Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The ability of biofilms to resist antimicrobial treatment, when planktonic microbes cannot, is of not only fundamental scientific interest, but also a concern in industrial and medical fields. The inability to control biofouling of water distribution networks and products, as well as recurrent infections of implanted medical devices, is not only costly, but also potentially lethal. Several mechanisms whereby biofilms are able to evade antibiotic and biocidal agents have been proposed and investigated, but no universally relevant characteristic has been identified. . Initial investigation, involving BacLightTh ! LIVEIDEAD viability probes, epifluorescence microscopy and image analysis into the ability of natural biofilm and planktonic populations, .cultured in situ in a cooling tower, to survive treatment with a commercial biocide was not conclusive. Subsequent laboratory experimentation with a bacterial isolate from the cooling tower water revealed that the ability of attached biofilms to resist antimicrobial treatment exceeded that of planktonic cells shed from the biofilm. The reduced ability of suspended cells to survive antimicrobial treatment was not statistically significant, compared to that of the biofilm (P = 0.05). This is in contrast to the wealth of literature published on the subject of biofilm antimicrobial resistance The dilution rate in the flowcells in which biofilms were cultivated was more than 100 times higher than the maximum specific growth rate of the test organism. Nevertheless, there was typically more than I x 108 cells/ml in the effluent, suggesting that a metabolically active, rapidly dividing layer of cells existed at the biofilm bulk-liquid interface, from where daughter cells continuously detached. Treatment with an antimicrobial agent resulted in a significant reduction in the viability and number of cells detached from the biofilm, suggesting that this metabolically active layer of the biofilm was more sensitive to antimicrobial treatment, possibly due to a higher specific growth rate. Antimicrobial resistance was shown to be affected by the growth rate for planktonic bacterial populations, with an increased ability to survive, correlated with a decrease in specific growth rate. This supports the contention that growth rate plays a role in the susceptibility of the active layer. The bacterial cells in the layers closest to the attachment surface of the biofilm has frequently been shown to be slow growing, due to nutrient and oxygen limitation, while the outer biofilm layer is more susceptible to unfavourable environmental conditions. It is possible that such differentiation, which results in a responsive outer biofilm layer, provides a mechanism for the protection of the cells in the deeper layers, and thus survival over time. The results presented here support several hypotheses put forth in literature to account for the increased resistance of biofilms towards antimicrobial agents. Future work will include an investigation into changes in the patterns of gene expression when a bacteria becomes attached to a surface, upon subsequent release from the biofilm, and the influence this has on the ability to resist antimicrobial treatment. / AFRIKAANSE OPSOMMING: Die vermoë van aangehegte mikrobes, in teenstelling met vrydrywende mikroorganismes, om behandeling met antimikrobiese middels te oorleef, is nie net van belang vanuit 'n fundamenteel wetenskaplike oogpunt nie, maar ook betekenisvol vir die industriële en mediese velde. Die beheer van bio-bevuiling van waterverspreidingsnetwerke en produkte, sowel as herhaalde infeksies van mediese inplantings, is nie net van kostebelang nie, maar ook potensieël lewensgevaarlik. Verskeie meganismes wat biofilms in staat stelom antimikrobiese behandeling te oorleef, IS voorgestel en ondersoek, maar geen alomteenwoordige eienskap is tot dusver geïdentifiseer nie. Aanvanklike ondersoeke na die vermoë van natuurlike biofilms en planktoniese 'gemeenskappe, om biosiedbehandeling in situ in 'n lugversorgingskoeltoring se water te oorleef, was onbeslis. Die eksperimentele metodes het gebruik gemaak van BacLight™ LIVE/DEAD lewensvatbaarheidkleurstof, epifluoressensie-mikroskopie en beeldanalise. Daaropvolgende ondersoeke met 'n bakteriese isolaat vanuit die koeltoring het daarop gedui dat biofilms beter in staat is om antimikrobiese behandeling te oorleef as selle wat vrygelaat word vanuit die biofilm. Die afname in the lewensvatbaarheid van vrydrywende selle, na afloop van biosiedbehandeling, was nie statisties beduidend in vergelyking met die van die biofilm nie (P = 0.05). Die bevinding is in teenstelling met wat algemeen aanvaar word in die literatuur. Die verdunningstempo waaronder die biofilms in die vloeiselle gekweek is, was meer as 100- voudig hoër as die maksimum spesifieke groeitempo van die toetsorganisme. Ten spyte hiervan was daar tipies meer as 1 x 108 selle/ml in die uitvloeisel teenwoordig. Dit dui op 'n metabolies aktiewe, vinnig verdelende laag selle in die boonste laag van die biofilm, naaste aan die vloeistof fase, waarvandaan dogterselle voortdurend vrygestel word. Behandeling met die antimikrobiese agent het 'n beduidende afname in die lewensvatbaarheid en aantal dogterselle tot gevolg gehad, wat lei tot die gevolgtrekking dat die metabolies aktiewe laag van die biofilm meer sensitief is vir antimikrobiese behandeling, moontlik weens 'n hoër spesifieke groeitempo. Daar is verder bewys dat die vermoë om die werking van die antimikrobiese middel teen te staan, afhanklik is van die spesifieke groeitempo van planktoniese populasies. 