Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer on earth. Agricultural waste is rich in cellulose and exploiting these renewable sources as a substrate for ethanol production can assist in producing enough bioethanol as a cost-effective replacement for currently used decreasing fossil fuels. Saccharomyces cerevisiae is an excellent fermentative organism of hexoses; however the inability of the yeast to utilize cellulose as a carbon source is a major obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or cellobiose phosphorylase, the latter having a possible metabolic advantage over -glucosidase. Recently, it has been showed that S. cerevisiae is able to transport cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has the advantage that end-product inhibition of the extracellular cellulases by glucose and cellobiose is relieved. Furthermore, the extracellular glucose concentration remains low and the possibility of contamination is decreased. In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium was cloned and expressed under transcriptional control of the constitutive PGK1 promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The enzyme was expressed intracellulary and thus required the transport of cellobiose into the cell. The fur1 gene was disrupted for growth of the recombinant strain on complex media without the loss of the plasmid. The recombinant strain, S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram cellobiose consumed. The recombinant enzyme had activity optima of 60°C and pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69 and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant protein was localized in the membrane fraction and no activity was present in the intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher CepA activity than whole cells indicating that the transport of cellobiose was inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass formation. Cellobiose utilization increased when combined with other sugars (glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This is possibly due to more ATP being available for the cell for cellobiose transport. However, no cellobiose was utilized when grown with fructose indicating catabolite repression by this sugar. To our knowledge this is the first report of a heterologously expressed cellobiose phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report also reaffirms previous data that cellobiose can be utilized intracellularly in S. cerevisiae. / AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol. Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut, het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag gehou en die moontlikheid van kontaminasie beperk. In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en ‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S. cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers (glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose was nie benut nie. Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het. Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on plant surfaces. Similarly, on fallen trees an ecological succession of different fungal species is known to occur during wood degradation. Some of these fungi may be pioneer fungi contributing to the initial degradation process, while others may be yeasts associated with the fruiting bodies of macro-fungi which in turn are able to utilize the more recalcitrant polymers in wood. Previously, it was revealed that an increase occurs in the wood degradation rate of certain white-rot fungi when co-cultured with selected yeast species. A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus sanguineus. It was found by some that this fungus is capable of selective delignification while growing on the wood of poplar trees, while other authors found a simultaneous delignification pattern on Eucalyptus grandis trees. In the latter case cellulose and lignin are degraded simultaneously. We were interested in how yeasts occurring on the surface of P. sanguineus fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood degradation by this white-rot fungus. Restriction Fragment Length Polymorphisms (RFLP) analyses were used to obtain an indication of the species composition of the culturable yeast community associated with fruiting bodies of P. sanguineus. The impact of the most dominant of these yeasts species, i.e. Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood degradation by P. sanguineus was then determined by analyzing the major wood components after growth of co-cultures on hot water washed E. grandis wood chips. Co-cultures of P. sanguineus with the other fungi were prepared by inoculating the wood chips, contained in solid state bioreactors and supplemented with molasses and urea, with the an appropriate volume of fungal inoculum, resulting in an initial moisture content of 60%. After two weeks of incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp industry, were then employed to determine the percentage cellulose, Klason Lignin, as well as polar and solvent-borne extractives in the chips. The resulting data were analyzed using box plots, as well as biplots. No degradation of Klason lignin was observed, while the percentage cellulose did decrease during fungal degradation. Taking into account the inherent shortcomings of the Klason Lignin determination, the results supported the findings of others that P. sanguineus shows a simultaneous delignification pattern while growing on E. grandis wood. In addition, it was found that the yeasts played no significant role in the degradation ability of P. sanguineus, while A. flavipes showed an antagonistic effect on P. sanguineus with respect to cellulose degradation. However, it was clear that the analytical methods used in this study were inadequate to accurately determine fungal degradation of wood. In addition, it was obvious that the methods used did not distinguish between fungal biomass and wood components. Nevertheless, the methods provided us with a fingerprint of each culture growing on E. grandis wood, allowing us to compare the chemical composition of the different cultures and the un-inoculated hot water washed wood chips. The question, therefore, arose whether the effect of a particular coculture, on the chemical composition of wood, differs between tree species. Consequently, chemical alterations in different tree species, induced by a P. sanguineus / A. flavipes co-culture, were investigated in the next part of the study. Wood chips originating from four tree species, i.e. Acacia mearnsii, Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with this co-culture. The culture conditions and subsequent analyses of the wood components were the same as in the first part of the study. From the box- and biplots constructed from the resulting data, it was clear that the chemical composition of each tree species were altered in a different manner by the coculture. Lignin content showed an apparent increase in A. mearnsii, while E. dunnii showed a decrease in cellulose content. The results indicate that wood of different tree species are degraded in a different manner and this phenomenon should be taken into account in selecting fungi for biopulping. / AFRIKAANSE OPSOMMING: Dit is algemeen bekend dat 'n verskeidenheid fungi, insluitend giste, op plantoppervlaktes mag voorkom. Dit is ook bekend dat 'n ekologiese opeenvolging van verskillende fungusspesies tydens hout-afbraak op omgevalle bome voorkom. Van hierdie fungi mag pionierfungi wees wat bydra tot die aanvanklike afbraakproses, terwyl ander giste mag wees wat geassosieer word met die vrugliggame van makro-fungi, wat op hul beurt weer in staat is om die meer weerstandbiedende polimere in hout te benut. Dit is voorheen bekendgemaak dat daar 'n toename plaasvind in die tempo van houtafbraak deur sekere witvrot-fungi wanneer dit in ko-kulture met geselekteerde gisspesies voorkom. 'n Bekende bewoner van verrottende bome is die wit-vrotfungus Pycnoporus sanguineus. Dit is gevind dat hierdie fungus tot selektiewe delignifikasie in staat is terwyl dit op die hout van populierbome groei, terwyl ander outeurs 'n gelyktydige patroon van delignifisering op Eucalyptus grandis bome gevind het. In laasgenoemde geval is sellulose en lignien gelyktydig afgebreek. Ons was geïnteresseerd in die effek van giste op die oppervlak van vrugliggame van P. sanguineus, en die pionierfungus Aspergillus flavipes, op die houtafbraak deur hierdie wit-vrotfungus. Restriction Fragment Length Polymorphisms (RFLP) analises is gevolglik gebruik om 'n aanduiding te kry van die spesiesamestelling van die kweekbare gisgemeenskap wat met die vrugliggame van P. sanguineus geassosieer word. Die impak van die mees dominante van hierdie gisspesies, naamlik Pichia guilliermondii en Rhodotorula glutinis, asook A. flavipes, op houtafbraak deur P. sanguineus is voorts bepaal deur die analise van die belangrikste houtkomponente na die kweek van ko-kulture op E. grandis houtskyfies wat met warm water gewas is. Ko-kulture van P. sanguineus met die ander fungi is voorberei deur die houtskyfies in vaste fase bioreaktore, aangevul met melasse en ureum, te inokuleer met 'n toepaslike volume van die fungus inokulum om 'n aanvanklike voginhoud van 60% te verkry. Na twee weke se inkubasie by 30°C met konstante belugting is die skyfies ge-oes. Standaard protokol (TAPPI Standard Methods), algemeen deur die papier en pulpindustrie gebruik, is ingespan om die persentasie sellulose, Klason Lignien, asook