## 2 Results
### Limonene Production Optimization  
Photosynthetic microorganisms can grow using solar energy (light), carbon dioxide (CO\({}_{2}\)), or organic carbon sources as the primary inputs to generate terpenids in a renewable manner [29]. _C. reinhardtii_ can undergo photoautotrophic growth in light, heterotrophic growth in darkness on acetate as a sole carbon source, and mixotrophic combinations of these growth modes [30]. In this study, the high-throughput cultivation, using mixotrophic acetate cultivations in a TAP medium (light + acetate) was necessary to compare the genetic constructs used for the limonene production. We carried out a scaled-up cultivation of the highest limonene-producing strains, V12-118, V12-203 and V12-209, in a TAP medium under both constant (24 h) light and 16:8 h light: dark (L:D) cycle conditions. We found that, after 5 d cultivation, the latter conditions, i.e., L:D cycling, gave the highest productivities in all three strains (Figure 5a). The limonene production of the V12-118, V12-203 and V12-209 strains was approximately 21, 27 and 39 \(\upmu\)g/L when grown under L:D cycling conditions, respectively, approximately 3-fold more than when the same strains were grown with constant light. This result is in accordance with a previous study of a sesquiterpenoid production, which indicated that L:D regimens promote a slower growth rate and therefore direct more metabolic activity towards the product formation [11]. Thus, L:D cycles can prolong the exponential growth phase, thereby increasing productivity regardless of the carbon regimen. _C. reinhardtii_ can use acetate as the sole carbon source for its heterotrophic growth, but can also use acetate and CO\({}_{2}\) for its mixotrophic growth. In the absence of CO\({}_{2}\), acetate will have an important effect on the biomass and heterologous protein production. [PERSON] et al. [31] attempted to optimize the growth medium to improve the zeaxanthin yield and found that the increased amounts of acetic acid (opt2 medium) could both promote cell growth and increase the zeaxanthin production in _C. reinhardtii_. We therefore used the opt2 medium with the best limonene-producing strains, V12-118, V12-203 and V12-209, to attempt to increase the limonene production and obtained titers of approximately 18, 19 and 24 \(\upmu\)g/L respectively, approximately 1.3-fold the levels achieved after growth in the TAP medium. Thus, compared with the TAP medium, growth in the opt2 medium increased the limonene production significantly in all three strains (Figure 5b), reflecting the experience of [PERSON] et al. [31] with zeaxanthin. Figure 5: Limonene yield in transgenic algae. (**a**) Strains were grown under constant (24 h) light (L) or 16:8 h light: dark cycles (LA) conditions. Strains were cultivated in 500 mL shake flasks containing 10  
a 300 mL TAP medium supplemented with 15 mM isoprenol at 25 \({}^{\circ}\)C and 120 rpm under constant (24 h) light or 16:8 h light: dark cycles, and using 5 mL dodecane as overlay for 5 d. (**b**) Strains were cultivated in 500 mL shake flasks containing a 300 mL TAP or opt2 medium supplemented with 15 mM isoprenol at 25\({}^{\circ}\)C and 120 rpm under constant (24 h) light, and using 5 mL dodecane as a solvent overlay for 5 d. Strains were cultivated in 500 mL shake flasks containing a 300 mL TAP medium supplemented with 10 mM isoprenol at 25\({}^{\circ}\)C and 120 rpm under 16:8 h light: dark conditions using 5 mL dodecane as a extraction overlay for 1, 2, 3, 4, 5, 6 or 7 d (e) or using 5, 10, 15 or 20 mL dodecane (**d**) for 7 d. (**e**) Strains were cultivated in 500 mL shake flasks containing a 300 mL TAP medium supplemented with 5, 10, 15, 20 or 25 mM isoprenol at 25\({}^{\circ}\)C and 120 rpm under 16:8 h light: dark conditions, and using 5 mL dodecane as an extraction solvent for 5 d. Three biological replicates were used to calculate the limonene yield averages and SDs.