Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Complex biofilm communities have extensively been studied in the past. Less work has been done on the early stages of biofilm formation. This study aimed to assess initial colonization patterns of biofilms on different surfaces and under different environmental conditions with application of novel methods describing biofilm surface profiles. Biofilms were cultivated on glass, polyvinylchloride (PVC) and polished stainless steel. Results from microscopy, followed by mathematical analysis and contact angle measurements proved that glass was the most appropriate substrate for this study. More extensive extracellular polymeric substances (EPS) production and apparently less cell attachment were observed on PVC and polished stainless steel surfaces. Two different series of experiments were conducted where biofilms were cultivated on the glass. Biofilm morphology was analysed under various conditions of temperature and nutrient concentration. Different temperature conditions were 8°C, 22°C and 37°C and different nutrient concentrations were 0.1%, 1% and 10% Tryptic Soy Broth (TSB). After obtaining samples after 1, 2, 3 and 4 days respectively, the biofilm surfaces were visualised using atomic force microscopy (AFM) and epifluorescence microscopy. Less cell attachment was displayed at lower temperatures and nutrient limitations. The roughness profile of the early stages of biofilm development was explored by the novel application of various existing statistical methods. Benoit software was applied for the statistical analysis of various data sets obtained from AFM imaging, using power spectrum, variogram and wavelet methods to determine the Hurst exponent. The variogram method proved to be the most suitable to describe biofilm surface profiles with consistent values of ± 0.9, indicating that biofilm growth behaviour will continue in a similar pattern. Fractal dimension values of images obtained from epifluorescence microscopy was determined by the box dimension method. The values described the self-affine patterns displayed by biofilms. Using the results of these investigations, a series of models concerning the initial stages of biofilm formation was compared to describe the development of colony patterns. This study showed that the AFM and epifluorescence microscopy can be used as analytical tools for raw data assembly. It also demonstrated a novel application of existing statistical methods in order to describe the early stages of biofilm formation. Using this approach it was shown that the early stages of biofilm formation display certain colony patterns that can be described and predicted. Such information may be used in efforts to control biofilm formation. / AFRIKAANSE OPSOMMING: Komplekse biofilmgemeenskappe is reeds breedvoerig m die verlede bestudeer. Minder werk is op vroeë stadiums van biofilmvorming gedoen. Hierdie studie het gepoog om die aanvanklike koloniseringspatrone van biofilmvorming op verskillende substrate en onder verskillende omgewingstoestande kwantitatief te bepaal met nuwe metodes om die oppervlakprofiele van biofilms te beskryf. Biofilms is gekweek op glas, polivinielchloried (PVC) en gepoleerde vlekvrye staal. Resultate van mikroskopie, gevolg deur wiskundige analise en kontakhoek-metings het getoon dat glas die mees geskikte substraat vir hierdie studie is. Die produksie van meer ektrasellulêre polimeriese substanse (EPS) en oënskynlik minder selaanhegting is waargeneem op PVC en gepoleerde vlekvrye staaloppervlaktes. Twee verskillende reekse eksperimente IS uitgevoer waar biofilms op glas gekweek IS. Biofilm-morfologie is geanaliseer onder verskillende toestande van temperatuur en nutriënt-konsentrasie. Verskillende temperatuur-toestande was goC, 22°C en 37°C en verskillende nutriënt-konsentrasies was 0.1%, 1% en 10% Tryptic Soy Broth (TSB). Nadat monsters onderskeidelik na 1, 2, 3 en 4 dae verkry is, is die biofilm oppervlaktes gevisualiseer deur atoomkrag mikroskopie (AFM) en epi-fluoressensie mikroskopie. Minder selaanhegting is waargeneem by laer temperature en nutriënt-beperkinge. Die grofheidsprofiele van die vroeë stadium van biofilm-ontwikkeling is ondersoek deur die nuwe toepassing van verskeie bestaande statistiese metodes. Benoit-sagteware is gebruik om die statistiese analise van verskeie data-stelle van AFM beelde te ondersoek deur power spectrum, variogram en wave/et -metodes te gebruik om die Hurst-eksponent te bepaal. Die variogram metode het voorgekom as die mees geskikte om biofilm oppervlakprofiele te beskryf met konstante waardes van ± 0.9, wat aandui dat biofilm groei sal aanhou in 'n soortgelyke patroon. Fraktale dimensie-waardes van beelde wat met epi-fluoressensie mikroskopie verkry is bepaal deur toepassing van Benoit-sagteware se box dimension metode. Die waardes beskryf die selfherhalende patrone wat deur biofilms gedemonstreer word. Deur die resultate van hierdie ondersoeke te gebruik, is 'n reeks modelle aangaande die aanvanklike stadiums van biofilmvorming vergelyk om die ontwikkeling van koloniepatrone te beskryf. Hierdie studie het getoon dat die AFM en epi-fluoressensie mikroskopie gebruik kan word as analitiese gereedskap vir rou data-versameling. 'n Nuwe toepassing van bestaande statistiese metodes om die vroeë stadiums van biofilmvorming te beskryf, is ook gedemonstreer. Deur hierdie benadering te gebruik, is getoon dat die vroeë stadiums van biofilmvorming sekere koloniepatrone aandui wat beskryf en voorspel kan word. Sulke inligting kan gebruik word in pogings om biofilmvorming te beheer.