Thesis (Ph. D.)--University of Hong Kong, 1984.

Thesis (doctoral)--Universität Strassburg, 1877. / Includes bibliographical references.

The tropical carnivorous pitcher-plant family, Nepenthaceae, is represented by the single dioecious genus Nepenthes. Whilst South East Asia represents the centre of diversity of the group several species have disjunct biogeographical distributions. Due to the highly polymorphic nature of Nepenthes and its distinct lack of discrete morphological characters no revision to date has been able to construct a phylogenetic classification. To address this issue, this study analyses nucleotide sequences from two independent regions, including chloroplast DNA (the trnL-F intron and intergenic spacer) and the nontranscribed spacer region from nuclear DNA (5S-NTS), together with a suite of morphological characters traditionally used to describe and group species. A total of 87 species are represented with the objectives of i) testing the monophyly of the genus and constructing a hypothesis of species relationships, ii) evaluating the taxonomic circumscription of poorly known species and iii) investigating character evolution and biogeographic patterns. In addition, for 25 species, multiple accessions were sequenced. Two artificial hybrids (of known sexual parentage), representing three geographically and taxonomically distinct species, were also included to test prevalent theories of hybrid origin and maternal inheritance. Cladistic analyses of these data strongly support the monophyly of the genus in which the disjunctly distributed species (including the easterly Nepenthes danseri from Waigeo Island and Halmahera) were found to form a grade while the South East Asian taxa form a clearly monophyletic group. Incongruence was found between the phylogenetic histories inferred from independent genetic markers and provides strong evidence to support natural hybridization as an important evolutionary process in the speciation of Nepenthes. The results support an ancient circum-Indian Ocean origin for Nepenthes, indicating the disjunct species to represent relictual taxa, with a geologically recent and rapid expansion into South East Asia. A phylogenetically consistent classification of the species is proposed.

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

Résumé confidentiel / Not available

Thesis (DTech (Environmental-health))--Cape Peninsula University of Technology, 2018. / The management of agricultural soil and its sustainable use, namely productivity, is paramount to the agricultural industry worldwide. Large-scale agricultural product producers and scientists emphasise using environmentally benign methods to increase agricultural production such as taking a green chemistry approach to agricultural activities and/or using cultivation techniques for the bio-augmentation of agricultural soil. Some of these agricultural products, such as cassava (Manihot esculenta), produce cyanogens which promote the infestation of a cyanogen-resistant microbial species known to produce mycotoxins during decomposition. Although cyanogens and mycotoxins are important components in the functioning of the earth system and agricultural soil, their cumulative effects can result in reduced soil productivity, hence degradation. Furthermore, the presence of mycotoxins in the environment and agricultural produce is hazardous to the environment, including the biotic communities in soil and humans. Therefore, an environmentally benign (green chemistry approach) method for the reduction of cyanogens and mycotoxins was proposed for this research study. The method investigated had to be applicable in-situ for the biodegradation of cyanogens and mycotoxins. Their reduction from decomposing cassava in cultivated soil, which can be used on a small and large scale, would mitigate deleterious effects of a less reported, unknown mycotoxins producer (fungal species), Cunninghamella bertholletiae (KT275316), found to be a free cyanide- (CN-) resistant isolate. The C. bertholletiae was isolated from decomposing cassava tubers and silt, subsequent to culturing on potato dextrose agar (PDA) and in an equivalent volume of nutrient broth (NB) containing KCN (4mg/40mL) at 30 °C for 120 hrs. The isolate demonstrated an ability to biodegrade CN- into NH3 and NO3. NH3 and NO3 are nitrogenous by-products produced when young cassava plants are cultivated in a controlled environment, with 80% of the initial CN- concentration being efficiently degraded to NH3/NO3 at a conversion rate of 77.5% and 72.5% (fungus from silt and cassava), respectively, within 120 hrs. From this research, it was observed that Sub-Saharan Africa is the largest contributor to the CN- load into the environment; from cassava cultivation as per FAO data. The quantity of CN- released was estimated at 0.025x10-3 to 6.71 ppq, with further increases of 60.5% being projected to be released into the environment by 2024. As such, it was hypothetically assumed that numerous species in cassava-cultivated soil become CN- resistant as they are exposed to CN- from decomposing cassava, becoming pathogenic thus antigonistic towards other biota in cassava-cultivated soil. Consequently, the pathogenicity of the isolate was investigated against organisms (n = 12) from cassava-cultivated soil. The isolate demonstrated inhibitory pathogenic activity against some soil bacterial communities such as Oligella ureolytica, Acinetobacter sp., Pseudomonas luteola and Sphingomonas paucimobilis. The isolate also demonstrated minor antagonistic effects against Myroides sp., Stenotrophomonas maltophilia, Candida lipolytica, Cryptococcus albidus and Rhodotorula sp.. Further research to identify extracellular metabolites produced by this organism, using a fermentation method was also carried out using a liquid state fermentation technique. 30 mL Erlenmeyer flasks containing 25 mL of NB/KCN (source of CN-) at 37 °C for 168 hrs, with a volume of (5 mL), extracts from the fermentation being filtered, centrifuged, mixed with chloroform for a liquid-liquid extraction procedure subsequent to a nitrogen-facilitated blow-down technique and reconstitution with 100% analytical grade methanol, for LC/MS-TOF 6230 analysis. The analysis revealed that the isolate was able to produce the mycotoxins/secondary metabolites, Fumonisin B1 (FB1) and Deoxynivalenol (DON). Though the isolate (KT275316) demonstrated the ability to biodegrade cyanide as well as produce mycotoxin, an environmentally benign strategy (green chemistry method) with a potential to biodegrade CN-/NH3/NO3/NO2 for the biodegradation of mycotoxins was evaluated, including the identification of biodegradation by-products post-biodegradation treatment. Thus, plant extracts from Nepenthes mirabilis were found to contain enzymes such as carboxylesterase, β-glucosidase, β-glucoronidase and phosphatidyl inositol phospholipase C (identified using both quantitative and qualitative methods). The plant extracts were used with treated samples from the fermentation and were subjected to biodegradation. Thus, resulting in biodegradation by-products such as Heptadecanone Octadecanamide, Octadecenal for FB1 and Tolmetin for DON, respectively. For future research, it is therefore recommended that plant extracts with similar properties to those observed for N. mirabilis extracts (juice) be sought for application of the proposed method.

