Effect of salinity on growth in 100 mL bubble tubes
The growth of K. antarctica in different salinities ranging from freshwater (0‰) to seawater (32‰) conditions is shown in Table 2 and Fig. 1S. Higher growth rates were attained under low salinity conditions (≤ 16‰). At salinity levels from 0 to 16‰, the maximum dry weight was in the range 2.69 to 2.90 g L−1. However, at salinity 32‰, the maximum biomass concentration was lower, reaching only 2.14 g L−1. Although growth rates at salinities 2 and 4‰ were similar, the highest productivity of 0.53 g L−1 day−1 was achieved at salinity 4‰. Hence, 4‰ was selected as the optimal salinity for further cultivations.
Effect of different stressors on growth in 350 mL bubble tubes
To select the best strategy that enables K. antarctica to produce high amounts of fatty acids and PUFAs, the impact of nitrogen (N−) starvation, phosphorus (P−) starvation, and high salinity (HS) conditions on growth, fatty acid, and protein assimilation was investigated. The growth curves of K. antarctica cultivated under these conditions are shown in Fig. 2a. In all conditions, the biomass concentration increased, but there were significant differences in the maximum productivity (F = 53.8, p < 0.001) and the maximum cell density (F = 311.5, p < 0.001) among the treatments (Table 1S). Control cultures displayed strong growth and achieved the highest dry weight of 3.73 g L−1 at day 5, significantly higher than each of the other treatments (Tukey’s HSD, p < 0.001). The lag phase in the HS treatment extended to 2 days, but the maximum growth rate of 0.69 g L−1 day−1 was slightly higher than the 0.57 g L−1 day−1 recorded in P-starved conditions, and eventually both achieved comparable maximum cell densities of 2.23 and 2.30 g L−1, respectively (Tukey’s HSD, p = 0.857). The maximum productivity of K. antarctica under N starvation was however significantly lower than each of the other treatments (Tukey’s HSD, p < 0.001), and the maximum cell density reached only a quarter of that achieved in the control medium (Table 3).
Figure 2b,d illustrates the nitrate and phosphate assimilation capacity of K. antarctica under the different stress conditions, while the corresponding nutrient uptake rates are presented in Table 3. Concordant with the growth rates, there were also significant differences in nitrate uptake (F = 47.0, p < 0.001) and phosphate uptake (F = 33.62, p < 0.001) between the treatments. Phosphate uptake in the N-starved cultures was 0.12 mM L−1 day−1, which was significantly lower than the uptake rate of 0.71 mM L−1 day−1 in the control cultures and 0.55 mM L−1 day−1 in the HS treatments (Tukey’s HSD, p ≤ 0.003). The rate of nitrate uptake in P-starved cultures was 2.46 mM L−1 day−1, which was significantly lower than that of the control (3.38 mM L−1 day−1) and HS groups (3.72 mM L−1 day−1) (Tukey’s HSD, p < 0.001). Due to rapid growth in the control treatments, nitrate and phosphate were each exhausted at day 3 (Fig. 2b,d). Similarly, the HS treatments also consumed the available nitrate by day 5. Hence, K. antarctica in the control medium and HS conditions still experienced N + P and N starvation, respectively, during the later stages of cultivation.
The A680/A540 ratio in both N- and P-starved media decreased throughout the experiment, but N-starved cells were characterized by lower ratios than P-starved cells, indicating more substantial loss of photosynthetic pigments (Fig. 2c). At the end of cultivation, the values for N-starved and P-starved cells were 0.95 and 1.04, respectively. Control cultures were characterized by an initial increase in A680/A540, reaching 1.47 at day 3, followed by a decline to 1.06 at day 5. This pattern is concordant with increased mutual shading during growth, followed by the effects of nutrient availability during the later stages of cultivation.
Effect of different stressors on fatty acid and protein production in 350 mL bubble tubes
The effects of N, P, and HS stress on the total fatty acid contents (CFA, % DW) are shown in Table 3. There were significant differences in CFA between the treatments (F = 33.9, p < 0.001), where both N− and P− conditions yielded significantly higher fatty acid concentrations (each 18% DW) than control and HS conditions (Tukey’s HSD, p ≤ 0.004). However, the reduced biomass productivity under N starvation ultimately led to the lowest overall fatty acid yield (YFA) of 165.4 mg L−1 in this treatment. In contrast, the control cultures achieved the highest YFA of 507.6 mg L−1.
In all treatments, the total fatty acid content increased from day 2 to day 5, mainly due to TAG accumulation (Table 4). The highest proportion of TAG was detected under N starvation at day 5, where it accounted for 79.5% of TFA (179.1 mg g−1 DW). In nutrient-replete conditions (control, day 2), the dominant fatty acids were C16:0, C16:4n−3, C18:3n−3 (α-linolenic acid, ALA), and C20:5n−3 (EPA). However, under N- and P-starved conditions, C16:0, C18:1n−9 (oleic acid, OA), C18:2n−6 (linoleic acid, LA), and ALA became dominant. TAG accumulation was attributed predominantly to a large increase in OA, comprising up to 52.7% of neutral lipids. However, the PUFAs LA, ALA, and C20:4n−6 (arachidonic acid, ARA) were also enriched in TAG, and together total PUFAs accounted for 33.9–45.8% of TAG at day 5 (Table 4). The highest PUFA content of 90.6 mg g−1 DW, as well as the highest ω−3 fatty acid content of 43.6 mg g−1 DW, was observed after 5 days of P starvation. The LA and ALA contents of P-starved cells amounted to 27.1 and 22.7 mg g−1 DW, respectively, which were also higher than the other treatments. The HS treatment induced the accumulation of TAG with a large proportion of OA at day 2. The proportion of TAG was higher in the HS treatment (35.0% TFA) than that of control cultures (20.2% TFA) at day 2.
