Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliography.

Thesis (DBA) -- Macquarie University, Graduate School of Management, 2007. / Bibliography: p. 209-223.

Thesis (Ph. D.)--Public Policy, Georgia Institute of Technology, 2008. / Herrera, Hector, Committee Member ; Cozzens, Susan, Committee Chair ; Rogers, Juan, Committee Member ; Shapira, Philip, Committee Member ; Bowman, Kirk, Committee Member.

Thesis (M.C.A.)--Victoria University of Wellington, 2009. / Includes bibliographical references.

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology in the Department of Biotechnology, Technikon Natal, 1999. / General removal of phosphorus (P) from wastewater was introduced in Scandanavia in the late 1960's. At that time it was believed that P alone was limiting to algal growth and that the sole removal of P would solve the problem of eutrophication. However, we now know that both P and nitrogen (N) contribute to this deleterious effect and as such, much research has been conducted concerned with both the biological and chemical removal of these nutrients from sewage effluents. Enhanced biological phosphorus removal (EBPR), which is basically the biological accumulation of soluble P (as polyphosphate or poly-P) from the bulk liquid in excess of normal metabolic requirements, still tends to be sensitive to many external parameters and, as such, is subject to fluctuations. This makes it extremely difficult for wastewater treatment installations to achieve and maintain full compliance with strict discharge regulations. A more comprehensive understanding of the microbial community within the mixed liquor of a wastewater treatment system is therefore required which will ultimately assist in improving system design and performance. Chemical and civil engineers, when designing biological wastewater treatment systems, consider only the processes (biological or chemical) taking place within the reactor/s with little or no regard for the individual microbial species or the entire microbial community involved. Process design appears to be tackled empirically from a 'black box' approach; biological reactions or processes occurring within a system such as wastewater treatment are all lumped together and attributed to a single surrogate organism ie., the response of the surrogate to certain stimuli accounts for the total system response. This is similar to an analogy which Professor George Ekama (Dept of Civil Engineering, UCT), a leading scientist in wastewater treatment and process design, refers to where engineers, if, for example, are confronted with modelling the dynamics of carbon dioxide utilisation ofa forest, would recognise the accumulative system response and not give cognisance to each individual tree's contribution. It is true that if one had to consider every microbial species present in a highly organised community such as activated sludge, process models, designed to make quantitative and qualitative predictions as to the expected effluent quality from a particular design, would become increasingly complex and superfluous. It is evident from the countless accomplishments that engineers have succeeded, to a certain degree, in modelling wastewater treatment systems. One only has to consider the tremendous success of biological P (bio-P) removal and nitrification/denitrification processes at full-scale. However, there are limitations to this empirical approach and EBPR processes occasionally deteriorate in phosphate removal efficiency. In order to further optimise biological processes, whether they be organics oxidation, bio-P removal, nitrification or denitrification, biological community analyses will have to play a more significant role in design. The better microbial community structure and function is understood, the better the control and management of the system. With the advent of improved microbial identification and enumeration (to a certain extent) techniques (in situ), it was considered significant to investigate the mechanism ofbio-P removal and to elucidate which bacteria are actively responsible for this process. To this end, experimental work was conducted in two phases: \xAE laboratory, where samples of mixed liquor were obtained from a full-scale wastewater treatment facility exhibiting biological nutrient removal (BNR) characteristics and @ pilot plant, where an enhanced culture ofpolyphosphate accumulating organisms (PAO's) was developed and probed using molecular identification and enumeration techniques (as well as a cultivation-dependent approach). During phase \xAE of experimentat / M

