Chan Yoo

Selection of microalgae for lipid production under high levels carbon dioxide

To select microalgae with a high biomass and lipid productivity, Botryococcus braunii, Chlorella vulgaris, and Scenedesmus sp. were cultivated with ambient air containing 10% CO(2) and flue gas. The biomass and lipid productivity for Scenedesmus sp. with 10% CO(2) were 217.50 and 20.65 mg L(-1)d(-1) (9% of biomass), while those for B. braunii were 26.55 and 5.51 mg L(-1)d(-1) (21% of biomass). With flue gas, the lipid productivity for Scenedesmus sp. and B. braunii was increased 1.9-fold (39.44 mg L(-1)d(-1)) and 3.7-fold (20.65 mg L(-1)d(-1)), respectively. Oleic acid, a main component of biodiesel, occupied 55% among the fatty acids in B. braunii. Therefore, the present results suggested that Scenedesmus sp. is appropriate for mitigating CO(2), due to its high biomass productivity and C-fixation ability, whereas B. braunii is appropriate for producing biodiesel, due to its high lipid content and oleic acid proportion.

Ettlia sp. YC001 showing high growth rate and lipid content under high CO2.

Over 100 green-colored colonies were isolated from environmental samples when cultivating on a BG11 agar medium, and 4 strains showing different morphologies were selected based on light microscopic observation. Among these strains, the microalgal species with the highest growth rate under 10% CO(2) was identified as Ettlia sp. YC001 using an 18S rDNA-based phylogenetic analysis and morphological comparison. The highest cell density of 3.10 g/L (based on dry cell weight) and biomass productivity of 0.19 g/L/d were obtained under 5% CO(2) after 16 days. The lipid content and productivity were also up to 42% of the dry cell weight and 80.0mg/L/d, respectively. The color of the Ettlia sp. YC001 culture changed from green to red after a month due to the accumulation of certain carotenoids. Therefore, it would seem that Ettlia sp. YC001 is appropriate for mitigating CO(2) due to its high biomass productivity, and a suitable candidate for producing biodiesel and high-value products.

Novel approach for the development of axenic microalgal cultures from environmental samples.

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.

Elucidation of the growth delimitation of Dunaliella tertiolecta under nitrogen stress by integrating transcriptome and peptidome analysis

Dunaliella tertiolecta LB 999 is an oleaginous microalgae species that produces large quantities of lipid and starch during nitrogen starvation; however, nitrogen starvation also limits the cell growth. In order to understand the underlying mechanisms of this phenomenon, the transcriptome and peptidome of D. tertiolecta LB 999 grown under different nitrogen and light conditions were analyzed. Integration of the de novo assembly of transcriptome sequencing reads with peptidome analysis revealed 13,861 protein-coding transcripts, including 33 transcripts whose expression patterns were significantly altered along with the growth phenotypes. Interestingly, 21 of these genes, which were highly enriched in the plastid region, were associated with chlorophyll synthesis and tetrahydrofolate-mediated C1 metabolism. Furthermore, intracellular glutamate levels are predicted to be the main factor that acts as a switch for the regulation of cell growth and carbon accumulation. These data provide the genetic information of D. tertiolecta for its future applications.

Simple processes for optimized growth and harvest of Ettlia sp. by pH control using CO2 and light irradiation.

Microalgae cultures show wide range of pH depending on the availability of light and CO2 for their strain specific photosynthesis. Thus, the modulation of light irradiation and CO2 supply can be applied for the pH control of microalgae cultures. The optimal pH of Ettlia sp. YC001, for phototrophic growth and auto‐flocculation was investigated by controlling light irradiation and 10% CO2 supply. Ettlia sp. YC001 showed the highest biomass productivity, 96.7 mg L−1 d−1, at pH 8.5. The flocculating activity of Ettlia sp. YC001 showed a sigmoid pattern with pH increase and was above 70% at pH 10.5. Based on these differentiated optimal pH regimes for the growth and flocculation, an integrated process consisting of cultivation and settling vessels was proposed. The integrated process demonstrated that high flocculation activity of Ettlia sp. YC001 could be achieved in the settling vessel with various hydraulic retention times by only irradiation of light to maintain high pH while maintaining the optimal growth in cultivation vessel with the light irradiation and CO2 supply at pH 8.5. Thus, the proposed strategy for pH control would provide a simple, cost‐effective, and flexible design and operation for microalgae cultivation‐harvest systems. Biotechnol. Bioeng. 2015;112: 288–296.

Establishment and maintenance of an axenic culture of Ettlia sp. using a species-specific approach

The establishment of an axenic culture of microalgae is essential step in understanding its physiology, genetics, and ecology. However, culturing of microalgae is usually accompanied by complex and variable associated prokaryotic and eukaryotic microorganisms. Conventional approaches used for obtaining axenic cultures of microalgae are time-consuming and often involve difficulties in maintaining and preserving axenicity. In this study, we developed a procedure for establishing an axenic culture of Ettlia sp. YC001 and demonstrate that we maintained the axenic culture through subculture in the long term. Three sequential treatments, an antibiotic cocktail, serial dilution, and plate spreading, were applied to strain YC001 and we confirmed axenicity using molecular and physiological methods. The bacterial community associated with strain YC001 was investigated to select antibiotics for their specific elimination. The xenic culture (1 × 106 cells/mL) was treated with the antibiotic cocktail-5 (AC-5), carbendazim, chloramphenicol, imipenem, rifampicin, and tetracycline for 3 days, followed by serial dilution up to 1 × 102 cells and spreading on agar plates. The pure colonies were analyzed using denaturing gradient gel electrophoresis (DGGE), fluorescence-activated cell sorting (FACS), and scanning electron microscopy (SEM). The procedure we developed can be applied to other strains of microalgae for the establishment of axenic cultures.

Light intensity as major factor to maximize biomass and lipid productivity of Ettlia sp. in CO2-controlled photoautotrophic chemostat

The optimal culture conditions are critical factors for high microalgal biomass and lipid productivity. To optimize the photoautotrophic culture conditions, combination of the pH (regulated by CO2 supply), dilution rate, and light intensity was systematically investigated for Ettlia sp. YC001 cultivation in a chemostat during 143days. The biomass productivity increased with the increase in dilution rate and light intensity, but decreased with increasing pH. The average lipid content was 19.8% and statistically non-variable among the tested conditions. The highest biomass and lipid productivities were 1.48gL-1d-1 and 291.4mgL-1d-1 with a pH of 6.5, dilution rate of 0.78d-1, and light intensity of 1500μmolphotonsm-2s-1. With a sufficient supply of CO2 and nutrients, the light intensity was the main determinant of the photosynthetic rate. Therefore, the surface-to-volume ratio of a photobioreactor should enable efficient light distribution to enhance microalgal growth.