polêre en oplosmiddel-gedraagde ekstrakte in die skyfies te bepaal. Die gevolglike data is geanaliseer deur gebruik te maak van box plots en biplots. Daar is geen afbraak van Klason Lignien bespeur nie, terwyl die persentasie sellulose wel toegeneem het tydens fungus degradasie. Met die inherente tekortkominge van die Klason Lignien bepaling inaggenome, het die resultate die bevindings ondersteun van andere wat getoon het dat P. sanguineus 'n gelyktydige delignifikasiepatroon openbaar terwyl dit op E. grandis hout groei. Daarby is dit gevind dat die giste geen beduidende rol in die afbraakvermoeë van P. sanguineus gespeel het nie, terwyl A. flavipes 'n antagonisiese effek ten opsigte van die sellulose degradering van P. sanguineus getoon het. Dit was egter duidelik dat die analitiese metodes wat in hierdie studie gebruik is, onvoldoende was om die degradering van hout akkuraat te bepaal. Daarby was dit duidelik dat die metodes nie tussen fungus biomassa en houtkomponente kon onderskei nie. Nogtans het die metodes 'n vingerafdruk verskaf van elke kultuur wat op E. grandis hout groei, wat ons toegelaat het om die chemiese samestelling van die verskillende kulture en die ongeïnokuleerde, met warm water gewasde houtskyfies te vergelyk. Die vraag het gevolglik ontstaan of die effek van 'n bepaalde ko-kultuur op die chemiese samestelling van hout van boomspesie tot boomspesie verskil. Gevolglik is die chemiese wisselinge in verskillende boomspesies, geïnduseer deur 'n P. sanguineus / A. flavipes ko-kultuur, in die volgende gedeelte van die studie ondersoek. Houtskyfies van vier boomspesies, naamlik Acacia mearnsii, Eucalyptus dunnii, E. grandis, en Eucalyptus macarthurii, is met hierdie ko-kultuur geïnokuleer. Die kultuurkondisies en daaropvolgende analises van die houtkomponente was dieselfde as in die eerste deel van die studie. Van die box- en biplots wat van die resultate getrek is, is dit duidelik dat die chemiese samestelling van elke boomspesie op 'n verskillende manier deur die ko-kulture verander is. Lignien-inhoud het ’n waarskynlike toename getoon in A. mearnsii, terwyl E. dunnii 'n afname in sellulose-inhoud getoon het. Die resultate toon dat hout van verskillende boomspesies op verskillende maniere afgebreek word en dat hierdie fenomeen in aanmerking geneem moet word wanneer fungi vir bioverpulping geselekteer word.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic, moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the same direction. An arsC (codes for an arsenate reductase), usually associated with ars operons, was absent from this ars operon. PCR and Southern-hybridization experiments revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located upstream of the arsRB operon. The intergenic region between the termination end of the kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp, suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As precursor gene in its native Sulfobacillus host. The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that processes involved in the production of functional proteins from the ars operon transcript were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to confer resistance to arsenic in the heterologous E. coli host. Eight Sulfobacillus strains isolated from different geographical areas were subjected to amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende, asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van ‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa) kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs 77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie. Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te verleen. Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al., 2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van ‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is. Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van restriksie ensiem herkenning setels, geïdentifiseer kan word.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host. / AFRIKAANSE OPSOMMING: ‘n Behoefte na ‘n koste-effektiewe en omgewingsvriendelike vervoer brandstof is besig om toe te neem. Lignosellulose word beskou as die volopste hernubare bron vir biobrandstof en lignosellulose bestaan uit sellulose, hemisellulose en lignien. Die gebruik daarvan vir die produksie van bio-etanol kan ’n voldoende alternatief vir fossielbrandstowwe bied. Verbruik van lignosellulose as bron vir die produksie van biobrandstof bied ’n oplossing vir die energie krises. Hemisellulose vorm ’n derde van lignosellulose substraat en bestaan uit xilaan en mannaan en word deur hemisellolitiese ensieme afgebreek wat algemeen by plantselwand-verterende organismes voorkom. Galaktoglukomannaan is die mees komplekse vorm van mannaan en benodig verskeie ensieme vir volkome hidroliese. Hierdie ensieme sluit in β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en galaktomanaan asetielesterases. Saccharomyces cerevisiae is ‘n bekende fermenterende organisme wat gereeld in verskeie industriële prosesse gebruik word en kan etanol van heksose suikers produseer. Die organisme beskik nie oor die vermoë om komplekse polisakkarides wat in lignosellulose voorkom te hidroliseer nie maar. DNS-manipuleringstegnieke en rekombinante tegnologie maak dit egter moontlik die probellm te oorbrug. S. cerevisiae het nogtans tekortkominge soos hiperglikosilering en daarom word ander nie-konvensionele giste (soos Kluyveromyces lactis) tans ook vir die produksie van rekombinante proteine ondersoek. Die mannanase geen (manI) vanaf Aspergillus aculeatus is in K. lactis GG799 en S. cerevisiae Y294 uitgedruk. K. lactis transformante was stabiel vir twee weke in opeenvolgende subkluture en het ‘n Man1 van 55 kDa geproduseer. Die rekombinante Man1 ensiem het ‘n temperatuur optimum van 70°C en pH optimum van 5.0 getoon in K. Lactis. Aktiwiteitsvlakke van 160 – 180 nkat/ml was bereik na 86 uur klutivering, In vergelyking met S. cerevisiae was aktiwiteitsvlakke eenders oor ‘n periode Die disrupsie van die ku80 geen het geen effek op die stabiliteit van die transformante in 10 dae opeenvolgende sub-kulture getoon nie. Die mannosidase geen (mndA) vanaf Aspergillus niger en die mannanase geen (man1) van Aspergillus aculeatus is konstitutief in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk. Uitdrukking van die MndA en Man1 proteïen in S. cerevisiae Y294 het onderskeidelik ‘n 140 kDa en 58 kDa spesie getoon met SDS-PAGE analisering. Die MndA ensiem het ‘n temperatuur optimum van 50°C and pH optimum van 5.0 getoon. Man1 het ‘n pH optimum van 6.0 en ‘n temperatuur optimum van 70°C getoon. MndA het lae hidrolitiese aktiwiteit op galaktoglukomannaan, maar geen β-mannosidase aktiwiteit getoon nie. Wanneer man1 and mndA saam in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk is, het die hidroliese van galaktoglukomannan dramaties afgeneem. ‘n Toename in die grootte van ‘n plasmied veroorsaak dikwels ‘n afname in kopiegetal wat die produksie van ManI verlaag. Die ko-uitdrukking van ManI en MndA kan ook tot ’n hoër metaboliese las lei en dus die laer produksie van ManI. Resultate in hierdie studie wys daarop dat meer navorsing benodig word in die soeke na alternatiewe gashere vir uitdrukking van mannanases. Ensiem mengsels vir industriële toepassings behoort eerder gebruik te word as die ko-ekspressie van verskeie ensieme in ’n enkel gasheer.

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

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

Thesis (MSc) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow spectrum of activity and are usually only active against bacteria from the same ecological niche. The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as natural food preservatives. The ideal would be to replace or reduce chemical preservatives such as sulfur dioxide, nitrates and nitrites. Bacteriocins are classified into four groups according to their structural and functional characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable, plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria monocytogenes. Production of bacteriocins may occur constitutively or may be regulated by a cell-density dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic growth, with no apparent change in production levels when the producer strain is cultured in the presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe that plantaricin 423 may be produced constitutively. A reporter system was constructed which consisted of the plantaricin 423 promoter, P423, fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B} Despite several repeats, no luciferase activity was recorded and no RNA homologous to the luxAB genes was detected. The region necessary for expression of plantaricin 423 may be located stream-up of the -80 region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins. Inclusion of the latter region in the reporter construct may result in the successful expression of luxAB. / AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met bakteriosiene. Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus cereus, Clostridium spp. en Listeria monocytogenes. Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids- afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423 moontlik konstitutief geproduseer word. 'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum (stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor word nie. Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot die suksesvolle uitdrukking van luxAB.