This thesis deals with the area of honeypots and honeynets. It defines their classification and contains detailed descriptions of their properties and features. It further elaborates on several freely available systems. The main focus is given to honeypot Nepethes that was being run for one month on an unfiltered Internet connection. A detailed analysis of the collected data is then given.

Thesis (Doctor of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Lignocellulosic biomass (agro-waste) has been recommended as the most promising feedstock for the production of bioalcohols, in the biofuel industry. Furthermore, agro-waste is well-known as the most abundant organic matter in the agricultural and forestry product processing industry. However, the challenge with utilizing agro-waste as a feedstock is its highly recalcitrant structure, which limits hydrolysis to convert the holocelluloses into fermentable sugars. Conventional pre-treatment methods such as dilute acid, alkaline, thermal, hot water and enzymatic, have been used in previous studies. The challenge with these conventional methods is the generation of residual toxicants during the pretreatment process, which inhibits a high bioalcohol yield, by reducing the microbial populations’ (fermenter) ability to be metabolically proficient during fermentation. Numerous studies have been developed to improve the engineered strains, which have shown to have an ability to reduce the inhibition and toxicity of the bioalcohols produced or by-products produced during pre-treatment, while enhancing the bioalcohol production. In the present study (chapter 5), evaluation of common conventional methods for the pretreatment of the mixed agro-waste, i.e. (˃45µm to <100µm) constituted by Citrus sinensis, Malus domestica peels, corn cobs from Zea mays and Quercus robur (oak) yard waste without a pre-rinsing step at a ratio of 1:1 at 25% (w/w) for each waste material, was undertaken, focusing on hot water pre treatment followed by dilute acid (H2SO4) pre-treatment. To further pretreat the mixed agro-waste residue, cellulases were used to further hydrolyse the pre-treated agro-waste in a single pot (batch) multi-reaction process. The TRS concentration of 0.12, 1.43 and 3.22 g/L was achieved with hot water, dilute acid and cellulases hydrolysis as sequential pretreatment steps, respectively, in a single pot multi-reaction system. Furthermore, a commercial strain was used to ascertain low (C1 to C3) and high carbon content (C4+) bioalcohol production under aerobic conditions. Multiple bioproducts were obtained within 48 to 72 h, including bioethanol and 1-Butanol, 3-methyl, which were major products for this study. However, undesirable bio-compounds such as phenolics, were detected post fermentation. Since multiple process units characterised by chemical usage and high energy intensivity have been utilized to overcome delignification and cellulolysis, a sustainable, environmental benign pretreatment process was proposed using N. mirabilis “monkey cup” fluids (extracts) to also reduce fermenter inhibitors from the delignification of mixed agrowaste; a process with minimal thermo physical chemical inputs for which a single pot multi-reaction system strategy was used. Nepenthes mirabilis extracts shown to have ligninolytic, cellulolytic and xylanolytic activities, were used as an enzyme cocktail to pretreat mixed agro-waste, subsequent to the furtherance of TRS production from the agro-waste, by further using cellulase for further hydrolysis. N. mirabilis pod extracts were determined to contained carboxylesterases (529.41±30.50 U/L), β-glucosidases (251.94±11.48 U/L) and xylanases (36.09±18.04 U/L), constituting an enzymatic cocktail with a significant potential for the reduction in total residual phenolic compounds (TRPCs). Furthermore, the results indicated that maximum concentration of TRS obtainable was 310±5.19 mg/L within 168 h, while the TRPCs were reduced from 6.25±0.18 to 4.26 ±0.09 mg/L, which was lower than that observed when conventional methods were used. Overall N. mirabilis extracts were demonstrated to have an ability to support biocatalytic processes for the conversion of agro-waste to produce fermentable TRS in a single unit facilitating multiple reactions with minimised interference with cellulase hydrolysis. Therefore, the digestive enzymes in N. mirabilis pods can be used in an integrated system for a second generation biorefinery.