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology : Biotechnology, Technikon Natal, 1998. / Waste activated sludge is a biological adsorbent whose potential to remove metals from solution and effluent has been demonstrated. Extracellular polysaccharides (EPS) as components of activated sludge are thought to contribute to activated sludge metal biosorption. During the present study characterisation and determination of the metal biosorptive capabilities of domestic and industrial extracellular polysaccharides (EPS) revealed similarities both in terms of chemical composition and metal adsorption potential. Extracellular polysaccharides were extracted from activated sludge, obtained from domestic and industrial sludge treatment plants, using chemical techniques which involved sodium hydroxide extraction and solvent precipitation. A purification technique, which involved precipitation of protein with chloroform and removal of nucleic acids was developed. To assess the efficiency of the purification method, the ratio of extracted polysaccharide to the amount of protein present was determined. This provided an indication of the magnitude of EPS extracted in relation to the degree of cellular disruption. The type of activated sludge being treated was shown to be of particular importance. The quantity of EPS present in the original sample was found to be higher in domestic sludge than in industrial sludge. Purified EPS was fractionated in a column of DEAE-Sepharose CL-6B using stepwise pH gradient elution. Molecular weight distribution was conducted on a column of Sepharose CL-4B. Component monosaccharides were identified by paper chromatography. Monomers identified were glucose, fructose, glucuronic acid and galactosamine. Ion-exchange chromatography results demonstrated the presence of a number of different polysaccharide fractions while gel filtration results indicated a wide molecular weight distribution range of EPS from both domestic and industrial activated sludge. This indicated potential for variety in the EPS content of the activated sludge. Metal adsorption studies were conducted to determine the capabilities of EPS to adsorb metals / M

Dissertation submitted in compliance with the requirements for the Master's Degree in Technol: Biotechnology, Technikon Natal, 1997. / As a result of rapid expansion of the industrial sector and increasing population, the environment has been under phenomenal stress. The volume of sewage and other effluents has increased tremendously in the last century. Globally, approximately .12 million tonnes of dry sludge biomass is produced and discarded of by landspreading, landfilling, incineration or dumping in lagoons and oceans. The discharge of industrial effluents into receiving waters has been documented to be the cause of severe environmental contamination. Heavy metals have been the cause of particular environmental concern. Their toxic and carcinogenic potentials at low concentrations, as well as the large quantities disposed to the environment, have prioritised them as leading contaminants. Current technologies of remediating heavy metal containing effluents are expensive and, in most cases, ineffective. Locally, most industries are merely diluting their effluents, thus resulting in the loss of valuable water resources. Waste sludges have shown the ability to adsorb heavy metals from their aqueous environment. Therefore, the current study attempted firstly, to compare biosorptive capacities of various waste sludges for a range of heavy metal ions, and secondly, to establish a relationship, if any, between biosorptive capacity and sludge surface charge. Finally, a laboratory scale biosorption process, encompassing desorption and recovery of metal ions from sludge surfaces, would have to be developed. Effluents used included pure, metal solutions of divalent zinc, cadmium, copper, nickel, trivalent and hexavalent chromium. In addition, synthetic effluents comprising a cocktail of the above-mentioned metal ions as well as an industrial effluent from a metal plating company were used. Five waste digested sludges were prepared and challenged against pure metal solutions to determine and compare their respective biosorptive capacities. Mechanisms of biosorption were elucidated using the Langmuir adsorption isotherm model. Sludge surface charge was determined using the millivolt quantification method. Upscaling of bioreactor trials to fully mixed laboratory scale was also investigated. These experiments encompassed the use of three sludges showing the greatest potential for biosorption and desorption using the selected mineral acid, H2S04, In addition, a simultaneous fully mixed biosorption and desorption process was designed and optimised. Subsequent trials involved comparing the latter process with a packed bed configuration whereby biomass was immobilised using poly sulfone resin. The overall comparative adsorptive capacities of the sludges (SI-SS) for metal ions in single solutions was S3 > S2 > S4 > SS > SI. Surface charge determination showed S3 to contain the most electronegative charge, with other sludges following in the same descending order as mentioned above. These findings supported the theory of a direct correlation between sludge surface charge and biosorptive potential. The affinity series of the sludges for metal ions followed the descending order of Cd2+ > Cu2+ > Ni2+ > Zn2+ > Cr6+ > Cr3+. Fully mixed studies, using mixed synthetic effluents, resulted in lower biosorptive capacities being recorded by the three selected sludges ie., S2, S3 and S4, as compared to single solution experiments. Biosorption studies with industrial effluent, containing Zn2+ as the most prevalent metal at 119.4 mg.F'. resulted in S3 biosorbing a maximum of 4.5 mg.g' of the cation. Sulphuric acid (H2S04) at O.2N, hydrochloric acid (HCI) at O.2N and acetic acid (CH3COOH) at O.4N were tested for their desorptive efficiencies. Sulphuric acid proved to be the most effective desorbing agent. Using S3 as biosorbent and O.2N H2S04 as desorbent, the manipulation and operation of a simultaneous process proved to be successful since both biosorption and desorption occurred concurrently, thus reducing time required for successful remediatio / M