Thesis (MSc) -- University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Rooibos tea plant (Aspalathus linearis) is indigenous to South Africa and occurs only in the Western Cape's Cedarberg region. Rooibos tea is produced from the leaves and fine stems of the plant. The tea is normally prepared by brewing the leaves and consuming the liquor. However, the Rooibos plant is not only used to prepare tea; the plant extracts are also used in various neutraceutical and pharmaceutical products, including health drinks, iced tea, soaps and moisturising creams. Although the tea plant contains native enzymes responsible for the colour and aroma development of Rooibos tea, the disruption and maceration of the plant material during processing is insufficient to allow these enzymes proper access to the substrates responsible for Rooibos tea's characteristics. The current processing of Rooibos tea is also time consuming and is done under uncontrolled conditions, leading to unnecessary loss in aroma and antioxidant content. The addition of enzymes could improve the maceration of the plant material, shorten the processing time and improve the extraction of aroma, colour and antioxidant components. During this study, 16 commercially available microbial enzymes were evaluated on three different Rooibos substrates for the improvement of aroma and colour development, as well as the extraction of soluble solids (SS) and total polyphenols (TP). Thirteen enzymes were evaluated on spent tea for the enhanced extraction of soluble solids and to determine the best candidates for further evaluation on fermented and green Rooibos tea. Seven of the enzymes improved the yield in SS from spent tea. Up to 232% improvement was obtained, depending on the type of enzyme and dosage applied. The best six enzyme preparations were further evaluated on fermented Rooibos tea. For Depol™ 670L at 20 ul/g tea, the laboratory treatment increased the yield in SS by 44%, while small-scale industrial simulations increased the SS by 26%. However, an increase in the yield in SS was usually accompanied by a decrease in the %TP/SS ratio, indicating that mainly inactive compounds were extracted. Based on the results with the commercial enzymes, twelve "synthetic" enzyme cocktails, consisting of different combinations of commercial enzymes were designed, of which three cocktails released increased amounts of SS without decreasing the %TP/SS ratio significantly. Thirteen enzymes were evaluated on dried and freshly cut green Rooibos tea, with three enzymes (Depol™ 670L, Pectinex Ultra SP-L and Depol™ 692L) increasing the yield in SS between 21% and 66%, and the TP content between 11% and 47%. Laccase was the best candidate in improving colour development from green tea, with the improvement being slightly better at 50°C than at 40°C. All the "synthetic" cocktails containing laccase improved the colour extract of all three substrates evaluated, but also significantly decreased the TP and antioxidant content. However, lower dosages of laccase resulted in colour development with little loss in the antioxidant content. Due to the promising results obtained with the treatments of Rooibos tea with laccases, it was decided to clone and express the laccase gene (lacA) of Pleurotus ostreatus into Aspergillus niger. The gene was successfully transformed into A. niger, but the expression of the recombinant gene was not effective. / AFRIKAANSE OPSOMMING: Die Rooibostee plant (Aspalathus linearisi is inheems tot Suid-Afrika en kom slegs in die Sederberg-omgewing in die Wes-Kaap voor. Rooibostee word van die blare en fyn stingels van die plant geproduseer. Die tee word normaalweg voorberei deur die blare in kookwater te laat trek en dan die aftreksel te drink. Die Rooibos plant