The increasing concern about water quality and energy demand promotes the development of innovative and low-cost processes to improve the nutrient uptake and energy efficiency of existing wastewater treatments (WWT). In this context, the inclusion of a microalgae system (MAS) in the flowsheet of a WWT plant represents a sustainable alternative to conventional technologies, as it combines a low-cost nutrient uptake system with the production of biomass suitable for biofuel production. However, at present, the energy required to cultivate and process the algae cells is often too high to justify their use. The adoption of a low energy harvesting system and an efficient energy conversion process are the sine qua non requirements to guarantee the sustainability of the process. In this thesis, current and innovative harvesting technologies for large scale applications have been reviewed to identify the optimal working conditions of each system and their link to the main characteristics of the algae suspension. In particular, the performance of the Ballasted Dissolved Air Flotation (BDAF) system was investigated using different algae and compared to the conventional Dissolved Air Flotation (DAF). BDAF was demonstrably a very viable harvesting method where the use of floating microspheres as ballasting agents allowed significant coagulant savings, reduced the level of energy dissipation within the flotation chamber, and lowered the overall carbon emissions and the process costs. Cont/d.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue. / AFRIKAANSE OPSOMMING: Maserasie ensieme vir die wynindustrie is ensiem mengsels wat hoofsaaklik pektinases, sellulases en hemisellulases bevat en word tydens wynbereiding gebruik om die druifkorrel se selwand af te breek, die polifenole en aroma molekules vry te stel en sodoende die wyn kwaliteit te verbeter. Hierdie soort ensieme word ook gebruik om inkomste-genererende molekules vanuit druiweprosesserings afval (doppe, pulp en pitte) te isoleer, en ook as prosesserings hulpmiddels in die produksie van bioetanol. Druifkorrel selwande is weerstandig teen ensiem afbraak en daarom is kennis oor die struktuur en samestelling van die selwand, asook hoe die selwand strukture deur die toediening van ensieme verander word noodsaaklik om sodoende hierdie prosesse te optimaliseer. Die doel van hierdie studie was om die huidige kennis uit te brei deur bestaande asook nuwe metodes te gebruik om die druifkorrel selwand te bestudeer met die fokus op die polimeerprofiel van die selwande. Selwand karakteriserings tegnieke wat in hierdie studie gebruik is sluit in die Comprehensive Microarray Polymer Profiling (CoMPP) metode wat monoklonale teenliggaampies en koolhidraat bindende modules (Carbohydrate binding modules, CBMs) wat spesifiek die selwandpolimere van die plant selwand herken, gebruik. Met hierdie metode het ons die vlakke van spesifieke polimere gemeet asook die skommeling in hulle vlakke soos dit beïnvloed is deur eksterne faktore soos ensiem hidroliese. Die CoMPP metode is tesame met monosakkaried profiel analise, met behulp van GC-MS, wat die boublokke van die selwand polimere bepaal, asook infrarooi spektroskopie om die veranderinge in die oorhoofse chemiese profiel te bepaal, gebruik. Datastelle wat met die selwand karakteriserings tegnieke gegenereer is, is ontleed met een- en multiveranderlike statistiese metodes om die hoof tendense in die data op te spoor. Hierdie studie het die selwand verskille, op die polimeervlak, van Pinotage druiwe uitgelig. Verskillende rypheidsgrade asook hoe dit verander tydens ‘n standaard wynfermentasie is gevolg. Laasgenoemde het die vrystelling van homogalakturonaan en die ontbloting van arabinogalaktoproteïene tot gevolg gehad. Met die byvoeging van maserasie ensieme was dit duidelik dat addisionele pektienverwydering plaasgevind het en dat die ensieme die selwand van die ryp druiwe ontrafel het. In oorryp druiwe was daar geen addisionele selwand afbreking sigbaar as gevolg van die aksie van maserasie ensieme nie, wat moontlik aandui dat die inherente druif ensieme reeds uitgebreide selwand afbraak versoorsaak het. Wanneer gesuiwerde ensieme met geïsoleerde selwande van Pinotage druiwedoppe en met Chardonnay druiweprosesserings afval geïnkubeer is onder gebufferde kondisies, is verskillende vlakke van ensiematiese hidroliese waargeneem en geklassifiseer. Die volgorde waarin die selwand polimere geëkstraheer is, asook die invloed van spesifieke ensieme in die bevordering van die ekstraksie proses, het belangrike inligting verskaf oor die toeganglikheid van spesifieke selwand polimere. Sinergistiese aksie tussen, byvoorbeeld ‘n endo-poligalakturonase (EPG) en ‘n endo-glukanase (EG) is geidentifiseer met behulp van die CoMPP data. Hierdie EPG en EG sinergisme is ook geïllustreer met ‘n gisras (‘n Saccharomyces paradoxus x S. cerevisiae hibried) wat in ‘n gebufferde druifprosesserings afval suspensie gefermenteer het. Hierdie gisras het ‘n endogene EPG en is ontwerp om ook ‘n rekombinante EG uit te druk vanaf ‘n genoom geïntegreerde kasset. Die selwande van die druiweprosesserings afval wat na die fermentasie geïsoleer is, was ontrafel en pektienverwydering het plaasgevind, soos bevestig met CoMPP data. In hierdie studie is bewys dat die selwand karakteriserings tegnieke vinnig en sensitief is. Dit het insigte verskaf oor die struktuur van die druifselwand en is gebruik om die veranderinge as gevolg van rypheidsverskille, wynfermentasie, ensiem hidroliese en hitte prosessering te evalueer. Buiten die bydraes tot kennis oor hierdie onderwerpe, is die bruikbaarheid van hierdie tegnieke ook aangetoon, veral in die evaluasie van verskillende ensieme en ensiemkombinasies, asook mikroörganismes vir die afbraak van druifweefsel.