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Basidiomycetous yeasts are found in a wide range of geographical areas ranging from tropical forests to desert regions. These yeasts are associated with different habitats such as soil, decaying vegetative debris, living plants and animals. Some may even be opportunistic human pathogens. In most of these habitats the yeasts may periodically be exposed to adverse conditions such as osmotic stress. Forty-one basidiomycetous yeast strains obtained from culture collections and isolated from nature were studied using various methods which includes the determination of different minimum water activities (aw; NaCl or sorbitol) for growth, survival in soil of varying moisture content, intracellular osmolytes accumulated and their release upon hypo-osmotic stress. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same level of water activity. Interestingly, there were no basidiomycetous strains that showed growth below 0.90aw. 13C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC) was used to analyze the osmolytes accumulated by all the strains of basidiomycetous yeasts when grown at 0.95 aw (NaCl). Glycerol was the major solute accumulated intracellulary by all the yeasts. Arabitol, mannitol or trehalose was accumulated in addition to glycerol in most yeasts whereas a number of yeasts only accumulated glycerol when grown at reduced aw. However, Cryptococcus laurentii US 1F was an exception by accumulating three solutes intracellularly when grown at reduced water activity. When exposed to hypo-osmotic shock all three solutes were rapidly released from the cells. Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 and Cryptococcus neoformans US I1 were further analyzed with 13C NMR spectroscopy to determine whether the type of osmolyte accumulated during different phases of growth at 0.95aw (NaCl) might change. No changes were observed as the same osmolytes were accumulated in all cases. Five yeast strains (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 and Sporidiobolus salmonicolor CBS 5937) were analyzed by HPLC for osmolytes released when exposed to hypo-osmotic shock. The strains differed in the pattern of response of osmolyte release. Only three strains released most of their osmolytes rapidly within 5 min, while C. macerans CBS 2206T and R. muculaginosa CBS 5951 retained most of the osmolytes intacellularly. This suggests that there might be different mechanisms of osmolyte release in basidiomycetous yeasts. A few strains of basidiomycetous yeasts (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F. floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, S. salmonicolor CBS 5937 and Filobasidium capsuligenum CBS 4381) were grown in soil cultures of different field capacity (100%, 25%, 10% and 5%) and evaluated for their survival in this environment. All the strains grew at 100% field capacity. Strains R. mucilaginosa CBS 5951, F. floriforme CBS 6240 and F. capsuligeum CBS 4381 also showed growth in soil at 25% field capacity. However, strains C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and C. laurentii CBS 0139 did not grow at this moisture content but survived up until the end of the experimental period. At lower soil moisture content (5% and 10% field capacity), the yeast strains either showed survival or decreased viability towards the end of the experimental period. Strain C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 and F. floriforme CBS 6240 showed survival at both 5% and 10% field capacity. However, strain F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 and S. salmonicolar CBS 5937 showed a decrease in viability after either 2 or 5 days of incubation. No relationship could be found between the type and number of intracellular osmolytes accumulated when exposed to osmotic stress (0.95aw NaCl) and the ability to grow and survive in soil with lower moisture content. Similarly, the ability of the yeasts to grow and survive in soil with lower moisture content did not correlate with their minimum aw for growth in a liquid medium. It was speculated that other factors, such as the physico-chemical composition of the soil, may also play a role in the survival of a particular yeast species in soil. This study has shown that the responses of basidiomycetous yeasts to reduced aw are physiologically similar to the ascomycetous yeasts. The types of osmolytes accumulated are similar but the basidiomycetous yeasts appear to be more sensitive to reduced aw and they tolerate NaCl better than sorbitol whereas the ascomycetous yeasts tolerate high sugar environments better. This is in agreement with the environments where these yeasts are usually found. / AFRIKAANSE OPSOMMING: Basidiomisete giste word aangetref in ‘n wye reeks geografiese areas, wat strek vanaf tropiese woude tot woestynstreke. Hierdie giste word geassosieer met verskillende habitatte soos grond, verrottende vegetatiewe reste, lewende plante en diere. Sommige mag selfs opportunistiese menslike patogene wees. By meeste van hierdie habitatte mag giste periodies blootgestel word aan moeilike toestande soos osmotiese stres. Een-en-veertig basidiomisete gisrasse, verkry vanaf kultuurversamelings en geisoleer vanuit die natuur, was bestudeer met verskeie metodes, waaronder die bepaling van verskillende minimum water aktiwiteite (aw; NaCl of sorbitol) vir groei, droë massa bepalings, akkumulasie van intrasellulêre osmoliete, asook hul vrystelling met hipo-osmotiese stres. Meeste rasse het meer weerstand teen NaCl as sorbitol gehad by dieselfde vlak van wateraktiwiteit. Dit was interessant om op te let dat geen basidiomisete stamme groei onder 0.90aw getoon het nie. Beide 13C kern magnetiese resonansie (KMR) spektroskopie en hoë uitset vloeistof chromatografie (HUVC) was gebruik om alle opgehoopde osmoliete te analiseer vir alle basidiomisete gisrasse tydens groei tot by 0.95 aw (NaCl). Vir alle giste was gliserol die opgelosde stof wat die meeste intrasellulêr opgehoop het. Arabitol of mannitol of trehalose het saam met gliserol in meeste giste opgehoop, terwyl ‘n aantal giste slegs gliserol opgehoop het tydens groei by verlaagde aw. Cryptococcus laurentii US 1F was daarenteen ‘n uitsondering deurdat dit drie opgelosde