Jaoon Young Hwan Kim

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.

Integrated Microfluidic Platform for Multiple Processes from Microalgal Culture to Lipid Extraction

For economically viable biofuel production from microalgae, it is necessary to develop efficient analytical platforms for quantitative evaluation of different lipid productivities of numerous microalgal species. Currently, microalgal culture, lipid accumulation, and lipid extraction depend on conventional benchtop methods requiring laborious and time-consuming processes. A poly(dimethylsiloxane) (PDMS)-based integrated microfluidic platform was developed to perform multiple steps in sample preparation on a single device for efficient and quantitative analysis of lipid from various microalgal strains. To achieve this goal, a simple microchannel with a micropillar array was integrated to connect the cell chamber and output reservoir, which act as a filtration unit that enables medium change and solvent extraction by fluid injection using a syringe pump. Multiple processes of cell culture, lipid accumulation, and lipid extraction were successfully accomplished using a single device without time-consuming and labor-intensive steps. Various conditions of solvent volume and temperature were investigated to optimize lipid extraction yield in the microfluidic device. The lipid extraction efficiency in the microfluidic system was higher than that in bulk using the same solvent. The lipid extraction efficiency achieved using less toxic aqueous isopropanol on the integrated device was 113.6% of that obtained with the conventional Bligh-Dyer method. Finally, lipid productivities of different microalgal strains grown in the microfluidic device were analyzed and compared. These results demonstrate that this simple integrated microfluidic platform can be applied as an alternative to conventional benchtop methods for efficient sample preparation in microalgal lipid analysis.

Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure

A perforated hemispherical microwell structure is shown to efficiently capture single Chlamydomonas reinhardtii (C. reinhardtii) cells, culture them to form colonies, and retrieve these colonies to serve as seeds for large-scale cultivation. This solution-phase formation and recovery of colonies could overcome the tedious and time-consuming process of selecting colonies from a solid-phase agar plate. The fabricated microdevice was composed of three layers: a top layer consisting of a cell solution for injection and recovery of a microalgal solution, a hemispherical perforated microwell array in the middle, and a bottom layer in which the solution is manipulated by controlling the hydrodynamic force. The microalgal (wild type and hygromycin B-resistant mutant) cells loaded in the top layer rapidly diffused into the microwell holes, and individual such cells were captured with high efficiency (>90%) and within 1 min by applying a withdraw mode in the bottom layer. Single-cell-based cultivation in a medium containing hygromycin B was then performed to generate colonies in the hemispherical microwell. While the wild type cells died, mutant cells resistant to hygromycin B survived well and grew into a colony within 2 days. The produced colonies in the microwells were recovered by applying a release mode in the bottom layer, so that a hydrodynamic force was exerted vertically to push out the colonies through the outlet in 10 s. The recovered cells were cultured on a large scale in medium by using a flask. The recovered C. reinhardtii was confirmed as a hygromycin B-resistant mutant by identifying the hygromycin gene in the polymerase chain reaction (PCR). The microdevice described here could in solution perform single-cell capture, colony formation, and retrieval of colonies for further large-scale cultivation, which could replace tedious and time-consuming solid-phase agar plate processes with a 7-fold reduction in the duration of the process.

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.

Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production

Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A2 knockout mutants have increased by up to 64.25% (to 80.92 g L-1 d-1). This study can provide crucial information for the biodiesel industry.

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.