Young Joon Sung

Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis

Microalgae possess great potential as a source of sustainable energy, but the intrinsic inefficiency of photosynthesis is a major challenge to realize this potential. Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report a microfluidic screening using competitive phototaxis of the model alga, Chlamydomonas reinhardtii, for rapid isolation of strains with improved photosynthetic efficiencies. We demonstrated strong relationship between phototaxis and photosynthetic efficiency by quantitative analysis of phototactic response at the single-cell level using a microfluidic system. Based on this positive relationship, we enriched the strains with improved photosynthetic efficiency by isolating cells showing fast phototactic responses from a mixture of 10,000 mutants, thereby greatly improving selection efficiency over 8 fold. Among 147 strains isolated after screening, 94.6% showed improved photoautotrophic growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in photoautotrophic cell growth and lipid production, respectively, a substantial improvement over previous approaches. We identified candidate genes that might be responsible for fast phototactic response and improved photosynthesis, which can be useful target for further strain engineering. Our approach provides a powerful screening tool for rapid improvement of microalgal strains to enhance photosynthetic productivity.

Multilateral approach on enhancing economic viability of lipid production from microalgae: A review

Microalgae have been rising as a feedstock for biofuel in response to the energy crisis. Due to a high lipid content, composed of fatty acids favorable for the biodiesel production, microalgae are still being investigated as an alternative to biodiesel. Environmental factors and process conditions can alternate the quality and the quantity of lipid produced by microalgae, which can be critical for the overall production of biodiesel. To maximize both the lipid content and the biomass productivity, it is necessary to start with robust algal strains and optimal physio-chemical properties of the culture environment in combination with a novel culture system. These accumulative approaches for cost reduction can take algal process one step closer in achieving the economic feasibility.

Quantitative analysis of the chemotaxis of a green alga, Chlamydomonas reinhardtii, to bicarbonate using diffusion-based microfluidic device

There is a growing interest in the photosynthetic carbon fixation by microalgae for the production of valuable products from carbon dioxide (CO2). Microalgae are capable of transporting bicarbonate (HCO3 (-)), the most abundant form of inorganic carbon species in the water, as a source of CO2 for photosynthesis. Despite the importance of HCO3 (-) as the carbon source, little is known about the chemotactic response of microalgae to HCO3 (-). Here, we showed the chemotaxis of a model alga, Chlamydomonas reinhardtii, towards HCO3 (-) using an agarose gel-based microfluidic device with a flow-free and stable chemical gradient during the entire assay period. The device was validated by analyzing the chemotactic responses of C. reinhardtii to the previously known chemoattractants (NH4Cl and CoCl2) and chemotactically neutral molecule (NaCl). We found that C. reinhardtii exhibited the strongest chemotactic response to bicarbonate at the concentration of 26 mM in a microfluidic device. The chemotactic response to bicarbonate showed a circadian rhythm with a peak during the dark period and a valley during the light period. We also observed the changes in the chemotaxis to bicarbonate by an inhibitor of bicarbonate transporters and a mutation in CIA5, a transcriptional regulator of carbon concentrating mechanism, indicating the relationship between chemotaxis to bicarbonate and inorganic carbon metabolism in C. reinhardtii. To the best of our knowledge, this is the first report of the chemotaxis of C. reinhardtii towards HCO3 (-), which contributes to the understanding of the physiological role of the chemotaxis to bicarbonate and its relevance to inorganic carbon utilization.

Arginine culture turns on the elusive nitrogen starvation signal during robust phototrophic growth in Chlamydomonas

Abstract Under nitrogen (N) starvation, microalgae increase carbon storage in the form of lipid droplets while also downregulating photosynthesis and eventually terminating growth. To improve lipid yield, we asked whether lipid droplets and N starvation responses can be induced without limiting growth or photosynthesis. In the chlorophyte Chlamydomonas reinhardtii, gametogenesis is induced either by N starvation or by growth with arginine as the sole N source. We showed that arginine cultures supported robust phototrophic growth, constitutively turned on N starvation-induced genes, and increased lipid droplets. The lipids accumulated in arginine cultures exhibited strong enrichment of saturated and monounsaturated fatty acids, a preferred characteristic of biodiesel precursors. The diatom Phaeodactylum tricornutum also accumulated lipid droplets in arginine culture without growth impairment. We document a system wherein N starvation responses are induced without compromising photosynthesis or growth, thereby suited to the producing valuable chemicals and biofuel precursors without requiring stressors in microalgae.Under nitrogen (N) starvation, photosynthetic organisms search for other N sources while slowing down photosynthesis by downregulating light harvesting and electron transport to balance the carbon/nitrogen ratio and eventually stopping growth due to N limitation. To investigate the elusive N starvation-specific signaling mechanisms, we sought a way to induce N starvation responses without limiting photosynthesis or cell growth. In the chlorophyte Chlamydomonas reinhardtii, gametogenesis is exclusively induced during N starvation except in arginine culture. We showed that the arginine-grown culture turned on N starvation responses including hundreds-fold induction of N starvation-induced genes, reduced chlorophyll content, and increased carbon storage in the form of lipid droplets. Arginine culture supported robust phototrophic growth but not heterotrophic growth, indicating that arginine catabolism contributes CO2 to Rubisco without directly fueling ATP synthesis. Based on in silico analysis, we propose the possible routes of arginine catabolism that may bypass critical steps for monitoring of cellular N status and thereby trigger N starvation responses. Our results describe a study system where the N starvation responses are constantly induced without compromising photosynthesis or growth, paving ways to discover the mechanisms that sense and respond to cellular N status in eukaryotic phototrophs. Highlights Arginine catabolism leads to the activation of nitrogen starvation responses while supporting robust photosynthesis and growth, presenting ways to investigate N starvation signaling mechanisms in photosynthetic organisms.

Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction

The aim of this work was to develop a new approach for simple and high-throughput selection of astaxanthin-hyperproducing Haematococcus mutants through a sequential combination method of azide-based colorimetric assessment and oil-based astaxanthin quantification. Randomly mutagenized cells were spotted on solid culture medium containing 50 µM of sodium azide to accelerate the biosynthesis of astaxanthin. After 3 days, highly-induced mutants were preliminarily isolated by visual inspection and their astaxanthin accumulations were rapidly quantified by soybean oil-based extraction method. On the whole, the selected mutants showed reduced vegetative growth rates but eventually exhibited higher astaxanthin productions than the parental strain owing to their improved inductive growths. Among them, M13 showed 174.7 ± 5.69 mg L-1 of the highest astaxanthin production, which is 1.59-times higher than that of wild-type. This wide-scope screening method expedites both upstream and downstream astaxanthin quantification, making it a useful tool for isolating microalgae with high astaxanthin production.