Enhanced microalgae growth and lipid production: a study of cytostatic inhibitors and glycerol assimilation

Abstract

Current microalgal cultivation strategies and the unrefined oil produced (priced at ~USD$294/barrel) (Benemann et al, 2012), have yet to produce globally significant amounts of commercially viable biofuels competitive with that of fossil derived crude oil currently at USD$45.22/barrel (WTI (Nymex) as of 28.08.2015) (Nasdaq, 2015). Underlying this issue is the economics of gross lipid production, a function of lipid content and cellular productivity. The survival response of microalgal strains that promote the accumulation carbon as lipids typically occurs under stress conditions (such as nutrient deficiency) at the expense of cellular growth. This study explored alternative strategies by inhibiting metabolic processes, cell growth and division, through the use of cytostatic inhibitors such as sodium oxamate and conazole fungicides to induce lipid production in the absence of low nutrient conditions. Additionally, the exogenous supply of glycerol as a potential source of additional carbon was also investigated to improve photoautotrophic microalgae growth. An inducible trigger for the rerouting of carbon towards lipid synthesis under nutrient replete conditions has great potential for research and commercial applications of microalgal biofuels. Photoautotrophic growth of locally collected strains was optimised through nutrient screens and subsequently dosed using the tested inhibitors. The testing of these inhibitors showed that sodium oxamate was able to inhibit cell division and increase the lipid content in most of the tested microalgae strains. On the other hand, conazole treatment was largely observed to decrease the cellular biomass of the microalgae, which suggests that carbon assimilation may have been reduced. The results indicate that both types of inhibitors were able to cytostatically affect most of the tested strains and that a number of strains responded well with increased lipid production. This process has allowed us to test the effectiveness of the compounds and to identify potential high lipid producers. Some species of microalgae are able to supplement CO2 fixation with the utilisation of exogenous carbon substrates, typically acetate or glucose. By exploring their facultative heterotrophic capabilities, photoautotrophic limitations such as light shading may be overcome. From a biotechnological perspective it would be advantageous if waste carbon streams from other processes could be used to supplement microalgal growth. One such byproduct is glycerol from the biodiesel industry. The radiolabelled results validated that glycerol is assimilated into central metabolism of Chlorella protothecoides and may be able to contribute towards cellular growth. The data presented in this thesis highlights that the microalgal metabolic diversity for particular biotechnological applications can be probed and manipulated by utilising inhibitors and substrates known to function on specific metabolic pathways. In chapter two, the hypothesis that sodium oxamate can induce lipid production in microalgae was tested. The key finding was that sodium oxamate was able to induce as well as increase lipid production in microalgae; and that despite sharing metabolic pathways not all strains responded equally to sodium oxamate treatment. In chapter three, it was postulated that highly conserved sterol synthesis pathways between microalgae and fungi suggested that conazole fungicide compounds should be cytostatic in all microalgae strains. Whilst published research results were unable to be replicated with Dunalliela tertiolecta, the key findings from this study reaffirmed that conazole treatment is cytostatic to microalgae and yielded increases in lipid production in Scenedesmus sp.. It has been reported within the literature that some microalgae species are able to grow heterotrophically on glycerol as a carbon source; which often contain other confounding carbon substrates. In chapter four, glycerol assimilation into microalgal metabolism was investigated using radiolabelled [14C]-glycerol in Chlorella protothecoides. Beta activity was detected in the biomass and a low level was observed in the Ca(OH)2 traps which validates that glycerol may be readily assimilated into metabolism. By identifying positively responding strains and utilising these seldom used modes of growth, alternate cultivation and lipid inducing strategies may be investigated to improve the biological economics of lipid productivity and improve the feasibility of microalgal biodiesel.