Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Galactooligosaccharides are produced by the transglycosylation activity of β-galactosidases (β-gal, EC 3.2.1.23) when utilising lactose as a substrate. They have emerged as important constituents used in the food and pharmaceutical industries owing to their prebiotic properties. Although transglycosylation was discovered in 1951 (Wallenfels 1951), and a number of β-gals have had their transglycosylation activity characterised, the activities of these enzymes are not optimal for industrial use. Their tendency to favour the hydrolytic reaction over the transglycosylation reaction, coupled with the production of shorter chain oligosaccharides has driven scientists to investigate altering protein structure both to increase chain lengths and the amount of oligosaccharide produced at lower substrate concentrations. In an attempt to alter the amount of oligosaccharide produced by a metagenomically derived β-gal belonging to the glycosyl hydrolase 2 family, random and site-directed mutagenesis were used. A randomly mutagenised library was screened on SOB agar plates containing 5% (w/v) lactose which should select for clones that synthesise oligosaccharides at relatively low concentrations. No such activity was detected. Site-directed mutagenesis was also utilised to alter protein structure. It was confirmed that the β-gal utilised in this study belonged to the glycosyl hydrolase 2 family through mutation of the predicted catalytic acid/base glutamic acid to a non-catalytic residue, thus removing activity. Another mutation was utilised to investigate if it was possible to increase the degree of polymerisation of oligosaccharides produced by the β-gal. This mutation was successful in increasing the degree of polymerisation. Biochemical characterisation of the β-gal revealed that it exhibited optimal activity at pH 8.0, with a temperature optimum of 30°C. The β-gal exhibited a Km and Vmax of 54.23 mM and 2.26 μmol/minute-1/mg protein-1 respectively, similar to kinetic parameters that have been determined for a number of previously characterised enzymes. / AFRIKAANSE OPSOMMING: Galaktooligosakkariede word geproduseer deur die transglikosileering aktiwiteit van β-galaktosidase (β-gal, EG 3.2.1.23) wanneer hulle laktose as 'n substraat gebruik. Hierdie oligosakkariede het na vore gekom as 'n belangrike bestandeel vir gebruik in die voedsel en farmaseutiese bedryf as gevolg van hulle prebiotiese eienskappe. Alhoewel transglycosylation al in 1951 ontdek is (Wallenfels 1951) en 'n aantal β-gals se transglycosylation aktiwiteit gekenmerk is, is hierdie ensieme nie ideaal vir industriële toepassings nie. Die geneigdheid om die hidrolitiese reaksie oor die transglycosylation reaksie bevoordeel, tesame met die produksie van korter oligosakkariede het wetenskaplikes ondersoek genoop om die proteïenstruktuur te verander om ketting-lengte en die kwantiteit van oligosakkaried geproduseer teen laer substraat konsentrasies te verhoog. In 'n poging om die opbrengs van die oligosakkaried wat deur 'n metagenomiese β-gal wat aan die glycosyl hidrolase 2 familie behoort te verander, is lukraak en terrein gerigte-mutagenese gebruik. Die mutagenese biblioteek is op SOB agarplate met 5% (w/v) lactose gekeur, om klone wat die fenotipe wat verband hou met die produksie oligosakkaried teen relatiewe lae konsentrasies te selekteer. Geen aktiwiteit is opgemerk nie. Terrein gerigte-mutagenese is ook gebruik om die proteïenstruktuur te verander. Deur ‘n bioinformatiese voorspelling, is dit bevestig dat die β-gal wat in hiedie studie gebruik word tot die glycosyl hidrolase 2 familie behoort. Dit is gedoen deur mutasie van die voorspelde katalitiese suur/basis glutamiensuur na 'n nie-katalitiese oorskot, dus die verwydering van aktiwiteit. Nog ‘n mutasie is gebruik om te ondersoek of dit moontlik was om die ketting-lengte van die oligosakkaried wat deur die β-gal geproduseer is te verhoog. Die mutasie was suksesvol in die verhoging van die oligosakkaried wat geproduseer was. Biochemiese karakterisering van die β-gal het getoon dat hierdie β-gal optimale aktiwiteit het by pH 8.0, met 'n optimum temperatuur van 30°C. Die β-gal het 'n Km en Vmax van 54.23 mM en 2.26 μmol/minute-1/mg proteïen-1 onderskeidelik, soortgelyk aan kinetiese parameters wat bepaal word vir ensieme wat voorheen gekenmerk is.

