Methods and systems for culturing algae

Abstract

This invention provides a novel method for supplying and recovering nutrients using membranes, along with a method and system for culturing algae that enables the practical application of low-cost and space-saving production of biofuels and bioenergy. [Solution] A reaction vessel is used that includes a digestion tank containing a digestion fluid with a high concentration of nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae. The algae in the culture tank consume the nutrients, maintaining a concentration difference between the digestion tank and the culture tank, and supplying the nutrients contained in the digestion fluid to the culture medium through the membrane by concentration diffusion, thereby cultivating the algae.

Description

Technical Field

[0001] The present invention relates to an algae culture method and an algae culture system.

Background Art

[0002] Various studies have been conducted on culturing algae such as microalgae (e.g., indigenous microalgae) and producing biofuels and bioenergy using the cultured algae as a raw material. To commercialize the production of biofuels and bioenergy using algae and carry out such production on a commercial scale, it is necessary to culture and recover a large amount of algae. However, culturing and recovering a large amount of algae requires high costs, and among these, the cost for recovering nutrient salts required for culturing algae is considered to be one of the causes of high costs. Therefore, for recovered nutrient salts, for example, nutrient salts in livestock manure and fermentation residues obtained after subjecting livestock manure to methane fermentation, economic rationality is emphasized, and in most cases, they have been used for spreading on farmland instead of being supplied for algae culture.

[0003] As methods for recovering nutrient salts from wastewater containing high-concentration nutrient salts, the hydroxyapatite (HAP) method, magnesium ammonium phosphate (MAP) method, and ammonia stripping method are known (Non-Patent Documents 1 to 3). The HAP method and the MAP method are methods for recovering phosphorus from sewage sludge or human waste as wastewater containing high-concentration nutrient salts. In these methods, phosphorus and nitrogen components can be recovered as precipitates (solids), but they are often used in wastewater treatment processes, and pretreatment such as removal of turbidity components from wastewater is required. The ammonia stripping method is a method in which high-concentration ammonia nitrogen contained in wastewater is expelled from a liquid phase to a gas phase as ammonia gas, and ammonia in the gas is recovered. This method is also often used in wastewater treatment processes, and the recovered ammonia has also been treated by catalytic decomposition.

[0004] In recent years, attempts have been made to develop methods for recovering nutrients from wastewater using membranes. These methods utilize MF membranes (microfiltration membranes), UF membranes (ultrafiltration membranes), and NF membranes (nanofiltration membranes) as the membranes, and nutrients can be recovered by diffusion driven by pressure. These membrane treatment methods have been used for livestock manure and its methane fermentation residues because they can be used in a small space and at a relatively low cost (Non-Patent Literature 4). However, applying static pressure due to the head difference causes clogging (fouling) on ​​the membrane surface, which slows down the movement of nutrients, making continuous use impossible. Furthermore, as a method utilizing membranes, a method for recovering nutrients by combining membrane separation, electrodialysis, and distillation processes is also known (Non-Patent Literature 5). However, a challenge with such methods is that they are costly because they require multiple processes. As a method for recovering nutrients from wastewater relatively efficiently with a low possibility of clogging, the use of FO membranes (forward osmosis membranes), which are an inverse application of the principle of RO membranes (reverse osmosis membranes) used in brine carbonization, is known. However, in order to utilize osmotic pressure, it is necessary to set the salt concentration on the recovery side high (3.5M sodium hydroxide), which limits the uses of the recovered nitrogen components.

[0005] Conventional methods for recovering nutrients have various problems and have not been sufficiently utilized for the cultivation and recovery of algae. Therefore, there is a strong need for a low-cost and useful method for supplying nutrients in algal cultivation. As a membrane-based method, there have been no reports of a method that uses a membrane with a pore size of 0.45 μm or less to supply nutrients separated by diffusion to the culture medium, driven by the concentration difference of nutrients in the digestate and culture medium, and then cultivating algae. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Takao Hagino, Tsuyoshi Hirashima: Development of a phosphorus recovery process from sewage sludge, Environmental Resource Engineering, 52:172-182, 2005. [Non-Patent Document 2] Hirokazu Shirage: Phosphorus Recovery and Resource Utilization System using MAP Method, Journal of Environmental Biotechnology, Vol. 4, No. 2, 109-115, 2005. [Non-Patent Document 3] Junichi Takahashi: Converting unused resources into animal feed by ammonia stripping of biogas plant fermentation digestate, Green Techno Information, Vol.3, No.33, 5-10, 2007. [Non-Patent Document 4] Mehta, CM, Khunjar, WO, Nguyen, V., Tait, S., & Batstone, DJ (2015, February 16). Technologies to recover nutrients from waste streams: A critical review. Critical Reviews in Environmental Science and Technology, Vol. 45, pp. 385-427 [Non-Patent Document 5] Mitsuyasu Yabe: Separation, concentration, and recovery of fertilizer components from methane fermentation digestate, AgriBio, Vol. 3, No. 4, 370-374, 2019. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a novel method for supplying and recovering nutrients using a membrane, and to provide a method for cultivating large quantities of algae at low cost, and an algae cultivation system for that purpose, which enables the practical application of biofuel and bioenergy production at low cost and in a small space by continuously cultivating algae, by supplying nutrients from a digestate containing high concentrations of nutrients into a culture medium at a supply rate suitable for algae cultivation. [Means for solving the problem]