'n Afname in groeitempo word geassosieer met 'n toename in oorlewing na antimikrobiese behandeling, wat die voorstel dat die groeitempo van die aktiewe laag 'n rol speel in die vatbaarheid daarvan, ondersteun. Dit is bekend dat die metaboliese aktiwiteit van bakteriese selle nader aan die aanhegtingsoppervlak van die biofilm verlaag is, weens 'n afname in diffusie van suurstof en nutriente in daardie deel van die biofilm. Dit is moontlik dat hierdie differensiasie, wat lei tot die vatbaarheid van die buitenste laag van die biofilm vir ongunstige omgewingstoestande, 'n oorlewingsmeganisme daarstel wat die onderliggende selle beskerm. Die resultate wat hier voorgelê word, ondersteun verskeie hipoteses wat die verhoogde weerstandbiedendheid van biofilms teen antimikrobiese middels beskryf. Toekomstige werk sluit ondersoeke in na veranderende patrone van geenuitdrukking wat plaasvind wanneer 'n bakterie in aanraking kom met 'n oppervlak, vasheg en ook weer vrygestel word, asook die invloed hiervan op die vermoë om antimikrobiese behandeling te oorleef.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Barley malt is the predominant raw material for beer brewing world-wide. To meet consumer demand, a constant high quality malt product is required. Malt quality is determined by the degree of substrate hydrolysis during germination and mashing which serves as fermentable substrates for alcoholic fermentation during brewing. It is often difficult to sustain malt of high quality due to inconsistent malt batches and poor germination capacities of dormant barley. External additives such as chemicals and gibberellic acid have been used to overcome these difficulties but are unwanted in the beverage industry. Maltsters are consequently always in search of alternative solutions. Microbes produce diverse enzymes which can contribute to substrate hydrolysis during germination. The development of such starter cultures might provide a natural and economically feasible alternative to augment barley germination. Starter culture technology has been employed in the malting industry, although the main focus has been to improve the microbial stability of malt. The exploitation of cultures with hydrolytic capabilities to augment barley germination is consequently largely unexplored. The aim of this study was to develop a starter culture which can contribute to the enzymatic degradation of barley polymers. Geotrichum spp. and Lactobacillus plantarum were isolated from substrates rich in polymers present in barley and screened for enzymatic capabilities. Geotrichum spp. produced cellulase, xylanase, protease and β-glucanase activities, while L. plantarum harboured cell-bound and extracellular α-amylase activities. These cultures were added in different combinations during the malting of Erica and SSG 564 cultivars, but did not enhance germination significantly. Improved malt parameters did not correlate with microbial enzyme activities and the data were not repeatable. Preliminary plate assays could thus not be used to predict enzyme production in a malting environment. Cell-free supernatants with known enzyme activities of Aspergillus sp., Trichoderma reesei and Rhizopus sp. significantly enhanced malt quality. To our knowledge, the use of fungal supernatant to augment malt modification is a novel concept. Supernatant is more convenient than starter cultures and will aid to deliver more constant malt products than live cultures, as known enzyme levels are added. / AFRIKAANSE OPSOMMING: Garsmout is wêreldwyd die oorheersende roumateriaal vir bier brou. Om die aanvraag van verbruikers te bevredig, word 'n konstante hoë gehalte mout produk vereis. Die kwailiteit van mout word bepaal deur die graad van substraathidrolise gedurende ontkieming, wat dien as fermenteerbare substraat vir alkoholiese fermentasie tydens verbrouing. Dit is dikwels moeilik om ʼn konstante, hoë gehalte, moutproduk te lewer as gevolg van variasie in mout en die swak ontkiemingsvermoë van dormante gars. Hierdie probleem kan oorbrug word met eksterne toevoegings soos chemikalieë en gibberelliensuur, maar dit is nie ʼn gewensde praktyk in die broubedryf nie. Vermouters is gevolglik gedurig op soek na alternatiewe oplossings. Mikroörganismes produseer diverse ensieme wat kan bydra tot substraathidrolise gedurende ontkieming. Die ontwikkeling van sodanige suurselkulture is moontlik 'n natuurlike en ekonomies praktiese alternatief om die ontkieming van gars te stimuleer. Suurselkulture is reeds in die moutindustrie gebruik, alhoewel die fokus hoofsaaklik was om die mikrobiese stabiliteit van mout te verbeter. Die konsep om kulture met hidrolitiese vermoëns te gebruik om garsontkieming aan te vul is gevolglik grootliks onverken. Die doel van hierdie studie was om 'n suurselkultuur te ontwikkel wat kan bydra tot 'n ensiematiese afbraak van die polimere in gars. Geotrichum spp. en Lactobacillus plantarum is uit substrate ryk aan polimere teenwoordig in gars