Le procédé de production d’éthanol à partir de biomasse lignocellulosique nécessite l’hydrolyse de cette dernière en sucres simples. Cette hydrolyse est le plus souvent réalisée par voie biologique grâce à des enzymes appelées cellulases. La production de ces enzymes représente cependant un verrou économique majeur au développement du procédé à grande échelle. Les cellulases sont généralement produites industriellement par le champignon filamenteux aérobie Trichoderma reesei, doté d’une forte capacité de sécrétion d’enzymes. Les cultures sont réalisées en bioréacteurs aérés et agités mécaniquement. Elles nécessitent de contrôler la concentration des substrats, ce qui requiert la maitrise de conditions hydrodynamiques et physicochimiques. En effet, le milieu de culture de T. reesei devient une suspension de cellules de champignons associées en filaments, de structure complexe, dont la viscosité augmente avec la concentration microbienne selon un comportement rhéofluidifiant. La viscosité est fonction de la morphologie du microorganisme qui peut, elle-même, varier avec les conditions de cultures. Cet accroissement de viscosité est un critère clef de l’extrapolation du procédé, car il affecte le transfert d’oxygène. Afin de maintenir une concentration en oxygène dissous suffisante, l’agitation et l’aération sont en général augmentées, entraînant un accroissement du cisaillement. Cet accroissement impacte en retour la morphologie du champignon, ralentit sa croissance puis diminue la production de cellulases. Ainsi, les conditions hydrodynamiques et rhéologiques engendrées au sein du bioréacteur sont complexes et variables dans le temps. L’interrelation entre conditions opératoires, morphologie, croissance du champignon et viscosité du moût de fermentation impose l’intégration de tous ces phénomènes pour l’optimisation du procédé, notamment à grande échelle. L’objectif de la thèse est de mettre en place une approche, visant à étudier au laboratoire la croissance de T. reesei et sa production d’enzymes, en reproduisant les contraintes hydrodynamiques associées aux conditions de fonctionnement des fermenteurs industriels. Pour ce faire, deux méthodologies originales ont été développées : une méthode de mesure de la viscosité du milieu, optimisée pour les champignons filamenteux, représentative des conditions rencontrées à grande échelle et qui s’appuie sur l’utilisation d’un rhéomètre rotatif équipé d’un rotor hélicoïdal ; une méthode d’analyse d’images associant un microscope motorisé et des algorithmes d’analyse d’images innovants, qui permet de générer des données sur la morphologie du champignon et d’identifier un critère morphologique pertinent basé sur le nombre de « trous » au sein d’un filament. Parallèlement à ces méthodes, différentes contraintes de cisaillement ont été mises en oeuvre en fermentation, afin de reproduire, à l’échelle du laboratoire, les conditions rencontrées à l’échelle industrielle. Ces outils ont été utilisés conjointement et validés lors de cultures non conventionnelles mimant les conditions industrielles en termes de cisaillement. Ils ont permis d’identifier un critère représentatif du cisaillement (EDCF) et d’établir, à partir de ce critère, des corrélations capables de prédire la viscosité du moût de fermentation, le taux de croissance maximum du microorganisme ainsi que certains paramètres morphologiques de la souche. De façon originale, ces corrélations déterminées à l’échelle du laboratoire ont été validées par des mesures effectuées à l’échelle industrielle. Au final, l’approche développée permet d’identifier au plus tôt les contraintes d’extrapolation à ne pas dépasser, afin d’orienter les choix technologiques des fermenteurs industriels impliquant des champignons filamenteux. / Ethanol production from lignocellulosic biomass requires its transformation into fermentable sugars before the alcoholic fermentation. This step called hydrolysis is catalyzed by cellulases and is often considered as the major technical and economic challenge for the process development. Cellulases are industrially produced by the filamentous fungus Trichoderma reesei, thanks to its high secretion capacity. This fungus is strictly aerobic and is thus cultivated in aerated and stirred bioreactors. The fermentation optimization requires control of physicochemical conditions. Actually the growth of fungi induces an increase of the broth viscosity with shear thinning behavior because of the formation of three-dimensional mycelial structures (from micrometer to millimeter). This viscosity increase has a negative impact on the oxygen transfer. In order to keep the dissolved oxygen concentration higher than a critical limit, it is necessary to increase the power input thereby increasing the shear stress, which may affect the morphology of the fungus as well as its growth and cellulose