word nie net gebruik om tee te maak nie; die tee ekstrak word ook gebruik vir verskeie neutraseutiese en farmaseutiese produkte, insluitende gesondheidsdrankies, ystee, seep en bevogtigingsrome. Ten spyte daarvan dat die teeplant sy eie ensieme vir die kleur en aroma ontwikkeling van Rooibostee bevat, is die verbreking en maserasie van die plantmateriaal tydens prosessering onvoldoende om die ensieme genoeg toe gang tot die substrate verantwoordelik vir die kenmerkende eienskappe van Rooibostee te gee. Die huidige prosessering van Rooibostee is ook tydrowend en geskied onder onbeheerde toestande, wat tot 'n onnodige verlies in aroma en antioksidante lei. Die toevoeging van ensieme kan die afbraak van die plantmateriaal verbeter, die behandelingsproses verkort en die aroma, kleur en antioksidant inhoud van ekstrakte verbeter. Tydens hierdie studie is 16 kommersieel-beskikbare mikrobiese ensieme op drie verskillende Rooibos substrate vir die verbetering van aroma, kleur en ekstraksie van oplosbare vastestowwe (SS) en totale polifenole (TP) getoets. Dertien ensieme is op oorskot tee vir die verbeterde ekstraksie van oplosbare vastestowwe geevalueer, waama die beste kandidate vir evaluering op gefermenteerde en ongefermenteeede Rooibostee gekies is. Sewe ensieme het die SS vanaf oorskot tee verhoog. Tot 232% verhoging is waargeneem, afhangende van die tipe ensiem en die dosis wat gebruik is. Die beste ensiern preparate IS verder op gefermenteerde Rooibostee geevalueer, Labarotoriurn behandelings met Depol™ 670L teen 20 ul/g tee het die SS inhoud met 44% verhoog, terwyl die kleinskaalse industriele simulasie die SS inhoud met 26% verhoog het. 'n Verhoging in SS het egter gewoonlik met 'n afname in die %TP/SS verhouding gepaard gegaan, wat aandui dat hoofsaaklik onaktiewe stowwe vrygestel IS. Na aanleiding van die resultate met die kommersiele ensieme, is twaalf "sintetiese" ensiemmengsels met verskillende ensiemkombinasies getoets, waarvan drie mengsels ook meer SS vrygestel het met byna geen verlaging in die %TP/SS verhouding nie. Dertien ensieme was op gedroogde en vars gekerfde groen Rooibostee getoets met drie ensieme (Depol™ 670L, Pectinex Ultra SP-L en Depol™ 692L) wat die SS met tussen 21% en 66%, en die TP inhoud met tussen 11% en 47% verhoog het. Lakkase was die beste kandidaat vir die verbetering van kleur ontwikkeling by groen Rooibostee met die verbetering effens beter by 50°C as by 40°C. Al die "sintetiese" ensiem mengsels wat lakkase bevat het, het die kleur by al die verskillende substrate verbeter, maar het ook die TP en antioksidant inhoud aansienlik verlaag. Laer lakkase dosisse het goeie kleurontwikkeling tot gevolg gehad met minimale verlies in die antioksidant inhoud. Vanwee die goeie resultate wat met die lakkase behandelings verkry is, is daar besluit om die lakkase geen (lacA) van Pleurotus ostreatus te kloneer en in Aspergillus niger uit te druk. Die geen is suksesvol in A. niger getransformeer, maar die uitdrukking daarvan was nie effektief nie.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: A severely polluted industrial effluent is generated by the local gelatinmanufacturing industry. Due to increasingly stringent restrictions on discharge qualities enforced by the National Water Act of 1998 and National Environmental Management Act of 1998, as well as increasing trade-effluent charges implemented via the Local Municipal Bylaws, the industry is compelled to consider a system to pre-treat the polluted effluent. A study was undertaken to examine the viability of anaerobic treatment of the gelatin-manufacturing effluent, since the anaerobic digestion technology is well recognised for the high success rate in the treatment of high-strength, complex wastewaters. Various laboratory and