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

In this study, basic cultivation experiments were performed on microalgae Desmodesmus sp, then, the experiment results were deductively expanded, and the use of paddy in the off-season for farmers is advocated. As a result of the continuous culture experiments using 100 mL test tubes, 101.3 mg dry biomass was harvested in the aeration culture using the BG-11 medium. In the aeration culture with mixing CO2, the maximum harvest was obtained under the 10%-CO2 concentration. Considering about CO2 concentration, the condition of 1%-CO2 was superior in CO2 use efficiency. Based on the changes in the pH of the medium, algae such as Desmodesmus sp exchanges ions between the inside of the cell and the surrounding environment, and changes the surrounding environment in order to adapt themselves. In the proforma calculation of second crops in paddy fields, 26.4 million tons dry biomass is harvested in 120 days long of culture a year. Considering that the harvested dry biomass amount, 187 × 10 GJ power energy is obtained by burning them. This amount of energy corresponds to about 5.2% of total electricity. DOI: Coming Soon Corresponding author: Naoto Kobayashi, Department of Innovative Energy Science and Engineering, Graduate school of Engineering, Chubu University, Japan. Email: n.kobayashi7010@gmail.com


Introduction
Photosynthesis is the root of life on the earth. This paper presents the concept of converting carbon dioxide by solar energy into a flammable energy source, with emphasis on the biological properties of green algae. These properties, acquired through long geological ages, effectively result in a photosynthetic reaction by greener lives by consuming minimum amount of energy. In the earliest era of terrestrial life, plants were starting to live in the water and they are presumed to have developed a mechanism of photosynthesis that uses solar energy. It's thought that the life had adopted the condition of that era's water component, temperature, and acidity. At the beginning, primitive algae like the cyanobacteria spread, and eventually the green algae based on chlorophyll was born. By and by, living organisms have evolved to maintain environment in the body that is close to the aquatic environment, and have extended the living environment to land. Green algae are known for having the ability to live in a wide range of pH environments.
In this study, we experimentally found that algae are forced to live according to the external environment in order to survive. This adaptability is explained as an ability to endure the living environment. This is a way of saying that primitive organisms only have the ability to accept the environment's pH and not to regulate their internal environment. We focus on the environmental controllability of algae that we experimentally found, and propose coexistent use of rice cultivation and algae cultivation in the paddy field. The paddy field, which is a freshwater area, already has examples of coexistence with rice cultivation and breeding duck and carp farming [1][2][3]. Algae cultivation in paddy fields is similar to rice production and suitable for the primary industry. By incorporating bioenergy production while maintaining rice production, it contributes to maintaining and improving food and energy self-sufficiency. Algae already have the environmental controllability under the clever mechanism of photosynthesis. The intuition that algae are undifferentiated is the haughtiness of Christian religious perspectives through which contemporary humanity has forgotten the awe of the respect of natural life.

Sample Algae Selection
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 1981 [4]. 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 1 [6]. 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.

Culture Experiment
Culture experiment was conducted by placing a 100 mL test tube (A-30, Maruemu, Osaka, Japan) in a  FCR-807K, Yutaka, Tokyo, Japan) and mixed so as to achieve the target CO 2 concentration. In the aeration culture with air only, the CO 2 cylinder wasn't used, so the CO 2 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 CO 2 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.

Filtration Recovery Experiment
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.

Fertilizer
In the culturing experiment, BG-11 was used as a fertilizer. At the NIES, C medium is used as the recommended medium for the culture. C medium is supplemented with vitamins, so it's suitable for most of green algae cultivation including Desmodesmus sp. The compositions of BG-11 medium and C medium are shown in Table 2.

Algae Growth Rate
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, CO 2 concentration is changed of 1%, 5%, 10%, and 20%, and the algae concentration became maximum under the condition of CO 2 concentration of 10%. Growth rate under low CO 2 concentration is somewhat slowed after 8 th day of cultivation.

pH Changes
Changes in pH over time, a chlorophyll-a amount under each culture condition are shown in Figure 3(b). The initial pH of the medium is 7.4 in the BG-11 medium, but at the stage when the algae is turbidified in the medium, the pH became around 8.0. After one day, it settles to a certain pH value depending on the conditions. After that, it changes to a slight alkaline with all conditions. Microalgae like Desmodesmus sp. is thought to exchange ions between the inside of the cell and the surrounding environment and adapt itself to the optimum environment in order to adapt to the surrounding environment. Normally, since the surrounding environment is infinitely large, there is not enough power to change the surrounding environment. However, in the closed narrow area like the test tube used in this experiment, there is enough power to protect itself from the surrounding environment. Desmodesmus sp. creates an optimum environment and grows freely.
Dry Algae Yield

Relationships Between Dry Biomass Yield and CO 2 Efficiency
Normalizing by the supplied CO 2 (wt%), the highest efficiency of CO 2 (dry mass (g)/total CO 2 (g) ×100%) was obtained under the natural air blowing in which CO 2 concentration is 0.04%. It suggests that biomass captures most effectively CO 2 from the natural air, as illustrated in Figure 4. At 0.04% of CO 2 , Desmodesmus sp. yielded dry weight of about 0.1 g in 14 days. If it got much more CO 2 of 10%, the product rate reached to the maximum value of about 0.37 g in 14 days. When CO 2 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.

Comparison of Fertilizer
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 fields [8] 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.

Calculation of Yield
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 Under the aeration condition of 1%-CO 2 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/m 3 /year. The total amount of paddy field area in Japan is 24,050 km 2 , of which approximately 12,000 km 2 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 12,000×10 6 (m 2 )×0.1 (m) = 1,200×10 6 m 3 . The total yield of algae will be, A total = A×W = 26.4×10 9 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)×26.4×10 6 (t/year) = 468×10 6 (GJ/ year). The power generation efficiency η of the thermal power plant is about 40%, the power energy obtained by burning algae is 187×10 6 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 = 10 12 kWh/year = 3,600×10 6 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.

Conclusions
Microalgae Desmodesmus sp. was cultured in the test tubes under several conditions. For estimating growth of algae culture, the algae concentration became maximum under the condition of CO 2 concentration of 10%. Growth rate under low CO 2 concentration is somewhat slowed after 8 th day of cultivation.
Normalizing by the supplied CO 2 , the highest efficiency of CO 2 was obtained under the natural air blowing in which CO 2 concentration is 0.04%. It suggests that biomass captures most effectively CO 2 content from the natural air.
Talking about pH changes, in the closed narrow area like this experiment, Desmodesmus sp shows enough power to protect itself from the surrounding environment.
However, since the surrounding environment is infinitely large, there is not enough power to change the surrounding environment. Even though it is a local paddy field, raising carbon dioxide concentration is inconsistent with the purpose of reducing greenhouse gas emissions in the environment.
In the calculation for the future use of algae as overseas dependence of primary energy like Japan, it's significant to be able to produce 5% of primary energy for electric power domestically. For industrialized countries like Japan, raising the domestic production rate of primary energy not only increases the domestic economic cycle index (GDP), but also has a positive impact on the world economy. Similarly, for developing countries, reducing fossil fuel consumption and mitigating deforestation will greatly contribute to the preservation of the global environment, and furthermore it will make an increasing the GDP in those countries.
Since the paddy field area of developing countries is larger than that of Japan, and the temperature of these countries is higher, so the supply of primary energy by algae is larger than that of Japan. Also, energy consumption of these countries is less than that of Japan, with the exception of China. Therefore, primary energy production in developing countries has greater effect on GDP of those countries than in Japan.