production. Actually, physico-chemical conditions generated inside the bioreactor are complex and vary with time. These interrelations, between process conditions, morphology, growth and viscosity, require the integration of all these parameters to optimize the full-scale process. The goal of the thesis work was to develop a scale-down approach at lab-scale to mimic hydrodynamic conditions of industrial bioreactors and to study their impact on T. reesei growth and cellulase production. For that purpose, two new tools were developed. The first one consists in a new rheological measurement set-up using a helical rotor dedicated to filamentous fungi preventing mycelium degradation during the measurements. The second one is an original image analyses method that uses specific algorithms. It was then possible to record various morphological data on fungi and to select the most relevant ones (like the number of holes). Meanwhile, a wide range of shear stress conditions were explored in the laboratory bioreactor to reproduce industrial conditions. The new tools we had developed, coupled to these unconventional cultures lead to identifying a shear stress relevant

Production of cellulase and polygalacturonase by Aspergillus niger and Aureobasidium pullulans was studied in submerged (SmF) and solid state fermentation (SSF) systems. Substrates used in fermentation systems were mandarin peels and grape pomace. With Aspergillus niger used on grape pomace as a sole carbon source, cellulase production was detected after 72 hours in SSF and after 24 hours in SmF systems. The activity of cellulase per gram of substrate was higher in a submerged than in a solid state fermentation system. The longer time for higher polygalacturonase production was necessary in submerged fermentation systems and polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 72 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. With Aureobasidium pullulans used on grape pomace as a sole carbon source, cellulase production was detected after 48 hours in SmF and SSF fermentation systems. The activity of cellulase per gram of substrate was higher in solid state system than in a submerged fermentation system. Longer time for higher polygalacturonase production was necessary in both fermentation systems. Polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 48 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. For both systems and both substrates manganese-dependent peroxidase was detected for the first time. Differences in the enzyme synthesis by Aspergillus niger and Aureobasidium pullulans depend on both the substrates used as well as on the fermentation system.

The aim of this thesis is to design a fermentation unit specially adjusted for household operating. Unit processes kitchen and garden biodegradable waste with requirements of low acquisition costs, operating costs and demands of service. Benefits of this unit are production of biogas, which will be utilized in household, and processed of biodegradable waste to nutritious manure. The first part of the work is aimed on description of anaerobic fermentation process and summary of operating conditions. The main part deals with potential of household and garden biodegradable waste for production of biogas and design of fermentation unit. Proposals for biogas utilization and economy of whole project are mentioned.

Les bactériophages sont de puissants prédateurs bactériens. Leur développement est généralement redouté dans les industries agro-alimentaires mettant en oeuvre des fermentations, car les phages sont responsables d’accidents de fabrication affectant la qualité finale du produit. Leur impact lors de la vinification est moins bien défini. Le procédé comporte une étape de fermentation malolactique (FML), qui est assurée par la bactérie lactique Oenococcus oeni. La maîtrise de la FML est un moyen efficace pour protéger la qualité et la typicité des vins, vecteurs de commercialisation. Jusqu’à présent, cette étape fondamentale du processus de vinification n’est pas toujours maîtrisée. L’explication majeure réside dans l’insuffisance de la biomasse bactérienne endogène, liée aux conditions physico-chimiques difficiles du milieu. Des solutions visant à conduire rapidement la FML sont disponibles, comme l’inoculation de souches commerciales de O. oeni. Ces stratégies n’offrent toutefois pas une totale garantie de succès, et des retards ou non déclenchements de FML sont toujours observés. Ces situations inexpliquées amènent à s’interroger sur l’impact d’autres paramètres sur la fermentescibilité malolactique. L’objectif de cette thèse était d’évaluer la présence et la diversité des bactériophages antagonistes de la bactérie lactique O. oeni présents dans l’écosystème. La diversité des prophages présents dans le pangénome de O. oeni a été explorée. La lysogénie est fréquente