No description available.

Diatom-diazotroph associations contribute extensively to vertical flux in the modern ocean, and are linked to extensive sedimentary layers in the paleo-record formed either by rapidly sinking aggregates or more slowly sinking cells and chains. Although diatom sinking rates have been extensively studied, the sinking rates of these diatom symbioses are poorly documented. The effect of silicate and phosphate limitation on the sinking rate of Hemiaulus hauckii and its symbiont Richelia intracellularis were determined in semi-continuous cultures using a SETCOL. TEP (transparent exopolymer particle) was measured concurrently for linkages to aggregate formation. Although cells occurred in chains of 1-20 cells long, aggregate formation did not occur in semi-continuous cultures. There was no significant effect of P or Si limitation on sinking rates compared to controls. Although H. hauckii possess a symbiont that lacks gas vesicles, sinking rates (0.04-0.72 m/day) were within the range predicted by size-rate relationships. A portion of each H. hauckii experimental measurement ascended as well with significant differences found between the lowered P 80% exchange and the lowered Si with 80% exchange (p=0.05) and between the lowered Si with 80% exchange and the Si control batch culture (p=0.039). TEP production did not change significantly between treatments. However, a fraction of TEP in each population was positively buoyant as well, with ascent rates between 0.32-0.49 m/day. This study confirms an earlier report of positive buoyancy in TEP as well as noting that a significant fraction of the Hemiaulus symbiosis can be ascending within the population. / text

Transparent exopolymer particles (TEP) have been reported as one of the main factors of membrane fouling in seawater reverse osmosis (SWRO) process. Research has been focused on algal TEP so far, overlooking bacterial TEP. This thesis investigated the effects of coagulation on removal of bacterial TEP/TEP precursors in seawater and subsequent reduction on TEP fouling in ultrafiltration (UF), as a pretreatment of SWRO. Furthermore, the performance of pretreatment (coagulation + UF) has been investigated on a bench-scale SWRO system. TEP/TEP precursors were harvested from a strain of marine bacteria, Pseudoalteromonas atlantica, isolated from the Red Sea. Isolated bacterial organic matter (BOM), containing 1.5 mg xanthan gum eq./L TEP/TEP precursors, were dosed in Red Sea water to mimic a high TEP concentration event. Bacterial TEP/TEP precursors added to seawater were coagulated with ferric chloride and aluminum sulfate at different dosages and pH. Results showed that ferric chloride had a better removal efficiency on TEP/TEP precursors. Afterwards, the non-coagulated/coagulated seawater were tested on a UF system at a constant flux of 130 L/m2h, using two types of commercially available membranes, with pore sizes of 50 kDa and 100 kDa, respectively. The fouling potential of coagulated water was determined by the Modified Fouling Index (MFI-UF). Transmembrane pressure (TMP) was also continuously monitored to investigate the fouling development on UF membranes. TEP concentrations in samples were determined by the alcian blue staining assay. Liquid chromatography-organic carbon detection (LC-OCD) was used to determine the removal of TEP precursors with particular emphasis on biopolymers. Finally, SWRO tests showed that TEP/TEP precursors had a high fouling potential as indicated by MFI-UF, corresponding to the TMP measurements. Coagulation could substantially reduce TEP/TEP precursors fouling in UF when its dosage was equal or higher than 0.2 mg Fe/L. The flux decline experiments showed that coagulation + UF pretreated water had a smaller fouling potential than MF pretreated water. This thesis also provides useful and practical information on controlling bacterial TEP/TEP precursors fouling in UF and RO systems.