Green microalgae are an important source of natural products such as β-carotene, and have recently become objects of intense study for producing biodiesel and valuable recombinant proteins. Application of chloroplast engineering in microalgae is limited by the availability of tools for genetically engineering the chloroplast of commercially important species. The phytoene desaturase gene of a previously isolated norflurazon tolerant mutant of Chlamydomonas reinhardtii was isolated and sequenced. A thymine to guanine transversion in exon 2 changes codon 131 resulting in a F131V mutation that is located in the NADP binding site domain on the primary structure. This mutation clusters with three conserved amino acids, whose substitution confers norflurazon tolerance in other species, in a pocket on a 3-D structure of the protein. The pocket identifies the target site of norflurazon. The side pocket is at the opening of a tunnel leading to the enzyme's NADP binding site. The mutant gene was cloned and used as marker for glass-bead mediated nuclear transformation of C. reinhardtii using direct selection with 5 μM norflurazon. Integration was by illegitimate recombination and transformants were able to grow in media containing 150 μM norflurazon. Transformants exhibited cross tolerance to fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wildtype. This allows mutant pds gene to act as a dual negative/positive selectable marker that is conditional on the herbicide used. The F131V mutation was introduced into a synthetic gene encoding a Dunaliella salina phytoene desaturase that contained codons used frequently in C. reinhardtii chloroplast genes. The 1.8 kbp CpPDS1 gene was assembled from 74 oligonucleotides by overlap PCR. The coding sequence was inserted into a Dunaliella tertiolecta chloroplast targeting vector that integrated the CpPDS1 sequence into the ycf3-trnL-rbcL region of the plastome. The resulting vector was transformed into D. salina and D. tertiolecta chloroplasts using particle bombardment with plasmid coated gold microprojectiles. Norflurazon tolerant colonies were isolated and the D. salina and D. tertiolecta clones were shown to contain a pds gene integrated in the plastome using PCR analyses. Transformation of the CpPDS1 gene into C. reinhardtii chloroplasts by rescue of an atpB mutation only gave rise to herbicide tolerant colonies if the presequence was removed. Industrial production of algae in large volumes is limited by the availability of light to drive algal growth. This problem was addressed by expressing fluorescent protein Katushka in the chloroplast of C. reinhardtii which converts yellow light to red light. The Katushka gene was transformed into chloroplasts using vector pB10, which was constructed to rescue a deletion in the chloroplast atpB gene in the mutant CC373 strain. The Katushka coding sequence was codon-optimised for expression in chloroplasts and expressed from three different promoter and 5' UTRs (atpA, atpB and psbD). C. reinhardtii wild type cells were able to grow under either blue or red LED lights but grew best when both were present. Wild type cell grew poorly in yellow LED lighting. Cells expressing Katushka in the chloroplast exhibited enhanced autotrophic growth in yellow light and under conditions where yellow light was present and red light was limiting. The improvement in growth was related to the levels of Katushka fluorescence detected in chloroplast transformants, which was highest for the atpA promoter and UTR.