dans l’espèce. Quatre groupes de prophages ont été identifiés sur la base de la séquence de l’intégrase, et du site de recombinaison site-spécifique utilisé. La pertinence de la classification établie a été vérifiée sur un panel de 40 phages isolés de moûts et de vins. Nos travaux suggèrent que la lysogénie est un moyen pour O. oeni de résister aux stress et aux phages, grâce à la présence de mécanismes de résistance sur les génomes prophagiques. La stabilité de la lysogénie lors de l’inoculation de souches lysogènes dans le vin et la possible libération de dérivés lytiques sont deux paramètres à prendre en compte lors des FML spontanées, et lors de l’inoculation de levains malolactiques. Ils sont susceptibles de moduler quantitativement et qualitativement la population. / Bacteriophages are responsible for the predation of bacteria. They pose an ever-present threat to the food industries because they invade and destroy the starter and affect production. Their destructive potential is currently difficult to establish during spontaneous production of wine. Malolactic fermentation (MLF) represents a main stage in the winemaking process, and is essentially driven by the lactic acid bacterium (LAB) Oenococcus oeni. A rapid and efficient FML is essential to optimize wine quality and typicity, as sales rely on top-quality products. Up to now, this essential step is not controlled, and this results from the limited growth of MLF bacteria in wine, due to stressing conditions. Inoculation of commercial bacterial starter cultures is a strategy to improve MLF control, and will allow a rapid and complete fermentation. However, despite these evolutions, sluggish or complete failures of MLF are still reported by wine farmers, either during spontaneous or directed fermentations. Such cases suggest that additional factors need to be identified. The outline of this thesis was to demonstrate the occurrence of phages infecting O. oeni in the ecosystem and provide essential information regarding phage diversity. We analyzed prophage diversity through comparative genomics and demonstrated that lysogeny is widespread. Four prophage groups were identified according to the integrase gene sequence and attachment site used for site specific recombination. The relevance of the classification scheme was verified through the analysis of a panel of 40 phages isolated from wine and must. We suggest that lysogeny helps O. oeni to cope with stress and phage attack, through the presence of specific anti-phage mechanisms harbored by the prophages. The stability of lysogens during inoculation in wine, and the possible release of lytic particules have to be considered during spontaneous or directed MLF. They are expected to shape the population.

Les bactériophages (ou phages) sont des prédateurs de bactéries et sont redoutés dans les productions agro-alimentaires mettant en œuvre des fermentations. En œnologie, la transformation du jus de raisin en vin fait appel à différents types de fermentation. La fermentation alcoolique est réalisée par des levures, et peut être suivie par une fermentation malolactique (FML), notamment pour les vins rouges, afin d’améliorer la stabilité microbiologique et les qualités organoleptiques du produit. La bactérie lactique OEnococcus oeni (O. oeni) appartient à la famille des Leuconostocaceae et est l’acteur principal de la FML. Des souches d’O. oeni sont utilisées comme levain malolactique, et inoculées dans le vin pour mieux maitriser les fermentations. O. oeni rencontre dans son environnement des phages spécifiques appelés oenophages. Toutefois, bien que la présence de ces oenophages ait été constatée, leur diversité reste à ce jour peu explorée, tout comme leurs implications dans l’élaboration du vin. Une fréquence élevée de la lysogénie a été observée dans l’espèce et parmi les levains commercialisés. Les risques associés a la présence de phages ou à la lysogénie sont des paramètres peu abordés dans la filière. Afin de répondre à ces interrogations, dans un premier axe, la diversité des oenophages a été étudiée en isolant des phages à partir d’une large collecte d’échantillons œnologiques menée dans le sud-ouest de la France. L’analyse d’échantillons de différents types de vin, de différents cépages, collectés à différentes étapes de la vinification nous a permis de mettre en lumière une diversité génomique des oenophages non suspectée. Nous avons initié le développement de nouveaux outils moléculaires pour étudier la dynamique des populations bactériennes et phagiques dans le contexte œnologique. Ainsi, une première approche par Digital Droplet PCR a été utilisée pour détecter et quantifier les populations lysogènes. Dans un deuxième axe, l’interaction phage-hôte en présence de composés phénoliques du vin a été étudiée. Les travaux suggèrent que la croissance d’O. oeni en présence de