Efficient open cultivation of cyanidialean red algae in acidified seawater

Abstract

Microalgae possess high potential for producing pigments, antioxidants, and lipophilic compounds for industrial applications. However, their open pond cultures are often contaminated by other undesirable organisms, including their predators. In addition, the cost of using freshwater is relatively high, which limits the location and scale of cultivation compared with using seawater. It was previously shown that Cyanidium caldarium and Galdieria sulphuraria, but not Cyanidioschyzon merolae grew in media containing NaCl at a concentration equivalent to seawater. We found that the preculture of C. merolae in the presence of a moderate NaCl concentration enabled the cells to grow in the seawater-based medium. The cultivation of cyanidialean red algae in the seawater-based medium did not require additional pH buffering chemicals. In addition, the combination of seawater and acidic conditions reduced the risk of contamination by other organisms in the nonsterile open culture of C. merolae more efficiently than the acidic condition alone.

Figure 1

Growth of C. merolae 10D, Cy. caldarium RK-1, and G. sulphuraria 074 W in the inorganic MA medium supplemented with different concentrations of NaCl (0–1000 mM). (a) A photograph and OD₇₅₀ of the cultures 7 days after inoculation. Cells cultured in MA (OD₇₅₀ of 1.0–2.0) without additional NaCl were collected by centrifugation and resuspended in the respective media to give an OD₇₅₀ of 0.1 and then cultured for 7 days in 24-well plates. Each data point represents the average and the error bar represents the standard deviation of three independent experiments. (b) A photograph and OD₇₅₀ of cultures 7 days after inoculation. Cells cultured in MA with 0.3 M NaCl (OD₇₅₀ of 1.0–2.0) were collected by centrifugation, resuspended in the respective media to give an OD₇₅₀ of 0.1, and then cultured for 7 days. Each data point represents the average and the error bar represents the standard deviation of three independent experiments. Cm, C. merolae 10D; Cc, Cy. caldarium RK-1; Gs, G. sulphuraria 074 W.

Figure 2

Growth of C. merolae in the acidified natural seawater supplemented with different inorganic nutrients. (a) Composition of respective media. (b) Cells cultured in MA with 0.3 M NaCl (OD₇₅₀ of 1.0–2.0) were collected by centrifugation, resuspended in the respective media to give an OD₇₅₀ of 0.1, and then cultured for 7 days. The photograph shows the cultures in the respective media 7 days after inoculation into a 24-well plate. (c) OD₇₅₀ of the cultures 7 days after inoculation. Each data point represents the average and the error bar represents the standard deviation of three independent experiments.

Figure 3

Growth of C. merolae, Cy. caldarium, G. sulphuraria cells in the seawater medium (SWM; the same as medium #15 in Fig. 2) supplemented with 20 mM (NH₄)₂SO₄, 40 mM NaNO₃, or 20 mM urea. (a) Cells cultured in MA with 0.3 M NaCl (OD₇₅₀ of 1.0–2.0) were collected by centrifugation, resuspended in the respective media to give an OD₇₅₀ of 0.1, and then cultured for 21 days. The image shows the cultures in the respective media 21 days after inoculation. (b) OD₇₅₀ of cultures 7, 14, and 21 days after inoculation. Each data point represents the average and the error bar represents the standard deviation of three independent experiments. Cm, C. merolae 10D; Cc, Cy. caldarium RK-1; Gs, G. sulphuraria 074 W.

Figure 4

Growth of C. merolae, Cy. caldarium, and G. sulphuraria cells in MA, MA with 0.6 M NaCl, and SWM with ammonium. (a) Growth curves of C. merolae, Cy. caldarium, and G. sulphuraria in the respective media. Each data point represents the average and the error bar represents the standard deviation of three independent experiments. (b) Micrographs of cells that were cultured in the respective media 14 days after inoculation. Images were obtained by differential interference contrast microscopy (DIC). Scale bar = 5 μm. (c) Algal dry weight and chlorophyll a and phycocyanin contents per algal dry weight 14 days after inoculation. Each data point represents the average and the error bar represents the standard deviation of three independent experiments.

Figure 5

Growth of C. merolae in SWM supplemented ammonium or nitrate at different pH ranges. (a) Cells cultured in MA with 0.3 M NaCl (OD₇₅₀ of 1.0–2.0) were collected by centrifugation, resuspended in SWM supplemented with ammonium (20 mM (NH₄)₂SO₄) and at different pHs (from 1.0 to 8.0) to give an OD₇₅₀ of 0.1, and then cultured for 14 days. The photograph shows the cultures 14 days after inoculation. The graph shows OD₇₅₀ (blue bar) and final pH (yellow bar) of cultures 14 days after inoculation. Each data point represents the average and the error bar represents the standard deviation of three independent experiments. (b) The same as in panel a, except that cells were inoculated into SWM with nitrate (40 mM NaNO₃) instead of SWM with ammonium.

Figure 6

Outdoor cultivation of C. merolae in MA and SWM supplemented ammonium. A preculture in the laboratory in MA or SWM with ammonium was inoculated into 7 L of nonsterile MA or SWM with ammonium. The culture was 13 cm in diameter and 53 cm in height as shown in (a) to give an OD₇₅₀ of 0.2. Cells were cultured for 28 days with aeration (7.5 L ambient air/min). The containers were open but the surface was covered with 4 mm mesh to avoid the contamination of relatively large materials, and placed into a water bath (without any temperature control) to moderate changes in culture temperature. The cultivation containers in the water bath were set inside a semi-open (one side was open) greenhouse without any temperature control. Experiment 1 was performed from July 31st to August 28th, 2019, and Experiment 2 was performed from August 14th to September 11th, 2019. In both cases, the highest intensity of sunlight was ~ 2,000 µmol m^–2 s^–1. (b) A photograph showing the cultures in MA and SWM supplemented with ammonium at day 0 for Experiment 1 and day 14 for Experiment 2. (c) Growth curves of C. merolae cultured in the respective media. Change in the temperature of the culture (temperature of the water in the water bath). (d) Micrographs of cells that were cultured in the respective media 14 days after inoculation. Images were obtained by phase contrast microscopy (PC), and the fluorescence images of chloroplasts were overlaid. Scale bar = 5 μm.