stowwe intrasellulêr versamel het tydens groei by verlaagde wateraktiwiteit. Al drie hierdie opgelosde stowwe is uit die selle vrygesel na blootstelling aan hipo-osmotiese skok. Verdere 13C KMR spektroskopie analise was gedoen op Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 en Cryptococcus neoformans US I1 om vas te stel of die tipe opgehoopde osmoliet tydens verskillende fases van groei by 0.95aw (NaCl) mag verander. Geen veranderinge was egter waargeneem aangesien dieselfde osmoliete in alle gevalle opgehoop het. Vyf gisrasse (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 en Sporidiobolus salmonicolor CBS 5937) was geanaliseer deur HUVC vir osmolietvrystelling tydens blootstelling aan hipo-osmotiese skok. Die rasse het verskil in hul responspatroon van osmolietvrystelling. Slegs drie rasse het meeste van hul osmoliete vrygestel binne die eerste 5 minute, terwyl ander, C. macerans CBS 2206T en R. muculaginosa CBS 5951 meeste van hul osmoliete intrasellulêr teruggehou het. Dit dui daarop dat daar dalk verskillende meganismes van osmolietvrystelling in basidiomisete giste mag voorkom. Etlike basidiomisete gisrasse (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, (B) S. salmonicolor CBS 5937, Filobasidium capsuligenum CBS 4381) was opgegroei in grondkulture van verskillende veldkapasiteit (100%, 25%, 10% en 5%) en ondersoek vir hul oorlewing in hierdie omgewing. Al die rasse kon groei by ‘n 100% veldkapasiteit. Die rasse R. mucilaginosa CBS 5951, F. floriforme CBS 6240 en F. capsuligeum CBS 4381 kon ook groei in grond met 25% veldkapasiteit. Alhoewel rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T en C laurentii CBS 0139 nie kon groei by hierdie voginhoud nie, het hulle nog steeds oorleef tot aan die einde van die eksperimentele tydperk. By verlaagde grond voginhoud (5% en 10% veldkapasiteit) het die gisrasse of oorleef of ‘n verlaagde lewensvatbaarheid openbaar teen die einde van die eksperimentele prosedure. Die rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 en F. floriforme CBS 6240 het oorleef by beide 5% en 10% veldkapasiteit. Die rasse F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 en S. salmonicolar CBS 5937 het egter ‘n verlaging in lewensvatbaarheid getoon na ‘n periode van of 2 of 5 dae inkubasie. Geen ooreenstemming kon gevind word tussen die tipe en aantal intrasellulêre opgehoopde osmoliete nie na blootstelling aan osmotiese druk (0.95aw NaCl), asook hul vermoë om te groei en oorleef in grond met ‘n laer voginhoud. Terselfdertyd het die giste se vermoë om te groei en oorleef in grond met ‘n laer voginhoud nie ooreengestem met hul minimum aw vir groei in vloeibare groeimedium nie. Ter spekulasie kan ander faktore, soos die fisio-chemiese samestelling van die grond ook ‘n rol speel in die oorlewing van ‘n spesifieke gisspesie in grond. Hierdie studie het getoon dat basidiomisete giste se respons tot verlaagde aw fisiologies dieselfde is as vir askomisete giste. Alhoewel die tipe versamelde osmoliete identies was, wil dit voorkom asof basidiomisete giste meer sensitief teenoor ‘n verlaagde aw is, tesame met ‘n beter bestandheid teen NaCl as sorbitol, terwyl askomisete giste hoë suiker omgewings beter kan verdra. Dit is in ooreenstemming met die tipe omgewings waar hierdie giste gewoonlik voorkom.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: A laboratory-scale model was used to simulate biological sulfate removal. The focus of the research was microbial community response, such as the relative abundance of functional groups to changes in influent medium composition. Specific oligonucleotide probes were obtained that recognised sulfate reducing bacteria (SRB) within the biofilm community. Terminal restriction fragment length polymorphism (T-RFLP) and BIOLOG™ Ecoplate analyses were used to study the SRB community when provided with sodium lactate, sucrose or ethanol as carbon sources in complex Postgate C broth. These two analyses, as well as conventional methods, were applied to follow succession patterns in the laboratory scale reactors, and to determine the possible presence and relative abundance of microorganisms other than bacteria under sulfate reducing conditions. T-RFLP and BIOLOG™ Ecoplate analyses indicated a few dominant organisms in the community and a slight decline after a shift to another carbon source. Fluorescent hybridization showed higher numbers of SRB relative to the total microbial community than conventional culturing techniques. Furthermore, microscopic observations showed that not only SRB and other bacteria, but also yeast and filamentous fungi were integrated in a biofilm under sulfate reducing conditions. These microscopic observations were verified with fluorescent in situ hybridization (FISH) and yeast Live I Dead viability probes. / AFRIKAANSE OPSOMMING: Biologiese sulfaat-verwydering is met behulp van "n laboratoriumskaalmodel gesimuleer. Die doel van die navorsing was om die respons van "n mikrobiese gemeenskap met byvoorbeeld die relatiewe hoeveelheid van funksionele groepe op veranderinge in invloeiende medium samestelling te bestudeer. Spesifieke oligonukleotiedpeilers wat sulfaatreduserende bakterieë (SRB) in "n biofilmgemeenskap kan opspoor is gebruik. Die SRB gemeenskap is bestudeer met behulp van terminale-restriksiefragmentlengtepolimorfisme (TRFLP) en BIOLOGTM Ecoplate analise waar natriumlaktaat, sukrose of etanol as koolstofbronne toegevoeg is. Hierdie twee tipes analise en konvensionele metodes is aangewend om suksessiepatrone in die laboratoriumskaalreaktor te volg en die moontlike teenwoordigheid en relatiewe hoeveelheid van organismes, uitsluitende bakterieë, onder sulfaatreduserende kondisies te bepaal. Analise van T-RFLP en BIOLOGTM Ecoplate het aangedui dat In paar dominante organismes in die gemeenskap teenwoordig was, wat effens afgeneem het na verskuiwing na 'n ander koolstofbron. Fluoresserende hibridisasie het hoër getalle van SRB relatief tot die totale mikrobiese gemeenskap aangedui as konvensionele kultuur tegnieke. Mikroskopiese analises het verder getoon dat benewens SRB en ander bakterieë ook giste en filamentagtige swamme onder sulfaatreduserende kondisies in "n biofilm geïntegreer was. Hierdie mikroskopiese waarneminge is bevestig deur fluoresserende in situ hibridisasie (FISH) en gis Lewe / Dood lewensvatbaarheid peilers.