certains flavonols et acides phénoliques réduit la capacité d’adsorption des phages sur leur hôte. / Bacteriophages are viral predators of bacteria and a major concern in fermentations involved in food processing industry. In winemaking, transformation of grape juice into wine involves different types of fermentations. Alcoholic fermentation is driven by yeasts, and can be followed by malolactic fermentation (MLF), especially for red wines, which can improve microbial stability and sensorial quality of wine. The lactic acid bacterium OEnococcus oeni (O. oeni), member of Leuconostocacae family, is generally responsible for the MLF process. Strains of O. oeni are also used as starters to master MLF. O. oeni encounters specific phages called oenophages. Even though the presence of oenophages has been observed, their diversity remains poorly investigated, just like their implications in winemaking. However, high frequency of lysogeny has been observed among O. oeni strains and starters. Risks linked with the presence of phages and lysogeny are questioned in the sector. In the first part of this thesis, oenophages diversity has been studied with the isolation of a large number of phages during the collection of a broad range of oenological samples in South West France. Analysis of samples coming from different wine types and varieties, collected at different steps of winemaking enabled us to highlight an underestimated genomic diversity. We also initiated the development of new molecular tools to study population dynamics among phages and hosts in the winemaking context. Thus, a first approach by Digital Droplet PCR has been used to detect and quantify lysogenic strains. In the second part, phage-host interactions in the presence of wine phenolic compound were investigated. Our results suggest that growth of O.oeni cells in the presence of particular flavonols and phenolic acids reduces adsorption capacities of phages on their host.

Soybean meal (SBM) is residua product after oil extraction, the SBM with 48% protein is used for poultry, cattle. The SBM contains significant amount of anti-nutritional factors. Degradation of most antigenic proteins and protease inhibitors in SBM fermented by fungal, yeast and bacterial strains. Soybean fermented products are used as feed for livestock or aquaculture. Recently, biofilm forming microorganisms were broadly applied for fermentation process using substrates such as rice bran, corn, soybean meal ... to produce probiotics. In this study, we isolated and selected beneficial microbial strains that are capable of well biofilm forming, produce digestive enzymes and resist pathogenic microorganisms to ferment of soybean meal. The result showed that, four microorganism strains including NA5.3; TB2.1; TB4.3 TB4.4 had ability of forming higher biofilm, producing digestive enzymes such as amylase, protease and cellulose. Among them, NA5.3 and TB 4.4 strains had anti-pathogenic bacteria capacity such as Vibrio parahaemolyticus; Enterococcus faecalis; Bacillus cereus and Escherichia coli. Four selected strains were checked effection of pH, temperature, NaCl and bile salt concentration to their biofilm formation. The result indicated suitable conditions for forming biofilm at pH 6-8 range; temperature range 30-37°C; NaCl concentration of 0-3%, bile salt concentration of 0.5-2%. The selected strains grew well during solid fermentation process, achieved 1011 CFU/gram. / Khô đậu nành là sản phẩm còn lại từ quá trình ép dầu chứa tới 48% protein thô và thường được sử dụng làm thức ăn cho gia cầm, gia súc. Nhưng trong khô đậu nành còn chứa một lượng đáng kể một số chất ức chế dinh dưỡng, các chất ức chế này lại được phân hủy bởi quá trình lên men nhờ một số loài vi khuẩn, nấm mốc hay nấm men. Sản phẩm lên men khô đậu tương được sử dụng làm thức ăn cho gia cầm, gia súc hay nuôi trồng thủy sản. Trong những năm gần đây, các vi sinh vật tạo màng sinh học đã được ứng dụng để lên men các cơ chất như cám gạo, ngô, khô đậu nành… tạo sản phẩm probiotics. Trong nghiên cứu này, chúng tôi đã phân lập và tuyển chọn một số vi sinh vật có lợi tạo màng sinh học cao, sinh các enzyme tiêu hóa và kháng lại một số vi khuẩn gây bệnh cho mục đích lên men khô đậu nành. Kết quả đã lựa chọn được 4 chủng vi khuẩn NA5.3; TB2.1; TB4.3 TB4.4 có khả năng tạo màng sinh học cao, sinh các enzyme như amylase, protease và cellulose.Trong đó,hai chủng NA5.3 và TB4.4 có khả năng kháng lại một số vi khuẩn gây bệnh như Vibrio parahaemolyticus; Enterococcus faecalis; Bacillus cereus và Escherichia coli. Bốn chủng vi khuẩn lựa chọn được nghiên cứu ảnh hưởng của các điều kiện lên khả năng tạo màng sinh học của chúng, chúng thích hợp ở pH 6-8; nhiệt độ 30-37°C; NaCl 0-3%, muối mật 0,5-2%. Sử dụng các chủng vi khuẩn này cho quá trình lên men rắn khô đậu tương, mật độ vi khuẩn sau khi lên men đạt 1011 CFU/gram.