Les exopolysaccharides (EPS) bactériens peuvent avoir une haute valeur ajoutée comme agents de rétention d’eau en cosmétologie. Cette étude s’est intéressée à l’EPS de Ramlibacter tataouinensis TTB310 (Rta). Cette bactérie, isolée d'un sol semi-aride, possède un cycle cellulaire particulier avec des bâtonnets mobiles sensibles à la dessiccation et des kystes non-mobiles produisant un EPS lui permettant de tolérer la dessiccation prolongée. La production d’EPS et la croissance ont été optimisées par le choix de lactate comme source de C et d'énergie, le maintien du pH pendant la croissance avec de l'acide lactique, l’augmentation des concentrations des autres substrats et l’ajout des micro- et macro éléments. Ainsi des quantités suffisantes d'EPS ont été obtenues. Une extraction de l'EPS fermement attachée à la surface des bactéries en utilisant de l’acide trichloroacétique a été élaborée et optimisée. La caractérisation biochimique de l'EPS a été réalisée en combinant différentes techniques : CPG, FTICR-MS, RMN et FTIR. L'unité répétée de l'EPS de Rta est un décasaccharide constitué de ribose, glucose, galactose, mannose, rhamnose, acide glucuronique, désoxyhexose et avec des substituants tels que acétyle, succinyle et méthyle. Enfin, grâce à la disponibilité du génome entièrement séquencé de Rta, le cluster de gènes impliqué dans la production d'EPS a été identifié à l’aide des approches de biologie moléculaire et d'imagerie. La connaissance de la structure de l'EPS de Rta permettra de mettre au point les modifications physico-chimiques nécessaires à sa solubilisation pour en étudier les propriétés rhéologiques. / Bacterial exopolysaccharides (EPS) can have a high added value when used as water retention agents in cosmetology. This work was focused on the EPS of Ramlibacter tataouinensis TTB310 (Rta). Rta, isolated from a semi-arid soil, has a special cell cycle with mobile rods sensitive to desiccation and non-mobile cysts producing an EPS allowing it to tolerate desiccation. EPS production and bacterial growth were optimized by : choosing lactate as a source of C and energy, maintaining the pH during the growth with lactic acid, increasing the concentrations of the other substrates, and additionning of micro- and macro elements. Thus sufficient amounts of EPS have been obtained. An extraction of EPS firmly attached to the bacteria surface using trichloroacetic acid was developed and optimized. The biochemical characterization of Rta EPS was performed by combining different techniques : CPG, FTICR-MS, RMN and FTIR. The repeated unit of Rta EPS is a decasaccharide consisting of ribose, glucose, galactose, mannose, rhamnose, glucuronic acid, deoxyhexose and with substituents such as acetyl, succinyl and methyl. Finally, thanks to the availability of the fully sequenced Rta genome, the gene cluster involved in the production of EPS has been identified, through molecular biology and imaging approaches. The elucidation of the structure of Rta EPS makes possible the future development of the physico-chemical modifications necessary for its solubilization in order to study its rheological properties.

Coccolithophores are unicellular microscopic algae (Haptophyta) surrounded by calcium carbonate plates that are produced during their life cycle. These species, whose contemporary contributor is Emiliania huxleyi, are mainly found in the sub-polar and temperate oceans, where they produce huge blooms visible from space. Coccolithophores are sensitive to ocean acidification that results from the ongoing accumulation of anthropogenic carbon dioxide (CO2) in the atmosphere. The response of these organisms to global change appears to be related to the reduction of their ability to produce calcium carbonate at the cellular level. At the community levels, one anticipates changes in the carbon fluxes associated to their blooms as calcification is reduced. However, the consequences of such environmental changes on this species are speculative and require improvements in the description of the mechanisms controlling the organic and inorganic carbon production and export. The first aspect of this work was to study the response of these organisms to artificially modified CO2 concentrations representative of the conditions occurring in the past (glacial) and those expected by the end of the century (2100). Two different levels were examined: the continuous monospecific cultures (chemostats) allowed us to work at the cellular level while the mesocosms gave light to the mechanisms taking place in an isolated fraction of the natural community. The second aspect of this work consisted of field studies carried out during four cruises (2002, 2003, 2004 and 2006) in the northern Bay of Biscay, where the occurrence of E. huxleyi blooms were observed in late spring and early summer. We describe the vertical profiles of biogeochemical variables (nutrients, chlorophyll-a, dissolved inorganic chemistry, particulate carbon, transparent exopolymer particles (TEP)) and study processes such as primary production, calcification and bacterial production. The properties of these blooms are compared with those reported in the literature and enriched with original measurements such as the abundance and concentration of TEP that could play an important role in carbon export to the deep ocean, modifying the properties of the settling ballasted aggregates.