[0008] The inventors of this invention conducted diligent research to solve the above problems and found that by installing a membrane with a pore size of 0.45 μm between the digestate tank and the culture tank in the reaction vessel, clogging of the membrane is prevented, and by maintaining the same volume of liquid in both the digestate tank and the culture tank, high-concentration nutrients in the digestate are supplied to the culture tank through the membrane by diffusion utilizing the concentration difference of nutrients in the digestate tank and the culture tank. This cycle of algae consuming the supplied nutrients is repeated, enabling continuous cultivation of algae. This invention was completed based on these findings.

[0009] In other words, the present invention provides the following embodiments. [Item 1] A method for culturing algae using a reaction vessel comprising a digestion tank containing a digestion fluid with high concentrations of nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae, A method for culturing algae, comprising the step of supplying nutrients contained in the digestive fluid to the culture medium via a membrane by diffusion utilizing the concentration difference of nutrients in the digestive fluid tank and the culture tank. [Item 2] The rate of nutrient supply is 177-188 g-N / m 2 The method described in item 1, which is / d. [Item 3] The film is 0.0193 m 2 The method according to item 1 or item 2, having the area described above. [Item 4] The culture rate of algae is 49-234 g / m². 3 The method described in any one of items 1 to 3, which is / d. [Clause 5] The method according to any one of Clauses 1 to 4, further comprising the step of the digestate tank and culture tank being further equipped with pumps, the pumps being used to circulate each liquid within the same tank and to maintain the same volume of the digestate and culture. [Item 6] The method according to any one of items 1 to 5, further comprising the step of supplying CO2 to a culture vessel. [Item 7] In the culture tank, phosphate ions (PO4 3- The method according to any one of claims 1 to 6, further comprising the step of supplying ). [Clause 8] The method according to any one of Clauses 1 to 7, wherein the digestate is a methane fermentation digestate. [Item 9] The method according to any one of items 1 to 8, wherein the culture medium is dechlorinated tap water, groundwater, or river / lake water. [Item 10] The method according to any one of items 1 to 9, wherein the algae are microalgae. [Item 11] The method according to any one of items 1 to 10, wherein the membrane is a microfiltration membrane (MF membrane). [Item 12] The method according to any one of items 1 to 11, wherein the nutrients include at least one selected from the group consisting of ammonia nitrogen, nitrate nitrogen, phosphate phosphorus, orthosilicic acid, potassium, calcium, magnesium, and sulfur. [Item 13] An algae cultivation system comprising a digestion tank, a membrane with a pore size of 0.45 μm or less, and a culture tank, wherein the digestion tank contains a digestion fluid containing high concentrations of nutrients, the culture tank contains a culture medium and algae, and the membrane is installed as a partition between the digestion tank and the culture tank. [Clause 14] The algae cultivation system according to Clause 13, characterized in that the algae in the culture tank consume nutrients, thereby maintaining a concentration difference between the digestive fluid tank and the culture tank, and supplying nutrients contained in the digestive fluid to the culture medium by concentration diffusion. [Clause 15] The algae cultivation system according to Clause 13 or Clause 14, wherein the digestion tank, the membrane, and the culture tank are arranged in a horizontal row in that order. [Clause 16] The algae cultivation system according to Clause 13 or Clause 14, wherein the digestion tank, the membrane, and the culture tank are arranged in a vertical line in that order. [Item 17] A method for supplying nutrients using a reaction vessel equipped with a digestate tank containing a digestate containing a high concentration of nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae, A method for supplying nutrients, characterized in that algae in the culture tank consume nutrients, thereby maintaining a concentration difference of nutrients between the digestive fluid tank and the culture tank, and supplying nutrients contained in the digestive fluid to the culture medium through a membrane by concentration diffusion. [Effects of the Invention]

