The large-scale purification of the major spinach thylakoid lipids by a combination of silica and carboxymethyl-cellulose chromatography is described. Yields of hundreds of milligrams of the lipids, representing 25-40% of the original lipid, have been obtained. In addition, routine purities in excess of 99.7% of the isolated lipids has been demonstrated. The structures of the purified lipids have been confirmed by fatty acid analysis, thin layer chromatography, and ¹³C-NMR. Some minor reassignments to previously published ¹³C-NMR for these compounds are described. In addition, the ¹H-NMR spectra for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), are shown. The resonance assignments for MGDG and SQDG have been obtained by a combination of off-resonance decoupling experiments and by two-dimensional COSY ¹H-NMR experiments. Similar experiments with DGDG have failed to resolve the proton assignments due to extensive overlapping of the proton resonances. Interbilayer interactions between large unilamellar vesicles of DGDG in aqueous salt solutions have been examined by light scattering, freeze-fracture electron microscopy, and X-ray diffraction. When suspended in aqueous salt solutions, vesicles of 100 nm diameter were found to aggregate in a rapid and reversible manner to yield aggregates greater than 1000 nm in diameter. Freeze-fracture electron microscopy showed these aggregates to consist of appressed, but not fused, vesicles. Quasi-elastic light scattering and turbidity experiments showed that aggregation was not due to charged impurities of the lipid behaving in accordance with electrostatic double layer theory. Experiments testing the efficacies of various chloride salts indicated a strong correlation existed between ionic radius and ability of the salt to promote aggregation. Similar experiments examining the effect of sodium salts, glycerol, and pH on vesicle aggregation implicate an interaction between the DGDG head group and structured water as underlying the aggregation process. The effect of additions of other lipids on the extent of DGDG aggregation has been examined. Addition of 0.5 to 5.0% of either anionic lipid phosphatidylglycerol (PG) or SQDG inhibited the aggregation of DGDG vesicles, probably by the development of an electrostatic potential. Different effects of PG and SQDG on the concentration of Mg²⁺ required for aggregation indicated that PG may form a bidentate ligand with Mg²⁺ at ≥ 5 mol% PG. SQDG did not show this behavior, indicating that its negatively charged sulfonate group is unavailable for cation complex formation. Addition of MGDG to DGDG up to 50 mol% had no effect on the Mg²⁺ requirement for aggregation, but at ≥ 25 mol% triggered irreversible vesicle aggregation. This suggests that the MGDG head group is as effective at causing aggregation as the DGDG head group. Further, MGDG probably triggers vesicle fusion at ≥ 25 mol%. The results suggest that the galactolipids may contribute to the close approach of thylakoids in higher plant chloroplasts. The permeability properties of large unilamellar vesicles of DGDG to ⁸⁶Rb⁺, ³⁶Cl⁻, and ³H-glucose have been determined. In addition, the permeabilities of binary, ternary, and quaternary mixtures of thylakoid lipids to ⁸⁶Rb⁺ have also been measured. Vesicles of DGDG were found to be 60-130 fold more permeable to Rb⁺ and 46-76 fold less permeable to CI⁻ than phosphatidylcholine vesicles. Vesicles of DGDG and PC were similar in glucose permeability. Electron spin resonance measurement of DGDG bilayer fluidity indicated that fluidity differences could not account for the observed differences in ion permeability. The addition of 50 mol% of MGDG to DGDG vesicles had no effect on Rb⁺ permeability, suggesting that the HII phase preference of MGDG does not increase bilayer permeability. The addition of SQDG led to a large increase of Rb⁺ permeability. The calculated permeability coefficient to Rb⁺ for a DGDG/MDGD/SQDG/PG (1/2/0.5/0.5) mixture similar to that of thylakoid membranes was 2.0-10⁻⁹ cm-s⁻¹. This value is three orders of magnitude higher than that for phospholipid systems, and ten-fold higher than that for vesicles of pure DGDG. It is concluded that the permeability properties of thylakoid lipids are dominated by oriented surface dipoles and not by bilayer fluidity or acyl chain packing considerations. It is also proposed that the high permeability of thylakoid lipids to cations is the main cause of low observed thylakoid membrane electrical potentials, and large proton gradients across thylakoid membranes. It has been