Le travail présenté porte sur le développement d’une nouvelle technologie innovante, nommée PAT Friday®, visant à produire des protéines recombinantes au sein des sécrétions extracellulaires de plantes carnivores génétiquement modifiées. Deux objectifs ont été fixés : Réaliser la preuve de concept de la technologie sur le modèle expérimental Drosera rotundifolia, en transformant la plante avec des gènes marqueurs et humains afin de mettre en évidence la présence des protéines recombinantes dans la glu ; et développer, après évaluation, la technologie sur un modèle potentiellement industrialisable, Nepenthes alata. Les résultats ont indiqué la présence des deux protéines marqueurs GFP et GUS dans les tissus et dans la glu de Drosera rotundifolia transformées. Les plantes ont également été transformées génétiquement avec les gènes humains de l’interféron gamma et du facteur intrinsèque. Les protéines recombinantes humaines ont été mises en évidence au sein des tissus végétaux. Le potentiel industriel du modèle Nepenthes alata a ensuite été étudié : 10 à 15 kg de protéines totales par hectare et par an peuvent être produits, grâce notamment à des récoltes successives non destructrices, et la possibilité de contrôler l’activité des protéases digestives naturelles. L’élaboration d’un protocole de régénération de la plante a été entreprise par embryogénèse somatique et organogénèse indirecte, en vue de sa transformation génétique. La technologie PAT Friday®, avec des étapes simplifiées d’extraction et de purification des protéines d’intérêt produites dans le liquide digestif, offre de nouvelles perspectives dans le domaine des protéines thérapeutiques produites à partir de plantes / The present work focuses on the development of a new innovating technology, called PAT Friday®, aiming at producing recombinant proteins into the extra-foliar fluid of modified carnivorous plants. Two objectives were assigned to this work : 1- to realize a proof of concept of the technology on the experimental model Drosera rotundifolia, transformed with marker and human genes, to confirm the occurence of the recombinant proteins into glu ; and 2 - to evaluate and develop, the technology on the model Nepenthes alata, more adapted to industrial scaling-up. The results indicate the presence of two marker proteins GUS and GFP inside the tissues and into the glu of modified Drosera rotundifolia plants. The same plant species has also been transformed with human gamma interferon and intrinsic factor genes. The corresponding human recombinant proteins have been detected into the plant tissues. Potential industrial scaling-up has been studied with the species Nepenthes alata. The results show a potential productivity of 10 to 15 kg of total proteins per hectare per year, thanks to non-destructive repeated harvests, and possibility to efficiently control the natural proteinase activity. The elaboration of a regeneration protocol has been undertaken through indirect organogenesis and somatic embryogenesis, with a view to transform genetically this plant. PAT Friday® technology, with simplified extraction and purification methods of the proteins of interest targeted into the liquid secretions, opens new perspectives in the field of therapeutical proteins produced in plants