In the human gut, bacterial fermentation of dietary fibers and proteins produces metabolites, primarily as short-chain fatty acids (SCFA), that are highly beneficial for host health. However, unlike dietary fiber, bacterial fermentation of protein additionally generates potentially toxic substances such as ammonia, hydrogen sulfide, amines, and indoles. It is believed that most gut bacteria favor utilization of dietary fiber over that of protein for energy. Therefore, when fermentable dietary fiber is readily available to colonic bacteria, protein fermentation, and its subsequent potentially toxic metabolites, remains relatively low. Dietary intake primarily determines the quantity of dietary fiber and protein substrate available to the gut microbiota and the resulting profile of metabolites produced. Increased protein consumption is associated with deleterious health outcomes such as higher risk of colorectal cancer and type II diabetes. Conversely, diets following US dietary recommendations are high in fiber, which promote a healthy microbiome and are protective against disease. Diets following the recommendation are also moderate in protein intake so that, ultimately, far more fiber than protein is available for colonic bacterial fermentation. On the contrary, dietary fiber intake is chronically low in a standard Western diet, while protein consumption is above dietary recommendations, which results in nearly equal amounts of dietary fiber and protein available for gut microbial fermentation. Furthermore, the popularity of high-protein diets for athletes, as well as that of high-protein low-carbohydrate diets for weight loss, may flip fiber and protein substrate proportions upside down, resulting in more protein than fiber available in the gut for fermentation. The objective of this study was to elucidate how substrate ratios in protein-fiber mixtures affect protein fermentation and metabolites, as well as examine the degree to which fiber source may influence these outcomes. Each dietary fiber source [fructooligosaccharides (FOS), apple pectin (Pectin), a wheat bran and raw potato starch mixture (WB+PS), and an even mixture of the three aforementioned fibers (Even Mix)] and protein were combined in three ratios and provided as substrate for in vitro fecal fermentation to understand how low, medium, and high fiber inclusion levels influence fermentation outcomes. They were compared to 100% protein and fiber (each different fiber) controls. Branched-chain fatty acids (BCFAs), metabolites produced exclusively from protein fermentation, were used as a measure of protein fermentation; the data were normalized based on the initial quantity of protein within the substrate. In protein-fiber substrate mixtures, only FOS and Even Mix inhibited BCFAs (mM/g protein basis) and only when they made up at least half of the substrate. Unexpectedly, the rate of protein fermentation was increased when the protein-fiber substrate contained 25% WB+PS fiber, possibly due to the starch component of the fiber. There was evidence that when pH drops during fermentation, as was the case for protein-FOS mixtures, it played a significant role in suppressing protein fermentation. Ammonia production was not largely affected by increasing the proportion of dietary fiber. A significant reduction did not occur until FOS made up at least 50% of the protein-fiber substrate; for Pectin, WB+PS, and Even Mix fibers, 75% inclusion was required for a significant decrease in ammonia. Interestingly, protein was butyrogenic. Protein as the sole substrate produced more butyrate than either Pectin or Even Mix as the sole substrates, and in fact, addition of Pectin to protein significantly reduced butyrate concentrations. However, the possible benefits of butyrate produced via protein fermentation needs to be tempered by the production of potentially toxic compounds and the association between protein fermentation and colorectal cancer. Overall, the thesis findings showed protein fermentation to be relatively stable and not easily influenced by increasing the availability of dietary fiber, and no clear evidence of microbial preference for carbohydrates over protein was found.