geïsoleer en vir hul ensiem aktiwiteite getoets. Geotrichum spp. het sellulase, xylanase, protease en β-glukanase aktiwiteit getoon, terwyl L. plantarum sel-gebonde en ekstrasellulêre α-amilase aktiwiteit getoon het. Hierdie kulture is in verskillende kombinasies tydens die vermouting van Erica en SSG 564 kultivars bygevoeg, maar het nie tot ʼn verbetering in die ontkieming van die gars gelei nie. Geen korrelasie is gevind tussen verbeterde mout parameters en mikrobiese ensiemaktiwiteit nie. Die resultate was ook nie herhaalbaar nie. Voorlopige plaattoetse kan dus nie as 'n maatstaf gebruik word om ensiem produksie deur suurselkulture in vermounting te voorspel nie. Sel-vrye supernatante van Aspergillus sp., Trichoderma reesei en Rhizopus sp., met bekende ensiem aktiwiteit, het die gehalte van mout aansienlik verbeter. Sover ons kennis strek is die gebruik van supernatante van fungi om die ontkieming van gars te stimuleer ʼn nuwe konsep. Supernatant is meer gerieflik as suurselkulture en sal help om konstante mout produkte te lewer aangesien ensiemvlakke beter beheer kan word.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Humanity is currently dependant on fossil fuels as an energy source. Increasing economic development and industrialization is, however, raising the demand for this unsustainable energy source. This increased pressure on dwindling reserves and growing concern over detrimental environmental effects associated with the use of these fuels have sparked great interest in the development of alternative sources. Bioethanol has surfaced as a good alternative to fossil fuels, as it can be produced from cheap, abundant, renewable, non-food sources. Bioethanol is also carbonneutral, i.e. utilisation thereof leaves the net level of carbon dioxide in the atmosphere unperturbed. Lignocellulose, more specifically its cellulose fraction, has been identified as a possible feedstock for the production of bioethanol. The use of lignocellulose as feedstock will allow for a more sustainable supply and much needed energy security. Lignocellulosic feedstocks can be divided into two main categories, i.e. wastes from processes other than fuel production and crops grown specifically for fuel production. Cereal crops such as triticale have been identified as good industrial crops for the production of energy. Triticale’s higher biomass yield, moderate water and nutrient requirements, steadily increasing area of cultivation and main use as an animal feed and not a human food source, makes it attractive as feedstock for the production of bioethanol. The combined activity of endoglucanases, exoglucanases and β-glucosidases is needed to hydrolyse crystalline cellulose to fermentable sugars. The high cost of these enzymes is, however, the most significant barrier to the economical production of bioethanol from cellulosic biomass. A promising strategy for a reduction in costs is the production of these cellulolytic enzymes, hydrolysis of biomass and fermentation of the resulting sugars to bioethanol in a single process step via a cellulolytic microorganism. The development of such a consolidated bioprocessing (CBP) organism can be achieved by the introduction of cellulolytic activity into a noncellulolytic microorganism that is able to ferment glucose to ethanol. Saccharomyces cerevisiae is a good host candidate for CBP as this yeast’s high tolerance towards ethanol and its use in industrial applications has been established. The enzymatic activities of endoglucanases and exoglucanases are, however, inhibited by the build-up of cellobiose during the hydrolysis of cellulose. This effect may be alleviated with the introduction of a better functioning β-glucosidase into the system. β-Glucosidases hydrolyse cellobiose to glucose, alleviating the inhibition on the enzymatic activities of endoglucanases and exoglucanases. Despite advances in enzyme production systems and engineering enzymes currently in use for higher stability and activity, there is still a demand to expand the current collection of enzymes. Bioprospecting for novel cellulolytic enzymes focuses on specific environment, with high turnover rates of cellulosic material or extreme conditions, such as the composting process. These enzymes are becoming more attractive compared to their mesophillic counterparts due to their potential industrial applications and the fact that they represent the lower natural limits of protein stability. / AFRIKAANSE OPSOMMING: Die mensdom is hoofsaaklik van fossielbrandstowwe as 'n energiebron afhanklik. Toenemende ekonomiese ontwikkeling en industrialisasie verhoog egter die aanvraag na hierdie onvolhoubare energiebron. Druk op kwynende reserwes en groeiende kommer oor die nadelige gevolge vir die omgewing wat met die gebruik van hierdie brandstowwe gepaard gaan, het tot groot belangstelling in die ontwikkeling van alternatiewe bronne gelei. Bio-etanol is 'n goeie alternatief vir fossielbrandstowwe, want dit kan van goedkoop, vollop, hernubare nievoedselbronne geproduseer word. Bio-etanol is ook koolstof-neutraal; die gebruik daarvan laat die netto vlak van koolstofdioksied in die atmosfeer onverstoord. Lignosellulose, en meer spesifiek die sellulose fraksie, is as moontlike grondstof vir die vervaardiging van bio-etanol geïdentifiseer. Die gebruik van