[0010] According to the present invention, nutrients can be supplied from digestate at a supply rate and in the required amount suitable for algal cultivation. Furthermore, since nutrients can be supplied without the need for pretreatment such as removal of turbidity components, low-cost algal cultivation can be achieved. Furthermore, according to the present invention, it becomes possible to cultivate algae on a large scale, and the practical application of biofuel and bioenergy production on a commercial scale can be expected. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram showing an example of an algae cultivation system according to the present invention. (a) is horizontal, and (b) is vertical. Qd indicates the amount of fluid flowing into the digestion tank, and Qc indicates the amount of fluid flowing into the cultivation tank. [Figure 2] The graph shows the time course of fluorescence intensity for each culture medium prepared from 20-fold, 50-fold, and 100-fold diluted digestate and CSi medium. [Figure 3] This is a schematic diagram of the experimental apparatus for cultivating indigenous microalgae by adding a mixed gas (CO2 gas) or by adding air. The experimental apparatus for cultivating by adding a mixed gas (CO2 gas) contains an aluminum gas bag (400 mL) containing a gas mixture of CO2 gas and air adjusted to a CO2 gas concentration of approximately 10%, connected by tubes and tube fittings to a vial bottle (volume 228 mL) with a butyl rubber aluminum seal stopper containing diluted digestate (100 mL) and microalgae solution (20 mL). The experimental apparatus for cultivating by adding air contains a vial bottle (volume 228 mL) with a breathable silicone stopper containing diluted digestate (100 mL) and microalgae solution (20 mL). [Figure 4] This is a schematic diagram of an experimental apparatus for separating turbidity components and nutrients in digested fluid. H represents the liquid volume (water level), and Φ40 represents the diameter of the opening (40 mm). [Figure 5]Figure 4 shows the change over time in the light transmittance (%) of the culture solution obtained using the experimental apparatus shown. ○ indicates the change over time in the light transmittance of the culture solution in the first experiment, and ● indicates the change over time in the light transmittance of the culture solution in the second experiment. The line with a transmittance of 34.9% represents the light transmittance of the 50-fold diluted digestate, which is considered optimal for culturing microalgae, as shown in Example 1. [Figure 6] The graphs show the time course of ammonium ions (NH4+) (g) in the digestate tank and culture tank. ◇ indicates the time course of ammonium ions (NH4+) in the digestate tank during the first experiment, ◆ indicates the time course of ammonium ions (NH4+) in the digestate tank during the second experiment, △ indicates the time course of ammonium ions (NH4+) in the culture tank during the first experiment, and ▲ indicates the time course of ammonium ions (NH4+) in the culture tank during the second experiment. [Figure 7] The graphs show the changes in potassium ions (K+) (g) in the digestion tank and culture tank over time. ◇ indicates the changes in potassium ions (K+) in the digestion tank during the first experiment, ◆ indicates the changes in potassium ions (K+) in the digestion tank during the second experiment, △ indicates the changes in potassium ions (K+) in the culture tank during the first experiment, and ▲ indicates the changes in potassium ions (K+) in the culture tank during the second experiment. [Figure 8] This shows the amount of fluid transferred per unit time and per unit area from the digestion tank to the culture tank (separation flux) and the amount of NH4+ transferred due to the movement of water from the culture tank to the digestion tank (transfer flux). [Figure 9] This chart shows the amount of microalgae in the culture tank from day 1 to day 28. Note that the concentration was not measured from day 8 to day 10. Within the culture period, ○ indicates the day distilled water was added, △ indicates the day the digestive fluid was replaced, and □ indicates the day nutrients were added. [Figure 10]The graph shows the amount of PO43- in the digestate and culture medium from day 1 to day 28. Note that the amount of PO43- on days 8 and 9 was not measured. In the graph, ■ indicates the amount of PO43- in the digestate, □ indicates the amount of PO43- in the culture medium, and within the culture period, ○ indicates the day distilled water was added, △ indicates the day the digestate was changed, and □ indicates the day nutrients were added. [Figure 11] The graph shows the amount of NH4+ in the digestate and culture medium from day 1 to day 28. Note that the amount of NH4+ on days 8 and 9 was not measured. In the graph, ■ indicates the amount of NH4+ in the digestate, □ indicates the amount of NH4+ in the culture medium, and within the culture period, ○ indicates the day distilled water was added, △ indicates the day the digestate was changed, and □ indicates the day nutrients were added. [Modes for carrying out the invention]

[0012] The present invention will be described in detail below.