Desalination is considered to be a major source of usable water in the Middle East, especially the Gulf countries which lack fresh water resources. A key and sometimes the only solution to produce high quality water in these countries is through the use of seawater reverse osmosis (SWRO) desalination technology. Membrane fouling is an economic and operational defect that impacts the performance of SWRO desalination technology. To limit this fouling phenomenon, it is important to implement the appropriate type of intake and pre-treatment system design. In this study, two types of systems were investigated, a vertical well system and a surface-water intake at a 9m depth. The purpose of this investigation is to study the impact of the different intake systems and pre-treatment stages in minimizing the concentrations of algae, bacteria, natural organic matter (NOM) and transparent exopolymer particles (TEP), in the feed water prior to pre-treatment, through the pre-treatment stages, and in the product water and concentrate. Water samples were collected from the surface seawater, the intakes (wells for site A, 9 m depth open ocean intake at site B), after the media filter, after the cartridge filter, and from the permeate and reject streams. The measured parameters included physical parameters, algae, bacteria, total organic carbon (TOC), fractions of dissolved NOM, particulate and colloidal TEP. The results of this study prove that the natural filtration and biological treatment of the seawater which occur in the aquifer matrix are very effective in improving the raw water quality to a significant degree. The results demonstrated that algae and biopolymers were 100% removed, the bacterial concentrations were significantly removed and roughly 50% or greater of TOC concentrations was eliminated by the aquifer matrix at site A. The aquifer feeding the vertical wells reduced TEP concentrations, but to differing degree. There is a slight decrease in the concentrations of, algae, bacteria, TOC, NOM, and TEP in the feed water at 9 m depth compared to the surface seawater at site B. The pre-treatment was of significant effectiveness and the improvements in reducing the membrane fouling potential were quite high and strong at this site. Investigation of the permeate stream showed some breakthrough of bacteria which is of concern because it may indicate a problem within the membrane system (e.g., broken seal and perforation). The aquifer feeding the wells in the subsurface system plays a main role in the improvement of water quality, so the pre-treatment seems less effective in site A plant. This proves that the subsurface intake is better than open ocean intake in terms of providing better raw water quality and ultimately reducing membrane biofouling.

Ce travail est une contribution à la connaissance de l’importance des particules de nature polysaccharidique, les TEP (Transparent Exopolymer Particles) ou particules d’Exopolymères Transparentes, en milieu pélagique lacustre.Les différentes études présentées dans ce mémoire se sont essentiellement focalisées sur la distribution de ces particules et leur relation avec le phytoplancton et les autres microorganismes du réseau trophique aquatique en milieu naturel (le lac Pavin, oligo-mésotrophe et le réservoir hypereutrophe de Grangent) et en conditions semi contrôlées(enclos limniques installés sur le lac de Créteil). Les résultats de l’étude printanière au lac Pavin indiquent que la majorité des TEP sont colonisées par les bactéries et que l’intensité de colonisation est fortement liée à la température et diminue avec l’augmentation en taille des particules. La distribution des nanoflagellés hétérotrophes (HNF) est fortement liée à la densité des TEP mais pas à l’intensité de colonisation de ces particules. L’abondance et la surface cumulée de TEP sont significativement plus élevées dans le lac oligo-mésotrophe que dans le réservoir hypereutrophe de Grangent. Les abondances et les concentrations élevées de particules dans le lac Pavin coïncident avec la présence de diatomées de grande taille au printemps et en automne et avec les chlorophycées à la fin de l’été.Dans le réservoir de Grangent les valeurs maximales de TEP coïncident avec le développement de la cyanobactérie Microcystis aeruginosa. Si les TEP augmentent avec la productivité de l’écosystème, la production de ces particules par unité de chlorophylle a dépend de la composition algale et tend à diminuer avec l’augmentation du niveau trophique du milieu. Les résultats issus de la biomanipulation en enclos limniques indiquent que la structure du réseau trophique aquatique (par la présence ou l’absence de poissons planctonophages) influence fortement la distribution,la dynamique et le spectre de taille des TEP. Dans le traitement poisson, l’abondance des TEP, la chlorophylle a et la biomasse des chlorophycées sont fortement corrélées. De par son broutage sur le phytoplancton, le zooplancton a un effet négatif sur les TEP dans le traitement sans poissons mais il contribue sans doute à la formation de TEP e tinfluence le spectre de taille de ces dernières dans ce traitement. Ce travail