Budding yeast are capable of existing in both a haploid and diploid state. Haploid cells have two mating types, MATa and MATα. When cells from the two mating types come in contact they signal using reciprocal pheromones and pheromone receptors, starting a regulated pheromone response that includes transcription of pheromone-response genes, polarization in the direction of highest pheromone concentration, and cell cycle arrest. Once cells have chosen a mating partner, they must fuse their cell walls, plasma membranes, and nuclei to form a single diploid cell.

Plant cell walls are complex structures that must not only constrain cellular turgor pressure but also allow for structural modification during the dynamic processes of cell division and anisotropic expansion. Cell walls are composed of highly glycosylated proteins and polysaccharides, including pectin, hemicellulose and cellulose. The primary cell wall polysaccharide is cellulose, a polymer composed of high molecular weight !- 1,4-glucan chains. Although cellulose is the most abundant biopolymer on Earth, there is still a lot to learn about its biosynthesis and regulation. This research began by applying a variety of analytical techniques in an attempt to understand differences in cell wall composition and cellulose structure within the plant body, between different plant species and as a result of acclimation by the plant to different environmental conditions. Next, a number of different Arabidopsis thaliana lines possessing mutations affecting cell wall biosynthesis were analyzed for changes in cellulose structure (crystallinity) and biomass saccharification efficiency. One of these mutants, isoxaben resistance1-2 (ixr1- 2), which contains a point mutation in the C-terminal transmembrane region (TMR) of cellulose synthase 3 (CESA3), exhibited a 34% lower biomass crystallinity index and a 151% improvement in saccharification efficiency relative to that of wild-type. The culmination of this research began with a chemical screen that identified the molecule quinoxyphen as a primary cell wall cellulose biosynthesis inhibitor. By forward genetics, a semi-dominant mutant showing strong resistance to quinoxyphen named aegeus was identified in A. thaliana and the resistance locus mapped to a point mutation in the TMR of CESA1. cesa1aegeus occurs in a similar location to that of cesa3ixr1-2, illustrating both subunit specificity and commonality of resistance locus. These drug resistant CESA TMR mutants are dwarfed and have aberrant cellulose deposition. High-resolution synchrotron X-ray diffraction and 13C solid-state nuclear magnetic resonance spectroscopy analysis of cellulose produced from cesa1aegeus, cesa3ixr1-2 and the double mutant shows a reduction in cellulose microfibril width and an increase in mobility of the interior glucan chains of the cellulose microfibril relative to wild-type. These data demonstrate the importance of the TMR region of CESA1 and CESA3 for the arrangement of glucan chains into a crystalline cellulose microfibril in primary cell walls.

No description available.

