Thesis (MSc)- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Wood is composed of three main components: cellulose, hemicellulose and lignin. Cellulose is the main structural polymer, whereas the function of lignin in plants is to impart rigidity to the cells, to waterproof the vascular system, and to protect the plant against pathogens. A group of microorganisms, called white-rot fungi, are able to selectively degrade the lignin and hemicellulose from wood leaving the cellulose virtually untouched. The most widely studied fungus of this group is the basidiomycete Phanerochaete chrysosporium, which has become a model organism in studies of lignin degradation. Lignin is a large, heterogenous and water insoluble polymer and therefore the enzymes needed to degrade it have to be extracellular and non-specific. There are a number of enzymes that are involved in the degradation of lignin, including lignin peroxidases, manganese dependent peroxidases and laccases. Laecases are blue copper oxidases that require molecular oxygen to function, whereas lignin peroxidases and manganese peroxidases are heme proteins that require hydrogen peroxide. Phanerochaete chrysosporium has all three of these enzymes, as well as a system for producing the hydrogen peroxide that is necessary for peroxidases to function. For both scientific and industrial purposes, it is important to obtain linkage maps of the positions of genes in the genome of an organism. Most fungi, including P. chrysosporium, lack easily identifiable phenotypical markers that can be used to map the position of genes relative to each other on the genome. Previous methods of mapping genes in P. chrysosporium involved auxotrophic mutants, radioactivity, or the use of hazardous chemicals. Here we describe an automated DNA-sequencing based mapping technique that eliminates many of the problems associated with previous techniques. Portions of the genes to be mapped were amplified from homokaryotic single basidiospore cultures using gene specific primers using the polymerase chain reaction (PCR) technique. The PCR products were sequenced to determine the segregation of alleles. Two previously mapped lignin peroxidases, lipA and lipC, were used to develop this method, and the results obtained corresponded to the known genetic linkage. A newly characterised 13-glucosidase encoding gene from P. chrysosporium was also mapped. Linkage was found between the 13-glucosidase gene and a histone (Hl) encoding gene. In P. chrysosporium the lignin peroxidase isozymes are encoded by a family of at least ten genes. Previous studies with P. chrysosporium BKM-F-1767 in defined media, wood and soil have shown differential expression of the lignin peroxidase isozymes. In this investigation the levels of expression of lignin peroxidases in P. chrysosporium ME446 cultures grown in nitrogen or carbon limited defined liquid media, as well as on aspen wood chips were determined by competitive reverse transcriptase polymerase chain reaction (RT-peR). These results were compared to those previously obtained from P. chrysosporium BKM-F-1767 to evaluate strain specific variation in the expression of lignin peroxidases. The results indicate that, although there were many similarities in the patterns of lignin peroxidase expression, there were also enough differences to conclude that there were strain specific variations in the temporal expression of the lignin peroxidases. To conclude, a fast and cost effective method for mapping genes in P. chrysosporium was developed. Also, we showed that strain specific variation in temporal expression of lignin peroxidases occurs. / AFRIKAANSE OPSOMMING: Hout bestaan uit drie hoof komponente nl. sellulose, hemisellulose en lignien. Sellulose is die hoof strukturele polimeer, terwyl die funksie van lignin in plante is om die selle te versterk, die vaskulêre sisteem waterdig te hou, en die plant teen patogene te beskerm. 'n Groep mikroërganisms, bekend as witvrotswamme, kan lignien en hemisellulose selektief uit die hout verwyder, terwyl die sellulosevesels oorbly. Vanuit hierdie groep swamme is die meeste navorsing op die basidiomiseet Phanerochaete chrysosporium gedoen Lignien is 'n groot, heterogene polimeer en is onoplosbaar in water. Die ensieme wat benodig word om lignien afte breek is daarom nie-spesifiek en kom ekstrasellulêr voor. 'n Aantal ensieme is by die afbraak van lignien betrokke, insluitend lignienperoksidase, mangaanperoksidase en lakkase. Lakkase is 'n blou koperoksidase wat suurstof benodig vir aktiwiteit. Lignienperoksidase en mangaanperoxidase is heemproteïene en benodig waterstofperoksied. Phanerochaete chrysosporium het al drie van hiedie ensieme, sowel as 'n sisteem wat waterstofperoksied produseer. Vir beide wetenskaplike en nywerheidsdoeleindes is koppelingskaarte wat die posisie van gene in die genoom van 'n organisme aandui noodsaaklik. Die meeste swamme, P. chrysosporium ingesluit, het geen fenotipiese merkers wat maklik van mekaar onderskei kan word nie, en dit is dus moeilik om 'n kaart van die ligging van gene op die genoom te bepaal. Vorige metodes om gene in P. chrysosporium te karteer het auksotrofiese mutante, radioaktiwiteit of gevaarlike chemikalieë gebruik. Ons beskryf 'n metode wat van automatiese DNA-volgordebepaling gebruik maak en wat baie van die tekortkominge van die ou metodes oorkom. Dele van die gene is met geen-spesifieke PKR-amplifikasie uit kulture van homokariotiese enkel basidiospore verkry en die DNA-volgorde is bepaal om die segregasie van die allele te ondersoek. Twee gene waarvoor 'n koppelingskaart alreeds uitgewerk is, fipA en lipt), was gebruik om hierdie metode te ontwikkel. Die resultate stem ooreen met die bekende genetiese koppeling tussen hierdie gene. 'n Geen wat onlangs in P. chrysosporium ontdek is, nl. I3-glucosidase, is ook met hierdie metode gekarteer. Koppeling is met 'n histoon (Hl) geen gevind. Die lignienperoksidase isoensieme in P. chrysosporium word deur 'n familie van ten minste tien gene gekodeer. Vorige navorsing met P. chrysosporium BKM-F-1767 in gedefineerde media, hout en grond het getoon dat 'n variasie in die uitdrukking van lignienperoxidase isoensieme voorkom. In hierdie ondersoek is 'n kultuur van P. chrysosporium ME446 in stikstof- of koolstof-beperkende vloeibare media opgegroei, as ook op aspen houtblokkies. Die vlak van uitdrukking van die lignienperoksidases is deur middel van die omgekeerde transkripsie polimerasekettingreaksie (RT-PKR) bepaal. Die resultate vir P. chrysosporium ME446 is vergelyk met vorige resultate van P. chrysosporium BKM-F-1767 om te bepaal of stamspesifieke variasies in die uitdrukking van lignienperoksidases voorkom. Daar is 'n aanduiding dat, alhoewel soortgelyke patrone in die vlakke van lignienperoksidase uitdrukking voorkom, daar ook noemenswaardige verskille is. Hieruit kan afgelui word dat stamverwante variasie van lignienperokisdase uitdrukking voorkom. Ten slotte, ons het 'n vinnige, goedkoop metode om die gene in P. chrysosporium te karteer ontwikkel. Ons het ook bewys dat stam-spesifieke variasie in die uitdrukking van die lignienperoxidase gene voorkom.