lignosellulose as grondstof sal meer volhoubare voorsiening en broodnodige energie-sekuriteit verseker. Sellulose grondstowwe kan in twee hoof kategorieë verdeel word, nl. Newe produkteafval van prosesse anders as brandstofproduksie en gewasse wat spesifiek vir brandstofproduksie gekweek word. Graangewasse soos korog is geïdentifiseer as 'n goeie industriële gewas vir die produksie van energie. Korog se hoër biomassa opbrengs, matige water en voedingstofvereistes, groeiende bewerkingsgebied en die gebruik as 'n veevoergewas eerder as 'n menslike voedselbron, maak dit aantreklik as 'n grondstof vir die vervaardiging van bio-etanol. Die gesamentlike aktiwiteit van endoglukanases, eksoglukanases en β-glukosidases is nodig om kristallyne sellulose tot fermenteerbare suikers te hidroliseer. Die hoë koste van hierdie ensieme is egter die grootste hindernis vir die ekonomiese produksie van bio-etanol vanaf sellulosiese biomassa. 'n Belowende koste verminderingstrategie is die produksie van hierdie sellulolitiese ensieme, die hidrolise van biomassa, en die fermentasie van die suikers na bio-etanol in 'n enkelstap-proses via 'n sellulolitiese mikro-organisme. Die ontwikkeling van so 'n gekonsolideerde bioprosesserings (CBP) organisme kan deur die uitdrukking van sellulolitiese aktiwiteite in 'n nie-sellulolitiese mikro-organisme wat wel in staat is om glukose na etanol om te fermenteer, gerealiseer word. Saccharomyces cerevisiae is 'n goeie kandidaat gasheer vir CBP, omdat hierdie gis ‘n hoë verdraagsaamheid teenoor etanol toon en sy gebruik in industriële toepassings gevestig is. Die ensiematiese aktiwiteite van endoglukanases en eksoglukanases word egter deur die ophoop van sellobiose gedurende die hidrolise van sellulose geïnhibeer. Hierdie effek kan met die byvoeging van meer effektiewe β-glukosidases verlig word. β-Glukosidases hidroliseer sellobiose na glukose en verlig dus die inhibisie op die endoglukanase en eksoglukanase ensiematiese aktiwiteite. Ten spyte van vooruitgang in ensiemproduksie stelsels en ensiemmodifiserings strategieë wat tans vir hoër stabiliteit en aktiwiteit in gebruik is, bestaan daar steeds 'n behoefte om die bestaande versameling van ensieme uit te brei. Bioprospektering vir nuwe sellulolitiese ensieme fokus op spesifieke omgewings, met hoë omsetkoerse van sellulose materiaal of omgewings met uiterste toestande, soos die komposterings-proses. Hierdie ensieme is besig om meer aantreklik in vergelyking met hul mesofieliese eweknieë te raak as gevolg van hul potensiele industriële toepassings en die feit dat hulle die laer natuurlike grense van proteïen-stabiliteit verteenwoordig. / Stellenbosch University and the Technology Innovation Agency for financial support

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Domestic rainwater harvesting (DRWH) refers to the collection and storage of rainwater for domestic purposes and in an effort to achieve the Millennium Development Goals (MGD), the South African government has started an initiative where DRWH tanks are financed in sustainable housing schemes in an aim to provide an additional water source directly to households. Although many provinces, including parts of the Eastern Cape and KwaZulu Natal, have been using harvested rainwater as a potable water source, there are a limited number of studies indicating the quality of harvested rainwater in South Africa. However, many studies, internationally, have indicated that while the practice of harvesting rainwater is gaining popularity, rainwater quality is not within potable standards (Chapter 1). During the first phase of the study, rainwater samples were collected from the Kleinmond Housing Scheme (Western Cape, South Africa). From a cluster of 411 houses, the DRWH tanks connected to 29 houses were selected for monitoring the microbial and physico-chemical properties of harvested rainwater. Drinking water guidelines stipulated by SANS 241 (2005), DWAF (1996), ADWG (NHMRC and NRMMC, 2011) and WHO (2011) were used throughout the study to monitor the quality of rainwater. Eight sampling sessions were then conducted from March to August 2012, during a high and low rainfall period. Overall, the physico-chemical parameters of the rainwater samples were within the respective drinking water guidelines. However, the microbiological analysis verified results obtained in international studies, and showed that the indicator bacteria numbers present in the DRWH samples exceeded the stipulated guidelines (Chapter 2 and 3). Species specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae and ipaH found in Enteroaggregative E. coli (EAEC), Enterohaemorrhagic E. coli (EHEC), Enteropathogenic E. coli (EPEC) and Enteroinvasive E. coli (EIEC), respectively, in the rainwater samples. The virulent pathogenic E. coli genes were then detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected routinely during the sampling period from ten DRWH tanks (Chapter 3). Bacterial isolates selected during the high rainfall period (June to August 2012), as well as PCR assays performed on total genomic DNA extraction from the rainwater samples, confirmed the presence of numerous pathogenic bacteria including Legionella spp. Klebsiella spp. and Shigella spp. Yersinia spp. were also isolated and detected for the first time in DRWH tanks (Chapter 