[0013] The present invention provides a method for cultivating algae using a reaction vessel equipped with a digestion tank containing a digestion fluid with high concentrations of nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae.

[0014] The present invention relates to a method for culturing algae, characterized by supplying nutrients contained in the digestive fluid to the culture medium via a membrane by diffusion, utilizing the concentration difference of nutrients between the digestive fluid tank and the culture tank, which is created when algae in the culture tank consume nutrients.

[0015] In this invention, "algae" refers to organisms that perform oxygen-evolving photosynthesis, excluding mosses, ferns, and seed plants that mainly inhabit the land. The algae of this invention may also be microorganisms that perform biosynthesis, such as Euglena. There are no particular restrictions on the algae, and they can be appropriately selected depending on the purpose.

[0016] The algae of the present invention are preferably microalgae (for example, indigenous microalgae). Here, microalgae refer to tiny algae that cannot be individually recognized by the naked eye. Microalgae may be either prokaryotes or eukaryotes.

[0017] Examples of microalgae include those belonging to any of the following taxonomic groups: green algae (Chlorophyta), glaucophyta, red algae (Rhodophyta), chlorarachniophyta, Euglenophyta, cryptophytes (Cryptophyta), brown algae (Phaeophyta), haptophytes (Haptophyta), heterokontophyta, dinoflagellates (Dinophyta), chromerida, and cyanobacteria. The taxonomic group to which microalgae belong is not yet determined; it is sufficient if they are included in or closely related to these taxonomic groups based on molecular phylogenetics.

[0018] In the cultivation method of the present invention, one type of algae can be used alone or in combination of two or more types. If algae are in a symbiotic relationship with other organisms, they may be used together with those organisms.

[0019] There are no particular restrictions on how to obtain microalgae; the method can be chosen appropriately depending on the purpose. For example, methods include collecting them from nature, using commercially available products, or obtaining them from conservation or depository institutions.

[0020] In the algae cultivation method of the present invention, the cultured algae can be recovered from the culture medium by commonly used methods such as centrifugation, sedimentation separation using a flocculant, and membrane separation. Alternatively, the biofilm formed on the surface of the culture medium can be deposited and recovered together.

[0021] In the present invention, "digestate" refers to the residue obtained after fermentation treatment at a biomass plant (BGP) using livestock excrement, food processing residues, waste cooking oil, food waste, sewage sludge, human waste, septic tank sludge, etc., as raw materials. The digestate of the present invention is, for example, methane fermentation digestate. Furthermore, the digestate of the present invention is preferably derived from livestock excrement (for example, cow manure) because it is easy to secure a large quantity of raw materials.

[0022] In this invention, "nutrients" refers to salts necessary as nutrients for algae (e.g., microalgae). Examples of nutrients include nitrogen such as ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and organic nitrogen; phosphorus such as phosphate phosphorus and organic phosphorus; silicon such as orthosilicic acid; potassium, calcium, magnesium, and sulfur. In this invention, nutrients can be used as a source of nutrients for algae growth.

[0023] In this invention, "culture medium" refers to a solution with high light transmittance. Examples of culture mediums include dechlorinated tap water, groundwater, and river / lake water. It is desirable that the culture medium of this invention has a light transmittance of 34.9% or higher. For example, in this invention, distilled water was used to inoculate the bottom layer water of a pond on the Hokkaido University campus with initial indigenous microalgae. In the culture medium of the present invention, for example, nutrients are supplied from a digestion tank, and indigenous microalgae consume these supplied nutrients and are cultured in a repeating cycle, thereby maintaining a low concentration of nutrients.

[0024] In this invention, the "membrane (filter)" is used as a partition between the digestate tank and the culture tank. Examples of membranes in this invention include microfiltration membranes (MF membranes), ultrafiltration membranes (UF membranes), and nanofiltration membranes (NF membranes). The membranes of this invention preferably have a pore size of 0.45 μm or less. If the pore size of the membrane exceeds 0.45 μm, turbidity components in the digestate also move into the culture medium, reducing the light transmittance of the culture medium and inhibiting algal biosynthesis. Furthermore, the membranes of this invention are suitable for tanks with a volume of 1 m³. 3 Hit, 0.0193m 2having an area equal to or larger than that, preferably 0.0256 m 2 having an area equal to or larger than that.

[0025] In the present invention, the term "turbidity component" refers to a substance that imparts turbidity to a liquid and has a particle size exceeding 0.45 µm. Examples of the turbidity component of the present invention include particulate organic substances, plankton, other microorganisms, and suspended solids.

