Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Members of the genus Sulfobacillus form an important part of the microbial consortia that are active in the biooxidation of sulphide ores in biomining processes, yet very little is known about these industrially important organisms. The study of sulfobacilli, and other biomining organisms, is hampered by the absence of effective gene cloning and inactivation systems. During this study, the groundwork was laid for the development of a genetic system for the genus Sulfobacillus. The plasmid diversity present in industrial and environmental isolates of sulfobacilli was assayed. Plasmids were plentiful in the assayed strains, providing the basis for development of cloning vectors for sulfobacilli. Plasmid DNA isolated from Sulfobacillus thermosulfidooxidans strain DSM 9293T was methylated at dam and dcm sites. Whether the methylase enzymes responsible for this methylation pattern form part of restriction-methylation systems or only play a regulatory role is unknown, but it does indicate the appropriate methylation state of DNA for the transformation of this strain. The DNA sequences of three plasmids originating from sulfobacilli were analysed and compared. There was no significant similarity between the three plasmid sequences, indicating diversity in plasmid genetic load and replication mechanisms. Plasmid pSulfBC1 was predicted to replicate via the rolling circle mechanism, while the replication mechanisms of pKara and pTHWX could not be predicted from sequence data. Two antibiotics, chloramphenicol and tetracyline, were found to be suitable for selection of Sulfobacillus transformants. E. coli – Sulfobacillus shuttle vectors were constructed using the Sulfobacillus plasmid, pKara, as the backbone with a Gram-positive chloramphenicol resistance marker and appropriate elements allowing replication in, and mobilization from, E. coli. These shuttle vectors were used in the evaluation of electroporation and conjugation as methods for the delivery of DNA to Sulfobacillus. Transformants of sulfobacilli could not be obtained by either transformation method, although some progress was made towards determining the optimal conditions for both methods. The most promising finding was that cells of E. coli and Sulfobacillus could be maintained on the same medium for a theoretically sufficient time to allow mating. It is likely that Sulfobacillus transconjugants can be obtained with the right combination of donor, mobilizable vector, selectable marker and treatment to neutralize restriction systems.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Lactobacillus plantarum JW3BZ and Lactobacillus fermentum JW15BZ isolated from boza, a Bulgarian cereal based fermented beverage, produce bacteriocins JW3BZ and JW15BZ active against a wide range of food spoilage and pathogenic bacteria. Strains JW3BZ and JW15BZ are resistant to low pH (pH 2.0–4.0). Both strains grow well in MRS broth with an initial pH ranging from 5.0 to 10.0. Strain JW3BZ displayed intrinsic resistance to bile salts. Strain JW15BZ, on the other hand, is sensitive to bile salts exceeding concentrations of 0.3% (w/v). Both strains are weakly hydrophobic and are resistant to a wide range of antibiotics, antiinflammatory drugs and painkillers. Strains JW3BZ and JW15BZ adhered at 4% to Caco-2 cells and they did not compete with Listeria monocytogenes Scott A for adhesion. A homologue of MapA, a gene known to play a role in adhesion, was detected in L. plantarum JW3BZ. Both strains have high auto- and co-aggregation properties. Bacteriocin JW15BZ was partially purified with ammonium sulfate, followed by separation on Sep-Pak C18 and reverse phase High Pressure Liquid Chromatography (HPLC). Two separate peaks with antimicrobial activity were recorded for bacteriocin JW15BZ, suggesting that it consists of at least two antimicrobial peptides. Lactobacillus plantarum JW3BZ contains genes homologous to plnE, plnF and plnI of the plnEFI operon that encode for two small cationic bacteriocin-like peptides with double-glycine-type leader peptides and its respective immunity proteins. The antimicrobial activity displayed by strain JW3BZ may thus be ascribed to the production of plantaricins E and F. Bacteriocin JW3BZ and JW15BZ displayed activity against herpes simplex virus (HSV-1) (EC50=200 μg/ml). Both strains were identified in boza after 7 days at storage at 4 oC and repressed the growth of Lactobacillus sakei DSM 20017, indicating that the bacteriocins are produced in situ. The sensory attributes of boza prepared with different starter cultures did not vary considerably, although statistical differences were observed for acidity and yeasty aroma. Encapsulation of strain JW3BZ and JW15BZ in 2% sodium alginate protected the cells from low pH (1.6) and 2.0% (w/v) bile. The rate at which cells were released from the matrix varied, depending on the conditions. Better survival of strains JW3BZ and JW15BZ encapsulated in 2% (w/v) alginate was observed during 9 h in a gastro-intestinal model. Highest release of cells was observed at conditions simulating colonic pH (pH 7.4), starting from 56-65% during the first 30 min, followed by 87%. Complete (100%) release was recorded after 2.5 h at these conditions. Strains JW3BZ and JW15BZ could be used as starter cultures in boza. The broad spectrum of antimicrobial activity of bacteriocins JW3BZ and JW15BZ is an added advantage, rendering the cells additional probiotic properties. Encapsulation of the cells in alginate gel increased their resistance to harsh environmental conditions and may be the ideal method to deliver viable cells in vivo.