3 % and 10 % (v/v). Dioic acid accumulation from C 12 and C 16 alkanes and alkane degradation intermediates was investigated using Y lipolytica wild type strain W29 as well as the POX deleted strains MTLY21 (∆POX2, POX3), MTLY35 (∆POX2, POX3, POX5) and MTLY37 (∆POX2, POX3, POX4, POX5). The quadruple-deleted strain MTLY37 was the only strain that was able to accumulate dioic acids from alkanes, alkanols and monocarboxylic acids. Dodecane was the best alkane substrate for dioic acid accumulation yielding 7 mg/ml dodecanedioic acid after 144h (23% w/v conversion). Lauric acid did not yield any dioic acid (probably due to toxicity), ·but 5 mg/ml hexadecanedioic acid was accumulated from hexadecanoic acid after 48h. All the strains accumulated dodecanedioic acid from the diterminal functionalised 1,12 dodecane diol and eo-hydroxy dodecanoic acid. The quadruple-deleted strain MTL Y37 accumulated a maximum concentration of 20 mg/ml dodecanedioic acid after 48h from 1,12 dodecanediol, while the triple-deleted strain MTL Y35 accumulated 18 mg/ml dodecanedioic acid after 48h from 12-hydroxydodecanoic acid.en_ZA
dc.description.abstractAfrikaans: Dikarboksielsure is waardevolle produkte wat vanaf alkane berei kan word via diterminale oksidasie deur bakterië en giste. Yarrowia lipolytica is een van die alkaanassimilerende giste waarvoor werktuie vir genetiese manipulering reeds ontwikkel is. Y lipolytica het 'n komplekse stel van 5 asiel ko-ensiem A (KoA) oksidase iso-ensieme (gekodeer deur POX1 tot POX5 gene) met verskillende substraat spesifisiteite. Baie inligting is beskikbaar rakende dikarboksielsuur produksie vanaf alkane deur Candida maltosa en Candida tropicalis stamme met onvoldoende β-oksidasie. In baie gevalle was β-oksidasie geblokeer op die vlak van die asiel. KoA oksidases. Daar was in verhouding min inligting beskikbaar rakende dikarboksielsuur produksie deur Y lipolytica stamme. Ons het danksy 'n samewerking met Dr. I.-M. Nicaud van die INRA-CNRS in Frankryk toegang gehad tot 'n reeks mutante met verskillende asiel-KoA oksidases uitgelaat. Dit was dus die doel van my studie om ophoping van dikarboksielsure deur hierdie Y lipolytica stamme te ondersoek. Ons het in die eerste instansie behoefte gehad aan 'n eenvoudige metode om groei te volg. Dit moes verkieslik 'n metode gebaseer op turbiditeitsmeting wees. Onoplosbare hidrofobiese substrate meng normaalweg in met turbiditeitsmetings. Hierdie probleem word vererger wanneer die substraat ook 'n vaste stof is. Y lipolytica selle kan nie d.m. v. sentrifugering geskei word van 'n hidrofobiese substraat omdat die selle hidrofobies is en 'n groot persentasie van die selle klou aan die water/oplosmiddel interfase. Ons het vasgestel dat wanneer die selle met 5M NaOH behandel word (pH 14) dit nie meer hidrofobies is nie. Gebaseer op hierdie waarneming het ons 'n prosedure vir monstervoorbereiding ontwikkel vir biomassa bepaling gebaseer op turbiditeit. In hierdie metode word monsters met SM NaOH en sikloheksaan behandel, voordat dit d.m.v. sentrifugering geoes word. Monstervoorbereiding kan in mikrosentrifuge buise gedoen word en turbiditeitsmetings met 'n mikrotiterplaatleser. Die nuwe metode om groei te volg is gebruik om die toksiese effek van dodekaan en heksadekaan asook hulle terminale en diterminale oksidasieprodukte op Y lipolytica H222 te bepaal. Die alkane en dikarboksielsure was nooit toksies vir Y lipolytica nie. Dodekanol het groei die meeste inhibeer in 'n gisekstrak-peptoon medium met glukose, terwyl dodekanal en dodekanoësuur groei die meeste inhibeer het in 'n semi-sintetiese medium. Heksadekanol en heksadekanoësuur was nie toksies vir Y lipolytica in 'n gisekstrak-peptoon medium met glukose nie, maar was wel toksies in 'n semi-sintetiese medium sonder glukose. Uiteindelik het onsvanaf Y lipolytica H222 en MTLA35, 'n stam met drie asiel-KoA oksidases uitgelaat, stamme berei wat kan groei in die teenwoordigheid van baie hoë konsentrasies dodekanol (10% v/v). Nie een van hierdie stamme het dikarboksielsuur opgehoop, wanneer dit in die teenwoordigheid van hoë konsentrasies dodekanol gegroei is nie (3% en 10% v/v). Dikarboksielsuurproduksie vanaf C 12 en C 16 alkane asook hulle geoksideerde derivate is vervolgens ondersoek. 'n Y lipolytica wilde tipe stam W29 en drie stamme met POX gene uitgelaat nl. MTLY21 (∆POX2, POX3), MTLY35 (∆POX2, POX3, POX5) en MTLY37 (∆POX2, POX3, POX4, POX5). is in hierdie ondersoek gebruik. Die stam met vier POX gene uitgelaat, MLTY37, was die enigste wat dikarboksielsure opgehoop het vanaf alkane en geoksideerde derivate. Dodekaan was die beste alkaan substraat vir die ophoping van dikarboksielsuur en het 7 mg/ml gelwer na 144h (23% omskakeling). Dodekanoësuur het geen dikarboksielsuur gelewer nie (waarskynlik omdat dit toksies was), maar heksadekanoësuur het 5 mg/ml dikarboksielsuur gelewer na A8h. Al die stamme het dikarboksielsuur opgehoop vanaf die diterminaal gefunksionaliseerde C 12 substrate. MTL Y3 7 het 20mg/ml dikarboksielsuur opgehoop vanaf 1, 12-dodekaandiol en MTL Y35 het 18 mg/ml opgehoop vanaf 12-hidroksie dodekanoësuur.af
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectDissertationen_ZA
dc.subjectCarboxylic acidsen_ZA
dc.subjectAcid-base chemistryen_ZA
dc.subjectHydrocarbonsen_ZA
dc.subjectYeasten_ZA
dc.subjectDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2003en_ZA
dc.titleDicarboxylic acid production by Yarrowia lipolytica strainsen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record