[0026] In the method for culturing algae of the present invention, 177 to 188 g-N / m 2 of nutrient salts can be supplied at a supply rate of / d.

[0027] In the method for culturing algae of the present invention, 49 to 234 g / m 3 of algae can be cultured at a culture rate (growth rate) of / d. In the present invention, the culture rate is 49 to 73 g / m 3 / d, 49 to 78 g / m 3 / d, 49 to 93 g / m 3 / d, 49 to 126 g / m 3 / d, 73 to 93 g / m 3 / d, 73 to 126 g / m 3 / d, 73 to 234 g / m 3 / d, 78 to 93 g / m 3 / d, 78 to 126 g / m 3 / d, 78 to 234 g / m 3 / d, 93 to 126 g / m 3 / d, 93 to 234 g / m 3 / d or 126 to 234 g / m 3 / d. In the method for culturing algae of the present invention, a phosphorus source, preferably a phosphate ion (PO4 3- ), is supplied to the culture tank at an appropriate ratio according to the amount of nitrogen supplied in the digestive juice, thereby increasing the culture rate of algae. For example, the ratio of the nitrogen supply amount in the digestive juice to the phosphate ion supply amount in the culture tank is 7:1.

[0028] The present invention's method for culturing algae involves circulating the digestate and culture solution in the digestate tank and culture tank, respectively, using a stirring device, preferably a pump, to maintain the same liquid level for both the digestate and culture solution and to maintain a concentration difference between nutrients in the digestate tank and culture tank.

[0029] The present invention provides a method for culturing algae, which enhances algal cultivation by supplying a carbon dioxide source, preferably CO2, to the culture tank.

[0030] The present invention relates to a method for culturing algae, wherein a phosphorus source, preferably phosphate ions (PO4), is supplied to the culture tank in accordance with the amount of nitrogen supplied in the digestate. 3- By supplying ) in an appropriate ratio, the cultivation of algae can be enhanced. For example, the ratio of nitrogen supply in the digestate to phosphate ion supply in the culture tank is 7:1. For example, in the algae cultivation method of the present invention, 1.05 mol / m³ is supplied to the culture tank. 3 By supplying phosphate ions at a specific concentration, the cultivation rate of algae increases, thereby enhancing algal growth.

[0031] The "algae cultivation system" in the present invention comprises a digestate tank, a membrane (filter), and a culture tank. The digestate tank contains a digestate containing high concentrations of salts, and the culture tank contains a culture medium and algae. The digestate tank and culture tank may have one or more devices such as a stirring device, a temperature control device, a pH adjustment device, a turbidity measuring device, a light control device, and a specific gas concentration measuring device such as CO2. The membrane is installed between the digestate tank and the culture tank, and its pore size is preferably 0.45 μm or less.

[0032] The algae cultivation system in the present invention may be arranged in a horizontal row with the digestion tank, membrane, and culture tank in that order, as shown in Figures 1(a) and (b), or it may be arranged in a vertical row.

[0033] The algae cultivation system of the present invention can be used by, for example, connecting a transparent polyvinyl chloride pipe with a diameter of 40 mm using a flange with a gasket and a 0.45 μm filter in between, and then filling a digestate tank and a culture tank, each with 600 mL of digestate and 600 mL of distilled water, respectively, with the water levels at the same level, and circulating the liquid from the bottom to the top of the tank at a rate of 400 mL / min using a pump to agitate the contents.

[0034] In the algae cultivation system of the present invention, the algae in the culture tank consume nutrients, thereby maintaining a concentration difference between the digestate tank and the culture tank, and supplying nutrients contained in the digestate to the culture medium through concentration diffusion.

[0035] The algae cultivation system of the present invention can supply nutrients to the cultivation tank at a supply rate suitable for algae cultivation, and can achieve low costs.

[0036] The algae cultivation system of the present invention can minimize the movement of turbidity components even when using a digestate containing a large amount of turbidity components and high concentrations of nutrients.

[0037] In the algae cultivation system of the present invention shown in Figure 1, the change in the concentration of nutrients in the digestate tank can be calculated using equation (1).

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[0038] In the algae cultivation system of the present invention shown in Figure 1, the change in the concentration of nutrients in the culture tank can be calculated using equation (2).

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[0039] In the algae cultivation system of the present invention shown in Figure 1, the change in the algae concentration in the culture tank can be calculated using equation (3).