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Microorganisms play an important role in the stability and maintenance of the ecosystem and in the condition of the soil. However, in their natural environment, microorganisms often experience changing and hostile conditions. They therefore need to be able to adapt physiologically and modify their micro-environment. Biofilm formation is one mechanism to establish favorable micro-environments. The extracellular polymeric substances (EPS) that are typically associated with biofilm formation may also have an impact on soil structure. The aim of this project was to evaluate the potential of microbial manipulation on EPS production and the possible impact thereof on soil structure in order to improve water retention. Specific objectives of this study included the screening of natural environments for EPS-producers, developing techniques to observe EPS production and accumulation in the pores between soil particles, measuring the effect of EPS production on soil water hydraulic gradient, as well as determining the fate and impact of EPS-producers when introduced to naturally-occurring soil microbial communities. Several environmental samples have been screened for EPS-producing microorganisms. Soil columns were then inoculated with these EPS-producers and the passage of 20 mlaliquots water through the columns measured at 3 or 4-day intervals. Microbes isolated from soil, through their EPS production capability proved to retain water more effectively than was the case for water-borne EPS-forming microbes. This phenomenon was further studied using flow cells, filled with soil and inoculated with the EPS-producers isolated from either soil or water. Fluorescence microscopy showed that the soil microbes produced EPS that clogged pores between sand particles more effectively. This clogging resulted in lowering the soil water hydraulic gradient. To evaluate the effect of EPS-producers on existing soil microbial communities, cell counts, Biolog™whole-community carbon utilization studies and T-RFLP (terminal-restriction fragment length polymorphism) analyses were performed. Shifts in the soil microbial community could not be readily seen by observing microbial numbers and T-RFLP-analysis, but was noticeable in carbon utilization patterns. / AFRIKAANSE OPSOMMING: Mikroorganismes speel 'n belangrike rol in die stabiliteit en instandhouding van die ekosisteem en in die kondisie van die grond. In hul natuurlike omgewing ervaar mikroorganismes dikwels veranderlike en ongunstige toestande. Mikroorganismes het dus nodig om hulself fisiologies aan te pas en verander hul mikro-omgewing daarvolgens. Biofilm-vorming is een meganisme om gunstige mikro-omgewings te skep. Die ekstrasellulêre polimeriese produkte (EPP) wat tydens biofilm-vorming gevorm word, mag ook 'n impak hê op die grondstruktuur. Die doel van hierdie projek was om die potensiaal van mikrobiese manipulasie op EPP-vorming te evalueer asook die moontlike impak daarvan op grondstruktuur wat sodoende waterretensie kon bevorder. Die spesifieke doelwitte van hierdie studie het ingesluit die isolasie van EPPproduseerders vanuit natuurlike omgewings, die ontwikkeling van verskeie tegnieke waarvolgens EPP-produksie en die akkumulasie daarvan in die porieë tussen gronddeeltjies bestudeer kon word, die effek van EPP-produksie op hidrouliese gradiënt van grondwater en om die lot en impak wat EPP-produseerders op natuurlike grondmikrobiese populasies te bepaal. Verskeie grond- en watermonsters was getoets vir die voorkoms van EPP-produserende mikroorganismes. Grondkolomme is geïnokuleer met EPP-produseerders en die vloei van 20 ml-volumes water deur die kolomme is gemeet met 3 of 4-dag intervalle. Grond-geïsoleerde mikrobes het beter waterretensie tot gevolg gehad as water- geïsoleerde mikrobes. Hierdie verskynsel was verder bestudeer deur die gebruik van vloeiselle, gevul met grond of sand en geïnokuleer met EPP-produseerders geïsoleer vanuit grond of water. Fluoressensie mikroskopie het aangetoon dat grondmikrobes EPP produseer wat die porieë tussen gronddeeltjies meer effektief verstop. Dié verstopping het gelei tot die verlaging van die grondwater se hidrouliese gradiënt wat bepaal is deur die gebruik van die konstante-vlak bepalingsmetode. Om die effek van EPP-produseerders op bestaande mikrobiese populasies te bepaal, is seltellings, Biolog™ heel-gemeenskap koolstofverbruik studies en T-RFLP (terminale-restriksie fragment-lengte polimorfisme) analises uitgevoer. Veranderinge in die mikrobiese populasie kon nie geredelik bloot deur die bepaling van mikrobiese getalle en T-RFLP-analise waargeneem word nie, maar wel met die koolstofverbruikspatrone.

Thesis (MSc)--Stellenbosch University, 1990. / ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will have to direct well-planned yeast strain-development programmes. However, the winemaker can only benefit from the extensive biochemical and molecular information of the yeast cell and the impressive arsenal of genetic techniques available, if the wine industry defines its requirements in genetic terms. The successful application of these genetic and recombinant deoxyribonucleic acid (DNA) techniques in breeding programmes depends on the availability of rapid and reliable techniques to differentiate between parental and hybrid strains. Ten strains of Saccharomyces cerevisiae used for commercial production of wine in South Africa, were characterised by electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA. Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95 and N97 were apparent in the number, position and intensity of the bands. Strains N93 and N181 originated from the same culture and, as expected, displayed the same characteristic protein, DNA restriction fragment and chromosomal banding patterns. Similar protein and DNA profiles were also obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain N96, cured of the K2 killer character. Results obtained by electrophoretic fingerprinting and karyotyping corresponded well, indicating that these techniques are valuable in the identification and quality control of industrial wine yeasts. The value of electrophoretic fingerprinting and karyotyping was also demonstrated in a breeding programme. The aim of this breeding programme was to obtain hybrids that combine the desired oenological characteristics of strains N76 and N96, and of strains N96 and N181. The protein banding patterns of hybrids USM21, USM22 and USM23 were identical and contained a combination of prominent unique bands present in the profiles of