Dissertations / Theses on the topic 'Applied Microbiology and Biotechnology'

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O presente estudo teve por objetivo analisar o impacto do óleo Diesel na microbiota nativa do solo de manguezal de Vila Velha- Itamaracá, analisando a capacidade biodegradadora natural dos microrganismos nativo presente nesta área. Os resultados mostraram que microrganismos solo de manguezal (fungos e bactérias) quando submetidos a impactos da substituição total de sua fonte de carbono sem um prévio contato com o poluente reduziram no número de colônias. Porém quando o solo foi submetido ao impacto com o poluente, durante trinta dias a reação microbiana foi de um aumento populacional, sendo observado a diminuição na diversidade das formas de colônias e surgimento de outras. Nos estudos realizados com a substituição total da fonte de carbono em meio aquoso sem estimulo a aeração confirmou-se através dos ensaios bioquímicos a presença do gênero Pseudomonas e sua participação no processo de biodegradação, através da redução de componentes do óleo Diesel analisado por cromatografia GCMS. Nos ensaios submetidos à agitação obteve-se um aumento populacional superior ao do impacto no solo, apresentando uma diminuição na tensão superficial em 40% no período de vinte dias, neste a redução do poluente em ensaios cromatográficos apresentou-se maior chegando a 99% no composto decano

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

This thesis purports to make two contributions to understandings of biotechnology. First, it presents a novel framework through which to view biotechnology as a complex series of fundamentally social and politically economic mediations rather than a decontextualised collection of technical and scientific phenomena. Second, the thesis presents a method for analysing contemporary discourses about biotechnology within this framework. The framework presented in the first content chapter of the thesis identifies what I see to be the four primary mediating "movements" that are central to seeing Biotechnology as Media: Alienation, Translation, Recontextualisation, and Absorption. The next chapter explicates these movements more fully using a combination of social practice and discourse theory. Using these four movements and the mediation framework as a guide, I then critically analyse a corpus of seventy two exemplary texts (approximately 700,000 words) about contemporary biotechnology. Mediation, in the sense I use it here, is not concerned with one particular media form or technology. Rather, it focuses on the process of mediation as the movement of meanings (Silverstone, 1999). I argue that seeing biotechnologies as mediations can provide a deeper and more critical understanding of how ways of seeing, being, acting, and describing (discourses) associated with contemporary biotechnology are moved from micro- and macro-biological and scientific contexts into the everyday lives of citizens and ecosystems. In particular, such a view highlights the forces and voices that currently determine the path and substance of political-economic movements in biotechnology and, consequently, how everyday perceptions of biotechnology are shaped or silenced in processes of mediation. A core assumption of the thesis is that processes of mediation are not neutral. Rather, they are always inherently interpretive, politically economic, and ethically significant. Any mediation involves "filtering" processes via which "content" is transformed into a form that is appropriate for a given medium by persons who have control over the medium, and by the nature of the medium itself. This applies as much in laboratory and scientific contexts as it does in the contexts of mass consumption, whether in newspapers, policy papers, movies (such as Gattaca), or consumer goods. The same is true in the mediation of biotechnology: there are technological and discursive restrictions on what and who can "contribute to" and "come out" of biotechnology and also what is construed as being a valuable and desirable outcome of biotechnology research and development. The three central analysis chapters of the thesis outline firstly how biotechnology can function as a time-based medium for the reproduction of already powerful discourses on, for example, the role of technology in human development and the consumer market as the moral medium between generators of new technologies and their "consumers". I identify exemplars of how the history of biotechnology and mediation (movement) is expressed in the corpus. This is followed by a more concentrated analysis of the ethical and social significance of the key "official" mediations presented in the corpus. I focus in particular on how the predominant policy evaluations of biotechnological mediations expressed in state, national, and international policy documents construct a "virtuous cycle" of product development that will ostensibly "deliver the benefits" of biotechnology to all citizens who, in the corpus, are framed predominantly as "consumers". The final chapter of the thesis reflects on the significance of biotechnology at the macro level of social practices and systems. Apart from its direct function as a technical medium for alienating hitherto inalienable aspects of life, such as configurations of DNA, and turning them into products for sale, I argue that, as a suite of mediating movements, biotechnology has the potential to effectively, and for the most part invisibly, mediate our more general understandings and experiences of ourselves, of other species, and of the world we live in. More specifically, I argue that biotechnological mediations actively, and often forcefully, promote a narrowing of the range of evaluative resources on offer to the general community, and indeed to biotechnologists themselves. Biotechnological mediations can therefore be described as part of a broader movement away from conditions of heteroglossia or dialogue (multi language, multi voice) toward conditions of monologia (one language, one voice). The thesis concludes with an important question: if we can identify these narrowing effects or mediations of biotechnology by using techniques such as Critical Discourse Analysis and by seeing biotechnology in a mediation framework, what can we do to interrupt them and generate movements that are more generative of heteroglossic and socially responsive ways of seeing, being, and acting? I offer a number of responses to the question in the conclusion.

This thesis purports to make two contributions to understandings of biotechnology. First, it presents a novel framework through which to view biotechnology as a complex series of fundamentally social and politically economic mediations rather than a decontextualised collection of technical and scientific phenomena. Second, the thesis presents a method for analysing contemporary discourses about biotechnology within this framework. The framework presented in the first content chapter of the thesis identifies what I see to be the four primary mediating "movements" that are central to seeing Biotechnology as Media: Alienation, Translation, Recontextualisation, and Absorption. The next chapter explicates these movements more fully using a combination of social practice and discourse theory. Using these four movements and the mediation framework as a guide, I then critically analyse a corpus of seventy two exemplary texts (approximately 700,000 words) about contemporary biotechnology. Mediation, in the sense I use it here, is not concerned with one particular media form or technology. Rather, it focuses on the process of mediation as the movement of meanings (Silverstone, 1999). I argue that seeing biotechnologies as mediations can provide a deeper and more critical understanding of how ways of seeing, being, acting, and describing (discourses) associated with contemporary biotechnology are moved from micro- and macro-biological and scientific contexts into the everyday lives of citizens and ecosystems. In particular, such a view highlights the forces and voices that currently determine the path and substance of political-economic movements in biotechnology and, consequently, how everyday perceptions of biotechnology are shaped or silenced in processes of mediation. A core assumption of the thesis is that processes of mediation are not neutral. Rather, they are always inherently interpretive, politically economic, and ethically significant. Any mediation involves "filtering" processes via which "content" is transformed into a form that is appropriate for a given medium by persons who have control over the medium, and by the nature of the medium itself. This applies as much in laboratory and scientific contexts as it does in the contexts of mass consumption, whether in newspapers, policy papers, movies (such as Gattaca), or consumer goods. The same is true in the mediation of biotechnology: there are technological and discursive restrictions on what and who can "contribute to" and "come out" of biotechnology and also what is construed as being a valuable and desirable outcome of biotechnology research and development. The three central analysis chapters of the thesis outline firstly how biotechnology can function as a time-based medium for the reproduction of already powerful discourses on, for example, the role of technology in human development and the consumer market as the moral medium between generators of new technologies and their "consumers". I identify exemplars of how the history of biotechnology and mediation (movement) is expressed in the corpus. This is followed by a more concentrated analysis of the ethical and social significance of the key "official" mediations presented in the corpus. I focus in particular on how the predominant policy evaluations of biotechnological mediations expressed in state, national, and international policy documents construct a "virtuous cycle" of product development that will ostensibly "deliver the benefits" of biotechnology to all citizens who, in the corpus, are framed predominantly as "consumers". The final chapter of the thesis reflects on the significance of biotechnology at the macro level of social practices and systems. Apart from its direct function as a technical medium for alienating hitherto inalienable aspects of life, such as configurations of DNA, and turning them into products for sale, I argue that, as a suite of mediating movements, biotechnology has the potential to effectively, and for the most part invisibly, mediate our more general understandings and experiences of ourselves, of other species, and of the world we live in. More specifically, I argue that biotechnological mediations actively, and often forcefully, promote a narrowing of the range of evaluative resources on offer to the general community, and indeed to biotechnologists themselves. Biotechnological mediations can therefore be described as part of a broader movement away from conditions of heteroglossia or dialogue (multi language, multi voice) toward conditions of monologia (one language, one voice). The thesis concludes with an important question: if we can identify these narrowing effects or mediations of biotechnology by using techniques such as Critical Discourse Analysis and by seeing biotechnology in a mediation framework, what can we do to interrupt them and generate movements that are more generative of heteroglossic and socially responsive ways of seeing, being, and acting? I offer a number of responses to the question in the conclusion.

Recent advancements in molecular biology such as next generation sequencing and more sensitive and rapid molecular detection methods like qPCR, have historically been developed for clinical applications in human genetics and for health care diagnostic purposes. The high demand for faster and more accurate molecular assays in the health care field has driven rapid development of inexpensive molecular techniques that when applied to the science of environmental microbiology, provides an unprecedented level of understanding of the microbial world around us. The goal of this dissertation is to begin to apply more advanced molecular technologies to problems in applied environmental microbiology. Appendix A is a brief literature review of next generation sequencing technologies for applications in environmental microbiology. Appendix B focuses on the development of a more robust virus nucleic extraction kit for the detection of viral genomes from environmental samples found to contain high concentrations of qPCR inhibitors, such as humic acids or heavy metals. Appendix C summarizes one of the largest virus surveys done in the US, using state of the art qPCR technologies in both wastewater influent and effluent from two wastewater treatment plants in the Southwest. Data suggests that traditional virus indicators may not be a viable tool to evaluate fecally impacted source water or virus removal during water treatment. The third study summarized in Appendix D, provides one of the first insights into the microbial ecology of biofilms utilized as biological treatment media using Roche 454 amplicon sequencing of the 16S rRNA gene.

