Saline microalgae for biofuels: Outdoor culture from small-scale to pilot scale

Three local isolates of the green alga Tetraselmis sp. identified as the most promising microalgae species for outdoor mass cultivation with high potential for biodiesel production due to high amounts of total lipids and high lipid productivity were employed in this study. The aim of the study was to compare three halophilic Tetraselmis strains (Tetraselmis MUR-167, MUR-230 and MUR-233) grown in open raceway ponds over long periods with respect to their specific growth rate and lipid productivity without additional CO2 and with CO2 addition regulated at pH 7.5 by using a pH-stat system. Attention also was given to the overall culture condition including contaminating organisms, biofilm development due to cell adhesion and cell clump formation. All tested Tetraselmis strains in this study were successfully grown outdoors in open raceway ponds in hypersaline fertilised medium at 7 % w/v NaCl over a period of more than two years. A marked effect of CO2 addition on growth and productivities was observed at high solar irradiance and temperatures between 15 – 33 oC. However, differences were identified between the three Tetraselmis strains in biofilm development, specific growth rate and lipid productivity in association with solar irradiance and temperature. Study of Tetraselmis MUR-167 was terminated after two years of outdoor cultivation due to stickiness and biofilm development on the pond walls, with a consequent decline in specific growth and productivities. Tetraselmis MUR-230 and MUR-233 showed quite similar annual specific growth and average productivities of 12 g (ash free dry weight) m-2 d-1 and 5.3±0.4 g m-2 d-1 in semi-continuous culture over a period of 365 days for biomass and lipid respectively; however MUR-233 had a higher specific growth rate and higher lipid productivity at higher temperatures in comparison with MUR-230. Further studies were carried out on Tetraselmis MUR-233 in 2 m2, 20 m2 and 200 m2 raceway ponds at a pilot plant in Karratha, West Australia. Comparison between Perth and Karratha showed that Tetraselmis MUR-233 had up to three times higher biomass productivities in winter and 40 % higher during summer in Karratha. Appreciable differences in specific growth during semicontinuous cultivation were observed in 2 m2 raceway ponds compared to the 20 m2 and 200 m2 ponds accompanied with a decline in biomass productivity of up to 20 % with increasing pond size. Highest biomass productivities of 30 g (ash free dry weight) m-2 d-1 and a lipid content of up to 43 % based on ash free dry weight was achieved at an optimum cell density of 60 x 104 cells . mL-1 combined with 0.4 daily dilution and CO2 addition even under hyper saline conditions. These results were obtained at 25 MJ . m-2 solar irradiance and temperatures between 15 – 31 oC. Tetraselmis MUR-230 and MUR-233 with CO2 addition and regulated pH out-competed the major contaminating organisms, the diatoms. Tetraselmis MUR-230 grew successfully with different N-sources such as ammonium chloride, urea and sodium nitrate, the highest cellular lipids obtained with urea and sodium nitrate. Cellular carbohydrates in Tetraselmis MUR-230 increased five times under N-depletion and CO2 addition. In summary, this study demonstrates the feasibility of outdoor large scale cultivation of the halophilic Tetraselmis strains MUR-230 and MUR-233 with a high potential as feedstock for biofuel and carbon dioxide mitigation. The sustainability is underlined by the fact that the Tetraselmis strains were able to grow in hot and dry areas using only seawater as water source.