parental strains, N76 and N96H (N96H is a haploid derived from N96). The DNA restriction fragment profiles of hybrids USM21, USM22 and USM23 contained slight variations, whereas their profiles were quite different from those of their parental strains, N76 and N96H. The contour clamped homogeneous electric field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical but differed from those of their parental strains, N76 and N96H. The protein profiles of hybrid USM30 and its parental strains, N96H and N181, were similar, whereas their DNA restriction fragment banding patterns and CHEF karyotypes showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after mass spore-cell mating. These four killer hybrids contain desirable oenological properties long sought after by the South African wine industry. Fermentation trials and evaluation of these hybrids were conducted independently by the Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers' Winery and they have now been released for commercial wine production. / AFRIKAANSE OPSOMMING: Om mededingend in die handel te bly, sal die Suid-Afrikaanse wynbedryf weloorwoe gisras-ontwikkelingsprogramme moet loads. Die wynmaker sal egter slegs voordeel kan trek uit die omvattende biochemiese en molekul...Lre inligting oor die gissel en die indrukwekkende arsenaal van genetiese tegnieke wat beskikbaar is, indien die wynbedryf sy vereistes in genetiese terme definieer. Die suksesvolle toepassing van hierdie genetiese en rekombinante deoksiribonuklei"ensuur (DNA) tegnieke in telingsprogramme sal afhang van die beskikbaarheid van vinnige en betroubare tegnieke om tussen ouerlike en hibried-rasse te onderskei. Tien rasse van Saccharomyces cerevisiae wat vir kommersiele wynproduksie in Suid-Afrika gebruik word, is met behulp van elektroforetiese bandpatrone van totale oplosbare selprotei"ene, DNA-restriksiefragmente en chromosomale DNA gekarakteriseer. Variasies in die protei"en- en DNA-profiele van rasse N6, N21, N66, N76, N95 en N97 het geblyk uit die aantal, posisie en intensiteit van die bande. Rasse N93 en N181 het uit dieselfde kultuur ontstaan en het, soos verwag, dieselfde karakteristieke protei"en-, DNA-restriksiefragmenten chromosomale bandpatrone getoon. Soortgelyke protei"en en DNA profiele is ook vir killerras N96 en ras N91 verkry. Ras N91 is 'n variant van ras N96 wat die K2 killerkenmerk verloor het. Resultate wat met behulp van elektroforetiese vingermerking en kariotipering verkry is, het goed ooreengestem en dui daarop dat hierdie tegnieke waardevol is vir die identifisering en beheer van industriele giste. Die waarde van elektroforetiese vingermerking en kariotipering in telingsprogramme is ook gedemonstreer. Die doel van hierdie telingsprogram was om hibriede te kry waarin die gewenste kenmerke van rasse N76 en N96, en van rasse N96 en N181, gekombineer is. Die protei"en-bandpatrone van hibriede USM21, USM22 en USM23 was identies en het 'n kombinasie van prominente unieke bande, teenwoordig in die profiele van hul ourlike rasse, N76 en N96H (N96H is 'n haploi"de afstammeling van N96), bevat. Die DNArestriksiefragment- profiele van hibriede USM21, USM22 en USM23 toon geringe onderlinge verskille, maar hul profiele het wesenlik van die van hul ouerlike rasse, N76 en N96H, verskil. Die kontoergeklampde-homogene-elektriese-veld (CHEF) elektroforetiese kariotipes van hibriede USM21, USM22 en USM23 was identies, maar het verskil van die van hul ouerlike rasse, N76 en N96H. Die protei"enprofiele van hibried USM30 en sy ouerlike rasse, N96H en N181, was soortgelyk, terwyl hul DNA-restriksiefragment-bandpatrone en CHEF-kariotipes diskrete verskille getoon het. Ten slotte is gevind dat prote'ien- en DNAvingermerkingstegnieke waardevol was in die seleksie van vier hibried-killerrasse na massa spoor-sel paring. Hierdie vier killerhibriede beskik oor gewenste wynkundige eienskappe waarna die Suid-Afrikaanse wynbedryf reeds lank soek. Fermentasie-proewe en evaluering is onafhanklik deur die Departement Wynkunde, Universitiet van Stellenbosch en deur Stellenbosch-Boerewynmakery gedoen en hulle is nou vir kommersiele wynproduksie vrygestel.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The ocean’s fish resources are declining mainly because of irresponsible exploitation. Fish is a vital source of protein for humans and growing world populations are threatening the sustainability of commercial fisheries. This has led to the rapid growth of aquaculture worldwide. In South Africa, aquaculture of both fresh and marine species is expanding and is now practised in all nine provinces of the country. One of the major problems in aquaculture is the economic losses as a result of diseases. Viruses, bacteria, fungi and parasites are well known to infect fish, with bacteria causing the majority of diseases. Antibiotics were commonly used to control diseases, however, due to their negative impact on the environment, the use of these agents is questioned. This has led to the search for probiotics as an alternative way to control bacterial diseases in aquaculture. Probiotics used in aquatic environments can be defined as live microbial supplements which have beneficial effects on the host by altering the microbial communities associated with the host and the immediate environment. Probiotics have a variety of different mechanisms of action, including competition with pathogens, production of beneficial compounds, enhancement of host immune response and antiviral effects. This study aimed to isolate potential probiotic bacteria from the gastrointestinal tract (GIT) of the South African abalone (Haliotis midae). Nine different bacterial species were isolated and identified as Corynebacterium variabilei, Staphylococcus carnosus, Staphylococcus equorum, Staphylococcus cohniii, Vibrio aestuarianus, Vibrio nigripulchritudo, Vibrio cyclitrophicus, Photobacterium leiognathi, and Paracoccus marcusii (Chapter 2). One of these isolates, P. marcusii (isolate 6.15), showed promising probiotic properties together with the potential to be used as a pigmentation source due to its production of the carotenoid astaxanthin. Aquatic animals are not able to synthesize astaxanthin and under aquaculture conditions do not come into contact with natural pigment sources. This results in dark grey meat which is unappealing for consumers. Therefore, astaxanthin is included in the feed of a variety of aquaculture species such as salmon, trout, red see bream and shrimp to give the meat a pink/orange colour. Astaxanthin also plays an important role in other essential biological functions of fish such as increasing the defence potential against oxidative stress and enhancing sexual maturity, embryo development, and egg survival. Mozambique tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), two important aquaculture species in South Africa, were used to evaluate