Salvia sensu lato includes more than about 9000 species and is considered one of the largest taxa of the Lamiaceae (Will et al., 2015). For a long time, Salvia species were known for a wide variety of medicinal used in folk medicine for the relief of pain, protecting the body against oxidative stress, free radical damages, angiogenesis, inflammation, bacterial and virus infection, etc… (Hamidpour et al., 2014). In vitro cultures have been considered as an alternative agricultural processes for producing secondary metabolites (Y. Kim et al., 2002). The research activity of this PhD project was aimed to the application of in vitro biotechnology techniques applied to aromatic plants for the production of bioactive compounds. The selected plants are species of Salvia showing an important antibacterial activity. The investigation study was carried out on S. corrugata and S. tingitana. Salvia corrugata Vahl. is an ornamental plant that grown easily along the Mediterranean coast. The aerial part is rich in terpenoid compounds like the diterpene quinones fruticuline A and demethylfruticuline A that present high antibacterial activity. Protocols of tissue culture and genetic transformation were set up with the aim to obtain in vitro shoot and hairy root biomass to grown in controlled condition for metabolite extraction. Two strains of Agrobacterium rhizogenes (wild type ATCC 15834 and hypervirulent LBA9402) were tested for their ability to induce hairy root on wounded leaves. The best response (75 %) was achieved by infection with ATCC 15834 about thirty days after the infection onto the hormone-free MS basal solid medium. Two hairy-root lines from ATCC 15834 treatment and one from LBA 9402 treatment were established. Transformation of selected clones was confirmed by polymerase chain reaction analysis of bacterial rolC and virC1 genes. The growth evaluation in TIS RITA® bioreactor put in evidence the best cultural conditions for the biomass production; the clone SCO-HR-FA8 had the best increase on Murashige and Skoog (1962) and ½ Woody plant medium (Lloyd et al., 1980) salt compositions in comparison to other tested media. 30 mg/L was the best sucrose concentration that guaranteed the highest biomass production. The growth curve of HR lie SCO-HR-FA8 was elaborated and then, three classes of elicitors and their combination were tested: a heavy metal ions (Ag+), the yeast extract and plant response-signaling compound (methyl jasmonate MJ). Among them, Ag+ and yeast extract (YE) at high concentration were most effective to stimulate the biomass production. The scale up of the biomass was performed using the bioreactor RITA®. The methanolic extracts of the biomass (16.8 g) was fractionated by Si gel MPLC to obtain 16 fractions. The methanolic extract and the semi-purified fractions were tested against several multidrug resistant clinical strains of various bacterial species: Staphylococci and Enterococci and E. coli. The total extract was poorly effective while the semi purified fractions displayed variable potency with MIC values ranging from 8 to >128 μg/mL and from 4 to >128 μg/mL respectively against the Staphylococci and Enterococci strains considered. Our results suggest that the application of biotechnology approach on S. corrugata, it is possible to induce hairy roots from leaf, optimize the condition to increase the production of biomass, scale-up using TIS bioreactor and produce secondary metabolites with antibacterial activity. The study carried out on S. tingitana aimed to develop an in vitro culture protocol to produce different plant tissues and investigate the antibacterial activity of the extract. The callus formation from sterilized leaves was evaluated on MS medium added with different PGRs in presence of ascorbic acid in light or in dark conditions. The result showed the importance of the presence 2,4-D and darkness for callus development. The high development percentage (94.4 %) was achieved by combining KIN and 2,4-D at the concentration of 0.5:0.5 or 1:1 mg/L respectively. The optimization of the medium for the callus growth was determined evaluating the type and concentration of cytokinin and auxin in dark. The suitable condition for callus proliferation was MS medium supplemented with 2,4-D 4.52 μM, KIN 2,32 μM and 10 mg /L of ascorbic acid. The elicitation with MJ or light intensity was investigated. The methanolic extract and fractions obtained by MPLC were inactive against Staphylococci species and E. coli. The fractions 11 and 12 displayed an antibacterial activity only against E. faecalis and E. faecium with MIC values ranging from 32 to 64μg/mL. In S. tingitana, a successful protocol for callus production was established; this in vitro culture system can be used as good method to obtain medicinally-useful secondary compounds from S. tingitana.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Wine production is a complex biotechnological process aiming at productively coordinating the interactions and outputs of several biological systems, including grapevine and many microorganisms such as wine yeast and wine bacteria. High-throughput data generating tools in the elds of genomics, transcriptomics, proteomics, metabolomics and microbiomics are being applied both locally and globally in order to better understand complex biological systems. As such, the datasets available for analysis and mining include de novo datasets created by collaborators as well as publicly available datasets which one can use to get further insight into the systems under study. In order to model the complexity inherent in and across these datasets it is necessary to develop methods and approaches based on network theory and multivariate data analysis as well as to explore the intersections between these two approaches to data modelling, mining and interpretation. Networks: The traditional reductionist paradigm of analysing single components of a biological system has not provided tools with which to adequately analyse data sets that are attempting to capture systems-level information. Network theory has recently emerged as a new discipline with which to model and analyse complex systems and has arisen from the study of real and often quite large networks derived empirically from the large volumes of data that have collected from communications, internet, nancial and biological systems. This is in stark contrast to previous theoretical approaches to understanding complex systems such as complexity theory, synergetics, chaos theory, self-organised criticality, and fractals which were all sweeping theoretical constructs based on small toy models which proved unable to address the complexity of real world systems. Multivariate Data Analysis: Principle components analysis (PCA) and Partial Least Squares (PLS) regression are commonly used to reduce the dimensionality of a matrix (and amongst matrices in the case of PLS) in which there are a considerable number of potentially related variables. PCA and PLS are variance focused approaches where components are ranked by the amount of variance they each explain. Components are, by de nition, orthogonal to one another and as such, uncorrelated. Aims: This thesis explores the development of Computational Biology tools that are essential to fully exploit the large data sets that are being generated by systems-based approaches in order to gain a better understanding of winerelated organisms such as grapevine (and tobacco as a laboratory-based plant model), plant pathogens, microbes and their interactions. The broad aim of this thesis is therefore to develop computational methods that can be used in an integrated systems-based approach to model and describe di erent aspects of the wine making process from a biological perspective. To achieve this aim, computational methods have been developed and applied in the areas of transcriptomics, phylogenomics, chemiomics and microbiomics. Summary: The primary approaches taken in this thesis have been the use of networks and multivariate data analysis methods to analyse highly dimensional data sets. Furthermore, several of the approaches have started to explore the intersection between networks and multivariate data analysis. This would seem to be a logical progression as both networks and multivariate data analysis are focused on matrix-based data modelling and therefore have many of their roots in linear algebra.
AFRIKAANSE OPSOMMING: Inleiding: Wynproduksie is 'n komplekse biotegnologiese proses wat mik op die produktiewe koördinering van verskeie interaksies en uitsette van verskeie biologiese sisteme. Hierdie sisteme sluit in die wingerd, wat van besondere belang is, asook die wyn gis en wyn bakterieë. Hoë-deurset data generasie word huidiglik beide globaal en plaaslik toegepas in die velde van genomika, transkriptomika, proteomika, metabolomika en mikrobiomika. As sulks is hierdie tipe datastelle beskikbaar vir ontleding, bemyning en verkening. Die datastelle kan de novo gegenereer word, met behulp van medewerkers, of dit kan vanuit die publieke databasisse gewerf word waar sulke datastelle dikwels beskikbaar gemaak word sodat verdere insig verkry kan word met betrekking tot die sisteem onder studie. Die hoë-deurset datastelle onder bespreking bevat 'n hoë mate van inherente kompleksiteit, beide ten opsigte van ditself asook tussen verskeie datastelle. Om ten einde hierdie datastelle en hul inherente kompleksiteit te modelleer is dit nodig om metodes en benaderings te ontwikkel wat gesetel is in netwerk teorie en meerveranderlike statistiek. Verdermeer is dit ook nodig om die kruisings tussen netwerk teorie en meerveranderlike statistiek te verken om sodoende die modellering, bemyning, verkening en interpretasie van data te verbeter. Netwerke: Die tradisionele reduksionistiese paradigma, waarby enkele komponente van 'n biologiese sisteem geontleed word, het tot dusver nie voldoende metodes en gereedskap gelewer waarmee datastelle, wat streef om sisteemvlak informasie te bekom, geontleed kan word nie. Netwerk teorie het na vore gekom as 'n nuwe dissipline wat toegepas kan word vir die model-skepping en ontleding van komplekse sisteme. Dit stem uit die studie van egte, dikwels groot netwerke wat empiries afgelei word uit die groot volumes data wat tans na vore kom vanuit kommunikasie-, internet-, nansiële- en biologiese sisteme. Dit is in skrille kontras met vorige teoretiese benaderings wat gestreef het om komplekse sisteme te verstaan met konsepte soos kompleksiteits teorie, synergetics , chaos teorie, self-georganiseerde kritikaliteit en fraktale. Al die bogeneomde is breë teoretiese konstrukte, gebasseer op relatief kleinskaal modelle, wat nie instaat was om oplossings vir die kompleksiteit van egte-wêreld sisteme te bied nie. Meerveranderlike Data-analise: Hoofkomponente-ontleding (PCA) en Partial Least Squares (PLS) regressie word dikwels gebruik om die dimensionaliteit van 'n matriks (en tussen matrikse in die geval van PLS) te verminder. Hierdie matrikse bevat dikwels 'n aansienlike groot hoeveelheid moontlikverwante veranderlikes. PCA en PLS is variansie gedrewe metodes en behels dat komponente gerang word deur die hoeveelheid variansie wat elke component verduidelik. Komponente is by de nisie ortogonaal ten opsigte van mekaar en as sulks ongekorreleerd. Doelwitte: Hierdie tesis verken die ontwikkeling van verskeie Computational Biology metodes wat noodsaaklik is om ten volle die groot skaal datastelle te benut wat tans deur sisteem-gebasseerde benaderings gegenereer word. Die doel is om beter begrip en kennis van wyn verwante organismes te kry, hierdie organismes sluit in die wingerd (met tabak as laboratorium-gebasseerde plant model), plant patogene en microbes sowel as hulle interaksies. Die breë mikpunt van hierdie tesis is dus om gerekenaardiseerde metodes te ontwikkel wat gebruik kan word in 'n geintergreerde sisteem-gebaseerde benadering tot die modellering en beskrywing van verskillende aspekte van die wynmaak proses vanuit 'n biologiese standpunt. Om die mikpunt te bereik is gerekenaardiseerde metodes ontwikkel en toegepas in die velde van transkriptomika, logenomika, chemiomika en mikrobiomika. Opsomming: Die primêre benadering geneem in hierdie tesis is die gebruik van netwerke en meerveranderlike data-ontleding metodes om hoë-dimensie datastelle te ontleed. Verdermeer, verskeie van die metodes begin om die gemeenskaplike grond tussen netwerke en meerveranderlike data-ontleding te verken. Dit blyk om 'n logiese progressie te wees, aangesien beide netwerke en meerveranderlike data-ontleding gefokus is op matriks-gebaseerde data modellering en dus gewortel is in liniêre algebra.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system.
AFRIKAANSE OPSOMMING: In hierdie studie is die chromosomale arseen weerstandbiedendheidsgene (ars gene), teenwoordig in alle Acidithiobacillus caldus isolate, gekloon en die DNA volgorde daarvan vanaf At. caldus #6 bepaal. Tien oopleesrame (ORFs) is geïdentifiseer op ‘n kloon wat arseen weerstandbiedend is, met drie homoloog aan ars gene, nl. arsC (arsenaat reduktase), arsR (reguleerder) en arsB (membraan-geleë pomp wat arseniet uitpomp). Die ars operon is gerangskik met die arsRC en arsB gene wat in teenoorgestelde rigtings getranskribeer word. Analise van die afgeleide aminosuurvolgorde van dié ars gene het getoon hulle is naverwant aan die ars gene van Acidithiobacillus ferrooxidans. Die ars gene was funksioneel na transformasie na ‘n E. coli ars mutant (ACSH50Iq), en het ‘n hoër vlak van weerstand teen arsenaat en arseniet gebied. ArsC was nodig vir weerstand teen arsenaat, maar nie vir weerstand teen arseniet nie. Geen van die ander ORFs het arseen weerstandbiedendheid in E. coli bevorder nie. Voorheen is ‘n ars operon, geleë op ‘n transposon (TnAtcArs), in ‘n hoogs arseen-weerstandbiedende stam van die middelmatige termofiliese, swawel-oksiderende, bio-ontgunning (“biomining”) bakterie Acidithiobacillus caldus #6 beskryf. In laasgenoemde studie is gevind dat TnAtcArs hoër vlakke van weerstand bied teen arsenaat en arseniet as die chromosomale operon. TnAtcArs is na ‘n lae arseen-weerstandbiedende At. caldus stam (C-SH12) gekonjugeer. Die resultaat was ‘n groot verhoging in arseen weerstandbiedendheid. RT-PCR analise het onthul dat arsR en arsC saam getranskribeer word. Benewens die feit dat ORF1 (kadmium induseerbare protein) en ORF5 (afgeleide integrase vir profaag CP-933R) nie betrokke is in weerstand teen arseniet and arsenaat nie, is ORF1 saam met arsRC getranskribeer en ORF5 saam met arsB. Deur gebruik te maak van die fusie-gene arsR-lacZ en arsB-lacZ is bewys dat die chromosomale ArsR reguleerder van At. caldus as ‘n inhibeerder van die arsR en arsB promoter uitdrukking funksioneer. Indusering van geen uitdrukking vind plaas wanneer arseniet of arsenaat bygevoeg word. Die chromosomaal-geleë ArsR is ook in staat om TnAtcArs te inhibeer, terwyl die transposon geleë ArsR nie daartoe in staat is om die chromosomale ars sisteem te reguleer nie.

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

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

While the production of mead, a fermented honey beverage, has declined in popularity around the world in recent centuries, a substantial mead industry continues to exist in Africa with an estimated annual production of 1 to 1.7 billion litres. This is largely an ‘invisible industry’, and has functioned outside the formal economy due to proscription of indigenous beverages during colonial times. The traditional African mead industry is, however, also now under pressure due to the environmental degradation of scarce natural ingredients, urbanisation and loss of indigenous knowledge systems (IKS) and, with time, the beverage will likely follow the declining trend of mead consumption observed elsewhere. An analysis of early reports of African mead production suggested that the Khoi-San, among the earliest inhabitants of the continent, are the originators of the mead making techniques which use fibrous plant materials derived from specific plant species, to facilitate mead fermentation in some way. The Eastern Cape represents a region with a large body of Khoi-San IKS preserved in their descendants among the Afrikaans and Xhosa populations. A survey to establish a baseline of mead-making technology in the Eastern Cape was undertaken, and involved interviewing traditional mead makers across an area of roughly 100 000 km2, showing that the mead, iQhilika(Xhosa) Kari (Khoi-San/Afrikaans), is produced using a very similar process throughout the region. This involves the roots of a Trichodiadema sp. plant (imoela – Xhosa, karimoer – Afrikaans/Khoi-San), honey, extract of brood and/or pollen and water. Various other fruit sugar sources were also found to be added at times producing seasonal beverages with unique organoleptic properties. A model traditional iQhilika production operation was investigated in order to describe the main features of the process. Biomass immobilised on Trichodiadema root segments was found to be distributed evenly through the profile of the bioreactor resulting in a well mixed fermentation and a productivity of 0.74 g EtOH/l/h. In the initial stages of fermentation, the ethanol yield was highest in the mid-regions of the bioreactor, but with time the regions closer to the surface, which had atmospheric contact had a higher yield. This phenomenon was attributed to aerobic fatty acid synthesis which allowed the yeast close to the surface to function more efficiently despite rising ethanol concentrations. The mead contained 44.25 g/l (7 % volume) ethanol produced in a fermentation time of 43.5 h. Yeast biomass in the traditional process was either immobilised in the form of flocs or attached to the Trichodiadema intonsum support. Electron microscopy revealed that the cells were covered in a layer of extra-cellular polymeric substance apparently assisting the immobilization, and which was populated by a consortium of yeasts and bacteria. Yeasts isolated from iQhilika brewed in two regions separated by 350 km were found to be very closely related Saccharomyces cerevisiae strains as determined by molecular genetic analysis. The traditional beverage was found to contain populations of Lactic acid bacteria (LAB), which are known spoilage organisms in other beverages. Spoilage characteristics of these organisms matched descriptions of spoilage provided by the IKS survey. Other possibly beneficial LAB, which may contribute useful flavour compounds, were also found to be present in the system. The basic functional aspects of the traditional process were used to design a continuous bench-scale tower bioreactor and process development was based on the IKS survey. This consisted of a packed bed bioreactor, consisting of 2 mm3 T. intonsum root segments, immobilising a novel Saccharomyces cerevisiae strain isolated from a traditional batch of iQhilika. The bioreactor performed well with a yield of close to the theoretical maximum and an ethanol productivity of 3.45 g EtOH/l/h. The parameters of the 5.6 l/d bench-scale bioreactor were used to design a full-scale production bioreactor with a planned maximum output of 330 l/d. This bioreactor had a productivity of 0.19 g EtOH/l/h. The organoleptic properties of the product produced were considered by a taste panel to be better than those of the product of the bench-scale tower bioreactor. This research was based on the development of IKS which imposed a number of constraints and obligations on the project to ensure environmental, and social, in addition to financial viability of the scale-up operation. Makana Meadery was established in partnership with Rhodes University as an empowerment company which, in addition to undertaking the commercialisation of the iQhilika process, would also develop methods for the production of scarce ingredients traditionally unsustainably sourced from fragile ecosystems, provide beekeeping training and the manufacture of beehives.

Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses, bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9, 4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control, although it was not possible to determine whether p-coumaroyl-CoA was produced. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast.
AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

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

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009.
Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.

The current world population is over 7.4 billion and expected to exceed 9 billion in 2040, causing a 70% increase in food demand. Global environmental degradation, in the form of salinization, pollution and global warming, has also reduced the availability of suitable arable land and water sources, contributing to promote crop improvement in order to increase the potential yields. Rice (Oryza sativa) is the most widely consumed staple food for a large proportion of the world population. Classical rice breeding programs use its natural variability to create new allelic combinations which are screened for selecting those presenting superior agronomic traits such as improved yield. Those improved lines are stabilized through inbreeding to maintain the phenotype in their progeny. Certified seed producers systematically select and propagate registered varieties year by year in order to maintain their uniformity and the original registered cultivar traits, since natural mutations, spontaneous breeding between varieties and alien grain contamination can introduce undesirable variability at this stage. Nowadays biotechnology is used to drive the improvement of rice traits such as increased yield and grain quality. Moreover it helps to rapidly bestow tolerance to biotic (diseases and insects) and abiotic (drought, salinity, cold temperatures, nutrients deficiency) factors. Some of the available biotechnological techniques applied for crop improvement are i) the genetic engineering, which allows the addition of foreign genes in the rice genome although being controversial due to the social and environmental concerns, ii) the anther culture, which fasten and improves the selection of new breeding lines, and iii) the Targeting Induced Local Lesions IN Genomes (TILLING) which combines the production of large mutant populations with the detection of mutants in genes of interest through molecular screening. The main aim of the thesis is to study different biotechnological tools and their applications to the improvement of Mediterranean rice varieties. To achieve this biotechnology is used to study the pollen dispersion of a genetically engineered rice line, to accelerate the stabilization process through anther culture technique and to introduce new variability using a mutagenesis protocol followed by molecular detection of mutants. In this thesis we first studied the pollen-mediated gene flow between wild rice, conventional rice and an herbicide resistant transgenic rice line in order to determine gene flow rates in relation to the distance and the prevailing wind speed and direction. Results showed that pollen dispersal is dramatically effected by the distances between rice plants and the speed and direction of the prevailing wind. Furthermore, the enhanced pollen dispersal capability of weedy rice can also play an important role in transgenic pollen dispersal, which unfortunately had been underestimated. Then, we adapted an anther culture protocol in order to efficiently obtain commercial dihaploid lines from a Mediterranean japonica variety. Furthermore, we described the greenhouse and field trials used to select the best lines for registration which are now being successfully commercialized. Finally, we developed a fast protocol for obtaining mutants with agronomic interest. This protocol is based on ethyl methanesulfonate mutagenesis of seed-derived calli. The in vitro regenerated mutant population plants were directly screened for senescence-related genes, allowing to shorten in more than eight months the common seed mutagenesis protocol. The molecular screening protocol was also optimized and several potential delayed senescence mutants were identified and tested.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005.
Plasmid pTC-F14 is a 14.2kb promiscuous, broad-host range IncQ-like mobilizable plasmid isolated from Acidithiobacillus caldus f. At. caldus is a member of a consortium of bacteria (along with Acidithiobacillus ferrooxidans and Leptospirilum ferrooxidans) that is used industrially for decomposing metal sulphide ores and concentrates at temperatures of 40ºC or below which is now a well-established industrial process to recover metals from certain copper, uranium and gold-bearing minerals or mineral concentrates. These biomining microbes are usually obligately acidophilic, autotrophic, usually aerobic iron- or sulphur-oxidizing chemolithotrophic bacteria. Their remarkable physiology allows them to inhabit an ecological niche that is largely inorganic and differs from those environments populated by the more commonly studied non-acidophilic heterotrophic bacteria. At. caldus, is a moderately thermophilic (45 to 50ºC), highly acidophilic (pH1.5 to 2.5) sulphur-oxidizing bacterium, and its role as one of the major players in the industrial decomposition of metal sulphide ores has become evident in recent years. At. caldus f from which pTC-F14 was isolated was found to be one of two dominant organisms in a bacterial consortium undergoing pilot-scale testing for the commercial extraction of nickel from ores.

A chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours.

The stream of new technological advancements and their integration into the field of microbiology have contributed significantly towards our understanding of life in the micro-scale world, making the fields of microbiology and biotechnology shine like never before. Since 1980, the recombinant protein-based therapeutics industry has become one of the fastest growing sectors in the biopharmaceutical market. Nearly 30% of commercially available recombinant proteins are produced in Escherichia coli, making this species one of the most commonly used bacterial expression systems for the production of recombinant biotherapeutics. However, when it comes to the production of enzymes and bioactive secondary metabolites (antibiotic, antifungal, antiviral and immunosuppressant), Streptomyces species remain the major producer within this sector. Meeting the high demand for such products requires a clear and in-depth understanding of the bioprocesses involved to achieve high yield and quality products, whilst keeping the process industrially attractive. It is generally accepted that the metabolome, as a down-stream process to the genome and proteome, may provide a clearer picture of a biological system. Thus, in this thesis a series of metabolomics approaches were adopted to obtain a deeper insight into the metabolic effects of recombinant protein production in E. coli and Streptomyces lividans. Furthermore, a Geobacter-based biomagnetite nanoparticle production system which displayed a prolonged lag phase upon scale-up was investigated by employing metabolic profiling and fingerprinting approaches combined with multivariate analysis strategies, to identify growth-limiting metabolites. The results of this analysis identified nicotinamide as the growth limiting metabolite. Nicotinamide-feeding experiments confirmed the above findings, leading to improved biomass yield whilst restoring the lag phase to bench-scale level. Raman and Fourier transform infrared spectroscopies combined with stable isotopic probing strategies were also employed to demonstrate the application of metabolic fingerprinting in providing detailed biochemical information for quantitative characterisation and differentiation of E. coli cells at community and single-cell levels. The single-cell approach proved promising, offering detailed biochemical information and perhaps accompanying other cultivation-free approaches such as metagenomics for further future investigations. It is hoped that the advances made in these studies have proved the potential applications of metabolomics strategies to aid the optimisation of microbially-driven bioprocesses.

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.

Fundamental processes involved in the microbial degradation of coal and its derivatives have been well investigated and documented over the past two decades. However, limited progress in industrial application has been identified as bottleneck in further active development of the field. The sporadic and unanticipated growth of Cynodon dactylon (Bermuda grass) has been observed on the surface of some coal dumps in the Witbank coal mining area of South Africa. Preliminary investigations showed the formation of a humic soil-like material from the breakdown of hard coal in the root zone of these plants. The potential of this system to contribute to industrial scale bioprocessing of hard coal was investigated. This study involved an investigation of the C. dactylon/coal rhizosphere environment and demonstrated the presence of fungal species with known coal bioconversion capability. Amongst these Neosartorya fischeri was identified and its activity in coal bioconversion was described for the first time. Cynodon dactylon plant roots were also shown to be colonized by mycorrhizal fungi including Glomus, Paraglomus and Gigaspora species. The role of plant photosynthate translocation into the root zone, providing organic carbon supplementation of fungal coal bioconversion was investigated in deep liquid culture with the N. fischeri isolate used as the biocatalyst. Organic acids, sugars and complex organic carbon sources were investigated and it was shown that glutamate provided significant enhancement of bioconversion activity in this system. The performance of N. fischeri in coal bioconversion was compared with Phanaerochaete chrysosporium and Trametes versicolor, both previously described fungal species in the coal bioconversion application. Fourier transform infrared spectroscopy indicated more pronounced oxidation and introduction of nitro groups in the matrix of the humic acid product of coal bioconversion in N. fischeri and P. chrysosporium than for T. versicolor. Macro-elemental analysis of biomass-bound humic acid obtained from the N. fischeri catalyzed reaction showed an increase in the oxygen and nitrogen components and coupled with a reduction in carbon and hydrogen. Pyrolysis gas chromatography mass spectroscopy further supported the proposal that the mechanism of bioconversion involves oxygen and nitrogen insertion into the coal structure. The C. dactylon bituminous hard coal dump environment was simulated in a fixed-bed perfusion column bioreactor in which the contribution of organic supplement by the plant/mycorrhizal component of the system was investigated. The results enabled the proposal of a descriptive model accounting for the performance of the system in which the plant/mycorrhizal component introduces organic substances into the root zone. The non-mycorrhizal fungi utilize the organic carbon supplement in its attack on the coal substrate, breaking it down, and releasing plant nutrients and a soil-like substrate which in turn enables the growth of C. dactylon in this hostile environment. Based on these results, the Stacked Heap Coal Bioreactor concept was developed as a large-scale industrial bioprocess application based on heap-leach mineral processing technology. Field studies have confirmed that bituminous hard coal can be converted to a humic acid rich substrate in a stacked heap system inoculated with mycorrhizal and N. fischeri cultures and planted with C. dactylon.

Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.

Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model.

Thesis (MScAgric.)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations.
AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

