The development and implementation of clean, economically viable and renewable energy sources has recently gained substantial interest, for two main reasons. Firstly, as we approach ‘peak oil’ production, oil prices have started to rise rapidly, raising the question of sustainable fuels to maintain current oil and fossil fuel-driven economies. Secondly, combustion of fossil fuels has led to increased greenhouse gas levels in the atmosphere. Together these issues have resulted in an economic and environmental problem which requires the urgent development of alternative energy sources.
Although a number of different techniques in energy conservation are now being explored, one that shows great potential is the use of microalga. Chlamydomonas reinhardtii (C. reihardtii) is a green unicellular microalga capable of producing biohydrogen from one of the most abundant energy sources; sunlight. This microorganism is able to convert carbon dioxide to a usable form of chemical energy (starch). The short doubling time and inexpensive culture requirements have resulted in C. reinhardtii being studied extensively as a model organism both at the molecular and the genetic level. There has been relatively limited research, however, into the metabolic pathways of this organism and how they affect its functionality as a biofuel producer. The implication of this is that microalgal biofuel systems still require much more improvement in order to bring them to commercial viability.
The aim of this thesis was to utilise the advancements in analytical and computational tools available today to initiate a global, metabolomic approach to understanding the underlying regulation of metabolism in C. reinhardtii. Deuterium, a stable hydrogen isotope, was examined in an attempt to trace its distribution into the metabolic network, as displayed by isotopic enrichment in metabolites. Multiple interconnected factors were shown to affect metabolite variation including: (i) cultivation (biomass), (ii) biomass sampling, (iii) isolation/ extraction method i.e. number of steps and solvents used and (iv) data acquisition. These made it difficult to draw conclusive reasons for the observed variance. However, this work provides a proof of concept for undertaking alga metabolomics using small (1 mL) sample sizes and remains promising as demonstrated in this work. The media optimisation results provide further understanding of growth conditions which can be manipulated to maximise growth for individual species or used to model growth rate, chlorophyll content, cell morphology amongst other desirable traits. The algal metabolomics (Nuclear magnetic resonance, NMR and Gas chromatography- mass spectrometry, GC-MS) coupled with isotopic tracer studies provides a platform and basis for future use during metabolic flux analysis in C. reinhardtii. Metabolomic and isotopic tracer studies can be used to better understand how C. reinhardtii cells develop and respond to their environment and ultimately how they can be metabolically engineered to enhance their output as a source of biohydrogen.