Tree-ring based climate reconstructions have been critical for understanding past variability and recent trends in climate worldwide, but they are scarce in Australia. This is particularly the case for temperature: only one tree-ring width based temperature reconstruction – based on Huon Pine trees from Mt Read, Tasmania – exists for Australia. Here, we investigate whether additional tree- ring parameters derived from Athrotaxis cupressoides trees growing in the same region have potential to provide robust proxy records of past temperature variability. We measured wood properties, including tree-ring width (TRW), mean density, mean cell wall thickness (CWT), and tracheid radial diameter (TRD) of annual growth rings in Athrotaxis cupressoides, a long-lived, high-elevation conifer in central Tasmania, Australia. Mean density and CWT were strongly and negatively correlated with summer temperatures. In contrast, the summer temperature signal in TRW was weakly positive. The strongest climate signal in any of the tree-ring parameters was maximum temperature in January (mid-summer; JanTmax) and we chose this as the target climate variable for reconstruction. The model that explained most of the variance in JanTmax was based on TRW and mean density as predictors. TRW and mean density provided complementary proxies with mean density showing greater high-frequency (inter-annual to multi-year) variability and TRW showing more low-frequency (decadal to centennial-scale) variability. The final reconstruction model is robust, explaining 55% of the variance in JanTmax, and was used to reconstruct JanTmax for the last five centuries (1530–2010 C.E.). The reconstruction suggests that the most recent 60 years have been warmer than average in the context of the last ca. 500 years. This unusually warm period is likely linked to a coincident increase in the intensity of the subtropical ridge and dominance of the positive phase of the Southern Annular Mode in summer, which weaken the influence of the band of prevailing westerly winds and storms on Tasmanian climate. Our findings indicate that wood properties, such as mean density, are likely to provide significant contributions toward the development of robust climate reconstructions in the Southern Hemisphere and thus toward an improved understanding of past climate in Australasia.

Research using microalgae Chlorella vulgaris was conducted in order to determine the maximum CO2 concentration under which algae can grow, within the emission range from oil and natural gas burning plants (0-20%). After choosing the optimal CO2 percentage, pH and alkalinity were determined; and finally, an electrochemical (EC) batch reactor connected to DC current was applied to achieve algae cell annihilation, and therefore, facilitate anaerobic digestion, methane production and energy recovery. It was determined that algae can grow under 20% CO2, being 15% CO2 the most effective (pH of 6.64 and alkalinity of 617.5 mg/L CaCO3). Electroporation using an electrochemical batch reactor is effective in breaking cells membranes, which simplifies anaerobic digestion process and methane production. The parameters found effective for completely breaking the algae cell are: detention time of 1 more or less 0.5 minutes, and minimum voltage and current of 65 Volts/285 ml and 3.9 Amps/285 ml, respectively

Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...

Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation.

Lentilhas d´água são as menores angiospermas aquáticas monocotiledôneas, as quais são classificadas em duas subfamílias (Lemnoideae e Wolffioideae) que contemplam trinta e sete espécies representando cinco gêneros: Spirodela, Landoltia, Lemna, Wolffiella e Wolffia (APPENROTH; BORISJUK; LAM, 2013; BORISJUK et al ., 2015; FAO, 1999; LANDOLT, 1992, LES et al ., 2002). Essas plantas apresentam rápida reprodução vegetativa o que implica no acúmulo acelerado de carboidratos e proteínas, que por sua vez, confere uma enorme aplicação industrial e biotecnológica, especialmente na bioenergia (APPENROTH; BORISJUK; LAM, 2013; FAO, 1999). O balanço de carbono no crescimento relaciona-se com à assimilação fotossintética, armazenamento e consumo desses compostos, que são influenciados pela intensidade de luz (SMITH; STITT, 2007; STITT; ZEEMAN, 2012). O maior dreno de carbono assimilado é para a síntese de parede celular e sacarose, portanto, conhecendo a composição dos polissacarídeos e suas interações junto com o conteúdo de carboidratos não estruturais impacta a produção de combustíveis alternativos. O presente trabalho visou quantificar e analisar os carboidratos estruturais (açúcares de parede celular apiose, arabinose, fucose, ramnose, glicose, xilose, galactose e manose) e não estruturais (açúcares solúveis e de armazenamento sacarose, frutose, rafinose, glicose e amido) de três espécies de Lemnoideae e duas Wolffioideae comparando com as taxas de crescimento dessas plantas. / Duckweeds are the smallest aquatic monocots classified in two subfamilies (Lemnoideae and Wolffioideae) that contemplates thirty-seven species representing five genera: Spirodela, Lemna, Landoltia, Wolffia and Wolffiella . These plants display fast vegetative reproduction, which implies a rapid accumulation of carbohydrates and protein, conferring them biotechnological and industrial applications, especially in bioenergy. Carbon balance among growth is related to photosynthetic assimilation, storage, and consume of compounds which are influenced by light intensity. The major sink of the carbon assimilated is to cell wall and sucrose synthesis, thereby knowing the polysaccharides composition and their interactions together with the non-structural carbohydrates content may impact the alternative fuels production. The present work aimed to quantify and comparative analyze structural (cell wall sugars apiose, arabinose, fucose, rhamnose, glucose, xylose, galactose, and mannose) and nonstructural (storage and soluble sugars starch, sucrose, glucose, raffinose, and fructose) carbohydrates of three Lemnoideae and two Wolffioideae species correlating to its growth rates.