souligne l’importance des particules de nature polysaccharidique en milieu pélagique lacustre qui doivent être considérées comme une part importante du carbone organique qui transite des producteurs primaires vers les décomposeurs et vers le sédiment. / This work adds to the knowledge of the significance of polysaccharidic detrital particles or TEP (= Transparent Exopolymer Particles) in freshwater pelagic environments. Studies in this thesis have mainly focused on the distribution of TEP and their relationships with phytoplankton and other microorganisms in natural environments (the oligo-mesotrophic Lake Pavin and the hypereutrophic reservoir of Grangent) and in limnetic enclosures (lake of Créteil). The intensity of bacterial colonization during spring in Lake Pavin was strongly related to temperature and decreased with particle size. The abundance of heterotrophic nanoflagellates (HNF) in this lake was more significantly related to the density of the particles than to the density of total bacteria and the intensity of bacterial colonization of TEP, suggesting that TEP is a more important factor for HNF development than attached and free bacteria. The abundance and the total surface area of the particles were significantly higher in the hypereutrophic Lake Grangent than in the mesotrophic Lake Pavin. Maximum TEP density in Lake Pavin was recorded during the spring diatom bloom, while TEP concentration peaked in late summer when the phytoplankton community was largely dominated by small-size chlorophytes with an abundant polysaccharide cell coating. In the hypereutrophic Lake Grangent,maximum values of TEP appeared during the summer development of the cyanobacterium Microcystis aeruginosa. Per cell production of TEP, expressed by the ratio between TEP concentration and chlorophyll a concentration, was significantly higher in the less productive lake and the analysis of the size spectra of the particles indicated a greater proportion of small particles in this lake. TEP therefore appear as more significant for microbial development and aggregates formation in the less productive environment. Results from limnetic enclosures (either dominated by planktivorous fish or fishless) indicated that food-web structure strongly influences the distribution and size spectra of TEP. TEP abundances were related to chlorophyll a concentrations and the biomass of chlorophytes in the fish treatment. As expected by the trophic cascades theory, zooplankton had an indirect negative effect on TEP abundance. Our results suggest, however, that metazoan probably influence the formation and the size spectra of the particles in the fishless treatment. TEP must be regarded as a major part of the organic carbon which is transferred from the primary producers to the microbial food web and the sediments in freshwater ecosystems.

Coccolithophores are unicellular microscopic algae (Haptophyta) surrounded by calcium carbonate plates that are produced during their life cycle. These species, whose contemporary contributor is Emiliania huxleyi, are mainly found in the sub-polar and temperate oceans, where they produce huge blooms visible from space. Coccolithophores are sensitive to ocean acidification that results from the ongoing accumulation of anthropogenic carbon dioxide (CO2) in the atmosphere. The response of these organisms to global change appears to be related to the reduction of their ability to produce calcium carbonate at the cellular level. At the community levels, one anticipates changes in the carbon fluxes associated to their blooms as calcification is reduced. However, the consequences of such environmental changes on this species are speculative and require improvements in the description of the mechanisms controlling the organic and inorganic carbon production and export.<p><p>The first aspect of this work was to study the response of these organisms to artificially modified CO2 concentrations representative of the conditions occurring in the past (glacial) and those expected by the end of the century (2100). Two different levels were examined: the continuous monospecific cultures (chemostats) allowed us to work at the cellular level while the mesocosms gave light to the mechanisms taking place in an isolated fraction of the natural community. The second aspect of this work consisted of field studies carried out during four cruises (2002, 2003, 2004 and 2006) in the northern Bay of Biscay, where the occurrence of E. huxleyi blooms were observed in late spring and early summer. We describe the vertical profiles of biogeochemical variables (nutrients, chlorophyll-a, dissolved inorganic chemistry, particulate carbon, transparent exopolymer particles (TEP)) and study processes such as primary production, calcification and bacterial production. The properties of these blooms are compared with those reported in the literature and enriched with original measurements such as the abundance and concentration of TEP that could play an important role in carbon export to the deep ocean, modifying the properties of the settling ballasted aggregates.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished