Author: AHM Mohsinul Reza
Reza, AHM Mohsinul, 2022 A novel approach to determine lipid inducing conditions in microalgae with aggregation-induced emission-based fluorophores, Flinders University, College of Science and Engineering
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The usage of microalgal lipids is ubiquitous, ranging from bioactive compounds to biofuel production. Identification of lipid inducing conditions and underlying biochemical changes can significantly improve lipid quantity and quality in algae. However, there is a lack of an appropriate tool in vivo to visualize lipid drops in algae. Traditional probes for lipid studies often suffer from various dye-specific limitations, such as aggregation-caused quenching and self-decomposition phenomena that hinder the accuracy of lipid identification. The recent emergence of aggregation-induced emission (AIE)-based nanoprobes has shown a promising attribute to overcome the limitations of conventional fluorophores for bioimaging the target molecules. This thesis aims to identify the lipid producing conditions using novel AIEgens as the lipid detecting probes in microalgae. Two morphologically distinctive algae were used Euglena gracilis, without a cell wall, and Chlamydomonas reinhardtii, with a carbohydrates-based cell wall.
Three experiments were included in this thesis. Experiment 1 evaluates the effects of light (70 mmol photons m-2 s-1), nutrient starvation and supplemented carbon source on lipid biosynthesis in E. gracilis cells. Cellular lipid drops (LDs) were detected with a lipid-specific AIE-probe, DPAS (C20H16N2O). Experiment 2 determines the lipid inducing conditions with AIE-probe, 2-DPAN (C24H18N2O), as a new tool to detect the LD formation in C. reinhardtii. In addition, hydrogen peroxide (H2O2) activity was measured as a component of the secondary messenger molecules and natural by-products. The novel H2O2 detection technique was established utilizing H2O2-specific AIEgen, TPE-BO (C38H42B2O4). Finally, experiment 3 determines the effects of external H2O2 supplementation on the lipid composition and content in C. reinhardtii.
The results of experiment 1 suggest that compared to the autotrophic condition (modified Cramer-Myers medium, MCM), nutrient-starved heterotrophic condition (nitrogen and calcium starved MCM, but with glucose supplementation in the dark) increased total lipid and health beneficiary LC-PUFAs production in E. gracilis by ~11.93% and ~14.36%, respectively. In experiment 2, C. reinhardtii cultured in the nitrogen and calcium starved, but sodium acetate (2.0 g/L) supplemented Woods Hole (MBL) medium under continuous light produced 4.32% more lipid than the autotrophic condition (MBL medium). Maximum amounts of health beneficiary α-linolenic acid (46.05 ± 2.31%) and H2O2 (38.57 ± 7.01 µM/g fresh cell) were detected in the lipid-induced cells. In experiment 3, supplementation of 0.4 mM and 0.6 mM H2O2 in the C. reinhardtii culture (MBL medium; continuous light) also increased the lipid accumulation to 12.67 ± 1.45% and 13.12 ± 1.04%, respectively, compared with that of the 6.77 ± 1.92% lipid in control (without H2O2).
Distinctive LDs labelled by DPAS in E. gracilis cells have confirmed the lipid enrichment conditions under confocal microscopy and flow cytometry. Confocal images labelled with 2-DPAN and TPEBO have also clearly demonstrated the LDs and H2O2, respectively, in the lipid-induced C. reinhardtii cells. Co-staining in the presence of traditional lipid-specific BODIPY dye and chlorophyll indicates that DPAS and 2-DPAN are suitable for multicolour imaging with red and green fluorophores. 2-DPAN showed better fluorescence intensity than DPAS and BODIPY in C. reinhardtii. Compared to BODIPY, 2-DPAN and DPAS are better probes and are recommended as a rapid and easy lipid screening tool to detect lipid drops in microalgae.
Keywords: Aggregation-induced emission, DPAS, Chlamydomonas reinhardtii, Euglena gracilis, fatty acid, hydrogen peroxide, lipid drops, TPEBO, 2-DPAN
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
Completed: 2022
School: College of Science and Engineering
Supervisor: Jian Qin