Die Effizienz von Biogasreaktoren hängt im wesentlichen Maße von der Substratverwertung durch die beteiligte Mikroflora ab. Die genaue Zusammensetzung der mikrobiellen Gemeinschaft ist jedoch bislang nur oberflächlich charakterisiert. In dieser Studie wird daher eine Übersicht über die mikrobielle Diversität in verschiedenen Biogasreaktorentypen (Rührkesselreaktor, Leach-bed-Reaktor, Festbett-Anaerobfilter) während der Fermentation verschiedener pflanzlicher Substrate (Mais-, Rüben-, Triticum-Ganzpflanzensilage, teilweise in Kofermentation mit Rindergülle) gegeben. Die Charakterisierung der Mikrobiologie erfolgte mittels der Entwicklung und nachfolgenden bioinformatischen Analyse von 16S rDNA Bibliotheken. Es wurden insgesamt sechs 16S rDNA Bibliotheken konstruiert. Insgesamt umfassten diese sechs 16S rDNA Bibliotheken 627 Klone. Mittels der zugehörigen fingerprint-Muster (amplified rDNA restriction analysis, ARDRA) wurden innerhalb der sechs 16S rDNA Bibliotheken 223 taxonomische Gruppen (operational taxonomic units, OTU) detektiert. Zur Erfassung der Archaea wurden 402 Klone analysiert. Für die Erfassung der Bacteria wurden 283 Klone untersucht. Damit wurden 114 Archaea OTU sowie 109 Bacteria OTU detektiert. Die Dominanz von hydrogenotrophen Methanbildnern in den Archaeaspezifischen 16S rDNA Bibliotheken sowie deren große Diversität sind Indizien für eine verstärkte Bildung von Methan durch Oxidation von CO2. In diesem Falle würde die Verwertung des Acetats überwiegend durch syntrophe Bacteria erfolgen. Die Analyse der Diversität innerhalb der Domäne Bacteria ergab für den Rührkesselreaktor bei der Kofermentation von Maissilage und Gülle im Normalzustand eine hohe Diversität unter den Vertretern der Phyla Firmicutes mit dem Genus Clostridium. / The efficiency of biogas reactors depends on the substrate utilisation by the involved microbial community. However, the exact composition of the microbial biocoenosis was rudimental characterized. In this study an overview of the microbial diversity in different anaerobic biogas reactor types (completly stirred tank reactor, leach bed reactor, fixed bed anaerobic filter) is given for the fermentation of different substrates (corn-, carrots-, triticale whole crop silage as renewable raw materials, partly in co-fermentation with cattle liquid manure). The characterisation of the microbial community was conducted via the construction of 16S rDNA libraries for both, methanogenic Archaea and fermentative Bacteria. Individual taxonomic groups within the 16S rDNA libraries were determined by means of amplified rDNA restriction analysis (ARDRA). The taxonomic classification of these groups was performed via a phylogenetic analysis of representative 16S rDNA sequences. In total six 16S rDNA libraries with 627 clones were developed. Together 223 taxonomic groups were detected; from these 114 was assigned to the domain Archaea and further 109 was assigned to the domain Bacteria. Within the examined biogas reactors a high diversity was found within the hydrogenotrophic methane producing Archaea, acetotrophic methane producing Archaea appears only with a comparatively small diversity. From the domain Bacteria fermentative species of phylum Firmicutes especially of the genus Clostridium were found to be dominant in the microbial community.