4). Based on the results obtained in the first phase and as many studies have indicated the poor quality of rainwater, the second phase of the project was aimed at designing and monitoring point of use treatment systems. Three polyethylene DRWH tanks (2000 L) were installed at the Welgevallen Experimental farm, Stellenbosch University, South Africa. Various treatment systems, such as activated carbon and slow sand filtration, solar pasteurization and a combined activated carbon/PVA nanofibre filtration column, were then intermittently connected to the three DRWH tanks during the high rainfall period (June to October 2013). Results for slow sand filtration and activated carbon filters indicated that the biological layer that had developed on the filtration media had not matured and for this reason chemical and microbial parameters were not reduced to within drinking water guidelines. A polyvinyl (alcohol) (PVA) nanofibre membrane without activated carbon in a column filtration system was analysed and results indicated that this system was also not effective in reducing the microbial numbers to within drinking water guidelines. Lastly, by utilising a PVA nanofibre membrane with activated carbon in a column filtration system, one litre of potable water was produced and all heterotrophic bacteria, E. coli and total coliform counts were reduced to zero and were within drinking water guidelines (Chapter 5). For the solar pasteurization system (Chapter 6), at treatment temperatures of greater than 72°C, all heterotrophic bacteria, E. coli and total coliforms were reduced to zero and were within drinking water guidelines. However, PCR assays confirmed the presence of Yersinia spp., Legionella spp., and Pseudomonas spp., at temperatures greater than 72°C. Results for chemical analysis also indicated all cations were within the international and national drinking water guidelines, with the exception of iron, aluminium, lead and nickel, which were detected in the pasteurized rainwater samples and were above the respective guidelines. It is hypothesised that these elements could have leached from the stainless steel storage tanks of the pasteurization system and it is therefore recommended that the storage tank of the pasteurization system be manufactured from an alternative material, such as a high grade polymeric material, which is able to withstand the high temperatures yet will not negatively influence the quality of harvested rainwater. / AFRIKAANSE OPSOMMING: Reënwater versameling vir huishoudelike gebruik verwys na die versameling en berging van reënwater vir huishoudelike doeleindes. In 'n poging om die Millennium Ontwikkelingsdoelwitte (MOD) te bereik het die Suid-Afrikaanse regering ‘n inisiatief begin om finansiële bystand aan huishoudings te verleen vir die implementering van reënwater tenke, in ‘n poging om ‘n addisionele water bron direk aan huishoudings te verskaf. Hoewel baie provinsies, insluitend dele van die Oos-Kaap en KwaZulu-Natal, reënwater gebruik as ‘n drinkbare water bron, is daar 'n beperkte aantal studies beskikbaar oor die gehalte van versamelde reënwater in Suid-Afrika. Baie studies internasionaal het egter al aangedui dat, alhoewel die praktyk van die versameling van reënwater besig is om in gewildheid toe te neem, die kwaliteit van reënwater nie binne drinkbare standaarde is nie (Hoofstuk 1). Tydens die eerste fase van die studie is reënwater monsters geneem van die Kleinmond Behuisings Skema (Wes Kaap, Suid Afrika). Van ‘n kompleks van 411 huise, is reënwater tenke van 29 huise geselekteer vir die monitering van die mikrobiese en fisiese-chemiese eienskappe van versamelde reënwater. Drinkwater riglyne soos gestipuleer deur SANS 241 (2005), DWAF (1996), AWDG (NHMRC en NRMMC, 2011) en WHO (2011) was regdeur die studie gebruik om die kwaliteit van reënwater te monitor. Reënwater monsters is tydens agt geleenthede geneem vanaf Maart tot Augustus 2012, tydens die hoë en lae rëenval periode. Oor die algemeen was die fisiese-chemiese parameters van die reënwater monsters binne die onderskeie riglyne vir drinkwater. Mikrobiese analises het egter die resultate van internasionale studies bevestig en het aangedui dat die getal indikator bakterieë teenwoordig in the reënwater die gestipuleerde riglyne oorskry (Hoofstuk 2 en 3). Spesies spesifieke inleiers was gebruik om die virulensie gene aggR, stx, eae en ipaH, teenwoordig in onderskeidelik Entero-aggregatiewe E. coli (EAEC), Entero- hemorragiese E. coli (EHEC), Entero-patogeniese E. coli (EPEC) and Entero-indringende E. coli (EIEC), in die reënwater monsters op te spoor. Die virulente pathogeniese E. coli gene was in 3% (EPEC en EHEC) en 16% (EAEC) van die 80 reënwater monsters, geneem van 10 reënwater tenke tydens die studie, waargeneem (Hoofstuk 3). Bakteriese isolate geselekteer tydens die hoë reenval periode (Junie tot Augustus 2012), so wel as PKR (polymerase ketting reaksie) ontledings uitgevoer op genomiese DNS wat van die reënwater monsters ge-ekstraheer was, het die teenwoordingheid van verskeie patogeniese bakterieë, insluitend Legionella spp., Klebsiella spp. en Shigella spp., bevestig. Dit is ook die eerste keer wat Yersinia spp. in versamelde reënwater waargeneem is (Hoofstuk 4). Die doel van die tweede fase van die projek was om water behandeling sisteme