Academic literature on the topic 'Applied Microbiology and Biotechnology'

Células transformadas de E.coli DH5-&#945; expressando a proteína verde fluorescente (GFPuv, pico de excitação e emissão de 394nm e 509nm) foram submetidas a extração pelo método de partição de três fases (TPP) e o extrato obtido purificado por cromatografia de interação hidrofóbica (HIC). O objetivo principal deste trabalho foi estudar a termoestabilidade da GFPuv extraída, para avaliar a sua possível utilização como indicador biológico econômico, de resposta rápida e precisa para processos térmicos de esterilização utilizando o calor úmido. A estabilidade térmica da proteína foi estudada em diferentes soluções-tampão (acetato, fosfato e tris-HCI 10mM) no intervalo de valor de pH de 5,O a 9,0 e, em temperaturas entre 75&#176; e 95&#176;C. Os parâmetros de resistência térmica determinados foram: o tempo de redução decimal (Valor D - min), valor z (&#176;C), coeficiente Q10 e valor de energia de ativação (kcal/mol). A termoestabilidade da GFPuv, expressa em valor D, mostrou correlação linear para valores de pH &#8805; 5,50, em tampão acetato. Em tampão fosfato, para valores de pH &#8805; 7,50 a estabilidade térmica da proteína foi independente do valor de pH da solução. Em tampão tris-HCI, o valor D mostrou-se inconstante ao aumento do valor de pH da solução. No intervalo de temperatura estudada, em tampão acetato a GFPuv apresentou melhor termoestabilidade (Ea de 19,27 kcal/mol) do que em tampão fosfato (Ea de 26,18 kcal/mol ao valor de pH 6,S) e em tampão tris-HCI (Ea 28,19 kcallmol ao valor de pH 7,0). Em tampão acetato e tris-HCI ao valor de pH 7,0, a termoestabilidade da proteína mostrou-se equivalente. Entretanto, em tampão fosfato aos valores de pH 7,5 e 8,0 e em tampão tris-HCI aos valores de pH 8,0 e 8,5 a GFPuv apresentou menor estabilidade térmica A GFPuv apresenta potencialidade para ser utilizada como indicador biológico em processos térmicos que utilizam calor úmido às temperaturas inferiores a 100°C.<br>Transformed cells of Escheríchía coli DH5-&#945; expressing recombinant green fluorescent protein (GFPuv, excitation and emission peaks at 394nm and 509nm), were subjected to the three-phase partitioning (TPP) method and the release extracts were eluted through methyl HIC column with a buffer solution (10 mM Tris-HCI, 10mM EDTA, pH=8.0). The purpose of this work was to study the thermal stability of the TPP-extracted recombinant protein, GFPuv, to determine its utility as a quick, accurate and economical biological indicator for moist heat-treatments. The thermal stability of the extracted GFPuv was studied in different buffer solutions (acetate, phosphate and tris-HCI 10mM) in the range of pH between 5.0 and 9.0 and at temperature between 75-95°C. The thermal resistance parameters determinated were: decimal reduction times (D-values, min), z-value (&#1776C), Q<sub<10 coefficient and Activation Energy (Ea, Kcal/mol). The thermal stability of GFPuv, expressed in D-values, showed linear correlation for pH &#8805; 5.50 in acetate buffer. In phosphate buffer, for pH &#8805; 7.50 the thermal stability was independent of pH value. In tris-HCI buffer the D-value was shown variable with the increase of pH value. In the studied temperature range, the acetate buffer at pH 6.0 presented better thermal stability for GFPuv (Ea 19.27kcal/mol) than phosphate (Ea 26.18 kcal/mol at pH 6.5) and tris-HCI buffer (Ea 28.19 kcal/mol at pH 7.0). In acetate and tris-HCI buffers at pH 7.0, GFPuv showed equivalent thermal stability. However, GFPuv showed lower thermal stability in phosphate buffer at pH 7.5 and 8.0 and in tris-HCI buffer at pH 8.0 and 8.5. The TPP-extracted GFPuv has great potential to be applied as a biological indicator in moist heat processes at temperatures below 100°C.

Made available in DSpace on 2014-06-12T15:51:12Z (GMT). No. of bitstreams: 2 arquivo4477_1.pdf: 2619648 bytes, checksum: c371ffaaab9afa32bc9f96397ed74514 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005<br>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.<br>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.<br>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.<br>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.<br>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.