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

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: The presence and growth of lactic acid bacteria (LAB) in wine and their influence on wine quality has received much attention in recent years. Lactic acid bacteria are responsible for conducting malolactic fermentation (MLF) in wine. The benefits associated with malolactic fermentation in terms of deacidification of wine and the contribution to wine flavour and complexity have also recently been the topic of research. It is impossible to describe malolactic fermentation as distinctly desirable or undesirable in terms of its influence on the final quality of wine. The benefits and disadvantages are dependent upon viticultural region, grape variety, wine composition, winemaking techniques and the style and objectives of the winemaker. Brandy production is a multi-stage process in which base wine production, distillation technique and wood maturation all have a large influence on the final chemical profile and organoleptic quality of the brandy. The volatile composition of the base wine, which basically undergoes a concentration process during the subsequent double distillation phase, is critical in determining the aroma and flavour quality of the final brandy product. Thus, the brandy is only as good as the base wine it is distilled from. The aims of this study were to determine the effect of lactic acid bacteria and spontaneous malolactic fermentation on the quality of brandy base wine and the resulting distillate, and to determine which LAB species had been responsible for the occurrence of spontaneous MLF. This study showed that LAB are present at high numbers and are able to conduct spontaneous MLF of brandy base wines. It was shown that the incidence of spontaneous MLF varied from year to year. In 1998, 50% of the commercially produced base wines had undergone partial MLF prior to distillation. In 1999 and 2000 respectively, 34% and 45% of the commercial base wines had undergone partial MLF prior to distillation. The occurrence of spontaneous MLF had an influence on the chemical composition and the sensory quality of the base wine and distillate. There was an increase in the concentrations of ethyl lactate, acetic acid and diethyl succinate in samples that had undergone MLF. There was also a decrease in the concentrations of esters, such as iso-amyl acetate, ethyl acetate, ethyl caproate, hexyl acetate and 2-phenethyl acetate in these same samples. Sensory evaluation of the base wines and distillates demonstrated that samples that had undergone MLF differed significantly from samples that had not undergone MLF. It was also shown that distillates that had not undergone MLF had a slightly better aroma profile than those that had. Sweet aromas, like chocolate and caramel, as well as negative aromas, like chemical or solvent, were more prominent in brandy distillates that had undergone MLF. Herbaceous and fruity aromas were more intense in distillates not having undergone MLF. Fifty-four strains, all Gram-positive and catalase negative, were isolated at different stages of brandy production. Seven strains were isolated from the grape juice, 15 strains were isolated from the base wine, 20 strains were isolated during MLF and 12 strains were isolated from the base wine after MLF had been completed. Based on C02 production from glucose and gluconate, 17 strains were classified as facultatively heterofermentative and 37 strains as obligately heterofermentative. Fifteen of the 37 obligately heterofermentative strains were rod-shaped and were regarded as lactobacilli. The remaining 22 strains were oval or cocci-bacilli shaped. The isolates were identified to species level by using numerical analysis of the total soluble cell protein patterns, 16S rRNAsequencing and polymerase chain reaction (PCR) with species-specific primers. The facultative heterofermentative lactobacilli were identified as Lactobacillus paracasei and Lactobacillus p/antarum. The fifteen obligately heterofermentative lactobacilli were identified as members of the species Lactobacillus brevis, Lactobacillus verrniforme, Lactobacillus buchneri and Lactobacillus hi/gardii. The 22 obligate heterofermentative isolates, with a coccoid morphology, could be grouped into two clusters and were identified as Oenococcus oeni. O. oeni was the species responsible for the occurrence of spontaneous MLF in most of the commercial base wines. Lb. brevis, Lb. hi/gardii and Lb. paracasei were also isolated from commercial base wines that had undergone spontaneous MLF. In nine out of 14 experimental base wine samples that had undergone spontaneous MLF, O. oeni was again the predominant species. Lb. brevis, Lb. hi/gardii and Lb. paracasei were identified in the remaining experimental base wine samples. This is the first report of the presence of Lb. perecese! and Lb. vermiforme in brandy base wine. It was shown that the occurrence of spontaneous MLF had a negative effect on the quality of brandy base wine, but that was shown to be due to the different species and strains performing MLF. In the non-preferred distillate samples, Lactobacillus spp. had performed MLF or had developed after or during MLF.
AFRIKAANSE OPSOMMING: Die teenwoordigheid en die vermoë van melksuurbakterieë (MSB) om in wyn te groei, is 'n onderwerp wat al heelwat nagevors is. Melksuurbakterieë is verantwoordelik vir die uitvoering van appelmelksuurgisting (AMG) in wyn. Die voordele verbonde aan appelmelksuurgisting, ten opsigte van die verlaging van die totale suurinhoud en die bydrae tot die verbeterde geur en kompleksiteit van die wyn, is ook al goed bestudeer. Wat die invloed op die finale wynkwaliteit betref, is dit byna onmoontlik om AMG as uitsluitlik gewens óf ongewens te beskou. Die voordele en nadele van AMG is afhanklik van verskeie faktore, nl. wingerdkundige streek, druifkultivar, wynsamestelling, wynmaakpraktyke, asook die styl en doelwitte van die wynmaker. Die produksie van brandewyn is 'n multistapproses waarin die bereidingsmetode van die basiswyn, die distillasietegniek en houtveroudering 'n groot invloed op die finale kwaliteit en chemiese samestelling van die brandewyn het. Die vlugtige verbindings van die basiswyn, wat tydens die dubbele distillasieproses gekonsentreer word, is van wesenlike belang in die bepaling van die aroma en geur van die finale brandewynproduk. Brandewyn is dus inderdaad net so goed soos die basiswyn waarvan dit gestook is. Die doelwitte van hierdie studie was om te bepaal wat die invloed van MSB en die voorkoms van spontane AMG op die kwaliteit van die basiswyn en die distillaat is, asook om die MSB wat vir die voorkoms van spontane AMG verantwoordelik was, te identifiseer. Hierdie studie het bewys dat MSB in hoë getalle teenwoordig was en dat dit in staat is om die spontane AMG van basiswyne uit te voer. Daar is bewys dat die voorkoms van spontaneAMG moontlik van jaar tot jaar kan verskil. In 1998 het 50%, in 1999 het 34% en in 2000 45% van die kommersieel-geproduseerde basiswyn gedeeltelike AMG spontaan voor distillasie ondergaan. Daar is ook gevind dat spontane AMG 'n invloed op die chemiese samestelling en sensoriese kwaliteit van die basiswyn en die distillaat gehad het. Daar was 'n toename in die konsentrasies van etiellaktaat, asynsuur en diëtielsuksinaat in monsters wat spontane AMG ondergaan het. In dieselfde monsters was daar ook 'n afname in die konsentrasies van iso-amielasetaat, etielasetaat, etielkaproaat, heksielasetaat en 2-fenielasetaat. Sensoriese evaluering van die basiswyne en distillate het getoon dat daar betekenisvolle verskille was tussen die monsters wat AMG ondergaan het en dié wat nie AMG ondergaan het nie. Daar is bewys dat die distillate wat nie AMG ondergaan het nie, 'n beter aromaprofiel gehad het as dié wat AMG ondergaan het. Soet geure, soos sjokolade en karamel, en negatiewe geure, soos "chemies" en "oplosmiddel", was prominent in distillate wat AMG ondergaan het. Kruidagtige en vrugtige geure was meer intensief in distillate wat nie AMG ondergaan het nie. Vier-en-vyftig bakteriese rasse, almal Gram-positief en katalase-negatief, is gedurende die verskillende stadia van brandewynproduksie geïsoleer. Sewe rasse is uit druiwesap, 15 rasse gedurende die alkoholiese fermentasie, 20 rasse gedurende AMG en 12 rasse na voltooiing van AMG geïsoleer. Op die basis van koolstofdioksied (C02)-produksie vanaf glukose en glukonaat is 17 rasse as fakultatief heterofermentatief en 37 rasse as obligaat heterofermentatief geklassifiseer. Vyftien van die 37 obligaat-heterofermentatiewe rasse was staafvormig en is as lactobacilli geïdentifiseer. Die oorblywende 22 het ovaal of kokkus-bacillusvormige selmorfologie getoon. Identifikasie tot op spesievlak is gedoen deur van numeriese analise van die totale oplosbare selproteïenprofiele, 16S-rRNAvolgordebepalings en spesie-spesifieke inleiers vir die polimerasekettingreaksie (PKR) gebruik te maak. Die fakultatief-heterofermentatiewe rasse is as Lactobacillus paracasei en Lactobacillus p/antarum geklassifiseer. Die 15 obligaat heterofermentatiewe stafies is as Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus hi/gardii en Lactobacillus vermiforme geïdentifiseer. Die 22 ovaal, obligaat heterofermentatiewe isolate kon in twee groepe ingedeel word en is as Oenococcus oeni geïdentifiseer. Daar is bevind dat O. oeni-isolate vir die voorkoms van spontane AMG in die meeste van die kommersiêle basiswyne verantwoordelik was. Lb. brevis, Lb. hi/gardii en Lb. paracasei is ook uit kommersiêle basiswyne wat spontane AMG ondergaan het, geïsoleer. In nege uit 14 van die eksperimentele basiswyne wat spontane AMG ondergaan het, was O. oeni die dominante spesie. In die oorblywende eksperimentele wyne is Lb. brevis, Lb. hi/gardii en Lb. paracasei aangetref. Hierdie is die eerste vermelding van die teenwoordigheid van Lb. paracasei and Lb. vermiforrne in brandewynbasiswyn. Daar is gevind dat die voorkoms van spontane AMG "n negatiewe invloed op brandewynkwaliteit het, maar dit is as gevolg van die verskeidenheid van MSB-spesies en rasse wat voorkom. In die distillate wat deur die proepaneel afgekeur is, het Lactobacillus spesies die AMG deurgevoer, of het dit tydens of na AMG ontwikkel.

To increase profitability for biogas production, new innovative substrates and condition of operations needs to be implemented. At the current state, fats, oils and greases (FOGs) represent a promising substrate even though it brings operational challenges to the anaerobic digestion process. By utilizing hydrodynamic cavitation (HC) as a pre-treatment of the FOGs, the efficiency of FOGs’ co-digestion with wastewater sludge can be significantly improved. Preliminary experiments conducted on oil and water demonstrates that the HC pre-treatment improves the oil solubilisation as well as forms stable oil and water emulsion that last for several hours. The pre-treatment also improved the soluble chemical oxygen demand (COD) of biosludge (BiSl) by up to 115% and the initial degradation rate by up to 35%. In a semi-continues system, this allowed a significant increment in the specific methane yield depending on the organic loading rate (OLR) applied1. With sufficient process optimization, the HC-pre-treatment may prove to be an energy efficient and effective pre-treatment of FOGs.

Limestone built heritage is at risk from the effects of biofilms, a microbial community encapsulated in a matrix of sugars, protein and extracellular DNA. Although biofilm research has been carried out in Mediterranean regions, few studies cover temperate Northern Europe climates, or the UK. This study concentrates on bacterial colonisation of Lincoln limestone, a highly vulnerable building material, and identifies the species, their role in biodeterioration and the efficacy of biocides against them. As part of this study the core species which comprise the bacterial component of the limestone microbiome have been characterised for the first time; this has allowed the identification of non-core species which are significantly associated with damaged and undamaged surfaces. Four mechanisms of biodeterioration have been identified, one previously unidentified, and isolated species have been characterised as to whether they are biodeteriorative and the mechanisms of biodeterioration that they employ. Two species, Curtobacterium flaccumfaciens and Solibacillus silvestris, have been characterised as producing biofilm matrix which actively causes biomechanical damage to the oolitic limestone structure as opposed to the passive enhancement of physical weathering which has been previously associated with biofilm matrix. Species capable of biodeterioration have also been shown to be present on both damaged and undamaged surfaces, something which has not been previously investigated. Environmental sampling, species identification and characterisation of species for biodeterioration have all combined to identify markers of biodeterioration, ie both physical markers and biomarkers. Specifically, a surface pH of 5.5 or lower and the presence of B. licheniformis is indicative of biodeterioration with a proportionally higher level of M. luteus when comparing damaged and undamaged stone. Finally this study brings the literature on conservation methods up to date by testing biocides which are in current usage, as many biocides in the literature are discontinued. This study is also the first in the field to show their efficacy against biofilm encapsulated bacteria and their propensity for chemically disrupting the biofilm matrix.

Dissertation (PhD Agric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the malolactic enzyme (MLE). The consequence of this conversion is a decrease in total acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of the bacteria the organoleptic profile of the wine is modified. In some wines MLF is considered as spoilage, especially in warm viticultural regions with grapes containing less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine colour, and biogenic amines may be produced. To induce MLF we provided s. cerevisiae with the enzymatic activities required for MLF, which is then conducted by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene (mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and expressed in S. cerevisiae. The activity of this enzyme was compared to two other malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni La11, expressed in the same yeast strain. All three recombinant strains of S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion rate and a significant lower final L-Iactate level were observed with the yeast expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the main organism responsible for MLF, could be safely repressed with a ribosomaly synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB 1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C, and ethanol or S02 does not affect its activity. The peptide was purified to homogeneity and sequence analysis suggests that the peptide is a member of the lantibiotic family of bacteriocins. The molecular mass was estimated by mass spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall biosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range, optimal activity was recorded at pH 5.0. The petide is, however, more stable between pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0. Pediocin PD-1 provides a safer biological alternative than chemical preservatives such as S02.
AFRIKAANSE OPSOMMING: Appelmelksuurgisting (AMG) word deur sekere melksuurbakterieë (MSB) uitgevoer en verwys na die dekarboksilering van L-malaat na L-Iaktaat, 'n reaksie gekataliseer deur die appelmelksuurensiem (AME). AMG verlaag die suurvlakke in wyn, speel 'n rol in mikrobiologiese stabiliteit, en verander die organoleptiese profiel van die wyn. In sommige wyne word AMG beskou as bederf, veral in warm wynbou streke met minder malaat in druiwe. AMG kan ongewenste organoleptiese veranderinge teweeg bring, die wyn se kleur beinvloed, en tot die produksie van biogene amiene lei. Vir die bevordering van AMG het ons S. eerevisiae met die ensiematiese aktiwiteit benodig vir AMG voorsien wat dan veilig deur die gis tydens alkoholiese fermentasie uitgevoer word. 'n AME-koderende geen (mIeD) is uit Pedioeoeeus damnosus NCFB 1832 gekloneer, gekarakteriseer en in S. Cerevisiae uitgedruk. Die aktiwiteit van die ensiem is vervolgens vergelyk met die aktiwitet van twee ander AME gene, mIeS van Laetoeoeeus laetis MG1363 en mleA van Oenoeoeeus oeni Lal1, uitgedruk in dieselfde gisras. AI drie rekombinante gisrasse het L-malaat binne die bestek van drie dae na L-Iaktaat omgeskakel en die finale L-malaat vlakke was minder as 0.3 gIL. Die tempo van omkakeling was egter laer in die gis wat die mIeD geen uitdruk en die finale L-Iaktaat vlakke was veel laer. Om AMG te inhibeer is die groei van O. oeni, die organisme hoofsaaklik verantwoordelik vir AMG, onderdruk deur die byvoeging van 'n ribosomaal gesintetiseerde antimikrobiese peptied, pediocin PD-1, geproduseer deur P. damnosus NCFB 1832. Gisgroei is nie geaffekteer nie. Pediocin PD-1 is stabiel in wyn by temperature wat wissel tussen 4°C en 100°C, en die aktiwiteit van die peptied word nie geaffekteer deur ethanol of S02 nie. Die peptied is gesuiwer volgens In eenvoudige metode wat amoniumsulfaat-presipitasie en katioon uitruilings-ehromatografie insluit. Aminosuur volgorde bepaling van gesuiwerde peptied dui daarop dat pediocin PD-1 tot die lantibiotiese familie van bakteriosiene behoort. Die molekulêre massa van die peptied, soos bepaal deur massa spektroskopie, is 2866.7 ± 0.4 Da. Pediocin PD-1 vorm porieë in selmembrane van sensitiewe selle soos aangedui deur die uitvloei van K+vanuit O. oeni selle. Die peptied kombineer hierdie aksie met die inhibisie van selwand biosintese wat lei tot sel lise. Verlies van sellulêre K+verminder by laer temperature, waarskynlik as gevolg van verandering in die lipied- en protein inhoud van die sitoplasmiese membraan. Alhoewel die peptied aktief is oor 'n breë pH grens, is die antimikrobiese aksie optimaal by pH 5.0. Die peptied is meer stabiel tussen pH 2.0 en 5.0 en toon die beste stabiliteit tussen pH 3.0 en 4.0. Peiocin PD-1 is 'n veilige biologiese alternatief vir chemiese preserveermiddels soos S02.