the probiotic and pigmentation effect of P. marcusii (isolate 6.15). Fish feed was coated with freeze dried bacterial cells (107 CFU/kg feed) and administrated to tilapia and trout. Because tilapia cannot incorporate astaxanthin into their meat, no pigmentation effect of P. marcusii (isolate 6.15) was evaluated for this species. However, tilapia showed significant improvement in growth and immune parameters. Fish supplemented with P. marcusii (isolate 6.15) had a higher percentage increase in body weight and a better feed conversion ratio for the duration of the trial. Enhanced lysozyme activity in the blood serum of the fish was also seen (Chapter 3). In contrast, P. marcusii (isolate 6.15) did not have any probiotic or pigmentation effect on rainbow trout. A possible reason for this may be that the concentration of P. marcusii (isolate 6.15) added to the feed was too low. More probably, it is suspected that no pigmentation was observed due to the destruction of the astaxanthin before being ingested by the trout, because astaxanthin is a very unstable molecule. Furthermore, the GIT microbial communities of trout were investigated over the duration of the trial for the different treatments. No similarities in community structures were observed betwee the different treatments, however, bacterial communities in the GIT of fish sampled at the same time were very similar (Chapter 4). / AFRIKAANSE OPSOMMING: Die oseaan se vis hulpbronne is besig om af te neem as gevolg van die onverantwoordelike gebruik daarvan. Vis is ‘n belangrike bron van proteïene vir mense en die toenemende wêreld populasie bedreig die volhoubaarheid van kommersiële visserye. As gevolg hiervan is daar ‘n drastiese toename in die akwakultuur industrie wêreldwyd. Ook in Suid Afrika brei die akwakultuur van beide vars water en mariene vis spesies uit. Een van die grootste probleme in akwakultuur is ekonomiese verliese as gevolg van siektes wat veroorsaak word deur virusse, bakterieë, fungi en parasiete. Bakterieë veroorsaak die meerderheid van die siektes en antibiotika word algemeen gebruik vir die beheer van bakteriële siektes. Die gebruik van antibiotika word egter bevraagteken omdat dit verskeie negatiewe implikasies vir die omgewing inhou Daarom word probiotika oorweeg as ‘n alternatief tot antibiotika om bakteriële siektes te voorkom en te behandel. Probiotika wat in akwatiese omgewings toegedien word kan gedefinieer word as a lewende mikrobiese aanvulling wat ‘n positiewe effek op die gasheer het, deur die mikrobiese gemeenskappe geassosieer met die gasheer en die ommidellike omgewing te verander. Hierdie mikrobiese aanvulling verbeter die gesondheid van die visse deur verskeie meganismes wat insluit kompetisie met patogene, produksie van voordelige chemiese verbindings, verhoging van die gasheer se immuniteit en antivirale effekte. Die doel van hierdie studie was om potensiële probiotika te isoleer uit die spysverterings kanaal (SVK) van die Suid Afrikaanse perlemoen spesie, Haliotis midae. Tydens die studie is daar nege verskillende bakteriële spesies geïsoleer en geidentifiseer as stamme verteenwoordegend van Corynebacterium variabilei, Staphylococcus carnosus, Staphylococcus equorum, Staphylococcus cohniii, Vibrio aestuarianus, Vibrio nigripulchritudo, Vibrio cyclitrophicus, Photobacterium leiognathi en Paracoccus marcusii (Hoofstuk 2). Een van die isolate, P. marcusii, het belowende probiotika en potensiële pigmentering eienskappe getoon a.g.v. die produksie van die karotenoïed astazantien. Akwatiese diere is nie daartoe instaat om hierdie pigment te produseer nie en onder akwakultuur toestande kom die visse ook nie in kontak met natuurlike bronne van hierdie pigment nie. Dit lei daartoe dat die vleis van visspesies soos forel en salm grys word wat dit onaantreklik vir verbruikers maak. Daarom word astazantien bygevoeg by visvoer om sodoende ‘n pienk/oranje kleur te verseker.Daar benewens speel astazantien ook ‘n rol in belangrike biologiese funksies van visse. Dit sluit in die verhoging in beskerming teen oksidatiewe stres, bevordering van seksuele volwassenheid, embrio ontwikkeling en eier oorlewing. Twee belangrike akwakultuur spesies in Suid Afrika, Mosambiek tilapia (Oreochromis mossambicus) en reënboog forel (Oncorhynchus mykiss), was in hierdie studie gebruik. Die probiotiese en pigmentasie effek van P. marcusii op reënboog forel was gëevalueer terwyl slegs die probiotiese effek op tilapia geëvalueer weens die onvermoeë van tilapia om die pigment in hul vleis te inkorpereer. Visvoer korrels was omhul met gevriesdroogde bakteriële selle (107 CFU/kg kos) en vir die visse gevoer. Daar was ‘n duidelike verbetering in groei en immuun parameters van tilapia. Visse toegedien met P. marcusii het ‘n hoër persentasie vermeerdering in liggaamsgewig en ‘n beter voedsel omsettings verhouding gehad tydens die verloop van die proef in vergelyking met die kontroles (Hoofstuk 3). In kontras hiermee kon daar geen probiotiese of pigmenterings effekte waargeneem word by die reënboog forel nie. ‘n Moontlike rede hiervoor kon wees dat die konsentrasie van P. marcusii wat by die kos gevoeg is te laag was. Dit is egter ook moontlik dat die astazantien vernietig was voordat dit deur die forel opgeneem is aangesien astazantien ‘n baie onstabiele molekuul is. Verder het ons die impak van verskillende visvoer behandelings op die mikrobiese gemeenskappe in die spysverteringskanaal (SVK) van forel tydens die verloop van die proef bestudeer. Geen ooreenkomste in mikrobiese gemeenskap strukture in die forel SVK is waargeneem tussen die verskillende voer behandelings nie, maar daar is wel ooreenkomste gevind tussen die mikrobiese gemeenskappe van visse by spesifieke tyd intervalle (Hoofstuk 4).

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lignocellulose, which is composed of cellulose, hemicellulose and lignin, is the main structural component of plant cell walls. Xylan is the main structural component of hemicellulose. Xylan is a complex heteropolysaccharide and, therefore, requires numerous synergistically acting enzymes for its complete hydrolysis. The focus of this study was on xylanases, which is a main chain cleaving enzyme required for xylan hydrolysis. Xylanases have numerous industrial applications and are commonly used in the biofuels, pulp and paper, food, animal feed and textile industries. Of particular interest is the use of xylanases in the biofuels industry due to the depletion of fossil fuels. A major bottleneck is, however, the low yield and high cost of the enzymatic hydrolysis process. In this study, three different xylanase genes from Aspergillus fumigatus, isolated from a triticale compost heap, were cloned and expressed in Saccharomyces cerevisiae. This yeast is an attractive host for the expression of these heterologous proteins, since A. fumigatus is considered a human pathogen