proposed previously that the high proportions of saturated phosphatidylglycerols (ie. DPPG) found in chilling-sensitive plants may promote the formation of gel phase lipid, and cause increased metabolite leakage, in the thylakoids of these species at chilling temperatures. The leakage of ⁸⁶Rb⁺ from large unilamellar vesicles of thylakoid lipids containing proportions of disaturated PG (as DPPG) mimicking those of chilling-sensitive and chilling-resistant plants has been measured. This data indicated that no increase in Rb⁺ permeation occurred between any of the tested vesicles systems between 7° and 30° C. Differential scanning calorimetry showed no heat flow indicative of gel to liquid- crystalline phase separation in any of the lipid mixtures, even with DPPG levels as high as 12 mol%. It is concluded that a direct effect of disaturated PG on chilling injury in sensitive plants by an increase of low-temperature thylakoid permeability is unlikely. / Science, Faculty of / Botany, Department of / Graduate

The effects of representative anaesthetics on the polymorphic phase behavior of phospholipids, particularly phosphatidylethanolamine, is studied. The series of normal alcohols and alkanes destabilize the bilayer structure of hydrated phosphatidylethanolamine causing the lipid to adopt hexagonal (HII) structures at lower temperatures than control By exception, ethanol and butanol stabilize the bilayer configuration. Chloroform also initiates bilayer destabilization, producing this effect at clinically relevant concentrations of anaesthetic. The charged anaesthetics tested show the ability to stabilize the bilayer configuration of phosphatidylethanolamine to higher temperatures than controls this occurs in a single or mixed lipid system (ie. phosphatidylethanolamine / phosphatidylserine). In all cases the ability of an anaesthetic to alter the polymorphic phase behavior of phosphatidylethanolamine is a function of its individual potency. The observed effects of anaesthetic agent on lipid polymorphism can be interpreted in relation to the molecular shapes of the lipids and anaesthetics involved. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Due to the numerous disadvantages associated with the use of fossil fuels, focus has been drawn on the environmentally friendly, renewable and carbon-neutral alternative, algalbased biofuels. Many microalgal species have been studied due to their ability to produce significant lipid yields which may be converted to biodiesel. In the present study three microalgal species were screened and a model organism that produced maximal lipid yields, had the greatest lipid productivity and showed potential to be used on a large scale basis, was selected. The selected species was identified, using both morphological and molecular methods, as Isochrysis galbana U4. Nitrogen (N) limitation and depletion studies showed that an internal N reservoir determines cell growth and eventual lipid accumulation in I. galbana when the external N reserves are deplete. Intracellular N depletion was associated with a decrease in the pyrenoid size and chlorophyll content, a breakdown of the chloroplast and the production of large lipid bodies which is advantageous in terms of lipid sequestration for biodiesel production. Cost reduction approaches for the mass culture of I. galbana were investigated. Factors that were proven to reduce costs, without altering the final lipid yield, included the use of urea as a N source and the supply of lower phosphorus (P) levels since this species is capable of growing optimally with as little as 0.25 ppm P. Furthermore, I. galbana cells demonstrated spontaneous flocculating abilities when cultured for prolonged periods. This is advantageous in the cost reductions of downstream harvesting processes. Both, 7 L and 16 L photobioreactors (PBR) were tested. Culture upscale resulted in the concomitant decrease in algal growth rate which was attributed to the limitations on the carbon dioxide and light supply in scaled up systems. Hence, it is suggested that multiple smaller units be used in an industrial setup. Overall, I. galbana is a promising candidate for biodiesel production, due to its ability to produce large amounts of lipid, its elevated growth rates and low P demand. The use of a two-phase PBR (The first phase being nutrient replete, promoting cell growth and division, and the second phase nutrient deplete, promoting lipid production) for the mass culture of this species in industry is recommended.

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