by die punt van gebuik te ontwerp en te monitor aangesien die resultate wat gedurende die eerste fase verkry is, sowel as vele ander studies, aangedui het dat die kwaliteit van versamelde reënwater swak is. Drie poli-etileen reënwater tenke (2000 L) was geïnstalleer op die Welgevallen Eksperimentele plaas van die Universiteit van Stellenbosch, Suid Afrika. Verskeie water behandeling sisteme, soos geaktiveerde koolstof en stadige sand filters, sonkrag pasteurisasie en ‘n gekombineerde geaktiveerde koolstof/PVA nanovesel filtrasie kolom, was met tye gekonnekteer aan verskeie reënwater tenke gedurende die hoë reënval periode (Junie to Oktober 2013). Resultate van die stadige sand en geaktiveerde koolstof filters het aangedui die biologiese laag nog nie ver genoeg ontwikkeld het nie en daarom was die chemiese en mikrobiese parameters nie verminder to binne die riglyne vir drinkwater nie. ‘n Polyviniel (alkohol) nanovesel membraan sonder geaktiveerde koolstof binne ‘n kolom filtrasie sisteem, was geanaliseer en resultate het aangedui dat hierdie sisteem ook nie die mikrobiese getalle verminder het tot drinkwater standaarde nie. Laastens, deur gebruik te maak van ‘n PVA nanovesel membraan met geaktiveerde koolstof in ‘n kolom filtrasie sisteem, was een liter drinkbare water geproduseer met alle heterotrofiese bakterieë, E. coli en totale koliform getalle verminder tot nul en was binne die riglyne vir drinkwater (Hoofstuk 5). Vir die sonkrag pasteurisasie sisteem (Hoofstuk 6), by behandelings temperature van groter as 72°C, was alle heterotrofiese bakterieë, E. coli en totale koliform getalle verminder tot nul en was binne die riglyne vir drinkwater. Maar PKR ontledings het die teenwoordigheid van Yersinia spp., Legionella spp., en Pseudomonas spp. by temperature groter as 72°C bevestig. Resultate van die chemiese analises het ook aangedui dat alle katione binne die drinkwater riglyne was, met die uitsondering van yster, aluminium, lood en nikkel. Hierdie elemente was in die gepasteuriseerde reënwater monsters waargeneem met konsentrasies wat die onderskeie riglyne oorskry het. Dit word gehipoteseer dat hierdie elemente uit die vlekvrye staal van die bergings tenk van die pasteurisasie sisteem kon uitgeloog het. Daarom word dit aanbeveel dat die bergings tenk van die pasteurisasie sisteem geproduseer moet word van ‘n alternatiewe materiaal, soos ‘n hoë graad polimeriese material, wat hoë temperature kan weerstaan maar nie ‘n negatiewe invloed sal hê op die kwaliteit van versamelde reënwater nie.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions. / NO AFRIKAANS SUMMARY AVAILABLE

Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Constructed wetlands have been widely used in the treatment of industrial and domestic wastewater to reduce biological and chemical oxygen demand (BOD and COD), to remove nitrate and enteric viruses as well as to generally improve water quality. Distillery wastewater has a complex character due to high concentrations of sugars, lignins, hemicellulose, dextrins, resins, polyphenols and organic acids, leading to a high COD that may exceed 100 000 mg/L. The potential application for the treatment of distillery wastewater by means of constructed wetlands is relatively unexplored. In 1999 a study was initiated at Distell Goudini distillery, Western Cape, South Africa, to explore the possibility of using constructed wetlands to treat distillery wastewater. It was found that constructed wetlands do have the ability to treat distillery wastewater providing that the influent COD does not exceed 15 000 mg/L for extended periods and the correct substrate material is used. The present study expanded on the above-mentioned study and specifically aimed to provide information on the microbiological controls in wetland systems in an applied sense that may contribute to improved treatment efficiency. Furthermore, this project aimed to contribute to our fundamental understanding of the microbial ecology of constructed wetlands used for the treatment of distillery wastewater. This study revealed that a highly dynamic microbial composition exists within wetlands. Furthermore it was found that wetlands can efficiently remove COD even though a low degree of similarity exists between microbial communities in various zones of the same wetland and those between different wetlands, as well as low similarity between communities sampled from the same zone over time. This demonstrates that it will be difficult to define the ‘ideal’ degradative community in terms of microbiological criteria and serves as a reminder that various indicators should be considered for monitoring system health. Furthermore the shifts in microbial community composition illustrate the ability of microbial communities to adapt to changes in the environment without compromising their functional efficacy. When studying the attached microbial communities within wetland systems it was found that different morphotypes are detected at certain stages of biofilm development while some organisms are present at most phases of biofilm formation. Measurement of CO2 production and dissolved organic carbon (DOC) removal in laboratory scale columns showed that grazing protists had a notable effect on overall microbial activity and that organic loading influenced these