pilot-scale studies were done, using different hybrid Upflow Anaerobic Sludge Blanket (UASB) and contact designs. Two mesophilic laboratory-scale hybrid UASB digester designs, fitted with polyethylene (AD-1) and polyurethane (AD-2), performed well at a hydraulic retention time (HRT) of 1.0 d. Chemical oxygen demand (COD) removal efficiencies of up to 90% (avg. 53%) for AD-1 and 83% (avg. 60%) for AD-2 at organic loading rates (OLR) of 9.56 and 4.62 kg COD.m-3.d-1, respectively, were obtained. High sulphate (S04) removal efficiencies of up to 96% (avg. 86%) for AD-1 and 98% (avg. 82%) for AD-2 were also achieved, respectively. A maximum total solid (TS) removal of 65% (avg. 25%) for AD-1 and 62% (avg. 28%) for AD-2 was reported. An average methane content of 80% (AD-1) and 79% (AD-2) with average methane yields per COD removed of 2.19 and 1.86 m3. kg CODremoved.df-o1r AD-1 and AD-2 were found, respectively. When the same digesters (AD-1 and AD-2) were combined in a muItiphase series configuration, a total COD removal efficiency of up to 97% (avg. 80%) at an OLR of 8.32 kg COD.m-3.d-1,was achieved. Excellent total S04 removals of 96% (avg. 69%) were accomplished. Up to 82% TS (avg. 29%) was also removed during this study and the biogas consisted of 89% methane (avg. 79%). For this multi-phase combination up to 92% volatile fatty acids (VFA) (avg. 48%) were removed, indicating possible selective phase separation of the respective fatty acid producing/utilising bacterial populations. The use of a laboratory-scale UASB bioreactor with recirculation, resulted in COD removal efficiencies of up to 96% (avg. 51%) at an HRT of 3.0 d, and 95% (avg. 54%) at a HRT of 1.0 d. Low performances were generally found, with average S04 and TS removals of 59% (max. 97%) and 26% (max. 67%), respectively at an HRT of 1.0 d. The biogas production was very low throughout the study (0.05 - 0.63 I,d-1 ). A pilot-scale UASB reactor (300 I) was constructed and performed satisfactory with a 58% average COD removal and maximum of 96%. S04 and TS removals up to 96% (avg. 44%) and 93% (avg. 63%), respectively, were obtained. The methane content of the biogas was 85%. The pilot-scale studies were conducted under actual field conditions, where various shock and organic loads had to be absorbed by the system. The pilot-scale contact configuration (300 I) did not perform satisfactory as a result of continuous blockages experienced in the feed and recirculation lines. Maximum COD, S04, VFA and TS removal efficiencies of 41% (avg. 27%), 62% (avg. 41%), 64% (avg. 27%) and 39% (avg. 21%), respectively, were obtained. The results of all the studies indicated acceptable COD removals with increasing OLR's. Indications of the presence of active methanogenic and sulphate-reducing bacterial populations were apparent throughout the studies. One possibility for the successful start-up and commissioning of the anaerobic reactors was the use of a well-adjusted biomass, which consisted of highly selected and adapted microbial consortium for the specific gelatinmanufacturing effluent. It was clear from this study that gelatin-manufacturing effluent can be treated successfully, especially with the use of the UASB design. A welldefined data base was constructed which could be of great value for further upscaling to a full-scale digester. / AFRIKAANSE OPSOMMING: 'n Hoogs besoedelde industriele uitvloeisel word gegenereer deur die plaaslike gelatien-vervaardigings industrie. As gevolg van toenemende streng beperkings op die kwaliteit van uitvloeisels wat bepaal word deur die Nasionale Water Wet van 1998 en Nasionale Omgewings Bestuurs Wet van 1998, asook toenemende munisipale heffings wat geimplementeer word via Plaaslike Munisipale Wette, word die industrie verplig om die uitvloeisel vooraf te behandel. 