Improvement of Energy Network by Naturally Farming of Chlorophyll-bearing Algae: Effects of Culture Condition Changes for the Yield of Microalgae

For culturing experiments, microalgae Desmodesmus sp. (NIES-96, Japanese name:Ikadamo) distributed from the National Institute for Environmental Studies (NIES) was used. It’s a freshwater microalgae which is in the shape of an ellipse or a rectangle with a body length of about ten and several micrometers. Desmodesmus sp. used in this study is a clone of a sample collected in Lake Shoji in Yamanashi Prefecture in 1981⁴. It’s universally living in the freshwater area around the world. Desmodesmus sp. has a chlorophyll-a/b which are photosynthetic pigment in the chloroplast. In principle, Desmodesmus sp. consists of a fixed number of cells, and it’s called Coenobium ⁵. An overview picture of Desmodesmus sp. is shown in Figure 1⁶. It’s easy to cultivate Desmodesmus sp. because it has accommodation ability to changes in habitat. In general, Desmodesmus sp. has about 10% to 50% oil and fat content.

Figure 1.Desmodesmus sp.

Culture experiment was conducted by placing a 100 mL test tube (A-30, Maruemu, Osaka, Japan) in a stainless steel test tube stand (SS30-10, Sanwakaken, Osaka, Japan) in the water tank (Dax C60, Kotobuki-kougei, Nara, Japan). For the turbid solution of algae and medium, an initial capacity of 75 mL was used, and five samples were prepared each conditions. All test tubes were stoppered with Biosilico (N-32, Shin-Etsu Polymer, Tokyo, Japan). Air in the test tube is always ventilated through biosilico. The water temperature was set at 25 ℃ with a heater (Metal heater SH80, Gex, Osaka, Japan) and a thermostat (NX003, Gex). Figure 2 shows the experimental apparatus diagram. As a light source, the white tape LED (5050 SMD, 3 m 43.5 W) was used and the quantity of photons was set up 100 μmol/m²/s by using a quantum meter (MQ-200, Apogee Instruments, Utah, United States). The measurement mode was ELEC. The 12 hours light period and the dark period were set by program timer (DT-01, Asone, Osaka, Japan). 6 o'clock to 18 o'clock is the light period, and 18 o'clock to 6 o'clock is the dark period. In the aeration culture experiment, in addition to the above-mentioned apparatus, a gas cylinder of air and CO₂ was used. Both of them were depressurized to 0.2 MPa with a regulator (for air: NPR-1B, Yamato, Osaka, japan, for CO₂: FCR-807K, Yutaka, Tokyo, Japan) and mixed so as to achieve the target CO₂ concentration. In the aeration culture with air only, the CO₂ cylinder wasn’t used, so the CO₂ concentration is about 400 ppm (0.04%) similar to that in the atmosphere. Thereafter, the flow rate was adjusted to 0.1 L/min with a flowmeter (RK1710, Kofloc, Kyoto, Japan), and the mixed gas was supplied to the cultivation test tube through a distributer, an air filter (13JP020AN, Advantec, Tokyo, Japan), and a glass tube (φ5 mm×φ3 mm). Aeration was done for many hours regardless of CO₂ concentration. The duration of the culture experiment is 14 days. 1 mL of algal turbid solution was collected once a day at regular time, then, pH (LAQUAtwin B-712, Horiba, Kyoto, Japan) and concentration (PD-303S, Apel, Saitama, Japan) was measured. The concentration was measured by wavelength of 620 nm and 678 nm. Table 1 shows the main conditions of the culture experiments.

Figure 2.Culture apparatus

After the 14-days cultivation experiment, the remaining amount of the algal turbid solution was measured with a measuring cylinder (Graduated cylinder Custom A, 100 mL, Shibata, Saitama, Japan), and cultured algae were collected by suction filtration. For vacuum filtration, a 500 mL filter flask (5340FK500R, AGC Techno Glass, Shizuoka, Japan), filter holder for this filtration (KG-47, Advantec) and a membrane filter (A500A047A, Advantec) were used. The collected algae was measured with a wet weight immediately after filtration, and then a dry weight was measured using an electronic balance (AUX220, Shimadzu, Kyoto, Japan) after air drying at room temperature for about 1 week. The amount of water contained in the algae after air drying was determined by a thermo-gravimetric analyzer (TGA-50, Shimadzu). The rate of temperature rise was set at 10 ℃/min and the sample was heated to 1,000 ℃ with a platinum cell. The initial temperature of the thermo-gravimetric measurement is about 25 ℃, which is equivalent to room temperature.

Figure 3 (a) shows the results of culture experiment of Desmodesmus sp., which is a typical example of the green algae containing chlorophyll. The graph shows the amount of transition of chlorophyll-a which is determined from the absorbance. In the 14-days cultivation, algae concentration continued increasing until experiments ended. It is thought that the algae in the test tube got stirred with bubbles generated by aeration, and light was supplied to the algae that was precipitated at the time of static culture to encourage the growth. For estimating growth of algae culture, CO₂ concentration is changed of 1%, 5%, 10%, and 20%, and the algae concentration became maximum under the condition of CO₂ concentration of 10%. Growth rate under low CO₂ concentration is somewhat slowed after 8th day of cultivation.

