Algae and cyanobacteria platforms for engineering biofuel production

Abstract

This thesis investigated the capacity of a model microalga and two cyanobacteria species to provide the feedstock for sustainable liquid biofuel production. In particular, genetic manipulations in conjunction with nutritional triggers were used to redirect carbon into lipid biosynthesis. Wild-type and starch mutants of Chlamydomonas reinhardtii, a well-established model green alga, were subjected to nitrogen starvation to examine carbon storage and partitioning between starch and lipid synthesis. The starch-less mutant, BAF-J5, was found to store up to 75% of dry cell weight as fatty acid when grown photoheterotrophically under nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. Fluorescent spectroscopy was used for semi-quantitative measurements of neutral lipid storage in lipid droplets stained with Nile Red. Oleosin-like and caleosin-like genes, which are associated with lipid droplets in plants were identified in the genome. However, proteomic analysis of isolated lipid droplets only identified a key lipid droplet associated protein. The fatty acids were identified and quantified by gas chromatography mass spectrometry. Long-chain saturated, mono- and polyunsaturated fatty acids were found. This study demonstrates it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid biofuels. Nutrient availability and temperature were used to improve the fatty acid profile for biodiesel, thereby, potentially reducing the requirement for costly refining. Two cyanobacterial model organisms; a wild-type and glycogen mutant of the mesophile Synechococcus sp. PCC 7942, and the thermophile wild-type Thermosynechococcus sp. BP-1 isolated from different environments and therefore likely to have different lipid metabolism and responses to nutritional triggers were selected for study. Synechococcus sp. PCC 7942 was grown phototrophically and subjected to nitrogen starvation to examine the effect of nutrient limitation on the fatty acid content and composition. Carbon was not found to be stored as fatty acid or hydrocarbons in either the wild-type or glycogen deficient mutant under nitrogen starvation. Thermosynechococcus sp. BP-1 was also grown phototrophically and subjected to nitrogen and phosphorus starvation. Again, nitrogen starvation did not result in carbon storage as fatty acids or hydrocarbons. Only trace levels of organic compounds were found in the culture medium under nitrogen sufficient and deficient conditions. These studies indicate that metabolic engineering would be required to enhance lipid production in these species. The experimentally determined fatty acid profiles from these organisms were used to calculate fuel properties and to determine if any of the strains or environmental treatments would produce a fuel that would meet the biodiesel specifications for either the EU or the USA. In conclusion, it was found that disruption of starch biosynthesis and nitrogen starvation, in conjunction with an optimised growth temperature, greatly improved the likely quality of a biodiesel fuel derived from Chlamydomonas reinhardtii. Show more