The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo.
xiv, 120 leaves : ill. ; 28 cm.

Dissertation (PhD)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours.
AFRIKAANSE OPSOMMING: L-Appelsuur en wynsteensuur is die mees prominente organiese sure in wyn en speel 'n kritiese rol in die wynbereidingsproses en organoleptiese wynkwaliteit, insluitende die fisiese, biochemiese en mikrobiese stabiliteit van wyn. Die produksie van hoë-kwaliteit wyne berus op die vermoë van 'n wynmaker om die suurinhoud korrek aan te pas om sodoende 'n gebalanseerde produk met optimale geur en kleur te produseer. Saccharomyces rasse kan gewoonlik nie appelsuur volledig tydens alkoholiese gisting benut nie en dra dus nie noemenswaardig tot 'n verlaging van die totale suurinhoud van wyn by nie. Die mate van appelsuur afbraak deur Saccharomyces wissel egter van ras tot ras. Sekere Saccharomyces rasse kan 'n groter persentasie appelsuur afbreek, maar die onderliggende rede vir hierdie verskynsel is onbekend. Die onderliggende meganismes vir hierdie verskynsel is gedurende hierdie studie uitgelig na afloop van voorlopige transkripsionele regulerings studies op die malaatensiemgeen. In teenstelling hiermee kan S. pombe tot 29 gIl appelsuur via die malo-alkoholiese padweg afbreek waartydens appelsuur na pirodruiwesuur en CO2, en uiteindelik na alkoholonder fermentatiewe toestande omgeskakel word. Verskeie redes vir die swak afbraak van appelsuur deur Saccharomyces cerevisiae is voorgestel. Eerstens beskik S. cerevisiae nie oor 'n meganisme vir die aktiewe transport van appelsuur, soos in die geval van S. pombe nie, en is aangewese op die stadige opname van appelsuur deur eenvoudige diffusie. Tweedens het die S. cerevisiae malaatensiem 'n baie laer substraataffiniteit vir appelsuur (Km = 50 mM) in vergelyking met die van S. pombe (Km = 3.2 mM), wat verder bydra tot die swak afbraak van appelsuur in S. cerevisiae. Laastens dra die mitochondriale ligging van die S. cerevisiae malaatensiem in teenstelling met die sitoplasmiese ligging van die S. pombe malaatensiem, verder by tot die swak afbraak van appelsuur, aangesien die mitochondria onder fermentatiewe toestande negatief gereguleer word. Die malaatpermease geen (maely en malaatensiem geen (mae2) van S. pombe is gevolglik gekloneer en heteroloog in 'n laboratoriumras van S. cerevisiae onder die beheer van die konstitutiewe 3-fosfogliseraat kinase (PGKI) promoter- en termineerdervolgordes uitgedruk. 'n Sterk malo-alkoholiese fenotipe was duidelik tydens fermentasies met die rekombinante gis in sintetiese en Chardonnay druiwemos, met 'n gepaardgaande verhoging in alkoholvlakke. Funksionele uitdrukking van die malo-alkoholiese gene van S. pombe in 'n S. cerevisiae laboratoriumras het die weg vir die genetiese modifisering van industriële wynrasse van S. cerevisiae vir kommersiële wynfermentasie gebaan. Om 'n integrale deel van die gis te word, moet die malo-alkoholiese gene stabiel in die genoom van industriële wynrasse geïntegreer word. Genetiese manipulering van industriële wynrasse word egter bemoeilik deur die homotalliese, multi-ploïediese en prototrofiese aard van industriële Saccharomyces rasse. Transformasie en integrasie van heteroloë gene in industriële Saccharomyces rasse vereis die gebruik van dominante merkers, bv. weerstandbiedendheid teen antibiotika of ander gifstowwe. Integrasie van hierdie merkers in die gisgenoom is egter nie vir kommersiële toepassing aanvaarbaar nie weens die afwesigheid van langtermyn risikobepalings en verbruikersweerstand. Tydens hierdie studie is daar dus gepoog om industriële wynrasse met 'n unieke strategie geneties te verbeter sodat slegs gis-DNA tydens die integrasie van die S. pombe mae1 en mae2 uitdrukkingskassette in die gisgenoom opgeneem word. Die Malo-alkoholiese integrasiekasset wat slegs die S. pombe mae1, mae2 oopleesrame en die S. cerevisiae PGK1 promoter en termineerdervolgordes bevat, is in die URA3 lokus van Saccharomyces bayanus ECll18 geïntegreer tydens parallelle transformasie met 'n 'phleomycin' weerstandbiedendheidsplasmied. Na seleksie van transformante op 'phleomycin' -bevattende media, is die S. bayanus EC 1118 transformante in nieselektiewe kondisies opgegroei sodat verlies van die 'phleomycin' plasmied kon plaasvind. Integrasie van die mae1 en mae2 uitdrukkingskassette is bevestig en kleinskaalse fermentasies in sintetiese en druiwemos het 'n vinnige en doeltreffende afbraak van appelsuur in die vroeë fases van die alkoholiese fermentasie getoon. Integrasie en uitdrukking van die malo-alkoholiese gene in S. bayanus ECl118 het geen nadelige effek op die fermentasievermoë van die gis getoon nie, terwyl sensoriese en chemiese ontleding van die Chardonnay wyne 'n verbetering in aroma relatief tot die kontrole wyne getoon het, met die afwesigheid van enige afgeure.

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 (MScAgric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: The production of quality wine is influenced by numerous factors of which grape quality is one of the most important factors. The production of quality wine, however, is not possible without good winemaking techniques and effective quality control. Critical control points (CCP) during the winemaking process must be identified to ensure optimum wine quality. Grape must is a complex medium that contains different micro-organisms which can be either beneficial or negative to wine quality, depending on the physical and chemical conditions that prevail in the must. Yeasts are responsible for alcoholic fermentation, lactic acid bacteria (LAB) for malolactic fermentation (MLF) and acetic acid bacteria (AAB) for the production acetic acid from ethanol. Yeasts and certain LAB can also produce acetic acid and thereby increasing the volatile acidity (VA) of wine. These micro-organisms can influence each other in complex fashions by competing for growth nutrients and by producing inhibitory substances. Most winemakers nowadays use commercial yeast strains to inoculate wine fermentations. This, however, does not assure a problem-free fermentation and cases of stuck and sluggish fermentations are annually reported worldwide. In these or most cases fermentation takes longer than 21 days to complete and the wine contains a residual sugar concentration of more than 4 g/L, which can be utilised by wine spoilage micro-organisms such as certain bacteria and other wild yeasts. Stuck and sluggish fermentations also increase the chances of oxidation due to the absence of the protective CO2 layer on the surface of the wine, which is formed during alcoholic fermentation. Another effect of stuck and sluggish fermentations is that valuable tank space is wasted due to the unexpected time consumption of these fermentation problems. Many factors during the winemaking process can be responsible for stuck and sluggish fermentations. In this thesis the different factors is discussed with the emphasis on the effect of the yeast strain. The way that certain yeast strains influence AAB and LAB numbers during fermentation and MLF through the production of inhibiting by-products such as medium chain fatty acids has not been investigated in detail in the past. Certain fungicides and pesticides that are used in vineyards to control pests (e.g. mildew) contain copper which can be inhibiting to yeast growth and alcoholic fermentation. Legal limits and withholding periods on these sprays are not always strictly obeyed and can lead to stuck and sluggish fermentations. This motivated us to evaluate the growth and fermentation activities of a selection of commercial wine yeasts in the presence of copper levels in the range of maximum legal limits. The effect of these commercial strains on the LAB and AAB numbers during alcoholic fermentation and MLF were also investigated. Our results showed that there was no significant difference on numbers of the AAB obtained from fermentations inoculated with different commercial wine yeast strains. However, with regards to the LAB numbers, one of the strains produced significantly more sulphur dioxide (SO2), which led to the inhibition of MLF in that wine. Our results further indicated which commercial yeast strains were capable of effectively fermenting high sugar musts and which strains were less effective. From the strains tested VIN13, N96 & L2056 were able to utilize fructose more effectively than NT50, RJ11 & D80. We could further distinguish between yeast strains that produced the lowest (VIN13 & RJ11) and the highest (WE372, NT50 & L2056) VA concentrations in must containing high sugar levels. Strains that were more tolerant against high copper levels were also identified. We tested six yeast strains in must with added copper (0.25 mM cu2+) in the form of CuSO4 .H2O. Three Cu2+-tolerant (D80, Collection Cepage Cabernet & NT50) yeast strains were distinguished from three less Cu2+-tolerant yeast strains (VIN13, NT112 & RJ11). This study made a valuable contribution in knowledge gained about commercially available wine yeast strains that can ferment effectively under certain stress conditions. Research such as this, where wine yeasts are evaluated to ferment more effectively during strenuous winemaking conditions, will be very beneficial to winemakers.
AFRIKAANSE OPSOMMING: Die produksie van gehalte wyn word deur verskillende faktore beïnvloed waarvan druifkwaliteit seker die belangrikste is. Die produksie van gehalte wyn is egter nie moontlik sonder goeie wynmaaktegnieke en effektiewe kwaliteitsbeheer nie. Kritieke kontrole punte (KKP) tydens die wynmaakproses moet dus geïdentifiseer word om sodoende ‘n verlaging in wynkwaliteit te vermy. Druiwemos het ‘n komplekse mikrobiologiese samestelling en bestaan uit verskillende mikroörganismes wat vooren nadelig vir wynkwaliteit kan wees, afhangende van die fisiese en chemiese toestande wat in die mos bestaan. Giste is verantwoordelik vir alkoholiese fermentasie, melksuurbakterieë (MSB) vir appelmelksuurgisting (AMG) en asynsuurbakterieë (ASB) vir die produksie van asynsuur vanaf etanol. Asynsuur word egter ook deur giste en MSB geproduseer en dra so by tot die vlugtige suurheid (VS) van ‘n wyn. Hierdie mikroörganismes kan mekaar op komplekse wyses beïnvloed deur o.a. te kompeteer vir voedingstowwe asook deur die produksie van inhiberende verbindings. Die meeste wynmakers maak gebruik van kommersiële gisrasse om alkoholiese fermentasies mee uit te voer. Gevalle van sogenaamde slepende en gestaakte alkoholiese fermentasies, waar suiker nie volledig na etanol en CO2 omgeskakel word nie, kom egter nog gereeld in die wynbedryf voor. In sulke gevalle neem die fermentasie gewoonlik langer as 21 dae om te voltooi met ‘n suiker konsentrasie van meer as 4 g/L wat in die wyn oorbly. Dit is nadelig vir wynkwaliteit aangesien dit nie net die kanse vir bederf deur bakterieë en giste verhoog nie, maar ook die kanse vir oksidasie verhoog a.g.v. die verlies van die beskermende CO2 lagie bo-oor die wyn. Hoe sekere gisrasse, ASB en MSB getalle gedurende fermentasie en AMG beïnvloed deur die produksie van inhiberende verbindings soos medium ketting vetsure en SO2, is ook nie baie in die verlede ondersoek nie. Sommige spuitstowwe wat gebruik word in die bekamping van swamsiektes bevat koper wat inhiberend kan wees vir gisgroei en alkoholiese fermentasie. Wetlike maksimum limiete en onthoudingsperiodes op spuitstofresidue word egter nie altyd gehoorsaam nie en kan lei tot slepende en gestaakte fermentasies. Dit het ons gemotiveer om ‘n seleksie van kommersiële gisrasse te evalueer in terme van gisgroei en fermentasie in die teenwoordigheid van kopervlakke naby die maksimum limiet. Ons resultate het gewys dat daar nie noemenswaardige verskille in AAB getalle tydens alkoholiese fermentasie tussen behandelings met verskillende kommersiële gisrasse was nie. Een van die gisrasse het wel noemenswaardig meer SO2 geproduseer wat gelei het tot inhibering van AMG in hierdie wyn. Ons het verder uitgewys watter kommersiële gisrasse instaat is om meer effektief in hoër suiker mos te fermenteer en watter van die rasse minder suksesvol was. Ons het ook rasse geïdentifiseer wat meer weerstandbiedend is teen hoë kopervlakke in mos en sodoende groter kans op ‘n suksesvolle fermentasie sal hê in mos wat koperresidue bevat wat afkomstig is van sekere spuitstowwe. Die effek van die ASB en MSB getalle gedurende fermentasie en AMG is ook ondersoek. Ons resultate het verder gewys watter kommersiële gisrasse instaat was om mos met hoë suikervlakke meer effektief te fermenteer. Vam die gisrasse wat getoets was het VIN13, N96 & L2056 fruktose meer effektief benut as NT50, RJ11 & D80. Ons kon verder onderskei tussen gisrasse wat die laagste (VIN13 & RJ11) en die hoogste (WE372, NT50 & L2056) vlakke van VS produseer in mos met hoë inisiële suikervlakke. Gisrasse wat meer tolerant was teen koperresidue in mos is ook geidentifiseer. Ons het ses gisrasse getoets in mos met bygevoegde koper (0.25 mM Cu2+) in die vorm van CuSO4 .5H2O. Daar is onderskei tussen drie Cu2+-tolerante (D80, Collection Cepage Cabernet & NT50) en drie minder Cu2+-tolerante gisrasse (VIN13, NT112 & RJ11). Hierdie studie lewer ‘n waardevolle bydrae in die invordering van kennis oor kommersieel beskikbare wyngisrasse wat meer effektief sal fermenteer onder sekere streskondisies wat in mos voorkom. Inligting soos hierdie is belangrik om die wynmaker se keuse uit die reeks bestaande kommersiële gisrasse te vergemaklik.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes.
AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend. Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie- Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was ‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123. Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie van hierdie ensieme bevestig nie.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Brettanomyces bruxellensis has become of increasing interest over the past few decades yet this complex red wine spoilage yeast is still poorly understood and strain variance also leads to the contradictory results reported in literature. This yeast is responsible for the production of phenolic compounds, associated with off-flavours that render wine unpalatable. Sulphur dioxide (SO2) is the most commonly used antioxidant and antimicrobial preservative instrumental in the control of spoilage yeasts such as B. bruxellensis. However, its diploid/triploid genome is enriched for genes that provide the yeast a fortuitous advantage, under conditions permissive for growth, with genotype-dependent SO2 tolerance phenotypes observed among numerous strains. This study investigates the metabolic, physiological and genetic responses associated with SO2 exposure. It also explores the environmental cues responsible for the onset of non-SO2 induced morphological characteristics. These morphological characteristics were investigated using fluorescent probes and microscopy in the presence of SO2 and in the absence thereof, in YPD media. Pseudohyphae formation was observed to be a highly strain dependent feature and less pronounced in the presence of 0.6 mg/L molecular SO2. This study also reports on the metabolic response observed over a 3-week period, following exposure to SO2, in a synthetic wine medium. The following metabolites were consistently monitored during the course of the experiment: acetic acid, acetaldehyde, D-glucose and D-fructose. Utilization of sugars was retarded in the presence of SO2 for up to 10 days in the presence of 1.2 mg/L molecular SO2 and overproduction of acetaldehyde was prominent, with a peak at day 10. The study further highlights the expression profiles observed for the SSU1 gene (referring to SO2 tolerance) and the PAD gene (referring to production of volatile compounds) under SO2 induced conditions in SWM, using qRT-PCR. The co-involvement of increased acetaldehyde production and elevated gene expression were indicative of B. bruxellensis yeast adapting to the presence of molecular SO2, allowing survival of this fascinating yeast. Sequencing of the SSU1 and PAD genes suggests the probable existence of different alleles of these genes that could explicate SO2 tolerance and phenolic compound production associated differences among strains of this species.
AFRIKAANSE OPSOMMING: Hoewel Brettanomyces bruxellensis oor die afgelope paar dekades toenemende belangstelling gewek het, word hierdie komplekse rooiwynbederfgis steeds swak verstaan en lei rasvariasie ook tot teenstrydige resultate in die literatuur. Hierdie gis is verantwoordelik vir die produksie van fenoliese verbindings, wat geassosieer word met afgeure, wat die wyn onsmaaklik laat. Swaweldioksied (SO2) is die algemeenste preserveermiddel wat, weens antioksidant- en antimikrobiese eienskappe, instrumenteel in die beheer van bederforganismes, soos B. bruxellensis, gebruik word. Nogtans is die diploïede/triploïede genoom vir gene verryk, wat die gis ‘n toevallige voordeel bied tydens ongunstige toestande, met genotipe-afhanklike SO2 weerstandbiedende fenotipes wat onder verskeie rasse waargeneem word. Hierdie studie ondersoek die metaboliese, fisologiese en genetiese reaksies tydens SO2-blootstelling. Dit bestudeer verder die omgewingsleidrade wat vir die aanvang van die nie-SO2 geassosiseerde morfologiese eienskappe verantwoordelik is. Hierdie morfologiese eienskappe is ondersoek met behulp van fluoresserende bakens en mikroskopie in die teenwoordigheid van molekulêre SO2 en, in die afwesigheid daarvan, in YPD-medium. Pseudohyphae-vorming is as ʼn baie rasspesifieke eienskap waargeneem en is minder prominent in die teenwoordigheid van molekulêre SO2. Hierdie studie rappoteer ook oor die metaboliese reaksies waargeneem oor ‘n 3-weke tydperk, na blootstelling aan SO2, in ‘n sintetiese wynmedium. Die volgende metaboliete was voordurend gemonitor tydens die verloop van die eksperiment: asynsuur, asetaldehied, D-glukose en D-fruktose. Benutting van die suikers is in die teenwoordigheid van SO2 vertraag en oorproduksie van asetaldehied is prominent waargeneem. Hierdie studie beklemtoon verder die uitdrukkingsprofiele vir die SSU1-geen (verwys na SO2-weerstandbiedendheid) en die PAD-geen (verwys na die produksie van vlugtige verbindings) in SO2-geïnduseerde toestande in SWM, met behulp van qRT-PCR. Die gesamentlike invloed van beide verhoogde asetaldehied produksie en verhoogde uitdrukking van gene, was beduidend van B. bruxellensis-gis wat aanpas in die teenwoordigheid van molekulêre SO2, wat die oorlewing van hierdie fassinerende gis verseker. Volgordebepaling van die SSU1- en PAD-geen dui daarop dat daar waarskynlik meer as een verskillende alleel vir dié gene bestaan, wat die SO2-verdraagsaamheid en produksie van fenoliese verbindings, wat tans tussen verskeie spesies teenwoordig is, kan verduidelik.

