Comparison of lipid productivity of Parachlorella kessleri heavy-ion beam irradiation mutant PK4 in laboratory and 150-L mass bioreactor, identification and characterization of its genetic variation

Parachlorella kessleri (formerly Chlorella kessleri) can accumulate high levels of both starch and lipids that can be used for the production of bioethanol and biodiesel, respectively. We mutagenized P. kessleri by heavy-ion beam irradiation and obtained a biotechnologically promising mutant strain, PK4, for further improvement of lipid productivity in comparison with wild type [1]. PK4 reached two times the optical density and accumulated 2.6 times as much starch in complete urea-phosphate (UP) media compared to tris-acetate-phosphate (TAP) media under laboratory conditions. Similarly to wild type, PK4 accumulated only negligible amounts of lipids in complete UP media. PK4 accumulated more lipids (WT: 1.17 g L⁻¹, PK4: 1.75 g L⁻¹) and accumulated lipids faster than WT after dilution of the UP media (WT; 0.22 g L⁻¹ day⁻¹, PK4; 0.43 g L⁻¹ day⁻¹). The productivity of PK4 was analyzed in mass culture using a 150-L thin-layer photo bioreactor housed in a temperature and light-uncontrolled glass greenhouse. The PBR consisted of two glass plates (each 6 m long and 1 m wide) at an inclination of 1.6%, arranged in a meandering path and connected with a trough. The culture was diluted four-fold with water on day 7 post-inoculation to ensure prompt nutrient limitation in the UP media. Lipid accumulation in PK4 was significantly induced so that it accumulated 66% of lipid per dry weight. Under 150-L mass cultivation conditions, PK4 showed high biomass productivity (0.82 g L⁻¹ day⁻¹) and high lipid productivity (0.59 g L⁻¹ day⁻¹). In addition, the whole genome of PK4 was sequenced to clarify the genetic variation upon heavy-ion-beam irradiation by comparison with the reference genome of WT. As a result, genetic differentiation of PK4 was found at three genes encoding endo-1,4-β-mannanase, an ATP/ADP transporter, and an elicitor response protein.