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Reliable energy resources could be considered as one of the cornerstones of the prosperity of the human race. The growing human population is constantly exerting more pressure on the world’s natural resources, which include natural fossil fuels that are non‐renewable. There are concerns regarding the use of fossil fuels due to its growing scarcity and its negative impact on the environment. There is thus a growing need in the world for energy sources that are renewable, more or less carbon neutral and therefore with a minimum environmental impact. Renewable energy is currently being harnessed from the wind, water and sun, but to a limited extent. These forms of natural resources are very attractive for the production of renewable energy, but these technologies are difficult to apply in the current transportation sector. Biofuels provide an alternative to the current use of liquid fossil fuels and it could be able to sustain the current fleet of automobiles worldwide in the intermediate to long term with minimal adjustment to the engines of these vehicles. Extensive research has been done on the production processes for biofuels. Previous processes included the use of high temperatures and acids that further increased the total production cost and thus making biofuels less attractive as an alternative energy source. Recent research has suggested a wide range of organic materials as substrate for the production of biofuels, which include lignin, hemi‐cellulose, cellulose and starch. Processes based on hemi‐cellulose, cellulose and lignin as substrate are still in its early research stages and commercial application of these processes will only occur over the medium‐ to long‐term. Starch is a very good alternative source for the production of biofuels, but there is a need for a microbial system for the conversion of starch to bio‐ethanol in a single step, referred to as Consolidated Bioprocessing (CBP). This would reduce the overall production cost of bio‐ethanol and thus making starch‐based substrates more attractive as an alternative energy source. The cost saving will be mainly due to the elimination of the pre‐treatment of raw starch at high temperatures and the addition of enzymes for the liquefaction and saccharification of starch to simple sugars. However, as there is no currently no known microbial organism known that can produce the required enzymes (i.e. amylases) as well as ferment the resulting sugars to ethanol, heterologous expression of these enzymes in a host strain able to ferment sugars could provide the best alternative system. In the first part of this study, 36 fungal strains known for the production of amylases were screened and compared for the highest extracellular enzyme activity on raw corn starch. The best two candidates, i.e. Aspergillus tubingensis (T8.4) and Mucor cincinelloides (1180), were then further evaluated to determine which organism has the highest efficiency when combined with a Saccharomyces cerevisiae laboratory strain. In fermentation experiments, A. tubingensis (T8.4) in combination with S. cerevisiae Y102 yeast strain resulted in the highest yield of ethanol. Literature on A. tubingensis is limited compared with other Aspergillii and it was previously accepted that A. tubingensis has the highest homology with Aspergillus niger. However, other reports – including the present study ‐ found that A. tubingensis is closer related to other Aspergillus spp. with regard to its amylolytic enzymes. The α‐amylase gene of A. tubingenis has a homology of 99.00% with that of Aspergillus kawachii whereas the glucoamylase gene has a homology of 99.26% with that of Aspergillus shirousami. In the second part of this study, two recombinant S. cerevisiae strains were constructed to express the wild type A. tubingensis α‐amylase (Atamy) and glucoamylase (Atglu), respectively. The combination of the two recombinant yeast strains was able to completely hydrolyse and also utilize raw corn starch for the production of bio‐ethanol, with a yield of 11.04 g/l of ethanol, which translates to 98% of the theoretical yield from starch with a 52% conversion of the total raw starch. This rate of conversion is lower than other reports which indicated up to 82% and 96% of the theoretical yield of ethanol from raw and soluble starch, respectively, by α‐ and glucoamylase. Furthermore, the combined expressed of the two genes was much more effective than when only one of the two genes were expressed, with a yield of 0.32 g/l ethanol for only Atamy and 2.52 g/l ethanol for Atglu. This proved that the combination of the A. tubingensis genes were best suited for the production of biofuels from raw starch. This also proved that the concept of constructing an amylolytic yeast strain capable of raw starch hydrolysis and fermentation was indeed feasible. / AFRIKAANSE OPSOMMING: Betroubare energiebronne kan as een van die boublokke vir die vooruitgang van die mensdom beskou word. Die groeiende menslike populasie is gedurig besig om meer druk op die wêreld se natuurlike hulpbronne te plaas, insluitende nie‐hernubare fossielbrandstowwe. Daar is kommer rakende die gebruik van fossielbrandstowwe weens ‘n afname in die beskikbaarheid en die negatiewe impak wat dit op die omgewing het. Daar is dus ‘n groeiende behoefte in die wêreld vir ‘n hernubare, min of meer koolstof‐neutrale energiebron wat ‘n minimale omgewingsimpak sal hê. Hernubare energie word tans tot ‘n beperkte