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[0040] In the algae cultivation system of the present invention shown in Figure 1, the nutrient separation flux (the amount of nutrients transferred from the digestate tank to the culture tank per unit time and unit area) can be calculated using equation (4).

number

[0041] In the above formula, V is the volume of the tank (V d V is the volume of the digestive fluid reservoir. c (where is the volume of the culture vessel), C s This is the nutrient concentration (C sd This refers to the nutrient concentration in the digestive fluid reservoir, C cd (This refers to the nutrient concentration in the culture tank), C x (Q) is the algae concentration, and Q is the water flow rate into the tank. d Q is the amount of water flowing into the digestive fluid tank. c (The amount of water flowing into the culture tank), r x This is the growth rate of microalgae, Y xs F is the nutrient consumption per microalgae, F is the nutrient separation flux, A is the nutrient consumption per microalgae. f k represents the filter area, and k represents the membrane migration velocity coefficient.

[0042] Assuming a steady state for each concentration, and that the growth rate of algae and the nutrient consumption per algae are constant in the steady state, the nutrient concentration in the digestate tank can be calculated using equation (5).

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[0043] Assuming a steady state for each concentration, and that the growth rate of algae and the nutrient consumption per algae are constant in the steady state, the nutrient concentration in the culture tank can be calculated using equation (6).

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[0044] Assuming a steady state for each concentration, and that the growth rate of algae and the nutrient consumption per algae are constant in the steady state, the concentration of algae can be calculated using equation (7).

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[0045] The present invention provides a method for supplying nutrients using a reaction vessel comprising a digestion tank containing a digestion fluid containing high-concentration nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae. The method is characterized in that the algae in the culture tank consume the nutrients, thereby maintaining the concentration difference of nutrients between the digestion tank and the culture tank, and supplying the nutrients contained in the digestion fluid to the culture medium through the membrane by concentration diffusion. [Examples]

[0046] Examples are shown below, but these are for the purpose of better understanding the present invention and do not limit the scope of the invention.

[0047] Example 1: Investigation of digestive fluids useful for algae cultivation A culture medium was prepared by adding 10 mL of environmental water (collected from the bottom layer of a pond on the Hokkaido University campus) to 50 mL of culture medium (bovine manure methane fermentation digestate and standard medium (CSi)). The bovine manure methane fermentation digestate was centrifuged and diluted 20, 50, and 100 times with distilled water to prepare 20-fold, 50-fold, and 100-fold diluted digestates. 4 mL of each prepared culture medium was taken, and the fluorescence intensity was measured for 12 days using a fluorescence spectrophotometer (FP-6600, JASCO) with an excitation wavelength of 436 nm and an emission wavelength of 684 nm. Furthermore, the light transmittance was measured for each diluted digestate. The wavelength of light was set to 784 μm, and the transmittance with distilled water in a 1 cm quartz cell was defined as 100%.

[0048] Figure 2 shows the changes in fluorescence intensity over time for each culture medium. In the 50-fold and 100-fold diluted digestates, a trend of increasing fluorescence intensity was observed, similar to the CSi medium. In the 20-fold diluted digestate, the number of days required for the increase in fluorescence intensity was delayed compared to the other digestates and the CSi medium, but the increase in fluorescence intensity was similar.

[0049] Table 1 shows the permeability of each diluted digestate. [Table 1]

[0050] These results suggest that microalgae can be cultured using digestive fluids with a light transmittance of 34.9% or higher. Furthermore, the 50-fold diluted digestive fluid with the highest fluorescence intensity is considered optimal for culturing microalgae.

[0051] Example 2: Investigation of factors that inhibit light transmission The digestate was filtered using a membrane filter with a pore size of 1 μM or 0.45 μM, and the light transmittance of the undiluted digestate and the filtrate filtered through each filter was measured using a spectrophotometer (U-1800, Hitachi High-Tech Science). Furthermore, 1 g or 0.25 g of granular activated carbon was added to 50 mL of digestate, shaken at 200 rpm for 0.5 hours or more, and then filtered in the same manner as above, and the light transmittance of the filtrate was measured. The wavelength of light was set to 684 μm, and the transmittance with distilled water in a 1 cm quartz cell was set to 100%.