Orientadores: Lucimara Gaziola de La Torre, Reinaldo Gaspar Bastos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A microfluídica é uma ciência que opera em pequenos volumes de fluídos dentro de canais em dimensões de micrômetros (10-6 m). Estes sistemas permitem controlar moléculas no espaço e no tempo, gerando resultados rápidos e confiáveis num sistema precisamente controlado e capaz de mimetizar ambientes celulares. Os dispositivos microfluídicos apresentam uma diversidade de geometrias aplicáveis para diversas áreas de pesquisas, sendo que a capacidade de formar gradientes permite avaliar as condições e o desempenho celular microbiano. Assim, este trabalho teve como objetivo desenvolver dispositivos microfluídicos capazes de formar gradiente de concentração difusivo e investigar sua aplicabilidade em bioprocessos. Diante disso, foram propostos três modelos de dispositivos usando materiais biocompatíveis: (i) dispositivo em base de vidro, denominado de Vidro-vidro; (ii) em base de vidro e poli dimetilsiloxano (PDMS), chamado de Vidro-PDMS e (iii) vidro e PDMS modificado quimicamente para tornar a superfície hidrofílica, Vidro-mPDMS. Os três dispositivos foram avaliados quanto à capacidade de formação de gradiente de concentração difusivo, os quais apresentaram um perfil linear. Além disso, validou-se o estudo do comportamento de Saccharomyces cerevisiae ATCC 7754 num gradiente de concentração de glicose de 0 a 40 g/L de glicose, sendo usado o dispositivo vidro-vidro. Foi observado que houve crescimento de células ao longo das câmaras microfluídicas, e isso possibilitou na determinação de parâmetros cinéticos, os quais não apresentaram diferença estatisticamente significativa com o cultivo em batelada convencional. As condições da microfluídica possibilitaram também a determinação da cinética de Monod, usando menores intervalos de gradiente. Portanto, este dispositivo microfluídico mostrou-se uma ferramenta com potencial para investigar comportamento celular frente à diferença de concentração e contribuirá para a otimização de bioprocessos através da determinação de parâmetros cinéticos
Abstract: Microfluidic is a science that operates in small amounts of fluids inside channels in dimensions of micrometers (10-6 m). These systems allow the precise control of molecules in space and time, generating fast and reliable results and it can also be used to mimics environment cellular . Microfluidic devices can be produced in diversity of geometries, it can be applied in several scientific areas and especially the formation of concentration gradients can be used to evaluate conditions and performance of microbial cell. Therefore, this work had the objective to develop microfluidic devices that are able to generate diffusive concentration gradients and investigate their applicability in bioprocesses. In this context, we propose three models of microfluidics devices using biocompatible materials: (i) Glass-based device, named glass-glass; (ii) glass and poli dimetilsiloxane (PDMS) based device, Glass-PDMS and (iii) glass and chemically modified PDMS (hydrophilic surface), Glass-mPDMS. The three devices were evaluated by their capacity of generating difusive concentration gradient, demonstrating linear concentration profile. Furthermore, the behavior of Saccharomyces cerevisiae ATCC 7754 inside of glucose concentration gradient ranging from 0 to 40 g/L were validated, using the glass-glass device . It was observed that cell growth along the microfluidic chambers, having determined the kinetic parameters, which was considered statistically similar to conventional batch cultivation. Conditions of microfluidics also allowed determination of the Monod kinetic, using smaller intervals gradient Therefore, the use of concentration gradient in microfluidic device is a potential tool for investigate of microbial cell behavior against the concentration difference and it can contribute to the optimization of bioprocesses through the determination of kinetic parameters
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestra em Engenharia Química

O óxido de etileno é um agente esterilizante dos mais importantes para materiais termo-sensíveis, dentre eles os artigos médico-hospitalares. Fios para sutura cirúrgica foram submetidos à exposição, em autoclave, a misturas gasosas com capacidade anti-microbiana, monitorando-se o processo através de Bacillus subtilis como indicador biológico. O objetivo deste estudo foi determinar a relação da letalidade do ciclo em função dos parâmetros de processo temperatura, tempo e pressão, expressando-a através de equações polinomiais decorrentes de modelo de ajuste pelo método da regressão linear múltipla. Os resultados analíticos e suas representações gráficas permitiram delimitar a região de letalidade, com simultânea confirmação da manutenção das características físicas farmacopeicas dos fios para sutura cirúrgica. Concluiu-se que o método estatístico empregado é adequado para a obtenção de equações de previsibilidade da atividade anti-microbiana em função dos parâmetros de processo. Além disto, a integridade física dos artigos médico-hospitalares foi preservada, assegurando-se sua utilização eficaz e segura.
Ethylene oxide is one of the most important sterilizing agents for thermo-sensitive materials, including the medical-hospital products. Strings for surgical sutures were exposed by autoclave to gaseous mixtures with anti-microbial properties, being the process monitored by Bacillus subtilis as a biological indicator. The purpose of this study was to determine the relation of cycle lethality in terms of process parameters (temperature, time and pressure) expressing them through polynomial equations decurrent of an adjustment model, by the multiple linear regression method. The analytical results and its graphic representations enabled us to delimit the lethal region having simultaneously the confirmation that the physical pharmacopeia requirements of the strings for surgical suture are maintained. It was concluded that the statistical method used is adequate to obtain equations of foreseeable anti-microbial activity in terms of process parameters. Besides, the physical integrity of the medical/hospital articles was preserved, so asserting its safe and effective use.

