Attached cultivation of green microalga ettlia sp. for lipid production

Abstract

An attached cultivation system has been receiving much attention as a breakthrough in microalgae cultivation technology. Attached cultivation generates a high concentration of biomass in the form of a biofilm, which significantly reduces the risk of contamination, the cost of harvesting and dewatering, and the amount of space required for cultivation. In this study, attached cultivation of green microalga species, Ettlia sp., was evaluated for lipid production. Firstly, an attached cultivation system, driven by diffusion of medium through a porous membrane where a biofilm is attached, was developed and used throughout the entire study. A novel wet measurement method for more accurate biofilm growth analysis was developed and successfully estimated the weight of dry biomass without sacrificing samples. The optimum initial conditions for seed culture age and inoculum density for attached cultivation were found to be day 4 for seed age and $2.5 \pm 0.5 g/m^2$ for inoculum density. By employing response surface methodology, optimization of light intensity and $CO_2$ concentration for surface biomass and lipid productivity was achieved. A maximum surface biomass productivity of $28.0 \pm 1.5 g/m^2$/day was achieved at 730 $\mu E/m^2$/s with 8% $CO_2$. The maximum surface lipid productivity was $4.2 \pm 0.3 g/m^2$/day at 500 $\mu E/m^2$/s with 7% $CO_2$. The lipid content of biofilm decreased as the light intensity increased due to proliferation of biomass with low lipid content in the middle of the biofilm at high light intensity. In order to understand the composition variation within the biofilm, layer by layer analysis of microalgal biomass was conducted using a different concentration of nutrients in the feeding media. The effect of nitrate and phosphate concentration on biomass and lipid productivity showed that a high concentration of nutrients did not improve biomass productivity, while a low phosphate concentration improved lipid content of biomass up to 30%. A nutrient starvation strategy was employed and evaluated to enhance lipid productivity. Although lipid content increased under nutrient depletion, overall lipid productivity under nutrient-replete conditions was highest with $4.9 \pm 0.5 g/m^2$/day on day 6. This was due to high lipid accumulation in the top layer of the biofilm even when a nutrient was sufficiently supplied. Ultimately, natural polarization of bio-composition is due to light and nutrient concentration gradients. In order to evaluate the feasibility of large-scale outdoor attached cultivation of Ettlia sp. for lipid production, solar irradiation on the Microalgae-Attached Panel (MAP) was simulated and a yearly average footprint biomass productivity was estimated and optimized. A double-side MAP design facing east/west offered the highest footprint biomass productivity with the 0.4m distance between the vertical panels during all season. Yearly average footprint biomass productivity of 25 g/$m^2$/day was achieved with the panel height of 3.4 m. This is equivalent to 60 tons of dry biomass/ha/yr, 9 to 15 tons of lipid/ha/yr (15% to 25% of lipid content), 420 to 600 kgs of lutein/ha/yr (0.7% to 1%), and 108 tons of $CO_2$/ha/yr (carbon content of 50% of biomass). In conclusion, attached cultivation of Ettlia sp. for lipid production has great potential for large-scale outdoor systems both economically and environmentally.