predator-prey interactions. Interestingly, increased clogging of pores occurred in the presence of protists, resulting in reduced flow through the porous matrix. Terminalrestriction fragment length polymorphism (T-RFLP) analysis of biofilms on gravel in experimental wetlands indicated that the presence of protists and algae had an effect on the microbial community composition. Scanning electron microscopy (SEM) showed that the presence of algae also had an influence on biofilm structure suggesting that the algae provided labile nutrients that were utilized by the bacterial and yeast members of the community. Finally, augmentation with a commercial mixture or microbial populations isolated from distillery effluent demonstrated that the concentration at which supplements are applied influence degradative efficiency. / AFRIKAANSE OPSOMMING: Kunsmatige vleilande word wêreldwyd gebruik in die behandeling van indusriële en huishoudelike afvalwater om biologiese en chemiese suurstof aanvraag (BSA en CSA) te verminder, om nitrate en ingewandsvirusse te verwyder asook om waterkwaliteit in die algemeen te verbeter. Distilleerafvalwater het komplekse eienskappe as gevolg van hoë konsentrasies suiker, lignien, hemisellulose, dekstrien, harpuis, polifenole en organiese sure, wat lei tot ‘n hoë CSA wat 100 000 mg/L kan oorskry. Daar is tot op hede relatief min studies gedoen oor die potensiële gebruik van kunsmatige vleilande vir die behandeling van distilleerafvalwater. In 1999 is ‘n studie by Distell Goudini distilleeraanleg in die Wes Kaap van Suid Afrika onderneem om die moontlikheid van kunsmatige vleilande vir die behandeling van distilleerafvalwater te bestudeer. Daar was bevind dat kunsmatige vleilande die vermoë het om distilleerafvalwater te behandel gegewe dat die invloeiende CSA nie 15 000 mg/L oorskry nie en dat die regte substraat materiaal gebruik word. Die huidige studie het by die bogenoemde studie aangesluit met die doel om informasie oor die mikrobiologiese kontroles in vleilandsisteme op ‘n toegepaste wyse te voorsien, wat tot verbeterde behandeling doeltreffendheid kan lei. Hierdie studie het verder beoog om by te dra tot ons fundementele kennis van die mikrobiese ekologie van kunsmatige vleilande wat gebruik word vir die behandeling van distilleerafvalwater. Dié studie het bevind dat daar ‘n hoogs dinamiese mikrobiese samestelling binne vleilande bestaan. Daar was verder bevind dat CSA steeds effektief deur vleilande verwyder kan word alhoewel daar ‘n lae graad van ooreenstemming is tussen mikrobiese gemeenskappe in verskeie sones van dieselfde vleiland en verskillende vleilande, asook ‘n lae graad van ooreenstemming tussen gemeenskappe wat in dieselfde sone oor tyd gemonster is. Dit demonstreer dat dit moeilik sal wees om die ‘ideale’ degraderende gemeenskap te vind in terme van mikrobiologiese kriteria en dien as ‘n herinnering dat verkeie indikatore in ag geneem moet word om die welstand van ‘n ekologiese sisteem te monitor. Die verskuiwings in mikrobiese gemeenskapsamestelling illustreer verder die vermoë van natuurlike sisteme om aan te pas by veranderinge in die omgewing sonder om funksionele doeltreffendheid te verminder. Die studie van aangehegte mikobiese gemeenskappe het aangedui dat veskillende morfotipes bespeur kan word tydens sekere fases van biofilm formasie terwyl sekere organismes tydens meeste van die fases teenwoordig is. Die bepaling van CO2 produksie en die verwydering van opgeloste organiese koolstof in laboratoriumskaal kolomme het geïlustreer dat voedende protiste ‘n waarneembare effek gehad op die algehele mikrobiese aktiwiteit en dat die organiese lading hierdie predator-prooi interaksie beïnvloed het. Dit was interessant om te vind dat die teenwoordigheid van protiste die verstopping van porieë aangehelp het en dus tot verlaagde vloei deur die poreuse matriks gelei het. Terminale-restriksie fragment lengte polimorfisme (T-RFLP) analiese van biolfilm op klipgruis in eksperimentele vleilande het aangedui dat die teenwoordigheid van protiste en alge ‘n effek gehad het op die mikrobiese gemeenskapsamestelling. Skandeerelektronmikroskopie (SEM) het bewys dat die teenwoordigheid van alge ook ‘n invloed op biofilm struktuur gehad het wat daarop dui dat alge maklik afbreekbare voedingstowwe aan die bakterieë en giste van die mikrobiese gemeenskap beskikbaar gestel het. Laastens was bewys dat die konsentrasie van toevoeging van ‘n kommersiële mikrobiese mengsel of mikrobiese populasies wat uit afvoer geïsoleer was, die effektiwiteit van degradering kan beïnvloed.

Dissertation (DPhil)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess. / AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is. Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe. Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en Afrika) te verminder. Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word, maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die alkoholbedryf gemaak. Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam, Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk, het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis. Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader: (i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk te wees. Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.