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2016.
Microbial spoilage has been reported in various food products and this has led to increased food, fruit and beverage losses, thereby threatening economic growth, food safety and security. Furthermore, statistics have shown that more than 30% of agricultural produce in developing countries, mostly in Africa, is lost owing to microbial spoilage. Beverages, food and fruits are predominant contributors to the South African export market. In recent years, contamination of these products resulting in spoilage has been a problem, although partial spoilage control has been achieved using chemical preservatives such as dimethyl dicarbonate, sodium benzoate, potassium sorbate, and sulphur dioxide (SO2). However, prolonged exposure to these chemical preservatives can cause human health problems such as skin and/or eyesight damage, muscle and stomach pain, cardiovascular disease and the impairment of brain function. To mitigate such health concerns, biologically benign alternatives are deemed suitable, providing the rationale for this study.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007.
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the quality and wholesomeness of the final product. Yeasts are primarily responsible for alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is conducted by lactic acid bacteria. These bacteria are important in winemaking and can have a positive or negative effect on the wine quality. Biogenic amines are one of the compounds produced by lactic acid bacteria, which affect the hygienic quality and wholesomeness of the wine negatively and directly pose a health risk to the consumer. The demand of consumers for higher quality and healthier foods has led to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide variety of food products, such as cheese, dried sausage, sauerkraut, fishery products, chocolates, wine and beer. This thesis focussed on the presence of biogenic amines in wine. The first objective of the study was to determine the ability of lactic acid bacteria isolated from South African wine to produce biogenic amines, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. The results obtained showed that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be the lactic acid bacteria responsible for tyramine and putrescine production and that it can contribute significantly to the overall biogenic amine content in wines. The results also suggest that amine production is strain dependent and not species specific. None of the lactic acid bacteria tested had the ability to produce histamine or cadaverine. It is important to remember that the ability of the lactic acid bacteria to produce biogenic amines has only been investigated in synthetic media and that it does not necessarily imply similar behaviour in wine. Wine represents a complex environment with a wide number of factors influencing microbial growth and decarboxylase activity and, thus, further investigation is necessary to determine if these amine-producing bacteria behave similarly in wine conditions. In addition, the polymerase chain reaction (PCR) amplification method was used for the identification of the tyrosine decarboxylase (TOe) gene in some of the tyramine-producing lactic acid bacteria. This was followed by the sequencing of the amplified products, which are partial TOe gene sequences, of two L. brevis strains and of a L. hilgardii strain. Only one tdc gene sequence has been described for bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis lOEB 9809 was described. An amino acid sequence alignment of the three TOe gene fragments, obtained in this study, with the known TOe gene fragment of L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of relatedness and conserved regions. To meet consumer demands, procedures are necessary to prevent the formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species involved in the winemaking process. Another possible way would be to develop a rapid detection method for bacteria carrying amino acid decarboxylase genes. The results of this study provide knowledge about which lactic acid bacteria in the winemaking process could contribute to the production of biogenic amines and the sequencing of additional partial TOe genes could possibly assist in the development of a rapid detection method for tyramine-producing lactic acid bacteria in food products.
AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê. Biogeniese amiene is een van die komponente wat deur melksuurbakterieë geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade, wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in wyn. Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid- Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde gedrag in wyn sal toon. Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809 en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van amiene in voedselprodukte voorkom word. Een manier van voorkoming is om amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte bydra.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or regarded as undesired yeasts in wine. However, these yeasts have recently come under investigation for their positive contribution towards wine aroma especially when used in sequential or co-inoculated fermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number of enzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases. The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction, as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content of the wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may have an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Although many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no attempts have been made to characterize these enzymes. This study set out to isolate and characterize genes encoding extracellular aspartic proteases from non-Saccharomyces yeasts. An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts, isolated from grape must, was performed. The aspartic protease-encoding genes of two non- Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, were isolated and characterized by in silico analysis. The genes were isolated by employing degenerate and inverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1. The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. The MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular weight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative protein with a predicted molecular weight of 39 kDa. These features are typical of extracellular aspartic proteases. The deduced protein sequences showed less than 40% homology to other yeast extracellular aspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the gene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced in media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was available. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape juice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in the presence of grape juice proteins, which was expected since these proteins are present in the natural habitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 other M. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was also visualized on plates. As far as we know, this is the first report on the genetic characterization of secreted aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the groundwork for further investigations.
AFRIKAANSE OPSOMMING: Nie-Saccharomyces giste is voorheen met wynbederf geassosieer en hul teenwoordigheid in wyn is ongewens. Hierdie giste is onlangs ondersoek vir hulle positiewe bydrae tot wyn aroma, in veral sekwensiële en ko-inokulerings met Saccharomyces cerevisiae. Sommige van die nie-Saccahromyces giste skei ‘n verskeidenhied ensieme af wat moontlik vir die wynmaker van nut kan wees. Een groep van hierdie ensieme is die aspartiese suurproteases. Soos deur sommige navorsers aangetoon word, kan die proteases die vorming van proteïenwaasverlaging, terwyl dit terselfdertyd die assimilerende stikstofinhoud van die wyn vir die gebruik en groei van fermentasie-mikroörganismes verhoog. Die proteases kan moontlik ook ‘n indirekte uitwerking op die aromaprofiel van die wyn hê deur die vrystelling van aminosure wat as aromavoorlopers dien. Alhoewel baie studies gedoen is wat die ekstrasellulêre teenwoordigheid van proteases bevestig in nie-Saccharomyces giste wat van druiwesap/wyn afkoms is, is daar geen dokumentasie oor die genetiese karakterisering van hierdie ensieme beskikbaar nie. Die doel van hierdie studie was om gene wat aspartiese proteases in nie-Saccharomyces giste enkodeer, te isoleer en gedeeltelik te karakteriseer. ‘n Versameling van 308 Saccharomyces en nie-Saccharomyces giste wat uit druiwe sap geïsoleer is, is gesif vir ensiematiese aktiwiteit deur plaattoetse uit te voer. Twee gene wat aspartiese protease enkodeer, is geïsoleer van twee nie-Saccharomyces giste. Dit hetpositief gedurende die aktiwiteitstoetse getoets en is deur in silico–analise gekarakteriseer. Die gene is deur die uitvoering van gedegenereerde en inverse PKR geïdentifiseer. Een geen is vanaf Metschnikowia pulcherrima IWBT Y1123 geïsoleer en is MpAPr1 genoem, terwyl die ander van Candida apicola IWBT Y1384 geïsoleer en CaAPr1 genoem is. Die MpAPr1-geen is 1137 bp lank en enkodeer ‘n proteïen wat uit 378 aminosure bestaan met ‘n voorspelde molekulêre massa van 40.1 kDa. Daar teenoor is die CaAPr1-geen 1101 bp lank en enkodeer vir ‘n proteïen wat uit 367 aminosure met ‘n molekulêre massa van 39 kDa bestaan. Hierdie eienskappe is kenmerkend van aspartiese protease. Die afgeleide proteïenvolgorde het minder as 40% homologie met ander ekstrasellulêre aspartiese proteases vertoon, wat dui op die nuwigheid van hierdie ensieme. Die MpAPr1-geen is heterologies in S. cerevisiae YHUM272 uitgedruk en dit het bevestig dat die geen inderdaad ‘n ekstrasellulêre aspartiese protease enkodeer. Die MpAPr1-geen is uitgedruk in media wat alleenlik proteïen as stikstofbron bevat het, terwyl dit onderdruk is in gevalle waar ‘n verkose stikstofbron beskikbaar was. Die geen is uitgedruk in die teenwoordigheid van kaseïen, BSA en proteïene afkomstig vanaf druiwesap en in die teenwoordigheid van ammoniumsulfaat onderdruk. Die hoogste uitdrukking was in die teenwoordigheid van druifproteïene. Hierdie proteïene is teenwoordig in die natuurlike habitat van die gis en is dus dalk ‘n bekende stikstofbron vir die gis. ‘n Genetiese sifting het die teenwoordigheid van die MpAPr1-geen in 12 ander M. pulcherrima–rasse, wat ook van wynkundige oorsprong is, bevestig. Die aspartiese protease-aktiwiteit van die 12 rasse is ook op agarplate waargeneem. Na ons wete, is dit die eerste verslag oor die genetiese karakterisering van afgeskeide aspartiese proteases van nie- Saccharomyces giste van wynkundige oorsprong en verskaf die grondslag vir verdere ondersoek.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important quality factors, but these can be influenced by many factors, such as grape-derived compounds that exist as free volatiles and also as glycosidically bound. The chemical composition of wine is determined by factors such as grape variety, geographic position, viticulture condition, microbial ecology of the grape and the winemaking process. The varietals aroma is determined by both the volatile and the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene derivatives, which are naturally present in the wine. Monoterpenes are very important in the flavour and aroma of grapes and wine. They can be found in grapes and wine either in the free, volatile and odorous form, or in the glycosidically-bound, non-volatile and non-odorous form. The ratio of glycosidically-bound compounds to free aroma compounds is very high in the Gewürztraminer, Muscat and Riesling cultivars in particular. The glycosidic bonds can be hydrolysed either by the acid method or by using enzymes. The acid method is disadvantageous because it can modify the monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying the aroma character. The enzyme method of breaking the glycosidic bonds occurs in two successive steps: initial separation of glucose from the terminal sugar by a hydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase, depending on the aglycone moiety), followed by the breaking of the bond between the aglycone and glucose by β-glucosidase. The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera), bacterial, yeast or fungal sources. Most of the enzymes produced by these sources are not functional under the winemaking conditions of low pH, low temperature, high glucose and high ethanol content. However, β-glucosidases from fungal origins, particularly from Aspergillus spp., are tolerant of winemaking conditions. The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii (BGLA), which is linked to the cell wall and the free β-glucosidase, was to determine if anchoring the enzyme to the cell wall will increase the activity of the enzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG 97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmids pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1 anchor domains. All the plasmids were genome-integrated and expressed in the reference strain Saccharomyces cerevisiae 303-1A. All the transformants were grown in 2% cellobiose and showed higher biomass production compared to the reference strain. β-Glucosidase activity was also assayed and transformed strain W16 showed a fourfold increase in activity compared to the reference strain. There was no significant increase in the activity of the other transformed strains, W24, W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was 40ºC. The recombinant strains together with the reference strain were used to make wine from Gewürztraminer grapes. The levels of numerous monoterpenes were enhanced in the resultant wines. The concentration of nerol was increased fourfold, that of citronellol twofold, and geraniol was 20% higher than in the wild type. There was also an increase in the levels of linalool and a-terpinol, but this was not significant. In wines produced with W97, W98 and W24, monoterpene levels did not show a significant difference. In future, the expression of the W16 expression cassette in an industrial wine yeast strain could be performed. In combination with the production of enzymes such as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which are involved in the first step of enzymatic hydrolysis, this wine strain could release the bound monoterpenes and enhance the aroma of the wine.