Normalizing by the supplied CO₂ (wt%), the highest efficiency of CO₂ (dry mass (g)/total CO₂ (g)×100%) was obtained under the natural air blowing in which CO₂ concentration is 0.04%. It suggests that biomass captures most effectively CO₂ from the natural air, as illustrated in Figure 4. At 0.04% of CO₂, Desmodesmus sp. yielded dry weight of about 0.1 g in 14 days. If it got much more CO₂ of 10%, the product rate reached to the maximum value of about 0.37 g in 14 days. When CO₂ concentrations increased to two orders (from 0.04 to 10%), the yields increased less than a half order only. By thermo-gravimetric analysis exhibit the moisture content of the algae to be about 10% after natural drying at room temperature for 1 week. For natural drying with the sunlight and air, we do not need expensive external energy from industrial processes.

Figure 4.CO2 capture efficiency vs. CO2 content in the feeding air. Desmodesmus sp. works with highest efficiency in CO2 content around 0.04%.

The microalgae Desmodesmus sp. was cultured in BG-11 medium in this study, because it has almost equal concentration of the constituents that present in the paddy field water. In paddy fields, rice cultivation is usually carried out. In the rice cultivation, nitrogen, phosphorus and potassium are used as fertilizer ⁷. Table 4 shows an excerpt from the results of measuring the quality of agricultural water passing through paddy fields⁸. NH₄⁺ and H₂PO₄^- are decreasing with the passage of time and thought to be used for the growth of rice plants. BG-11 medium contains three major nutrients such as nitrogen, phosphorus and potassium as well as plant fertilizer. Comparing the representative value of the ingredients between BG-11 medium and paddy water, the types of main components are similar, but the component’s concentration is lower in paddy water. For example, NaCO₃ contains nitrate ion 12.84 mmol/L in BG-11 medium (Table 5). On the other hand, nitrate ion contains 0.12 mmol/L in paddy water, thus actual nitrogen concentration in the paddy water is about 1/100 or less.

Water filled in paddy fields after rice cultivation was collected, then preliminary experiments were carried out in glass containers larger than test tubes. As a result, algae cultivated in paddy water showed about half the growth rate as compared with BG-11 medium. This test water was collected after filling the water more than 100 days after draining the water post harvesting the rice. Therefore, it's assumed that the content of ingredients is lower than the actual paddy field water. It clearly indicates that the BG-11 medium is excessive in nutrition, and the algae requires no high concentration nutrients such as BG-11 medium.

In this study, we propose that the case of using paddy field in Japan be considered as an example of the cultivation of Desmodesmus sp. in the future and calculated for calculations of the yield. The most important condition is that algae should be cultivated in the more natural state. The harvested algae is not supposed to extract fuel at all, it is assumed that it is burned fully after sun drying. In Japan, rice cultivation is carried out from May to September. Therefore, for the algae cultivation in paddy fields, we assume about 120 days in the period from October to April. During for this period, it should be considered making soil for rice cultivation and giving time to recondition of the paddy fields. Wide shallow paddy fields are about 10 cm water deep and suitable for cultivation of algae. When the concentration of algae increases due to the growth light does not reach into the water, and photosynthesis is inhibited. In some previous studies, there are cases reported that the effective penetration depth of sun light is about 30 cm in the paddy field.

In the main cultivation experiment, Desmodesmus sp. was cultivated in a 100 mL test tube. Under the aeration condition of 1%-CO₂ concentration, the biomass was about 260 mg in 14 days as described in Section 3.3. As an off-season crop of paddy fields, it is assumed that Desmodesmus sp. can grow in the paddy field for 120 days/year. So this yield is calculated, A = 22 kg/m³/year. The total amount of paddy field area in Japan is 24,050 km², of which approximately 12,000 km² excluding Hokkaido, Tohoku, Hokuriku and Sanin area ⁹. Assuming that they can be cultivated at depths of up to 0.1 m in these paddy fields, the effective water volume W (total culture volume) of paddy fields is 12,000×10⁶ (m²)×0.1 (m) = 1,200×10⁶ m³. The total yield of algae will be, A_total = A×W = 26.4×10⁹ kg/year. Production volume of rice is 8 million tons per year in Japan ¹⁰. Rice straw and paddy are added, it is about 15 million tons per year yield, which was invented by photosynthesis during the 5 months from May to September in summer. Algae yield is about 6 million tons per month which is about twice compared with rice.

Next, the total calorific value C due to burning of algae is obtained. The fever amount of green algae like Desmodesmus sp. is about 20 GJ/t ¹¹. On the other hand, since the harvested algae contains about 10% of moisture, it is necessary to consider subtracting the latent heat of evaporation of water. The latent heat of evaporation of water at 100 ℃ is 2.257 kJ/g ¹², so the net calorific value of algae is about 17.75 GJ/t. As mentioned earlier, considering that the harvested Desmodesmus sp. burns entirely, the total calorific value C_total = 17.75 (GJ/t)×26.4×10⁶ (t/year) = 468×10⁶ (GJ/year). The power generation efficiency η of the thermal power plant is about 40%, the power energy obtained by burning algae is 187×10⁶ GJ/year. According to the Energy White Paper 2018 of the Ministry of Economy, Trade and Industry, the total energy generated in 2016, is about 1 trillion kWh ¹³. In other words, P = 10¹² kWh/year = 3,600×10⁶ GJ/year. Desmodesmus sp.'s replaces the thermal energy production by burning of fossil fuels that corresponds to 5.2 % of electric power consumed in Japan in a year.

In this calculation, laboratory-level figures are deductively converted into a large area of paddy fields. As future research subjects, we must conduct demonstration experiments using real paddies. In the demonstration experiment, the difference between the spectrum of sunlight and artificial light and the photon number density (illuminance) becomes clear. Demonstration experiment reveals the difference from uniform irradiation. Also, the influence of water depth and the effect of stirring by wind waves will become clear.