The global demand for energy is projected to increase by more than thirty percent by 2035, but the supply of fossil fuels is finite and alternative energy sources must be explored. The search for the ultimate bioenergy source, one that is both sustainable and cost-effective, has attracted the attention of the fuel industry and governments worldwide. In particular, interest in microalgal-derived biofuels has increased rapidly and microalgal production systems have been suggested as a viable solution to the issue of fuel supply, as well as contributing to a reduction in carbon emissions to minimize climate change effects (Stephens et al. 2010a; Wijffels and Barbosa, 2010). The gap between microalgae technology and oil-based energy is mainly represented by production costs such as algae cultivation, harvesting and extraction. An improvement in microalgae-to-biomass conversion efficiency is therefore essential to make the system profitable (Dekker and Boekema, 2005; Jansson, 2000). Many efforts to improve microalgae biomass yield have focused on photosynthetic efficiency, though progress has been slow. This is mainly due to the many parameters that need to be optimised such as exposure to light, availability of nutrients, bioreactor design, strain selection and so on. Current screening capacities are limited due to large volume culture methods and the lack of accurate techniques for the high-throughput phenotypic analyses. It is therefore critical that better technologies for screening and optimisation of high-value strains are developed in order to meet the urgent need for alternative fuel supplies. To this end, this project has examined the feasibility of strain development of
Chlamydomonas reinhardtii grown in microculture format and explored the potential of high-throughput methods to analyse important parameters such as antenna size, energy loss and biomass yield. Chapter one provides an overview of the photosynthesis generally as well as a more detailed introduction concerning components of the light reaction and how these may be altered to increase biomass yield. The specific aims and significance of the thesis are also presented. Chapter two investigates the physical limitations on maximal growth rate in microculture, including light penetration and intensity, path length, shaking speed, cell density and volume. From this, it became clear that the existing technique for the estimation of biomass yield was not suitable for small volumes. Hence, chapter three presents a novel method based on flow cytometry to accurately estimate biomass in small volume cultures (Chioccioli et al., in review). This method is amenable to high-throughput analyses and is therefore suitable for the large-scale screening and optimisation of microalgae strains. Chapter four explores the use of flow cytometry to analyse a range of other important parameters. Small antennae mutants were identified based on low fluorescence as a means to quickly screen large numbers of putative mutants following mutagenesis. Dynamic regulation of chlorophyll content and growth cycle in response to different light intensities was also analysed via flow cytometry in order to identify the optimal light intensity for biomass accumulation. Finally, we applied these data to investigate the response of wild-type and small antennae mutant Stm3LR3 to varying light intensities over time and again used flow cytometry as well as quantitative real time PCR in order to analyse the regulation of the light capture and biomass yield. Chapter five provides a summary of the key conclusions of the thesis. Overall, this thesis has provided a proof-of-concept for the scale-up of microalgae phenotypic analysis based on flow cytometry as the principal assay. This technique has provided an accurate high-throughput method to monitor a range of biological processes including biomass yield, antennae regulation, growth cycle dynamics and light regulation. The application of these findings lies in large-scale microalgae strain development and will therefore serve to advance the identification of novel, high quantity biomass-producing strains. The optimisation of biomass production is important not just for the development of biofuels such as oil, methane, ethanol and hydrogen, but also for the development of other high-value biomass products, biomass animal and fish feeds, and for targeted protein expression.