and would not be suited for large-scale enzyme production. The recombinant xylanases obtained in this study were functional after expression in the yeast host and yielded high levels of enzyme activity, ranging from 100 to 300 nkat/mg dry cell weight (DCW). Higher enzyme yields will reduce the overall cost of the enzymatic hydrolysis process, making these enzymes attractive to the biofuels industry. The recombinant xylanases obtained in this study were also free of other cellulases. This characteristic makes these enzymes attractive to the pulp and paper industry as cellulose fibres are required to remain intact. Two of the recombinant xylanases, F10 and F11, were relatively stable at a temperature of 50°C with pH optima at pH 6, while the recombinant xylanase G1 only maintained half of its activity at this temperature and displayed pH optimum at pH 5. No synergistic effect was observed between the recombinant xylanases in this study. Future studies could investigate the synergistic interaction between these recombinant xylanases and other accessory enzymes used for the degradation of xylan, such as the esterases. Xylan hydrolysis levels could increase significantly due to a synergistic effect, which would further reduce the overall cost of the lignocellulose enzyme hydrolysis process. / AFRIKAANSE OPSOMMING: Lignosellulose, saamgestel uit sellulose, hemisellulose en lignien, vorm die hoof strukturele bestanddeel van plantselwande. Xilaan is die hoof strukturele komponent van hemisellulose. Xilaan is ʼn komplekse hetero-polisakkaried en verskeie saamwerkende ensieme vir volledige hidroliese hiervan word benodig. Die fokus van hierdie studie is op xilinases, die hoof kettingbrekende-ensiem vir xilaan hidroliese. Xilinases het verskeie industriële toepassings onder meer in die biobrandstof-, papier en pulp-, voedsel-, dierevoeding- en tekstielindustrieë. Weens die uitputting van fossielbrandstofreserwes word xilinases in die biobrandstof industrie van groot waarde geag. Lae opbrengste en hoë kostes van die ensiemhidroliese proses bly egter ʼn knelpunt. In hierdie studie is drie verskillende xilinase gene vanuit ʼn tritikale komposhoop Aspergillus fumigatus isolaat gekloneer en in Saccharomyces cerevisiae uitgedruk. Gis is ʼn aanloklike gasheer vir die uitdrukking van hierdie heteroloë proteïne aangesien A. fumigatus as menspatogeen nie vir grootskaalse ensiemproduksie geskik is nie. Die rekombinante xilinases verkry in hierdie studie is funksioneel in die gis gasheer uitgedruk en hoë vlakke ensiemaktiwiteit is verkry, van 100 tot 300 nkat/mg droë sel massa (DSM). In die lig van hoër ensiemopbrengste wat die totale koste van die ensiem hidroliese proses verlaag, word die ensieme in hierdie studie aanloklik vir die biobrandstof industrie. Die rekombinante ensieme in hierdie studie verkry is ook vry van ander sellulases, ʼn eienskap wat van waarde is vir die papier en pulp industrie waar die sellulose vesels intak moet bly. Twee van die rekombinante xilinases, F10 en F11, was relatief stabiel by ʼn temperatuur van 50°C met ‘n pH optimum van pH 6, terwyl die rekombinante xilinase G1 slegs die helfte van sy aktiwitieit by hierdie temperatuur kon behou met ʼn pH optimum van pH 5. Geen samewerkende effek kon tussen die drie rekombinante xilinases waargeneem word nie. Toekomstige studies kan die samewerkende effek tussen hierdie rekombinante xilinases en bykomstige ensieme betrokke by xilaanafbraak, soos byvoorbeeld die esterases, ondersoek. Xilaanhidroliese vlakke kan aansienlik as gevolg van hierdie samewerkende effek verhoog, wat die koste van ensiem hidroliese van lignosellulose verder kan verlaag. / Stellenbosch University and the Technology Innovation Agency for financial support

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The argument of whether to use food for biofuel (bioethanol) production prompted the search for an alternative non-food biomass, such as lignocellulose, as feedstock for bioethanol production. However, a hindrance in producing bioethanol from lignocellulose on an industrial scale is the cost associated with hydrolysing the lignocellulose to its respective sugar monomers. Improving enzyme production and enhancement of enzyme cocktails for efficient lignocellulose hydrolysis is, therefore, a necessary prerequisite. In this study, a yeast culture collection from the Wine and Fermentation Technology Division (ARC Infruitec- Nietvoorbij, Stellenbosch, South Africa), isolated from fruit from various regions in South Africa, was screened for β-glucosidase and β-xylosidase enzyme activities. β-glucosidases catalyse the hydrolysis of cellobiose and by doing so prevents end-product inhibition of cellobiohydrolases and endoglucanases during cellulose degradation. Similarly, β-xylosidases hydrolyse xylobiose and prevents end-product inhibition of endoxylanases during hemicellulose degradation. After initially screening 2180 non- Saccharomyces yeasts, two yeast isolates were selected that could potentially serve as enzyme source for lignocellulose hydrolysis; one as a producer of a β-glucosidase and another as a β-xylosidase producer. The yeasts were identified as a β-glucosidase producing Rhodotorula slooffiae-like yeast isolate 131B2 and a β-xylosidase producing Aureobasidium pullulans isolate 23B25, respectively. The production of β-glucosidase by Rhodotorula slooffiae-like yeast isolate 131B2 and of β-xylosidase by Aureobasidium pullulans isolate 23B25 was optimised using response surface methodology according to a central composite design. Subsequently, the crude and partially purified enzymes were characterised based on molecular mass, pH optima and stability, temperature optima and stability and inhibition by lignocellulose hydrolysis end-products, such as glucose, xylose and ethanol. The crude β-glucosidase from Rhodotorula slooffiae-like yeast isolate 131B2 was also compared to the commercial Aspergillus niger βglucosidase preparation (Novozyme 188) based on the characteristics mentioned above and as βglucosidase supplement during Avicel (microcrystalline cellulose) hydrolysis by the commercial cellulase preparation (Celluclast). The crude β-xylosidase by Aureobasidium pullulans isolate 23B25 could not be compared to a commercial β-xylosidase as none was available at the time of the study. During the study, the crude β-glucosidase 131B2 and β-xylosidase 23B25 showed potential as lignocellulose hydrolytic enzymes. Attempts were made to obtain the β-glucosidase and β-xylosidase genes from the respective yeast isolates using PCR-based approaches and by constructing cDNA libraries. However, cloning the β-glucosidase and β-xylosidase genes using these methods proved after several attempts to be unsuccessful, although, during this section of the study valuable information was obtained about the obstacles involved with using these approaches when