AFRIKAANSE OPSOMMING: Wyn is ‘n komplekse medium. Wynaroma, -geur en -kleur is belangrike kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan wees of glikosidies gebind is. Die chemiese samestelling van wyn word bepaal deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande, mikrobiese ekologie van die druif en die wynbereidingsproses. Die variëteitsaroma word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene, norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom. Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn. Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in glikosidiesgebinde, nie-vlugtige en nie-geurige vorms. Die verhouding van glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die Gewürztraminer-, Muscat- en Riesling-kultivars. Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode gehidroliseer word. Die nadeel van die suurmetode is dat dit monoterpene kan modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter modifiseer nie. Die ensiemmetode waarmee die glikosidiese verbinding afgebreek word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose van die terminale suiker deur ‘n hidrolase (a-L-arabinofuranosidase, a-Lramnosidase of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur die verbreking van die verbinding tussen die aglikoon en glukose deur β- glukosidase. Die β-glukosidase-ensiem kan vanaf ‘n verskeidenheid plant- (Vitis vinifera), bakterie-, gis- en swambronne verkry word. Die meerderheid van die ensieme wat deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie. β- Glukosidase vanaf ‘n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter wynbereidingstoestande verdra. Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand geanker is. Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie studie gebruik. BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is gekoppel aan CWP2, en in pDLG 98 is dit aan AGa1-ankergebiede gekoppel. Al die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A genoomgeïntegreer en uitgedruk. Al die transformante is in 2% sellobiose gegroei en het hoër biomassaproduksie as die verwysingsras getoon. β-Glukosidaseaktiwiteit is ook geëssaieer en die getransformeerde ras W16 het ‘n viervoudige verhoging in aktiwiteit in vergelyking met die verwysingsras getoon. Daar was geen noemenswaardige verhoging in die aktiwiteit van die ander getransformeerde rasse, W24, W97 en W98, nie. Ensimatiese karakterisering vir optimum-pH en - temperatuur is gedoen – vir al die rasse was die optimum-pH 4 en die optimumtemperatuur 40ºC. Die rekombinante rasse, tesame met die verwysingsras, is gebruik om wyn met Gewürtztraminer-druiwe te maak. Die vlakke van talryke monoterpene is in die gevolglike wyne verhoog. Die konsentrasie van nerol is viervoudig verhoog, dié van sitronellol tweevoudig, en geraniol was 20% hoër as in die wilde tipe. Daar was ook ‘n verhoging in die vlakke van linaloöl en a-terpinol, maar hierdie verhoging was nie noemenswaardig nie. In wyne wat met W97, W98 en W24 gemaak is, het die monoterpeenvlakke nie ‘n noemenswaardige verskil getoon nie. In die toekoms sal die uitdrukking van die W16-uitdrukkingskasset in ‘n industriële wyngisras uitgevoer kan word. In kombinasie met die produksie van ensieme soos a-arabinofuranosidase, a-ramnosidase, β-apiosidase, wat in die eerste stap van ensimatiese hidrolise betrokke is, sal hierdie wyngisras die gebonde monoterpene kan vrylaat en die aroma van die wyn kan verbeter.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Fermentation is a complex process in which raw materials are transformed into high-value products, in this case, grape juice into wine. In this modern and economically competitive society, it is increasingly important to consistently produce wine to definable specifications and styles. Process management throughout the production stage is therefore crucial to achieve effective control over the process and consistent wine quality. Problematic wine fermentations directly impact on cellar productivity and the quality of wine. Anticipating stuck or sluggish fermentations, or simply being able to foresee the progress of a given fermentation, would be extremely useful for an enologist or winemaker, who could then take suitable corrective steps where necessary, and ensure that vinifications conclude successfully. Conventional methods of fermentation monitoring are time consuming, sometimes unreliable, and the information limited to a few parameters only. The current effectiveness of fermentation monitoring in industrial wine production can be much improved. Winemakers currently lack the tools to identify early signs of undesirable fermentation behaviour and to take preventive actions. This study investigated the application of Fourier transform mid infrared (FT-IR) spectroscopy in transmission mode, for the quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The major research objectives were firstly to establish a portfolio of quantitative calibration models suitable for quantification of the major quality determining parameters in fermenting must. The second major research objective focused on a pilot study aimed at exploring the use of off-line batch multivariate statistical process control (MSPC) charts for actively fermenting must. This approach used FT-IR spectra only, for the purpose of qualitative monitoring of alcoholic fermentation in industrial wine production. Towards these objectives, a total of 284 industrial-scale, individual, actively fermenting tanks of the seven major white cultivars and blends, and nine major red cultivars, of Namaqua Wines, Vredendal, South Africa, were sampled and analysed with FT-IR spectroscopy and appropriate reference methods during vintages 2007 to 2009. For the quantitative strategy, partial least squares regression (PLS1) calibration models for determination of the classic wine parameters ethanol, pH, volatile acidity (VA), titratable acidity (TA) and the total content of glucose plus fructose, were redeveloped to provide a better fit to local South African samples. New PLS1 models were developed for the must components glucose, fructose and yeast assimilable nitrogen (YAN), all of which are frequently implicated in problem fermentations. The regression statistics, that included the standard error of prediction (SEP), coefficient of determination (R2) and bias, were used to evaluate the performance of the redeveloped calibration models on local South African samples. Ethanol (SEP = 0.15 %v/v, R2 = 0.999, bias = 0.04 %v/v) showed very good prediction and with a residual predictive deviation (RPD) of 30, rendered an excellent model for quantitative purposes in fermenting must. The models for pH (SEP = 0.04, R2 = 0.923, bias = -0.01) and VA (SEP = 0.07 g/L, R2 = 0.894, bias = -0.01 g/L) with RPD values of 4 and 3 respectively, showed that the models were suitable for screening purposes. The calibration model for TA (SEP = 0.35 g/L, R2 = 0.797, bias = -0.004 g/L) with a RPD of 2, proved unsatisfactory for quantification purposes, but reasonable for screening purposes. The calibration model for the total content of glucose plus fructose (SEP = 0.6.19 g/L, R2 = 0.993, bias = 0.02 g/L) with a RPD of 13, showed very good prediction and can be used to quantify total glucose plus fructose content in fermenting must. The newly developed calibration models for glucose (SEP = 4.88 g/L, R2 = 0.985, bias = -0.31 g/L) and fructose (SEP = 4.14 g/L, R2 = 0.989, bias = 0.64 g/L) with RPD values of 8 and 10 respectively, also proved fit for quantification of these important parameters. The new calibration models of ethanol, total glucose plus fructose; and glucose and fructose individually, showed an excellent relation to local South African samples and can be easily implemented by the wider wine industry. Two calibration models were developed to determine YAN in fermenting must by using different reference methods, namely the enzyme-linked spectrophotometric assay and Formol titration method, respectively. The results showed that enzyme-linked assays provided a good quantitative model for white fermenting must (SEP = 14.10 mg/L, R2 = 0.909, bias = -2.55 mg/L, RPD = 6), but the regression statistics for predicting YAN in red fermenting must, were less satisfactory (data not shown). The Formol titration method could be used successfully in both red- and white fermenting must (SEP = 16.37 mg/L, R2 = 0.912, bias = -1.01 mg/L, RPD = 4). A minor, but very important finding was made with respect to the storage of must samples that were taken from tanks, but that could not immediately be analysed with FT-IR spectroscopy or reference values. Principal component analysis (PCA) of frozen samples showed that must samples could be stored frozen for up to 3 months and still be used to expand the calibration sample sets when needed. Therefore, samples can be kept frozen to a later stage if immediate analyses are not possible. For the purpose of the pilot study that focused on the use of FT-IR spectroscopy for qualitative off-line monitoring of alcoholic fermentation, a total of 21 industrial-scale fermentation tanks were monitored at 8- or 12-hourly intervals, from the onset of fermentation to complete consumption of the grape sugars. This part of the work excluded quantitative data, and only used FT-IR spectra. MSPC charts were constructed on the PLS scores of all the FT-IR spectra taken at the various time intervals of the different batches, using time as the y-variable. The primary aim of this research objective was to evaluate if the PLS batch models could be used to discriminate between normal and problem alcoholic fermentations. The models that were constructed clearly showed the variations in patterns over time, between red- and white wine alcoholic fermentations. One Colombar tank that was fermented at very low temperature in order to achieve a specific wine style, was characterised by a fermentation pattern that clearly differed form the rest of the Colombar fermentations. This atypical fermentation was identified by the batch models constructed in this study. PLS batch models over all the Colombar fermentations clearly identified the normal and problem fermentations. The results obtained in this study showed that FT-IR spectroscopy showed great potential for effective quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The work done in this project resulted in the development of a portfolio of calibration models for the most important quality determining parameters in fermenting must. The quantitative models were subjected to extensive independent test set validation, and have subsequently been implemented for industrial use at Namaqua Wines. Multivariate batch monitoring models were established that show good discriminatory power to detect problem fermentations. This is a very useful diagnostic tool that can be further developed by monitoring more normal and problem fermentations. Future work in this regard, will focus on further optimisation and expansion of the quantitative and qualitative calibration models and implementation of these in the respective wineries of Namaqua Wines.
AFRIKAANSE OPSOMMING: Fermentasie is ‘n komplekse proses waartydens rou material getransformeer word na produkte van hoë waarde, in hierdie geval, druiwesap na wyn. In die huidige ekonomies-kompeterende samelewing, is dit al hoe meer belangrik om volhoubaar wyn te produseer wat voldoen aan definieerbare spesifikasies en style. Goeie prosesbestuur tydens die wynproduksie stadium is baie belangrik om herhaalbaarheid en gehaltebeheer te verseker. Problematiese wynfermentasies het ’n direkte impak op beide kelderproduktiwiteit en wynkwaliteit. Die voorkoming van slepende- of steekfermentasies, of selfs net om probleme te voorsien, sou uiters bruikbaar wees vir ‘n wynkundige of wynmaker, wat dan die toepaslike regstellende stappe kan neem waar nodig, om te verseker dat die wynbereiding suksesvol voltooi word. Konvensionele metodes van monitering van alkoholiese fermentasie is tydrowend, soms onbetroubaar en die inligting beperk tot ‘n paar parameters. Die huidige effektiwiteit van fermentasie monitering in industriële wynproduksie kan heelwat verbeter word. Wynmakers ervaar tans ’n behoete aan tegnologië wat die vroeë tekens van ongunstige fermentasiepatrone kan identifiseer, en hul doeltreffendheid om moontlike regstellende aksies te neem, is dus beperk. Hierdie studie het die toepassing van Fourier transformasie mid-infrarooi (FT-IR) spektroskopie in transmissie, ondersoek met die oog op kwantitatiewe en kwalitatiewe monitering van alkoholiese gisting tydens industriële wynproduksie. Die vernaamste navorsingsdoelwitte was eerstens om ’n portefeulje van kwantitatiewe kalibrasiemodelle te vestig, wat geskik is om die belangrikste kwaliteitsbepalende parameters in gistende mos te kwantifiseer. Die tweede hoofnavorsingsdoelwit was ’n loodsstudie wat ondersoek ingestel het na die opstel van multiveranderlike statistiese proseskontrole grafieke van aktief-gistende mos, met die oog op aflyn-kwalitatiewe monitering van alkoholiese gisting in industriële wynproduksie. Hiervoor is slegs FT-IR spektra gebruik. Vir die doel van hierdie studie is monsters van ’n totaal van 284 individuele, aktief-gistende tenke van die sewe hoof wit kultivars en hul versnydings en nege hoof rooi kultivars van Namaqua Wyne, Vredendal, Suid Afrika, geneem. Al die monsters is met toepaslike chemiese metodes en FT-IR spektroskopie analiseer tydens die parsseisoene van 2007 tot 2009. Vir die kwantitatiewe strategie is parsiële kleinste kwadraat (PKK1) kalibrasiemodelle vir die bepaling van die klassieke wynparameters etanol, pH, vlugtige suur (VS), titreerbare suur (TS) en die totale konsentrasie van glukose plus fruktose herontwikkel, om beter te pas op plaaslike Suid-Afrikaanse monsters. Nuwe PKK1 kalibrasiemodelle is ontwikkel vir die komponente glukose, fruktose en gis-assimileerbare stikstof, aangesien hierdie komponente gereelde aanduidings van probleemgisting is. Die regressiestatistieke het die standaardvoorspellingsfout (SVF), bepalingskoëffisiënt (R2) en sydigheid ingesluit en was gebruik om die prestasie van die herontwikkelde kalibrasiemodelle vir plaaslike Suid-Afrikaanse monsters te evalueer. Etanol (SVF = 0.15 %v/v, R2 = 0.999, sydigheid = 0.04 %v/v) het baie goeie regressiestatistiek getoon en met ‘n relatiewe voorspellingsafwyking (RVA) van 30, was dit ‘n uitstekende model vir kwantifisering in gistende mos. Die modelle vir pH en VS met RVA waardes van 4 en 3 onderskeidelik, is geskik vir semi-kwantitatiewe toepassings. Die kalibrasiemodel vir TS met ‘n RVA waarde van 2, was nie geskik vir akkurate kwantifisering nie, maar wel vir semikwantitatiewe analises. Die kalibrasiemodel vir die totale glukose plus fruktose inhoud in gistende mos, met ‘n RVA waarde van 13, het uitstekende regressiestatistiek gegee en is geskik vir akkurate kwantifiseringsdoeleindes. Die nuut-ontwikkelde kalibrasiemodelle vir glukose en fruktose, met RVA waardes van onderskeidelik 8 en 10, is geskik vir akkurate kwantifisering van hierdie belangrike parameters. Die kalibrasiemodelle vir etanol, totale glukose plus fruktose, en glukose en fruktose afsonderlik, het uitstekende korrelasies getoon met plaaslike Suid-Afrikaanse monsters en is gereed om toepassing te vind in die wyer wynindustrie. Twee kalibrasiemodelle is ontwikkel om gis-assimileerbare stikstof in gistende mos te bepaal, deur gebruik te maak van verskillende verwysingsmetodes van analise; hierdie metodes was ‘n ensiem-gekoppelde spektrofotometriese toets en die Formoltitrasie metode. Resultate het getoon dat goeie regressiestatistiek vir FT-IR spektroskopie-gebaseerde kalibrasiemodelle waar data wat met die ensiem-gekoppelde toetse verkry is, as verwysingwaardes gebruik is, in wit gistende mos (SVP = 14.10 mg/L, R2 = 0.909, sydigheid = -2.55 mg/L, RVA = 6), maar nie in rooi gistende mos nie. Die Formoltitrasie metode as verwysingsmetode, was geskik vir die ontwikkeling van goeie kalibrasiemodelle in beide rooi- en wit gistende mos (SVP = 16.37 mg/L, R2 = 0.912, sydigheid = -1.01 mg/L, RVA = 4). ’n Sekondêre, maar baie belangrike bevinding is gemaak met betrekking tot die stoor van mosmonsters wat geneem is van tenke, maar wat nie dadelik met die verwysingsmetodes en FT-IR spektroskopie analiseer kon word nie. Multiveranderlike hoofkomponentanalise op vars en gevriesde sapmonsters het getoon dat gevriesde monsters gebruik kan word om die kalibrasie datastel uit te brei, wanneer benodig. Dus, sapmonsters kan gevries word tot ’n later stadium as onmiddelike analises nie moontlik is nie. Vir die doel van die tweede navorsingsdoelwit van die studie, naamlik kwalitatiewe af-lyn monitering van alkoholiese fermentasie met FT-IR spektroskopie, is ‘n totaal van 21 industriëlegrootte fermentasietenks ge-monitor deur sapmonsters met 8- tot 12-uurlikse intervalle te trek, vanaf die begin van fermentasie, totdat al die druifsuiker gemetaboliseer is. Vir hierdie deel van die werk is die kwantitatiewe data nie gebruik nie; slegs die FT-IR spektra. Multiveranderlike statistiese proseskontrole grafieke is opgestel op grond van die PKK tellings wat bereken is op al die FT-IR spektra wat gemeet is by die verskillende tydsintervalle. Vir hierdie analise is tyd as y-veranderlike gebruik. Die vernaamste doel van hierdie ondersoek was om te evalueer of die PKK-gebaseerde modelle kon onderskei tussen normale en slepende gistings. Die modelle wat verkry is, het die variasie oor tyd in die fermentasiepatrone tussen wit- en rooiwyn fermentasies tydens alkoholiese gisting, duidelik uitgewys. Een Colombar tenk wat teen baie lae temperatuur gefermenteer is om ‘n spesifieke wynstyl te verkry, se fermentasiepatroon het aansienlik verskil van die ander Colombar tenks wat gemonitor is, en hierdie atipiese patroon is ook deur die kwalitatiewe modelle identifiseer. ‘n PKK model oor al die Colombar fermentasies kon duidelik tussen normale en slepende gistings onderskei. Die resultate wat in hierdie studie verkry is, het getoon dat FT-IR spektroskopie baie goeie potensiaal toon vir die aanwending van kwantitatiewe en kwalitatiewe monitering van alkoholiese fermentasie tydens industriële wynproduksie. Die werk wat in hierdie projek gedoen is, het gelei tot die vestiging van ‘n portefeulje van kalibrasiemodelle vir die belangrikste kwaliteitsbepalende parameters in fermenterende mos. Die kwantitatiewe modelle is baie deeglik getoets met onafhanlike toets datastelle, en daarna is die kalibrasiemodelle geimplementeer vir industriële gebruik by Namaqua Wyne. Multiveranderlike statistiese proseskontrole grafieke wat baseer is op data wat vanaf 21 verskillende fermentasietenks verkry is, het baie goeie potensiaal getoon om probleemfermentasies vroeg te identifiseer. Dié grafieke is ‘n baie nuttige diagnostiese hulpmiddel wat verder ontwikkel kan word om verskillende tipes probleemfermentasies te monitor. Toekomstige navorsing in hierdie konteks, sal toegespits word op die optimisering en uitbreiding van die kwantitatiewe en kwalitatiewe modelle, sowel as toepassing van die tegnieke in die onderskeie kelders van Namaqua Wyne.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006.
Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.