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The genus Fusarium contains members that are phytopathogens of a number of agricultural commodities causing severe diseases such as wilts and rots. Fusarium species also secrete mycotoxins that have devastating effects on humans and animals. The ability of Fusarium species to change their genetic makeup in response to their immediate environment allows these fungi to exist in diverse habitats. Due to the ubiquitous nature of Fusarium, it forms part of the fungal communities in both agricultural and native soils. Fynbos is the major vegetation type of the Cape Floristic Region (CFR), which is a region that is renowned for its high plant species diversity and endemism. In this study, the occurrence and distribution of Fusarium species in indigenous fynbos soils and associated plant debris is investigated. In addition, the phylogenetic relationships between Fusarium species occurring in this particular habitat are evaluated. Fusarium isolates were recovered from soils and associated plant debris, and identified based on morphological characteristics. The morphological identification of isolates was confirmed using Polymerase Chain Reaction (PCR) based restriction fragment length polymorphism (RFLP) analyses of the translation elongation factor 1 alpha (TEF-1α) and internal transcribed spacer (ITS) regions. Furthermore, phylogenetic relationships between Fusarium species were based on the TEF-1α, ITS and β-tubulin gene regions. One-hundred-and-twenty-two (122) Fusarium strains were isolated from the fynbos soils in the Cape Peninsula area (Western Cape). Based on both morphological and molecular identification, the most prevalent Fusarium species in the fynbos soils were F. oxysporum Schlecht. emend. Snyd. and Hans., F. solani (Martius) Appel and Wollenw. emend. Snyd. and Hans., F. equiseti (Corda) Sacc. and an undescribed Fusarium species. Fusarium oxysporum was the dominant species in fynbos soils and strains of this species displayed significant genetic variability. Some strains of both F. oxysporum and F. solani showed close phylogenetic affinities to formae speciales (strains pathogenic to specific plant hosts) in the phylogenetic analyses. However, no diseased plants were observed in and within the vicinity of our sampling sites. In the third chapter, the undescribed Fusarium strains are described as Fusarium peninsulae prov. nom. Morphologically these strains are characterized by falcate macroconidia produced from brown sporodochia. The macroconidia are pedicellate, falcate to curved with hooked apical cells. Also, this fungus produces apedicellate mesoconidia on polyphialides in the aerial mycelium and forms microconidia sparsely. Chlamydospores are formed abundantly on aerial mycelium and submerged hyphae. All these morphological characteristics closely relate this fungus to F. camptoceras species complex in Fusarium section Arthrosporiella. However, phylogenetic analysis based on the ITS sequences differentiate these strains from F. camptoceras and other related species in section Arthrosporiella. Considering the fact that both as phytopathogens and saprophytic fungi, Fusarium species secrete a variety of cell wall degrading enzymes such as cellulases and xylanases. These enzymes allow the fungi to degrade the plant cell wall components to obtain nutrients. In Fusarium, notably endoxylanases play a role in phytopathogenesis of these fungi. Endoxylanase enzymes from F. oxysporum f. sp. lycopersici, F. verticillioides and F. graminearum have been characterized. In this final chapter, the use of the endoxylanase encoding gene, as a molecular marker in phylogenetic analysis was evaluated using F. graminearum (Fg) clade species as model. Degenerated primers were designed and the endoxylanase region amplified by PCR, cloned and sequenced. PAUPgenerated neighbour-joining analysis of the endoxylanase (XYL) region enabled all species to be distinguished and was as informative as the analysis generated with UTPammonia ligase (URA), phosphate permase (PHO), reductase (RED) and trichothecene 3- О-acetyltransferase (TRI101). Furthermore, the results of the phylogenetic analysis of XYL showed better species resolution in comparison to the analysis of the structural genes (TEF-1α and histone H3). Overall, the results demonstrated that phylogenetic analysis of XYL combined with other functional genes (URA, PHO, RED and TRI101) clearly distinguished between the Fg clade species far better than the analysis of structural genes (TEF-1α and histone H3).

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.

Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: More than sixteen isolates of iron-oxidizing bacteria belonging to the genus Leptospirillum were included in this study, with the finding that they were clearly divisible into two major groups. Group I leptospirilla had mol% G+C ratios within the range 49-52%, three copies of rrn genes and based on 16S rRNA sequence data, clustered together with the Leptospirillum ferrooxidans type strain (DSM2705or LI5). Group II leptospirilla had mol% G+C ratios of 55-58%, two copies of rrn genes and based on 16S rRNA sequence form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present amongst the leptospirilla tested with two DNA-DNA hybridization similarity subgroups being found within group I. The two groups could also be distinguished based on the sizes of their 16S-23SrRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a new species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and carrying out restriction enzyme digests of the products. Several but not all isolates of the group II leptospirilla, but none from group I (L. ferrooxidans) were capable of growth at 45°C. Plasmid DNA was isolated from strain ATCC49879 (L. ferrooxidans). Restriction endonuclease mapping of what appeared to be about 60 kb of plasmid DNA, established that two plasmids of approximately 30.0 kb and 27.0 kb were present. These were named p49879.1 and p49879.2 respectively. Attempts to isolate the plasmids separately were not successful. Partial sequencing of the two plasmids was carried out and sequence analysis of p49879.1 and p49879.2 indicated that the plasmids shared regions of homology. Total plasmid DNA was DIG-labelled and used as a probe in Southern hybridization experiments with genomic DNA from all sixteen original leptospirilla isolates as the target DNA. All leptospirilla belonging to Group I gave a positive signal, little or no homology to Group II leptospirilla was obtained. The region of homology present in all L. ferrooxidans strains was localized to an area on plasmid p49879.2 showing high amino acid identity to a transposase/putative transposase of Methanosarcina acetivorans and plasmid CPl from Deinococcus radiodurans Rl respectively. Whether these regions of homology indicate that complete, functional transposons are present in all L. ferrooxidans isolates still remains to be determined. Preliminary sequence analysis of both plasmids resulted in the identification of regions with amino acid sequence identity to the TnpA and TnpR of the Tn2l-like transposon family, and the mobilization regions of IncQ-like plasmids (particularly that of pTFl from At. ferrooxidans). Another potentially interesting ORF was identified in p49879.2 with high amino acid sequence identity to an ArsR-like protein that belongs to a second atypical family of ArsR transcriptional regulators. Whether this protein is functional in the regulation of arsenic resistance genes has not yet been determined, nor have other arsenic resistance genes been identified. Future work includes further sequence analysis of these plasmids to better understand their contribution to the isolates in which they are found. / AFRIKAANSE OPSOMMING: Meer as sestien isolate van die yster-oksiderende bakterieë, wat aan die genus Leptospirillum behoort, is in die studie ingesluit en die resultate het getoon dat dié groep verder in twee hoof groepe verdeel kan word. Groep I het "n mol% G+C van tussen 49% en 52% gehad, sowel as drie kopieë van die ribosomale gene (rrn). Hiermeesaam het die 16SrRNA volgorde data getoon dat hierdie isolate groepeer saam met Leptospirillum ferrooxidans (DSM2705T en LI5). Groep II leptospirilla het "n mol% G+C van tussen 55% en 58% gehad sowel as twee kopieë van die rrn gene en saam met die 16SrRNA volgorde data het hierdie isolate "n aparte groep gevorm. Genoom DNA-DNA hibridisasie eksperimente het gewys dat daar drie subgroepe onder die Leptospirillum wat getoets was is, met twee naverwante groepe wat onder Groep I val. Daar kan ook tussen die twee hoof groepe onderskei word op grond van die grootte van hul 16S- 23SrRNA intergeniese gebiede. Ons stel dus hier voor dat die Groep II leptospirilla as "n nuwe spesie beskou word naamlik, Leptospirillum ferriphilum sp, nov. Die twee spesies kan maklik onderskei word deur die PKR amplifikasie produk van die 16SrRNA te verteer met restriksie ensieme. Vele, maar nie al van die Groep II isolate kan by 45°C groei nie, terwyl geen van die Groep I leptospirilla (L.ferrooxidans) kan nie. Plasmied DNA was geisoleer uit Leptospirillum ferrooxidans ATCC49879. Aanvanklike analise het gedui op die teenwoordigheid van een 60.0 kb plasmied. Verdere restriksie ensiem kartering het wel getoon dat hierdie, in teen deel, twee plasmiede van ongeveer 30.0 kb en 27.0 kb in grootte is: p49879.1 en p49879.2. Pogings om die twee plasmiede apart te isoleer was onsuksesvol. Totale plasmied DNA is gemerk met die Random primed DNA labelling kit (Roche diagnostics) en gebruik as peiler in Southern klad eksperimente met genoom DNA, van al sestien isolate, as teiken. Alle leptospirilla wat aan Groep I behoort het "n positiewe sein gegee terwyl geen sein teen Groep II DNA opgemerk was nie. Die area wat, tussen die plasmiede en Groep I homologie getoon het, is gelokaliseer tot "n area op plasmied p49879.2 wat hoë amino suur identiteit toon aan "n transposase geen van Methanosarcina acetivorans, en "n voorgestelde transposase geen op plasmied CPI van Deinococcus radiodurans Rl. Dit moet nog vasgestel word of hierdie area van homologie dui op die teenwoordigheid van "n volledige, funksionele transposon in alle L. ferrooxidans isolate. Gedeeltelike DNA volgorde bepalings van beide plasmiede het gelei tot die identifikasie van areas met hoë amino suur volgorde identiteit aan die TnpA en TnpR gene van die Tn21-tipe transposon familie, sowel as aan die mobilisasie gene van IncQsoortige plasmiede (veral die van pTFI uit Acidithiobacillus ferrooxidans). "n Oop lees raam van belang, wat op plasmied p49879.2 geidentifiseer was, het hoë amino suur volgorde identiteit aan "n ArsR-tipe geen getoon wat aan "n tweede atiepiese familie van ArsR transkripsionele reguleerders behoort. Op die stadium is dit nog onbekend of hierdie protein funksioneel is in die regulering van arseen weerstandbiedenheidsgene.

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.