Hyperosmosis and its combination with nutrient-limitation are novel environmental stressors for induction of triacylglycerol accumulation in cells of Chlorella kessleri

Abstract

Triacylglycerols of oleaginous algae are promising for production of food oils and biodiesel fuel. Air-drying of cells induces triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, therefore, it seems that dehydration, i.e., intracellular hyperosmosis, and/or nutrient-limitation are key stressors. We explored this possibility in liquid-culturing C. kessleri cells. Strong hyperosmosis with 0.9 M sorbitol or 0.45 M NaCl for two days caused cells to increase the triacylglycerol content in total lipids from 1.5 to 48.5 and 75.3 mol%, respectively, on a fatty acid basis, whereas nutrient-limitation caused its accumulation to 41.4 mol%. Even weak hyperosmosis with 0.3 M sorbitol or 0.15 M NaCl, when nutrient-limitation was simultaneously imposed, induced triacylglycerol accumulation to 61.9 and 65.7 mol%, respectively. Furthermore, culturing in three-fold diluted seawater, the chemical composition of which resembled that of the medium for the combinatory stress, enabled the cells to accumulate triacylglycerol up to 24.7 weight% of dry cells in only three days. Consequently, it was found that hyperosmosis is a novel stressor for triacylglycerol accumulation, and that weak hyperosmosis, together with nutrient-limitation, exerts a strong stimulating effect on triacylglycerol accumulation. A similar combinatory stress would contribute to the triacylglycerol accumulation in air-dried C. kessleri cells.

Figure 1. Effects of sorbitol-induced hyperosmotic, nutrient-limiting or combinatory stress on cell growth and TG accumulation in C. kessleri.

The cells were cultured for 2 days under hyperosmotic conditions for measurement of (a) OD₇₃₀ values (open bars) or Chl contents (closed bars), and (b) the contents of TG (closed bars), TL (see top values of open bars), and therefore polar lipids (open bars) in the cultures. The cells were cultured for 2 days under nutrient-limiting or combinatory stress conditions for measurement of (c) OD₇₃₀ values and (d) the contents of TG (closed bars) and TL (see top values of open bars) in the cultures. (e) TG contents relative to TL, on the basis of fatty acids, estimated from data of (b) or (d). The values shown are the averages ± SE for three distinct groups of data. ‘Initial’ in (a–e) indicates the initial level. The initial levels of OD₇₃₀ were adjusted to 0.3 for (a,c) whereas those of TG or TL are the same for (b,d). The significance of differences was evaluated by Student’s t test. *P < 0.05. **P < 0.1. In (b,d), see differences in the TG or TL content between 0.0, and 0.3, 0.6 or 0.9.

Figure 2. Effects of NaCl-induced hyperosmotic, nutrient-limiting or combinatory stress on cell growth and TG accumulation in C. kessleri.

The cells were cultured for 2 days under hyperosmotic conditions for measurement of (a) OD₇₃₀ values (open bars) or Chl contents (closed bars), and (b) the contents of TG (closed bars), TL (see top values of open bars), and therefore polar lipids (open bars) in the cultures. The cells were cultured for 2 days under nutrient-limiting or combinatory stress conditions for measurement of (c) OD₇₃₀ values and (d) the contents of TG (closed bars) and TL (see top values of open bars) in the cultures. (e) TG contents relative to TL, on the basis of fatty acids, estimated from data of (b) or (d). The values shown are the averages ± SE for three distinct groups of data. ‘Initial’ in (a–e) indicates the initial level. The values shown are the averages ± SE for three distinct groups of data. The values of ‘initial’ and control are the same as those in Fig. 1. The significance of differences was evaluated by Student’s t test. *P < 0.05. **P < 0.1. In (b,d), see differences in the TG or TL content between 0.0, and 0.3, 0.6 or 0.9.

Figure 3. Additive or synergistic effects of hyperosmotic and nutrient-limiting stress on TG accumulation.

(a,b) Open bars indicate TG contents in the cultures (a) or those relative to TL (b) under combinatory stress with 0.3 M sorbitol whereas red, blue, and closed bars indicate TG contents under single stress conditions with nutrient-limitation, 0.3 M sorbitol, and 0.6 M sorbitol, respectively. (c,d) Open bars indicate TG contents in the cultures (c) or those relative to TL (d) under combinatory stress with 0.15 M NaCl whereas red, blue, and closed bars indicate TG contents under single stress conditions with nutrient-limitation, 0.15 M NaCl, and 0.3 M NaCl, respectively. The values were from Figs 1b,d,e and 2b,d,e.

Figure 4. Effects of hyperosmotic or nutrient-limitation stress on cell growth and TG accumulation in C. reinhardtii.

(a) Photograph of 2-day cultures under the respective conditions. (b) OD₇₃₀ values (open bars) or Chl contents (closed bars). The initial levels of OD₇₃₀ were adjusted to 0.2. (c) Accumulated levels of TG (closed bars) or TL (see top values of open bars) in the culture. The values shown are the averages ± SE for three distinct groups of data.

Figure 5. Effects of hyperosmotic stress, with the use of seawater, on cell growth and TG accumulation in C. kessleri.

The cells were grown for 3 days in 1/3 MASF (triangles) or MASF (squares) medium, or 1/3 SW (diamonds), and then subjected to the following measurements. (a) OD₇₃₀ values of the culture. The initial levels of OD₇₃₀ were adjusted to 0.2. (b) Dry cell weights relative to the initial level. (c) TG contents in the culture. (d) TL contents in the cultures. TG contents relative to TL (e) or dry cell weight (f). Our previous data for the air-drying cells (closed circles) are also included in (b,e,f). The values in (e,f) were estimated from data of (c,d), and those of (b,c), respectively. The measurements were performed every other day for the cultures in 1/3 MASF or MASF medium, and once on Day 3 for the culture in 1/3 SW. (g) Nile-red stained lipid droplets in cells stressed with or without the use of seawater. White bars represent 10 μm. The values shown are the averages ± SE for three distinct groups of data. The significance of differences was evaluated by Student’s t test. *P < 0.05. **P < 0.1. In (e,f), see differences in the TG content between air-drying and seawater-stressed cells, at respective time points.