the desired gene sequence is unknown and novel. / AFRIKAANSE OPSOMMING: Die debat oor die toepaslikheid van voedsel vir bio-brandstofproduksie (bio-etanol), het daartoe gelei dat alternatiewe nie-voedsel grondstowwe, soos lignosellulose, as voermateriaal vir bio-ethanol ondersoek word. Die koste geassosieer met die hidrolise van lignosellulose na die onderskeie suiker monomere belemmer industriële-skaal toepassing van lignosellulose vir bio-etanolproduksie. Verbeterde ensiemproduksie en verhoogde doeltreffendheid van ensiemmengsels vir lignosellulose hidrolise is dus ‘n noodsaaklik voorvereiste. In hierdie studie is 'n giskultuurversameling geisoleer vanaf vrugte van verskillende streke in Suid-Afrika deur die Wyn en Fermentasie Tegnologie Afdeling (ARC Infruitec-Nietvoorbij, Stellenbosch, Suid-Afrika) vir β-glukosidase en β-xilosidase ensiemaktiwiteite gesif. β-glukosidases wat die hidrolise van sellobiose kataliseer voorkom eindprodukinhibisie van sellobiohidrolases en endoglukanases tydens sellulose afbraak. β-xilosidases, op hul beurt, hydroliseer xilobiose en voorkom eindprodukinhibisie van endoxilanases tydens hemisellulose afbraak. Na afloop van die aanvanklike sifting van 2180 nie-Saccharomyces giste, is twee giste wat potensiëel as 'n ensiembron vir lignosellulose hidrolise kan dien geselekteer; een vir β-glukosidase en ‘n ander vir β-xilosidase produksie. Die giste is as ʼn β-glukosidase-produserende Rhodotorula slooffiaeagtige gisras 131B2 en ʼn β-xilosidase-produserende Aureobasidium pullulans gisras 23B25 onderskeidelik geïdentifiseer. Die Rhodotorula slooffiae-agtige gisras 131B2 se produksie van β-glukosidase en die Aureobasidium pullulans gisras 23B25 produksie van β-xylosidase was geoptimiseer met behulp van “response surface methodology” volgens 'n “central composite design”. Daarna was die gedeeltelik-gesuiwerde kru-ensieme volgens molekulêre massa, pH optima en stabiliteit, temperatuur optima en stabiliteit, en inhibisie deur lignocelluloses hidrolise end-produkte soos glukose, xylose en etanol, gekarakteriseer. Die kru βglukosidase van die Rhodotorula slooffiae-agtige gisras 131B2 is ook met die kommersiële Aspergillus niger β-glukosidase (Novozyme 188) volgens die eienskappe vroeër genoem vergelyk en as β-glukosidase aanvulling tydens die kommersiële sellulase (Celluclast) se hidrolise van Avicel (mikrokristalline sellulose). Die kru β-xylosidase van die Aureobasidium pullulans gisras 23B25 kon nie vergelyk word met 'n kommersiële β-xylosidase nie, aangesien daar nie een beskikbaar was tydens die studie nie. Gedurende die studie het altwee, die kru β-glukosidase 131B2 en β-xylosidase 23B25, potensiaal getoon as lignosellulose hidrolitiese ensieme. Pogings was aangewend om die β-glukosidase en β-xilosidase gene vanuit die onderskeie gis isolate met behulp van PKR-gebaseerde tegnieke en die opstel van cDNA biblioteke te kloneer. Hierdie klonering strategieë was egter na verskeie pogings onsuksesvol, maar waardevolle inligting oor die struikelblokke betrokke by die gebruik van hierdie benaderings wanneer die gewenste geen se DNS basispaarvolgorde onbekend en uniek is, was verkry.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aim of this study was to develop a ZnO nanowire-array biosensor that would detect immunoglobulins and record changes in the concentration of an antibody. Early detection of disease-causing agents is essential for an early response. In contrast to conventional methods, biosensors may detect disease-associated agents much faster and more accurate, which holds specific benefits to rural communities. The development of such a biosensor would be favourable for diagnostics in underprivileged communities without infrastructure. The hypothesis was that binding of antibodies to the surface of ZnO nanowires would result in the generation of a piezoelectric potential that, when channelled through a Schottky barrier, would produce a constant voltage reading. Piezoelectricty would be generated due to the bending of the nanowires, or tensile stress applied to the nanowires due to binding of the antibodies. The performance of such a device largely depends on the methods used to construct the ZnO nanowires and methods used to funtionalize the sensor surface. The biggest challenge was thus to chemically modify the self-assembled monolayers (SAMs) and create intermediate monolayers that would react to primary amino groups of lysozyme and form a covalent amide bond. Lysozyme was selected as model antigen, since its structure and reaction with antibodies has been well studied. Alkanethiol and dialkyl disulphides were used to form SAMs. Different SAMs were compared to select the absorbate that would bind the highest concentration of lysozyme. Lysozyme was best immobilized onto Au film layers in the presence of SAM 3-mercaptopropionic acid. Weakest immobilization was in the presence of combined SAM 11-mercaptoundecanoic acid/1-nonanethiol. The sensitivity of the constructed ZnO nanowire biosensor was tested in vitro, in the presence of different concentrations of lysozyme antibodies. An increase in the dimension of the ZnO seed layer led to an increase in the mean diameter of the ZnO seed grains, and subsequently an increase in the mean diameter of the synthesized ZnO nanowires. Deposition of the ZnO seed layer, using the RF cylindrical magnetron sputtering technique, improved the c-axis alignment of the nanowires and produced nanowires with similar dimensions. However, deposition of the ZnO seed layer using the sol-gel spin coating technique, produced nanowires with irregular c-axis alignments and irregular diameters. An increase in the Au film thickness led to a decrease in the mean diameter of the synthesized ZnO nanowires and worsening of the c-axis alignment. In contrast to single crystalline Au (111) film layers, polycrystalline Au layers increased the mean diameter of the synthesized nanowires. The crystal orientation of the Au film layer had no effect on the c-axis alignment. Increased voltage readings were recorded with an increase in antibody binding, indicating that the ZnO nanosensor may be used to record changes in immunoglobulin levels. Antibody concentrations ranging from 10 ng/ml to 20 μg/ml were sensed. This is the first study showing that ZnO nanowires, conformed into piezoelectric transducers, may be used in the detection of antibodies. The current size of the chip with ZnO nanowires is approximately 1 cm², which is too big to incorporate into a compact monitoring device. Apart from the challenge to produce smaller nanowire-arrays, highly sensitive sensors and miniature amplifiers will have to be developed to increase the strength of the signals generated by the nanowires. The biosensor will also have to be optimised to detect a variety of immunoglobulins. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om ‘n ZnO nanodraad biosensor te ontwikkel wat immunoglobuliene kan opspoor en veranderinge in konsentrasies van die teenliggaampies sal reflekteer. Vroë deteksie van siekte veroorsaakende agente is belangrik vir n vroeg tydige respons. In teenstelling tot konvensionele metodes, kan biosensors siekte veroorsaakende agente vining en akkuraat opspoor, wat veral voordele vir gemeenskappe in landelike gebiede inhou. Die hipotese was dat binding van teenliggaampies aan die ZnO nanodrade ‘n piëzo-elektriese potensiaal sal skep, wat dan ‘n konstante leesbare spanningspotensiaal sal lewer nadat dit deur ‘n Schottky versperring gestuur is. Piëzo-elektrisiteit word gegenereer deur die buiging van die nanodrade, of deur spanning wat op die nanodrade geplaas word deur binding van die teenliggaampies. Die sukses van die ontwerp hang grootliks af van die metode wat gebruik word om die ZnO nanodrade te konstrueer en metodes wat gebruik word om die sensor oppervlak te funksionaliseer. Die grootste uitdaging was dus om die monolae wat outomaties saam groepeer (SAMs) chemies so te verander dat intermediêre monolae vorm wat aan primêre aminogroepe van lisosiem bind ten einde kovalente amied-bindings te vorm. Lisosiem is as model antigeen geselekteer omdat die struktuur en reaksie daarvan met teenliggaampies reeds goed bestudeer is. Alkaantiol en di-alkiel disulfied is gebruik om SAMs te vorm. ‘n Verskeidenheid SAMs is vergelyk ten einde die anker te selekteer waaraan die hoogste konsentrasie lisosiem sal bind. Lisosiem is die effektiefste aan Au film lae ge-immobiliseer in die teenwoordigheid van SAM 3-merkapto-propanoësuur. Die swakste immobilisasie is in die teenwoordigheid van kombineerde SAM 11-merkapto-dekanoësuur/1-nanotiol waargeneem. Die sensitiwiteit van die ZnO nanodrade is in vitro getoets, in die teenwoordigheid van verskillende konsentrasies van lisosiem teenliggaampies. ‘n Toename in die dimensie van die ZnO grondlaag het die gemiddelde deursnit van die ZnO grein verhoog en so ook die gemiddelde deursnit van die gesintetiseerde ZnO nanodrade. Toediening van die ZnO grondlaag deur gebruik te maak van die RF silindriese mikrogolf-verstuiwings tegniek het die orientasie van die c-aslyn van die nanodrade verbeter. Toediening met die sol-gel draai-bedekkings tegniek het ‘n onreëlmatige orientasie van die c-aslyn teweeg gebring, asook ‘n variasie in die afmetings van die nanodrade. ‘n Toename in die Au laag het ‘n afname in die gemiddelde afmetings van die nanodrade en ook ‘n onreelmatige oriëntasie van die c-aslyn veroorsaak. In teenstelling met enkel-kristallyne Au (111) het poli-kristallyne Au lagies ‘n toename in die gemiddelde deursnit van die nanodrade veroorsaak. Die kristal-oriëntasie van die Au laag het geen effek op die belyning van die nanodrade gehad nie. Die spanningspotensiaal het verhoog met ‘n toename in teenliggaampie binding. Hiervolgens kan die ZnO nanosensor gebruik word om veranderinge in immunoglobulien vlakke te monitor. Teenliggaampie konsentrasies wat wissel van 10 ng/ml tot 20 μg/ml is opgespoor. Hierdie is die eerste studie wat toon dat ZnO nanodrade, omskep tot piëzo-elektriese transduseerders, gebruik kan word in die opsporing van teenliggaampies. Die grootte van die skyfie met die ZnO nanodrade is tans ongeveer 1 cm² en is te groot om in ‘n kompakte biosensor in te bou. Benewens die uitdaging om kleiner nanodraad skyfies te ontwikkel, sal hoogs sensitiewe sensors en seinversterkers ontwikkel moet word om die sein afkomstig van die nanodrade te versterk. Die biosensor sal ook ge-optimiseer moet word om ‘n verskeidenheid immunoglobuliene op te spoor.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main vectors for the spread of antibiotic and heavy metal resistance genes in the environment. Functional and taxonomic characterization of novel plasmids is, therefore, central to our general understanding of plasmid biology and their contribution to microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring resistance to tetracycline were isolated from the opportunistic fish pathogens Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed the presence of 18 putative open reading frames (ORFs), of which the tetAR tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs with significant similarity to known sequences within the NCBI database. Putative functions were assigned to 10 of the ORFs based on their distant homology with proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to comprise the minimal region required for replication (minimal replicon) by means of deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were found to be essential for plasmid replication. Inactivation of ORF3 resulted in an increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7 days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown that ORF3 autoregulated its own promoter. These results, therefore, suggested that although ORF3 was not essential for replication, it may be involved in plasmid copy number regulation and control. Host range analysis indicated that p31T1 was able to replicate in two other members of the γ-proteobacteria group (Escherichia coli and Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus suggesting a limited host range. Furthermore, p31T1 was mobilized only at low frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability, host range and mobilization characteristics determined for p31T1 and that no other plasmid replication and mobilization systems with significant sequence similarity to these plasmids have yet been identified, it is likely that these two plasmids are the first representative members of a new family of plasmids found within aquacultureassociated Aeromonas species and which are involved in the spread of tetracycline resistance. / AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede (slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het. Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7- kb minimale replikon fragment wat benodig word vir replisering en is deur middel van delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie (5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem. Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding van tetrasiklien weerstandbiedendheid.