mate uit wind, water en die son verkry. Hierdie vorms van natuurlike energie hulpbronne is baie aanloklik vir die vervaardiging van hernubare energie, maar hierdie tegnologië is moeilik toepasbaar in die huidige vervoersektor. Biobrandstowwe voorsien ‘n alternatief vir die huidige gebruik van fossielbrandstowwe en kan moontlik die huidige voertuigvloot wêreldwyd oor die medium‐ tot langtermyn onderhou met minimale enjinaanpassings van hierdie voertuie. Deeglike navorsing is alreeds op die vervaardigingsprosesse vir biobrandstowwe gedoen. Vorige prosesse het die gebruik van hoë temperature en sure ingesluit wat produksiekostes verder verhoog en gevolglik die gebruik van biobrandstowwe as ‘n alternatiewe energiebron minder aantreklik gemaak het. Onlangse navorsing het die gebruik van organiese materiaal as substraat vir die produksie van biobrandstowwe voorgestel, wat lignien, hemi‐sellulose, sellulose en stysel insluit. Prosesse met die gebruik van hemi‐sellulose, sellulose en lignien as substraat is nog in die beginfase van ontwikkeling en kommersialisering van hierdie prosesse sal eers oor die medium‐ tot langtermyn plaasvind. Stysel is ‘n baie goeie alternatiewe bron vir die produksie van biobrandstowwe, maar ‘n mikrobiese sisteem word vir die omskakeling van stysel in bio‐etanol in ‘n enkele stap benodig, bekend as gekonsolideerde bioprosessering (GBP). Dit sal die algemene produksiekoste van bio‐etanol verlaag en dus styselsubstrate as ‘n alternatiewe energiebron meer aantreklik maak. Die kostebesparing sal hoofsaaklik realiseer omdat die vooraf‐behandeling van rou stysel byhoë temperature en die toevoeging van ensieme vir die vervloeiing en versuikering van stysel tot eenvoudige suikers, uitgeskakel word. Aangesien daar tans geen bekende mikrobe organisme is wat die nodige ensieme (nl. amilases) kan produseer en ook die suikers wat daardeur vrygestel is, na etanol kan fermenteer nie, kan die heteroloë uitdrukking van hierdie ensieme in ‘n gasheer‐ras wat die suikers kan fermenteer, moontlik die beste alternatief verskaf. In die eerste deel van hierdie studie is 36 fungi rasse wat bekend is vir hul amilase produksie geevalueer en met mekaar vergelyk vir die hoogste ekstrasellulêre ensiemaktiwiteit op rou mieliestysel. Die beste twee kandidate, naamlik Aspergillus tubingensis en Mucor cincinelloides, is verder ge‐evalueer om te bepaal watter organisme het die hoogste effektiwiteit in kombinasie met ‘n Saccharomyces cerevisiae laboratorium gisras. In fermentasie‐eksperimente het A. tubingensis in kombinasie met S. cerevisiae Y102 gisras die hoogste etanol opbrengs gelewer. Inligting rakende A. tubingensis is beperk relatief tot ander Aspergillii en dit was voorheen aanvaar dat A. tubingensis die hoogste homologie met Aspergillus niger het. Ander verslae – insuitende die huidige studie ‐ het egter gevind dat A. tubingensis nader verwant aan ander Aspergillus spp. in terme van amilolitiese ensieme is. Die α‐amilase geen van A. tubingensis het ‘n homologie van 99.00% met dié van Aspergillus kawachii en die glukoamilase ‘n homologie van 99.26% met dié van Aspergillus shirousami getoon. In die tweede gedeelte van hierdie studie is twee rekombinante S. cerevisae gisrasse gekonstrueer om onderskeidelik die α‐amilase (Atamy) en glukoamilase (Atglu) van A. tubingensis uit te druk. Die kombinasie van die twee rekombinante gisrasse was in staat om die volledige hidrolise en benutting van rou mieliestysel vir die produksie van bio‐etanol deur te voer met ‘n opbrengs van 11.04 g/l wat gelykstaande is aan 98% van die teoretiese opbrengs vanaf stysel met ‘n omskakeling van 52% van die totale rou stysel. Hierdie omskakelingskoers is laer as ander studies wat onderskeidelik 82% en 96% van die teoretiese opbrengs van rou en oplosbare stysel vir α‐ en glukoamilase getoon het. Verder was die kombinasie van die twee gene meer effektief as wanneer slegs een gebruik is, met ‘n 0.32 g/l opbrengs vir Atamy en 2.52g/l vir Atglu. Hierdie het bewys dat die kombinasie van die A. tubingensis meergeskik vir die produksie van bio‐etanol was. Dit het ook bewys dat die beginsel van ‘n amilolitiese gisras wat in staat is om rou stysel te hidroliseer en te fermenteer, inderdaad moontlik is.

Thesis (DSc (Microbiology))--University of Stellenbosch, 2005. / The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Aflatoxins are mycotoxins predominantly produced by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1), the most abundant aflatoxin, is highly mutagenic, toxic, carcinogenic and teratogenic to humans and animals and is particularly correlated with the incidence of hepatocellular carcinoma in parts of Africa, China and South East Asia. In this regard aflatoxin is classified as a type I human carcinogen by the International Agency for Research on Cancer. Furthermore, aflatoxin contamination of food and feed is responsible for extensive economic losses due to loss of crops and farm animals. In spite of regulations regarding acceptable levels of aflatoxin in food, aflatoxin contamination remains a serious worldwide problem, especially in developing countries where it occurs predominantly in dietary staples. Inactivation of aflatoxin by physical and chemical methods has not yet proved to be effective and economic. However, biological detoxification offers an attractive alternative for eliminating toxins as well as safe-guarding the desired quality of food and feed. In this study, the biological degradation of AFB1 by bacteria and fungi was investigated. Several bacteria, including Rhodococcus spp., as well as white rot fungi have the potential to degrade a wide range of polycyclic hydrocarbon compounds due to the large repertoire of enzymes they produce and therefore the ability of some of these microorganisms to degrade AFB1 was investigated. Effective degradation of AFB1 by intracellular extracts of Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia corynebacterioides DSM 20151 and N. corynebacterioides DSM 12676 was demonstrated. Furthermore, AFB1 was effectively degraded by liquid cultures as well as intra- and extracellular extracts of Rhodococcus erythropolis DSM 14303. Significant (P<0.001) reduction in AFB1 was observed following treatment with R. erythropolis extracellular extracts with only 33.20% residual AFB1 after 72 h. Results indicated that the degradation by R. erythropolis DSM 14303 is enzymatic and that the enzymes are constitutively produced. The degradation of AFB1 when treated with R. erythropolis DSM 14303 extracellular extract coincided with a total loss of mutagenicity. In addition, treatment of AFB1 with culture fractions containing recombinant 2,3-dihydroxybiphenyl dioxygenase, which was produced through extracellular expression of the bphC1 gene of R. erythropolis DSM 14303 in Escherichia coli BL21, resulted in significant (P<0.0001) degradation (49.32%) and reduced mutagenic potency (42.47%) of the molecule. Significant (P<0.0001-0.05) degradation of AFB1 was obtained following treatment with culture extracts containing laccase enzyme produced by white rot fungi (17.10- 76.00%), purified fungal laccase from Trametes versicolor (1 U/ml, 87.34%) as well as with recombinant laccase produced by Aspergillus niger (118 U/L, 55.00%). Furthermore, treatment of AFB1 with purified fungal laccase enzyme (1 U/ml) resulted in loss of the mutagenic potency of the molecule. The decrease in the fluorescence and mutagenic properties of AFB1 following treatment with the microbial preparations imply changes to the furofuran- and/or lactone rings of the molecule. The current study contributes towards developing genetic engineered microbial strains which could be applied as an important bio-control measure. Such strains could exhibit multifunctional technological properties including degradation of AFB1, to significantly improve the quality, safety and acceptability of food. / AFRIKAANSE OPSOMMING: Aflatoksiene is mikotoksiene wat hoofsaaklik deur die filamentagtige fungi, Aspergillus flavus en Aspergillus parasiticus geproduseer word. Die algemeenste aflatoksien, aflatoksien B1 (AFB1), is hoogs mutagenies, toksies, karsinogenies en teratogenies vir mense en diere. Veral in sekere dele van Afrika, China en Suid-Oos Asië bestaan daar `n korrelasie tussen aflatoksien en die voorkoms van hepatosellulêre karsinoom en gevolglik word aflatoksiene as `n tipe I menslike karsinogeen deur die Internasionale Agentskap vir Kankernavorsing geklassifiseer. Aflatoksien kontaminasie in voedsel het ook `n ekonomiese impak as gevolg van verlies aan landbougewasse en diere. Ten spyte van maatreëls betreffende die toelaatbare vlakke van aflatoksiene in voedel, is aflatoksien kontaminasie steeds `n groot probleem wêreldwyd, veral in ontwikkelende lande waar dit hoofsaaklik in stapelvoedsel voorkom. Huidiglik is die inaktivering van aflatoksiene deur fisiese en chemiese metodes nie effektief en ekonomies nie. Daarteenoor bied biologiese tegnieke `n gunstige opsie vir die eliminering van die toksiene, terwyl die organoleptiese eienskappe van die voedsel steeds behoue bly. Hierdie studie fokus op die biologiese afbraak van AFB1 deur bakterieë en fungi. Verskeie bakterieë, insluitend Rhodococcus spp., sowel as witvrot fungi produseer `n verskeidenheid ensieme wat hulle in staat stel om `n wye reeks polisikliese hidrokoolstofverbindings af te breek en gevolglik is afbraak van AFB1 deur sommige van hierdie mikroörganismes bestudeer. Effektiewe afbraak van AFB1 deur intrasellulêre ekstrakte van Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia corynebacterioides DSM 20151 en N. corynebacterioides DSM 12676 is aangetoon. AFB1 is ook effektief in vloeibare kulture sowel as intra- en ekstrasellulêre ekstrakte van Rhodococcus erythropolis DSM 14303 afgebreek. `n Beduidende (P<0.001) afbraak van AFB1 is waargeneem na behandeling met R. erythropolis DSM 14303 ekstrasellulêre ekstrakte, met slegs 33.20% oorblywende AFB1 na 72 h. Resultate het getoon dat die afbraak deur R. erythropolis DSM 14303 ensimaties is en dat die ensieme konstitutief geproduseer word. Afbraak van AFB1 deur R. erythropolis DSM 14303 het ook tot `n totale verlies aan mutagenisiteit gelei. Verder het behandeling van AFB1 met rekombinante 2,3-dihidroksiebifenieldioksiginase fraksies wat geproduseer is deur ekstrasellulêre uitdrukking van die bphC1 geen van R. erythropolis DSM 14303 in Escherichia coli BL21, beduidende (P<0.0001) afbraak (49.32%) en vermindering in mutagenisiteit (42.47%) van die molekuul teweeggebring. Beduidende (P<0.0001-0.05) afbraak van AFB1 is verkry na behandeling met witvrot fungus kultuurekstrakte wat lakkase-ensiem bevat (17.10-76.00%), gesuiwerde lakkase geproduseer deur Trametes versicolor (1 U/ml, 87.34%), sowel as rekombinante lakkase geproduseer deur Aspergillus niger (118 U/L, 55.00%). Verder het die behandeling van AFB1 met gesuiwerde lakkase-ensiem (1 U/ml) gelei tot verlies aan mutagenisiteit van AFB1. Die afname in fluoressensie en mutageniese eienskappe van die AFB1-molekuul na behandeling met die onderskeie mikrobiese preparate dui op struktuurveranderings aan die furofuraan- en/of laktoonringe van die molekuul. Hierdie studie lewer `n bydrae tot die ontwikkeling van geneties gemanipuleerde mikrobiese rasse wat as `n belangrike biokontrole kan dien. Sulke rasse met multifunksionele tegnologiese eienskappe, insluitend die afbraak van AFB1, kan die kwaliteit, veiligheid en aanvaarbaarheid van voedsel verbeter.