The highest amount of EPA was 6.7 mg g−1 DW (4.9% TFA) in high-density control cultures (day 5) but slightly lower during P and N starvation at 5.4 and 3.8 mg g−1 DW, respectively (Table 4). The lowest EPA content of 2.1 mg g−1 DW was recorded for the HS group at day 2. In contrast to EPA, the total ARA content was enhanced by N starvation. ARA increased from 1.4 mg g−1 DW in nutrient-replete conditions to 7.4 and 4.6 mg g−1 DW (4.1 and 2.6% TFA) in N and P treatments, respectively, primarily accumulating in the TAG fraction.
The total protein content (CPRO) also varied significantly between the different treatments (F = 269.9, p < 0.001). Under P starvation, the protein content of K. antarctica at day 5 was 31.4% DW, which was significantly higher than that of N− (12.2% DW), HS (27.0% DW), and control treatments (21.9% DW) (Tukey’s HSD, p ≤ 0.001). Cells in N-starved conditions therefore had the highest C:N ratio of 19.8, approximately twice that of the other treatments, which ranged from 7.9 to 10.7 (Table 3).
Effect of light intensity on growth in 380 mL flat-plate photobioreactors
The effect of light intensity on the growth of K. antarctica was studied in flat-plate photobioreactors. In the first experiment (Li-Ex I), the maximum biomass productivity increased from 0.48 to 1.04 g L−1 day−1 when the PPFD was raised from 70 to 250 μmol photons m−2 s−1, but there was no further increase in growth rate when light was increased to 500 μmol photons m−2 s−1 (Table 5, Fig. 3a). The extracellular nitrate in all cultures was exhausted by day 8 (Fig. 3c). In cultures supplied with 250 and 500 μmol photons m−2 s−1, the A680/A540 ratio decreased rapidly after day six, reaching 0.95 to 0.93 after nine days. In cultures supplied with 70 μmol photons m−2 s−1 though, the effect was dampened, and the A680/A540 ratio reached a lowest value of 0.98 at day 16 (Fig. 3b). Under all light conditions, the initial pH 6.5 increased to pH 7.8–7.9 by day 7 or 8, then decreased slightly to pH 7.6–7.7 toward the end of cultivation, as the growth rate slowed (Fig. 3d). Together, the attenuated growth under 500 μmol photons m−2 s−1 coupled with the nitrate and pH data (Table 5) indicated that nutrient availability and possibly CO2 supply might have constrained the maximum growth rate. To test whether we could obtain higher yields, the nutrient and CO2 concentrations were subsequently increased and the experiment was repeated at 500 μmol photons m−2 s−1. In this second experiment (Li-Ex II), the maximum biomass productivity was indeed substantially higher, reaching a maximum of 2.37 g L−1 day−1, with the highest cell density measured at 11.68 g L−1 at the end of cultivation (Fig. 4c, Table 5). In Li-Ex II, the absorbance ratio A680/A540 remained stable between 0.95 and 1.09 throughout the experiments.
Photosynthetic yield in 380 mL flat-plate photobioreactors
To characterize the photosynthetic efficiency of K. antarctica under different light intensities, the maximum biomass yield per mol photons PAR (Yx/mol, g mol−1 photons) was calculated for each treatment (Table 5). The cultures under low irradiance used light more efficiently than those at higher light intensities, averaging 1.05 g mol−1 photons at 70 μmol photons m−2 s−1, and reducing to 0.27 g mol−1 photons at 500 μmol photons m−2 s−1. In Li-Ex II, however, K. antarctica showed improved response at 500 μmol photons m−2 s−1, with strong growth throughout the duration of the experiment (Fig. 4a). In this second experiment, the photosynthetic efficiency at 500 μmol photons m−2 s−1 was improved to 0.73 g mol−1 photons, and the maximum areal productivity of 31.58 g m−2 day−1 was the highest recorded (Table 5).
Fatty acid production in 380 mL flat-plate photobioreactors
In the flat-plate photobioreactors, K. antarctica illuminated with 70, 250, and 500 μmol photons m−2 s−1 was able to accumulate considerable amounts of fatty acids, with a large increase in TAG and a slight decrease in polar lipid fatty acids toward the stationary phase (Fig. 5, Fig. 2S). The highest TFA content of 271.9 mg g−1 DW was recorded under 250 μmol photons m−2 s−1 at day 18, where 90.1% TFA was found in TAG (Fig. 2S). Total PUFAs at day 10 (late exponential phase) ranged from 52.9 to 53.9 mg g−1 DW and consistently increased up to 88.5 mg g−1 DW at day 18 in the late stationary phase. The highest total PUFA content was recorded at day 18 when cultures were supplied with 250 μmol photons m−2 s−1, where 86.2% of total PUFAs were found in TAG. In all Li-Ex I treatments, a time-dependent increase in ARA and LA content was observed, mostly in the TAG fraction (Fig. 5a,b). The highest ARA content was obtained under 250 μmol photons m−2 s−1 at 9.6 mg g−1 DW (3.5% TFA) in the late stationary phase. The total EPA content was mostly conserved throughout growth, ranging between 2.9 and 4.1 mg g−1 DW (Fig. 5a). However, the percent share of EPA consistently decreased from 3.6% of TFA during the late exponential phase to its lowest value of 1.5% of TFA in the late stationary phase. The ratio of ω−3 to ω−6 fatty acids was the highest at day 10, but it decreased thereafter to 0.5, irrespective of the PPFD (Fig. 2S), due to a large increase in the abundance of LA (Fig. 5b).