'n Studie is onderneem om die lewensvatbaarheid van anaërobe behandeling van gelatien-vervaardigings uitvloeisel te ondersoek, aangesien anaërobe verterings tegnologie alombekend is vir die goeie sukses behaal in die behandeling van hoë-sterkte, komplekse uitvloeisels. Verskeie laboratorium- en loods-skaal studies is gedoen, met verskillende hibried Opvloei Anaërobe Slykkombers (OAS) en kontak ontwerpe. Goeie werksverrigting was verkry by 'n hidroliese retensie tyd (HRT) van 1.0 d met twee mesofiliese laboratorium-skaal hibried OAS verteerder ontwerpe wat uitgevoer was met poli-etileen (AD-1) en poli-uretaan (AD-2) materiaal. Chemiese suurstof behoefte (CSB) verwyderings van so hoog as 90% (gem. 53%) vir AD-1 en 83% (gem. 60%) vir AD-2 by organiese ladingstempo's (OLT) van 9.56 en 4.62 kg CSB.m-3.d-1,was onderskeidelik verkry. Hoë sulfaat (S04) verwyderings van tot 96% (gem. 86%) vir AD-1 en 98% (gem. 82%) vir AD-2 was ook onderskeidelik verkry. 'n Maksimum totale vaste stof (TVS) verwydering van 65% (gem. 25%) vir AD-1 en 62% (gem. 28%) vir AD-2 is gerapporteer. 'n Gemiddelde metaan inhoud van 80% (AD-1) en 79% (AD-2) met 'n gemiddelde metaan opbrengs per CSB verwyder van 2.19 en 1.86 m3.kg CSBverwyder.dv-i1r AD-1 en AD-2, was onderskeidelik gevind. Met die aanwending van dieselfde twee verteerders (AD-1 en AD-2) in 'n series gekoppelde multi-fase konfigurasie, is 'n totale CSB verwydering so hoog as 97% (gem. 80%) verkry by 'n OLT van 8.32 kg CSB.m-3.d-1. Uitstekende totale S04 verwydering van 96% (gem. 69%) is behaal. Tot 82% TVS (gem. 29%) was vewyder gedurende die studie en die biogas het uit 89% metaan (gem. 79%) bestaan. Vir die multi-fase kombinasie is 'n maksimum van 92% vlugtige vetsure (WS) (gem. 48%) verwyder, wat dui op die moontlike skeiding van selektiewe fases van die onderskeie vetsuur produserende/verbruiker bakteriële populasies. CSB verwydering van tot 96% (gem. 51%) by 'n HRT van 3.0 d en 95% (gem. 54%) met 'n HRT van 1.0 d was verkry, tydens die gebruik van In laboratorium-skaal OAS bioreaktor met hersirkulasie. Lae werksverrigting was oor die algemeen waargeneem, met gemiddelde S04 en TVS verwyderings van 59% (maks. 97%) en 26% (maks. 67%) by In HRT van 1.0 d. Die biogas produksie was baie laag gedurende die studie (0.05 - 0.63 I,d-\ In Loods-skaal OAS verteerder was opgerig en bevredigende resultate was verkry met In gemiddeld van 58% CSB verwydering en maksimum van 96%. S04 en TVS verwyderings so hoog as 96% (gem. 44%) en 93% (gem. 63%) is onderskeidelik verkry. Die metaan inhoud van die biogas was 85%. Die loods-skaal studie was uitgevoer gedurende ware veld kondisies, waartydens verskeie skok en organiese ladings deur die sisteem geabsorbeer is. Die loods-skaal kontak konfigurasie (300 I) het nie bevredigende resultate getoon nie, as gevolg van voortdurende blokkasies wat ondervind is in die toevoer en hersirkulasie pype. Maksimum CSB, S04, WS en TVS verwyderings van 41% (gem. 27%), 62% (gem. 41%), 64% (gem. 27%) en 39% (gem. 21%) was onderskeidelik verkry. Die resultate van al die studies het aanvaarbare CSB verwydering aangedui by toenemende OLT's. Indikasies van aktiewe metanogene en sulfaat-reduserende bakteriële populasies was ook teenwoordig gedurende die studies. Die suksesvolle aansit-prosedure en begin van die anaërobe verteerders kan toegeskryf word aan die gebruik van In goed aangepaste biomassa, wat uit hoogs selektiewe en aangepaste mikrobiese populasies vir die spesifieke uitvloeisel bestaan. Hierdie studie het getoon dat gelatien-vervaardigings uitvloeisel suksesvol met die OAS ontwerp behandel kan word. In Goed gedefinieerde data basis kan voorsien word, wat van groot waarde sal wees vir verdere opgradering na In volskaalse verteerder.