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005. / Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Research on probiotics has increased over the past years, which led to commercialization of a number of probiotic supplements and functional foods. In vitro assays such as tolerance to acid and bile, adhesion to mucus and epithelial cells, antimicrobial activity and antibiotic resistance tests are performed to screen lactic acid bacteria for probiotic properties. Enterococcus mundtii ST4SA produces an antimicrobial peptide (peptide ST4SA) with activity against Gram-positive and Gram-negative bacteria. Lactobacillus plantarum 423 produces plantaricin 423, a typical class II bacteriocin, active against a number of Gram-positive bacteria. A gastro-intestinal model (GIM) simulating the gastro-intestinal tract (GIT) of infants, was developed to study the survival of E. mundtii ST4SA and L. plantarum 423 and evaluate them as possible probiotics. Growth of the two strains in the GIM was compared to the growth of commercially available probiotics. Infant milk formulations were used as growth medium. Changes in pH, the addition of bile salt and pancreatic juice, and intestinal flow rates were controlled by peristaltic pumps linked to a computer with specifically designed software. Strain ST4SA was sensitive to low pH and high concentrations of bile salts. Growth of strain ST4SA was repressed in the first part of the GIM, however, the cells recovered in the ileum. Strain 423 was also sensitive to acidic conditions. However, the cells withstood the presence of bile and pancreatin in the first part of the GIT. Neither of the two strains displayed bile salt hydrolase (BSH) activity. Both strains were resistant to amoxicillin, ampicillin, chloramphenicol, cefadroxil, roxithromycin, meloxicam, doxycycline, erythromycin, novobiocin, rifampicin, tetracyclin, bacitracin, oflaxacin and cephazolin, anti-inflammatory drugs Na+- diklofenak and ibuprofen, and painkillers codeine terprim hydrate aminobenzoic acid, metamizole aspirin and paracetamol. Strain 423 was resistant to ciprofloxacin. Genes encoding cytolysin, non-cytolysin β-hemolysin and cell aggregation substances were detected on the genome of strain ST4SA but they were not expressed. L. plantarum 423 does not contain genes encoding gelatinase, cell aggregation, enterococcus surface protein, hemolysin, non-cytolysin β- hemolysin and enterococcus endocarditis antigen. Both strains inhibited the growth of Listeria monocytogenes ScottA in the GIM. Survival of the strains improved when used in combination and compared well with the survival of commercially available probiotics. Adhesion to epithelial cells is an important prerequisite for bacterial colonization in the GIT. The adhesion of E. mundtii ST4SA and L. plantarum 423 was studied using Caco-2 (human colon carcinoma epithelial) cells. Both strains revealed good adhesion compared to other probiotic strains. No correlation was found between hydrophobicity, auto-aggregation and adhesion to Caco-2 cells. Antibiotics and anti-inflammatory medicaments had a negative effect on adhesion. Different combinations of proteins were involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. E. mundtii ST4SA, L. plantarum 423 and L. monocytogenes ScottA were stained with fluorescent dyes to visualize adhesion to Caco-2 cells. Adhesion of L. monocytogenes ScottA to Caco-2 cells was not reduced in the presence of strains ST4SA and 423. Cell-free culture supernatants of both strains inhibited the invasion of L. monocytogenes ScottA. The cell structure of Caco-2 cells changed in the presence of L. monocytogenes ScottA. Strains ST4SA and 423 protected Caco-2 cells from deforming. / AFRIKAANSE OPSOMMING: Navorsing op probiotika het die afgelope tyd drasties toegeneem en aanleiding gegee tot die kommersialisering van ‘n groot hoeveelheid probiotiese supplemente en funksionele voedselsoorte. In vitro studies, soos bv. weerstand teen suur en gal, vashegting aan mukus en epiteelselle, antimikrobiese aktiwiteit en weerstand teen antibiotika word uitgevoer om te bepaal of melksuurbakteriëe aan probiotiese standaarde voldoen. Enterococcus mundtii ST4SA produseer ’n peptied met antimikrobiese werking teen Grampositiewe en Gram-negatiewe bakteriëe. Lactobacillus plantarum 423 produseer ‘n tipiese klas II bakteriosien, plantarisien 423, met aktiwiteit teen sekere Gram-positiewe bakteriëe. ’n Gastro-intestinale model (GIM) wat die spysverteringskanaal (SVK) van babas simuleer, is ontwikkel om die oorlewing van E. mundtii ST4SA en L. plantarum 423 te bepaal en hul eienskappe met dié van kommersiële probiotiese stamme te vergelyk. Babamelk formules is as groeimedium gebruik. Verandering in pH, byvoeging van galsoute en pankreassappe, en intestinale vloei is met behulp van peristaltiese pompe gereguleer wat seine vanaf ‘n spesiaal ontwikkelde rekenaarprogram ontvang. E. mundtii ST4SA was sensitief vir lae pH en hoë galsoutkonsentrasies en groei is in die eerste deel van die GIM onderdruk. Selgetalle het wel in die ileum herstel. Stam 423 was ook sensitief vir lae pH, maar het die galsout- en pankreatienvlakke in die laer deel van die SVK weerstaan. Geen galsout-hidrolase aktiwiteit is by enige van die twee stamme gevind nie. Beide stamme het weerstand getoon teen amoksillien, ampisillien, chloramfenikol, cefadroksiel, roksitromisien, meloksikam, doksisiklien, eritromisien, novobiosien, rifampisien, tetrasiklien, basitrasien, oflaksasien, kefazolien, die anti-inflammatoriese medikamente Na+-diklofenak en ibuprofen, en die pynstillers kodeïenterprimhidraataminobensoësuur, metamisoolaspirien en parasetamol. L. plantarum 423 was bestand teen ciprofloksasien. Gene wat kodeer vir sitolisien, nie-sitolisien β-hemolisien III en sel-aggregasie is op die genoom van E. mundtii ST4SA gevind, maar word nie uitgedruk nie. L. plantarum 423 besit nie die gene wat vir gelatinase, selaggregasie substansies, enterokokkus selwandproteïen, hemolise, nie-sitolisien β-hemolisien en enterokokkus endokarditis antigeen kodeer nie. Albei stamme inhibeer die groei van Listeria monocytogenes ScottA in die GIM. Die twee stamme in kombinasie het tot beter oorlewing in die GIM gelei. Stamme ST4SA en 423 vergelyk goed met kommersieël beskikbare probiotika. Vashegting van probiotiese stamme aan epiteelselle is belangrik vir kolonisering in die SVK. Vashegting van E. mundtii ST4SA en L. plantarum 423 is bestudeer deur van Caco-2 (kolon epiteel) selle van die mens gebruik te maak. Die aanhegting van beide stamme aan Caco-2 selle het goed vergelyk met kommersieël beskikbare probiotiese stamme. Geen korrelasie is gevind tussen hidrofobisiteit, aggregasie en vashegting aan Caco-2 selle nie. Antibiotika en antiinflammatoriese medikamente het ‘n negatiewe effek op vashegting gehad. Verskillende kombinasies van proteïene is betrokke in die vashegting van E. mundtii ST4SA en L. plantarum 423 aan Caco-2 selle. E. mundtii ST4SA, L. plantarum 423 en L. monocytogenes ScottA is met fluoreserende kleurstowwe gemerk om vashegting aan Caco-2 selle te monitor. Vashegting van L. monocytogenes ScottA aan Caco-2 selle is nie deur die teenwoordigheid van stamme ST4SA en 423 beïnvloed nie. Sel-vrye kultuursupernatante van beide stamme het die binnedring van L. monocytogenes ScottA verhoed. Die selstruktuur van Caco-2 selle het in die teenwoordigheid van L. monocytogenes ScottA van vorm verander. E. mundtii ST4SA en L. plantarum 423 het die Caco-2 selle teen vervorming beskerm.