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the major hemicellulose component in plant cell walls, is the most abundant polysaccharide after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second most abundant renewable monosaccharide in nature. Very few hemicelluloses are either homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their hydrolysis is more complex than the enzyme group responsible for the hydrolysis of cellulose. Although the ability to degrade xylan is common among bacteria and filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of fermenting polymeric xylan directly to ethanol. However, it was shown that the natural xylanolytic ability of this yeast is very weak. In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast to produce heterologous proteins, and to determine the enhancement of xylan utilisation by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase (PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not function in P. stipitis . When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene (xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity declined over time due to the presence of extracellular proteases, secreted by P. stipitis. Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02) with the S. cerevisiae mating factor secretion signal (MFal) under control of the P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124 nkatlml, respectively. The expression of the fungal xylanolytic genes in P. stipitis also led to an increased biomass yield when the recombinant strains were cultured on birchwood xylan as sole carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass level than the control strain. Biomass levels of the recombinant strains were further improved on average by 85% by growing them in a fermentor under conditions of high oxygenation. The strains were also tested for direct conversion of xylan to ethanol and the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield over the reference strain. These strains represent a step towards the efficient degradation and utilisation of hemicellulosic materials by ethanol-producing yeasts. / AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of heeltemal linieêr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom etanol vanafpolimeriese xilaan te produseer. In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie funksioneel in P. stipitis nie. Ekstrasellulêre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii ~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulêre proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulêre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen (xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon ekstrasellulêre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word. Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik. Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik. Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6- voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en benutting van hemisellulose deur etanolproduserende giste.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is commonly associated with the wine industry. However, this yeast was also isolated from soils not associated with vines. Despite the fact that S. cerevisiae is not perceived as an autochthonous soil yeast, its interaction with other soil microbiota suggests the contrary. Aside from a few in vitro studies, the fate of S. cerevisiae in soil is largely unknown. This may partly be ascribed to the lack of reliable methods to enumerate fermentative yeasts in soil. Consequently, we evaluated an enumeration procedure for fermentative yeasts in soil, whereby yeast malt extract (YM) agar plates containing selective agents, were incubated in anaerobic jars before the colonies were enumerated. This procedure proved to be selective for fermentative yeasts, such as industrial strains of S. cerevisiae. We then commenced studying the growth and survival of S. cerevisiae in soil differing in moisture content and nutrient levels, using S. cerevisiae strain S92 and the genetically modified strain S. cerevisiae ML01, as well as two autochthonous soil yeasts, Cryptococcus laurentii and Cryptococcus podzolicus. The yeast strains were each inoculated into three series of microcosms containing sterile soil with a moisture content of ca. 30% (v/w), a moisture content of ca. 15% (v/w), or a moisture content of ca. 30% supplemented with nutrients used in agriculture. Growth of each strain was monitored for a period of 48 days and all the yeasts were found to grow or survive under these conditions, up until the end of the incubation period. Generally, the cryptococci reached larger population sizes in the soil than the Saccharomyces strains, which may be due to their ability to utilize a wider range of carbon sources and to survive in semi-arid soils. Aside from cell numbers observed in nutrient supplemented soil, in which S. cerevisiae ML01 reached higher numbers than S92, there was no significant difference between the growth and survival of the Saccharomyces strains. In all the microcosms, metabolic rates, as determined by measuring CO2 emissions from soil, reached a maximum within the first day and then declined over the remainder of the trial, possibly due to depletion of nutrients. Differences in CO2 emissions from the different series of microcosms were attributed to different metabolic rates and energy expenditure needed to maintain yeast populations under different conditions. Each of the above-mentioned yeasts was subsequently inoculated in a microcosm prepared from non-sterile soil and monitored using selective enumeration procedures. The Saccharomyces strains were enumerated using the above-mentioned soil dilution plates incubated in anaerobic jars. The presence of natural soil biota caused a decrease in viable yeast numbers for all strains and this was ascribed to competition with and predation by other soil borne organisms. Further evidence for competition and/or amensalism impacting on Saccharomyces populations in soil was obtained when monitoring co-cultures of Saccharomyces with C. laurentii 1f and C. podzolicus 3f in soil microcosms, revealed a significant reduction in Saccharomyces numbers during a 28 day incubation period. However, when the two Saccharomyces