[0052] Table 2 shows the light transmittance of the undiluted digestive fluid and the filtrate. [Table 2]

[0053] As shown in the results above, the light transmittance of the undiluted digestive fluid was zero, and the light transmittance of the filtrate filtered through a 1 μm filter did not change regardless of the amount of activated carbon. On the other hand, in the filtrate filtered through a 0.45 μm filter, the light transmittance was greatly improved by adding activated carbon compared to the undiluted digestive fluid. This suggests that coloring components (0.45 μm or smaller) are adsorbed onto the granular activated sputum. Furthermore, since the light transmittance of the filtrate filtered through a 1 μm filter did not improve, it was shown that even if the coloring components are removed, the light transmittance will not improve if particles smaller than 1 μm are present. These results indicate that in digested fluid, turbidity components, which have a larger particle size than coloring components, inhibit light transmission, and that separation of turbidity components and nutrients is necessary in algal cultivation. Furthermore, it was shown that using filters with a pore size of 0.45 μm or less is useful for separating turbidity components and nutrients.

[0054] Example 3: Mixed gas (CO) in the cultivation of indigenous microalgae 2 Effects of gas addition Digestion from a bovine manure biomass plant (BGP) was centrifuged and diluted 50 times with distilled water to achieve a transmittance of 28% at a wavelength of 684 nm. KH2PO4 was added as a phosphorus source to a concentration of 60 mg / L to prepare the diluted digestate. For the microalgae solution, indigenous microalgae collected from the bottom of Ohno Pond on the Hokkaido University campus were cultured in the diluted digestate for about 10 days. Diluted digestate (100 mL) and microalgae solution (20 mL) were added to a vial bottle (228 mL volume) with a butyl rubber aluminum seal stopper. Then, 400 mL of an aluminum gas bag (GL Sciences) containing a mixture of CO2 gas and air, adjusted to a CO2 gas concentration of approximately 10%, was added. This bag was then connected to the vial bottle (left diagram in Figure 3) using tubing and tube fittings to investigate the effect of CO2 gas addition on culturing indigenous microalgae. As a control, the effect of air exposure on culturing indigenous microalgae was investigated using vials (right figure in Figure 3) sealed with permeable silicone containing diluted digestate (100 mL) and microalgae solution (20 mL). Each vial was cultured for 3 days under the culture conditions shown in Table 3. [Table 3]

[0055] The results of atmospheric culture and CO2 culture are shown in Table 4. [Table 4]

[0056] As shown in Table 4, the concentration of indigenous microalgae in CO2 gas culture using a mixed gas was higher than that in atmospheric culture. This suggests that the CO2 gas supply from the mixed gas was greater than that from the atmosphere. Therefore, it was suggested that supplying CO2 gas activates algal cultivation.

[0057] Example 4: Separation test of turbidity components and nutrients in digested fluid Using the experimental apparatus shown in Figure 4, the light transmittance of the culture medium and the ammonium ions (NH4) of the digested fluid and culture medium were measured. + ) and potassium ions (K + The concentrations were measured to confirm that turbidity components and nutrients were separated. Specifically, a transparent PVC pipe with a diameter of 40 mm was connected with a flange, sandwiching a gasket and a 0.45 μm microfiltration (MF) membrane. The membrane separated the two tanks, creating a digestate tank and a culture tank. 600 mL each of digestate and distilled water were added to each tank, and the water levels were made the same. To agitate the contents, the liquid was circulated from the bottom to the top of the tank at a rate of 400 mL / min using a pump. The water level H was measured over time, 5 mL of liquid was taken from each tank, and the light transmittance of the culture solution was measured using a fluorescence spectrophotometer with a fluorescence wavelength of 684 nm (n=2). The concentrations of ammonium ions and potassium ions in both solutions were also measured (n=2). The duration of this experiment was 7 days.

[0058] Figure 5 shows the change in light transmittance of the culture medium over time. While light transmittance decreased over time, the rate of change gradually slowed. This suggests that coloring components smaller than 0.45 μm permeated the membrane, and their migration speed decreased as the concentration difference decreased. The light transmittance of the culture medium was higher than that of the 50-fold diluted digestate (34.9%) shown in Example 1, which is considered optimal for culturing microalgae. This indicates that the movement of turbidity components that inhibit algal cultivation was minimized.

[0059] The time-dependent changes in ammonium ion and potassium ion concentrations in the digestion tank and culture tank are shown in Figures 6 and 7, respectively. + and K + Both ions and the culture medium concentration increased over time, and the rate of increase slowed as time passed. Conversely, the concentration in the digestate decreased. This result indicates that nutrients were supplied from the digestate tank to the culture tank.