Dissertation (PhD)--Stellenbosch University, 2007. / PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology. / AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC 3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek of dit op te neem en intrasellulêr te benut. Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/ transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen 0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te fermenteer. Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1 ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium. Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende anaerobe geassosieer word. Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut. Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg word.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: This study strives to improve two current industrial processes by making them more cost effective through the use of hydrolytic enzymes or microbial systems. The first process targeted is the industrial conversion of starch to ethanol. In the second process, hydrolytic enzymes are applied to the manufacturing of instant coffee. The engineering of microbial systems to convert starch to bio-ethanol in a one-step process may result in large cost reductions in current industrial processes. These reductions will be due to decreased heating energy requirements, as well as a decrease in money spent on the purchase of commercial enzymes for liquefaction and saccharification. In this study, a recombinant Saccharomyces cerevisiae strain was engineered to express the wild-type Aspergillus awamori glucoamylase (GA I) and α-amylase (AMYL III) as well as the Aspergillus oryzae glucoamylase (GLAA) as separately secreted polypeptides. The recombinant strain that secreted functional GA I and AMYL III was able to utilise raw corn starch as carbon source, and converted raw corn starch into bio-ethanol at a specific production rate of 0.037 grams per gram dry weight cells per hour. The ethanol yield of 0.40 gram ethanol per gram available sugar from starch translated to 71% of the theoretical maximum from starch as substrate. A promising raw starch converter was therefore generated. In the second part of this study, soluble solid yields were increased by hydrolysing spent coffee ground, which is the waste generated by the existing coffee process, with hydrolytic enzymes. Recombinant enzymes secreted from engineered Aspergillus strains (β-mannanase, β-endoglucanase 1, β-endo-glucanase 2, and β-xylanase 2), enzymes secreted from wild-type organisms (β-mannanases) and commercial enzyme cocktails displaying the necessary activities (β-mannanase, cellulase, and pectinase) were applied to coffee spent ground to hydrolyse the residual 42% mannan and 51% cellulose in the substrate. Hydrolysis experiments indicated that an enzyme cocktail containing mainly β-mannanase increased soluble solids extracted substantially, and a soluble solid yield of 23% was determined using the optimised enzyme extraction process. Soluble solid yield increases during the manufacturing of instant coffee will result in; (i) an increase in overall yield of instant coffee product, (ii) a decrease in amount of coffee beans important for the production of the product, and (iii) a reduction in the amount of waste product generated by the process. / AFRIKAANSE OPSOMMING: Hierdie studie poog om twee huidige industriële prosesse te verbeter deur die prosesse meer kosteeffektief met behulp van hidroltiese ensieme en mikrobiese sisteme te maak. Die eerste industrie wat geteiken word, is die omskakeling van rou stysel na etanol, en die tweede om hidrolities ensieme in die vervaardiging van kitskoffie te gebruik. Die skep van mikrobiese sisteme om rou-stysel in ’n ’een-stap’ proses om te skakel na bio-etanol sal groot koste besparing tot gevolg hê. Hierdie besparings sal te wyte wees aan die afname in verhittingsenergie wat tydens die omskakelingsproses benodig word, asook ’n afname in die koste verbonde aan die aankoop van duur kommersiële ensieme om die stysel na fermenteerbare suikers af te breek. In hierdie studie is ’n rekombinante Saccharomyces cerevisiae-gis gegenereer wat die glukoamilase (GA I) and α-amilase (AMYL III) van Aspergillus awamori, asook die glukoamilase van Aspergillus oryzae (GLAA) as aparte polipeptide uit te druk. Die rekombinante gis wat die funksionele GA I en AMYL III uitgeskei het, was in staat om op die rou-stysel as koolstofbron te groei, en het roustysel na bio-etanol teen ’n spesifieke tempo van 0.037 gram per gram droë gewig biomassa per uur omgeskakel. Die etanolopbrengs van 0.40 gram per gram beskikbare suiker vanaf stysel was gelykstaande aan 71% van die teoretiese maksimum vanaf stysel as substraat. ’n Belowende gis wat roustysel kan omskakel na bio-etnaol was dus geskep. In die tweede deel van hierdie studie is die opbrengs in oplosbare vastestowwe vermeerder deur die koffie-afval wat tydens die huidige industrieële proses genereer word, met hidrolitiese ensieme te behandel. Rekombinante ensieme afkomstig vanaf Aspergillus-rasse (β-mannanase, β-endoglukanase 1, β-endo-glukanase 2 en β-xilanase 2), ensieme deur wilde-tipe organismes uitgeskei (β-mannanase), asook kommersiële ensiempreparate wat die nodige ensiemaktiwiteite getoon het (β-mannanase, sellulase en pektinase) is gebruik om die oorblywende 42% mannaan en 51% sellulose in koffie-afval te hidroliseer. Hidrolise eksperimente het getoon dat ’n ensiempreparaat wat hoofsaaklik mannanase bevat, die oplosbare vastestofopbrengs grootliks kan verbeter, met ’n verhoogde opbrengs van 23% tydens geöptimiseerde ensiembehandelings. ’n Verhoogde opbrengs in oplosbare vastestowwe tydens die vervaardiging van kitskoffie sal die volgende tot gevolg hê: (i) ’n toename in totale opbrengs van kitskoffie produk, (ii) ’n afname in die hoeveelheid koffiebone wat vir die produksie ingevoer moet word, en (iii) ’n afname in die hoeveelheid afval wat tydens die vervaardigingsproses produseer word.