Lee Chi-Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-167). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.II / Abstract --- p.IV / 摘要 --- p.VI / Content --- p.VIII / Abbreviations --- p.VIX / List of figures and tables --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.1 / Chapter 1.1 --- How are the thermophilic proteins stabilized? --- p.2 / Chapter 1.1.1 --- Hydrophobic interactions --- p.2 / Chapter 1.1.2 --- Hydrogen bonds --- p.4 / Chapter 1.1.3 --- Electrostatic interactions --- p.6 / Chapter 1.1.4 --- Reduction in ΔCP --- p.9 / Chapter 1.2 --- Models of study: Thermococcus celer and yeast L30e --- p.12 / Chapter 1.2.1 --- Thermococcus celer ribosomal protein L30e --- p.12 / Chapter 1.2.2 --- Yeast ribosomal protein L30e --- p.13 / Chapter 1.2.3 --- Comparison between the two proteins --- p.13 / Chapter 1.3 --- Objective of this study --- p.20 / Chapter Chapter 2 - --- Materials and Methods --- p.21 / Chapter 2.1 --- General techniques --- p.21 / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5α and BL21(DE3)pLysS --- p.21 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.22 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.24 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.24 / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) --- p.25 / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.26 / Chapter 2.1.7 --- Ligation of DNA fragments into vector --- p.26 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.28 / Chapter 2.1.9 --- Native-PAGE electrophoresis --- p.32 / Chapter 2.2 --- Protein Engineering of Proteins --- p.35 / Chapter 2.2.1 --- Polymerase chain reaction (PCR) --- p.35 / Chapter 2.2.2 --- Site-directed mutagenesis of T. celer L30e --- p.37 / Chapter 2.2.3 --- Protein engineering of yeast L30e --- p.42 / Chapter 2.3 --- "Sub-cloning of mutation PCR fragment into expression vector, pET8c" --- p.45 / Chapter 2.4 --- Expression of recombinant proteins --- p.45 / Chapter 2.5 --- Purification of T. celer and its mutants --- p.46 / Chapter 2.5.1 --- Extraction of proteins by sonication --- p.46 / Chapter 2.5.2 --- Purification by ion-exchange chromatography --- p.47 / Chapter 2.5.3 --- Purification by affinity chromatography --- p.48 / Chapter 2.5.4 --- Purification by size exclusion chromatography --- p.49 / Chapter 2.6 --- Purification of yeast L30e and its mutants --- p.49 / Chapter 2.6.1 --- Extraction of proteins by sonication --- p.49 / Chapter 2.6.2 --- Purification yeast L30e variants by washing the inclusion bodies --- p.50 / Chapter 2.6.3 --- Purification by column chromatography --- p.51 / Chapter 2.7 --- Thermodynamic studies of proteins --- p.52 / Chapter 2.7.1 --- Guanidine-induced denaturation --- p.52 / Chapter 2.7.2 --- Thermal-induced denaturation --- p.53 / Chapter 2.7.3 --- Determination of protein stability curves by denaturant unfolding --- p.54 / Chapter 2.7.4 --- ΔCP and protein stability curve determination by thermal unfolding --- p.55 / Chapter 2.8 --- Media and buffer recipes --- p.56 / Chapter 2.8.1 --- Medium for bacterial culture --- p.56 / Chapter 2.8.2 --- Reagents for competent cell preparation --- p.58 / Chapter 2.8.3 --- Nucleic acid electrophoresis buffers --- p.58 / Chapter 2.8.4 --- Buffers for T. celer L30e variants purification --- p.59 / Chapter 2.8.5 --- Buffers for yeast L30e variants purification --- p.59 / Chapter 2.8.6 --- Reagents of SDS-PAGE --- p.60 / Chapter Chapter Three - --- Purification of T. celer and Yeast L30e --- p.63 / Chapter 3.1 --- Purification of T. celer L30e and its mutants --- p.63 / Chapter 3.2 --- Purification of yeast L30e and its mutants --- p.72 / Chapter Chapter Four - --- Thermodynamic Studies of T. celer and Yeast L30e --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Result --- p.79 / Chapter 4.3 --- Discussion --- p.85 / Chapter Chapter Five- --- Mutagenesis Study of a Charge Cluster in T. celer L30e --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Result --- p.92 / Chapter 5.3 --- Structure determination of T. celer L30e mutants --- p.99 / Chapter 5.4 --- Discussion --- p.105 / Chapter Chapter Six - --- Alanine Scanning Mutagenesis of Charge Residues of T. celer L30e --- p.114 / Chapter 6.1 --- Introduction --- p.114 / Chapter 6.2 --- Result --- p.114 / Chapter 6.3 --- Discussion --- p.121 / Chapter Chapter Seven - --- Protein Engineering of T. celer and Yeast L30e --- p.132 / Chapter 7.1 --- Introduction --- p.132 / Chapter 7.2 --- Result --- p.136 / Chapter 7.3 --- Discussion --- p.138 / Chapter Chapter Eight - --- Concluding Remarks --- p.141 / Appendix --- p.143 / Reference --- p.154

Thesis (J.S.D.)--Stanford University, 1996. / Includes bibliographical references (p. 361-373). Also available online.