Thesis (MScEng (Process Engineering))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Recently, improved technologies have been developed for the biobutanol fermentation process: higher butanol concentrations and productivities are achieved during fermentation, and separation and purification techniques are less energy intensive. This may result in an economically viable process when compared to the petrochemical pathway for butanol production. The objective of this study is to develop process models to compare different possible process designs for biobutanol production from sugarcane molasses. Some of the best improved strains, which include Clostridium acetobutylicum PCSIR-10 and Clostridium beijerinckii BA101, produce total solvent concentrations of up to 24 g/L. Among the novel technologies for fermentation and downstream processing, fedbatch fermentation with in situ product recovery by gas-stripping, followed by either liquid-liquid extraction or adsorption, appears to be the most promising techniques for current industrial application. Incorporating these technologies into a biorefinery concept will contribute toward the development of an economically viable process. In this study three process routes are developed. The first two process routes incorporate well established industrial technologies: Process Route 1 consist of batch fermentation and steam stripping distillation, while in Process Route 2, some of the distillation columns is replaced with a liquid-liquid extraction column. The third process route incorporates fed-batch fermentation and gas-stripping, an unproven technology on industrial scale. Process modelling in ASPEN PLUS® and economic analyses in ASPEN Icarus® are performed to determine the economic feasibility of these biobutanol production process designs. Process Route 3 proved to be the only profitable design in current economic conditions. For the latter process, the first order estimate of the total project capital cost is $187 345 000.00 (IRR: 35.96%). Improved fermentation strains currently available are not sufficient to attain a profitable process design without implementation of advanced processing techniques. Gas stripping is shown to be the single most effective process step (of those evaluated in this study) which can be employed on an industrial scale to improve process economics of biobutanol production. / AFRIKAANSE OPSOMMING: Onlangse verbeteringe in die tegnologie vir die vervaardiging van butanol via die fermentasie roete het tot gevolg dat: hoër butanol konsentrasies en produktiwiteit verkry kan word tydens die fermentasie proses, en energie verbruik tydens skeiding-en suiweringsprosesse laer is. Hierdie verbeteringe kan daartoe lei dat biobutanol op ʼn ekonomiese vlak kan kompeteer met die petrochemiese vervaardigings proses vir butanol. Die doelwit van die studie is om proses modelle te ontwikkel waarmee verskillende proses ontwerpe vir die vervaardiging van biobutanol vanaf suikerriet melasse vergelyk kan word. Verbeterde fermentasie organismes, wat insluit Clostridium acetobutylicum PCSIR-10 en Clostridium beijerinckii BA101, het die vermoë om ABE konsentrasies so hoog as 24 g/L te produseer. Wat nuwe tegnologie vir fermentasie en skeidingprosesse behels, wil dit voorkom of wisselvoer fermentasie met gelyktydige verwydering van produkte deur gasstroping, gevolg deur of vloeistof-vloeistof ekstraksie of adsorpsie, van die mees belowende tegnieke is om tans in die nywerheid te implementeer. Deur hierdie tegnologie in ʼn bioraffinadery konsep te inkorporeer sal bydra tot die ontwikkeling van ʼn ekonomies lewensvatbare proses. Drie prosesserings roetes word in die studie ontwikkel. Die eerste twee maak gebruik van goed gevestigde industriële tegnologie: Proses Roete 1 implementeer enkellading fermentasie en stoom stroping distillasie, terwyl in Proses Roete 2 van die distilasiekolomme vervang word met ʼn vloeistof-vloeistof ekstraksiekolom. Die derde proses roete maak gebruik van wisselvoer fermentasie met gelyktydige verwydering van produkte deur gas stroping. Die tegnologie is nog nie in die nywerheid bewys of gevestig nie. Om die ekonomiese uitvoerbaarheid van die proses ontwerpe te bepaal word proses modellering uitgevoer in ASPEN PLUS® en ekonomiese analises in ASPEN Icarus® gedoen. Proses Roete 3 is die enigste ontwerp wat winsgewend is in huidige ekonomiese toestande. Die eerste orde koste beraming van die laasgenoemde projek se totale kapitale koste is $187 345 000.00 (opbrengskoers: 35.96%). Die verbeterde fermentasie organismes wat tans beskikbaar is, is nie voldoende om ʼn proses winsgewend te maak nie; gevorderde proses tegnologie moet geïmplementeer word. Gasstroping is bewys as die mees effektiewe proses stap (getoets in die studie) wat op industriële skaal geïmplementeer kan word om die winsgewendheid van die biobutanol proses te verbeter. / Centre for Renewable and Sustainable Energy Studies

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.