strains were cultured in soil microcosms inoculated with a protistan predator, populations of both strains increased and remained at these high levels for the duration of the trial. These findings point to a possible symbiosis between Saccharomyces and the protista whereby the predators ensure continuous nutrient cycling within the soil microcosms. In the final part of the study, epifluorescence microscopy revealed that, similar to known soil cryptococci, the two Saccharomyces strains were able to form biofilms in oligotrophic conditions. The results of this study showed that in the presence of natural soil microbes, no differences exist between the growth and survival of S. cerevisiae S92 and S. cerevisiae ML01. Also, the findings point to a natural niche for this species somewhere in the soil habitat. / AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae word algemeen met die wynindustrie geassosieer. Hierdie gis is egter ook uit grond geïsoleer wat nie met wingerd geassosieer word nie. Ten spyte van die feit dat S. cerevisiae nie as ‘n outogtoniese grondgis beskou word nie, dui sy interaksie met ander grondmikrobiota op die teendeel. Behalwe vir ‘n paar in vitro studies, is die lot van S. cerevisiae in grond grootliks onbekend. Dit mag gedeeltelik aan die gebrek aan betroubare metodes om fermenterende giste in grond te tel, toegeskryf word. Ons het gevolglik ‘n tellingsmetode vir fermenterende giste in grond geëvalueer waarin gis-mout ekstrak (GM) agar plate, bevattende selektiewe agente, in anaërobiese flesse geïnkubeer is voordat die kolonies getel is. Hierdie metode was selektief vir fermenterende giste, soos die industriële stamme van S. cerevisiae. Hierna is die groei en oorlewing van S. cerevisiae bestudeer in gronde met verskillende vog- en nutriëntvlakke deur gebruik te maak van S. cerevisiae stam S92 en die geneties gemodifiseerde stam S. cerevisiae ML01, asook twee outogtoniese grondgiste, Cryptococcus laurentii en Cryptococcus podzolicus. Die gisstamme is elk geïnokuleer in drie reekse van mikrokosmosse bestaande uit steriele grond met ‘n vogvlak van ca. 30% (v/w), ‘n vogvlak van ca. 15% (v/w), of ‘n vogvlak van ca. 30% aangevul met landbounutriënte. Die groei van elke stam is waargeneem vir ‘n tydperk van 48 dae en al die giste het onder hierdie omstandighede tot aan die einde van die inkubasietydperk gegroei of oorleef. Oor die algemeen het die cryptococci groter populasies in die grond gevorm as die Saccharomyces stamme, wat toegereken kan word aan hul vermoë om ‘n wyer reeks koolstofbronne te benut en om in droë gronde te oorleef. Behalwe dat S. cerevisiae ML01 ‘n hoër aantal selle in nutriënt aangevulde grond behaal het as S92, was daar geen beduidende verskil tussen die groei en oorlewing van die Saccharomyces stamme nie. In al hierdie mikrokosmosse het die metaboliese tempo, soos bepaal deur CO2 vrystellings vanuit grond te meet, ‘n maksimum bereik binne die eerste dag en dan het dit afgeneem oor die res van die toetsperiode, waarskynlik as gevolg van die uitputting van die nutriënte. Verskille in die CO2 vrystellings wat vir die verskillende reekse van mikrokosmosse aangeteken is, is te wyte aan die verskillende metaboliese tempo’s en energiegebruik benodig om gispopulasies onder verskillende omstandighede in stand te hou. Elk van bogenoemde giste is vervolgens geïnokuleer in ‘n mikrokosmos wat voorberei is van nie-steriele grond, en waargeneem deur selektiewe enumerasie prosedures toe te pas. Die Saccharomyces stamme is getel deur gebruik te maak van bogenoemde grondverdunningsplate wat in anaërobiese flesse geïnkubeer is. Die teenwoordigheid van natuurlike grondbiota het in alle stamme ‘n afname in lewensvatbare gisgetalle veroorsaak en is toegeskryf aan die kompetisie met en predasie deur ander grondorganismes. Verdere bewys van die impak van kompetisie en/of amensalisme op Saccharomyces populasies in die grond, is die beduidende afname in Saccharomyces getalle tydens ‘n 28 dag inkubasie tydperk, waartydens ko-kulture van Saccharomyces stamme met C. laurentii 1f en C. podzolicus 3f in grond mikrokosmosse ondersoek is. Toe die twee Saccharomyces stamme egter in grond mikrokosmosse opgekweek is wat met ‘n protistiese predator geïnokuleer is, het populasies van albei stamme gegroei en om hierdie hoë vlakke gebly tot aan die einde van die toets. Hierdie bevindings dui ‘n moontlike simbiose tussen Saccharomyces en die protista aan waardeur die predatore deurlopende nutriëntsiklering binne die grondmikrokosmos verseker. In die laaste deel van die studie toon epifluoressensie mikroskopie aan dat, net soos bekende grond cryptococci, die twee Saccharomyces stamme in staat is om biofilms in oligotrofiese omstandighede te vorm. Die resultaat van die studie toon aan dat in die teenwoordigheid van natuurlike grondmikrobe daar geen verskil tussen die groei en oorlewing van S. cerevisiae S92 en S. cerevisiae ML01 is nie. Die bevindings dui ook aan dat daar ‘n natuurlike nis vir hierdie spesie iewers in die grondhabitat is.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol. / AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Intraspecific diversity among yeasts, including basidiomycetous yeasts has mostly been studied from a taxonomic point of view. The heterobasidiomycetous genus Cryptococcus is no exception and it was found to contain species that display heterogeneity both on a genetic and physiological level, i.e. diversity among strains originating from different geographical areas. It was stated that this diversity within yeast species is possibly caused by intrinsic attributes of the different habitats the strains of a particular species originate from. However, little is known about the diversity of a species within a specific habitat. Thus, in this study intraspecific diversity among selected cryptoccoci isolated from a single soil sample originating from pristine Fynbos vegetation , was investigated.