[0060] The amount of fluid transferred per unit time and unit area from the digestion tank to the culture tank (separation flux) and the amount of NH4 transferred from the culture tank to the digestion tank. + The amount of fluid transferred (transfer flux) is shown in Figure 8. The separation flux was calculated from the concentration difference between the two tanks and the concentration change in the culture tank, and the transfer due to the concentration difference was observed. The transfer flux was calculated by determining the amount of water transferred from the difference in water head between the two tanks and multiplying it by the concentration in the culture tank. It was confirmed that the water head tended to increase on the culture tank side over time, reaching 3-4 cm. This is thought to be because the solute concentration on the digestate side is higher than that on the culture medium side, creating an osmotic pressure difference between the two tanks, and causing water to move to the digestate side. As shown in Figure 8, the transfer flux was significantly smaller than the separation flux to the culture medium side, suggesting that the separation flux to the culture tank side is primarily due to diffusion caused by the concentration difference between the two tanks. Furthermore, although there is some variability, a linear relationship is observed between the concentration difference and the separation flux, indicating that the separation flux is dependent on the concentration difference. Using a linear approximation, the slope was 0.087 m / d and the correlation coefficient was 0.908.

[0061] Example 5: Investigation of large-scale cultivation of microalgae In the algae cultivation system shown in Figure 1, one unit volume is defined as one unit, and the volume of the tank is 1 m³. 3Based on the parameters related to digestate generation shown in Table 5, the growth rate of microalgae was predicted using digestate obtained from a 100-head dairy cow farm. The manure generation volume and digestate water content were based on parameters described in the New Energy Foundation: Biomass Technology Handbook, p.240 (2008), Ohmsha, and Heinz Schulz, Barbara Eder: Practical Biogas Technology, p.135 (2002), respectively. The NH4 concentration parameter of the digestate was based on experimental values ​​obtained using an ion chromatography analyzer (DIONEX DX-120, Thermo Fisher Scientific KK). [Table 5]

[0062] In a biogas plant (fermentation tank; BGP) with a capacity of 100 dairy cows, the daily output is 5.5 m³. 3 It is predicted that a certain amount of digestive fluid will be generated, and if 1% of it is used, the amount of fluid flowing into the digestive fluid tank (Q d ) is 0.055m 3 / d. Next, using the average growth rate and NH4 consumption per microalgae in atmospheric culture and CO2 gas culture shown in Table 4, the following equation is used:

number

number

number

[0063] Based on the results above, by maintaining a culture tank concentration that allows microalgae to grow, and by maintaining a constant concentration difference between the two tanks, the microalgae cultivation rate can be increased to 49 or 73 g / m². 3 / d can be achieved. However, 1m 3 The membrane area per unit is 0.0193 or 0.0256 m². 2 The above is required, and the separation rate of NH4 is 188 or 177 g / m³. 2 The value is / d. The larger the membrane area, the lower the separation rate. Therefore, the algae cultivation system and method of algae cultivation of the present invention can achieve a general algae cultivation rate while also achieving a nutrient supply rate, thereby enabling continuous algae cultivation and large-scale cultivation and harvesting of algae.

[0064] Example 6: Effects of nutrients on microalgae cultivation In this experiment, a system was used to cultivate algae, consisting of a digestate tank, a microfiltration membrane with a pore size of 0.45 μm, and a 10 L tub (culture tank). The concentrations of nutrients and microalgae were measured and analyzed to investigate the effects of nutrients on microalgae cultivation. Specifically, follow these steps to determine the concentration of microalgae in the culture tank and the NH4 in the digestate and culture medium. + and PO4 3- The concentration of [substance] was measured. In this experiment, indigenous microalgae were used as the microalgae. (1) Preparation of culture medium 113 mL of bovine manure methane digestate was filled into the digestate tank, and 5000 mL of distilled water was added to the tub (culture tank). The apparatus, including the digestate tank and membrane, was then placed in the culture tank. Next, the temperature in the culture tank was set to 26°C, and the mixture was stirred at 190 rpm for 5 days using a stirrer (NZ-1200, Tokyo Rikakikai) to prepare the culture medium. Furthermore, the entire culture tank was placed on an electronic balance to measure the evaporation rate of distilled water, and distilled water was added irregularly to maintain a liquid volume of 5000 mL. (2) Microalgae concentration in the culture tank and ions (NH4) in the digestate / culture medium + and PO4 3- ) Measurement of concentration (1) The culture medium prepared in (1) was inoculated with pre-cultured microalgae. The inoculated culture medium was cultured for 28 days at 250-300 rpm using a stirrer (NZ-1200, Tokyo Rikakikai) under the culture conditions shown in Table 6. Every 24 hours after culturing, 1 mL of the digestate tank and 10 mL of the culture tank were sampled to measure the microalgae concentration in the culture tank and the PO4 concentration in the digestate and culture medium. 3- The concentration was measured. Also, NH4 in digestive fluids and culture media + The concentration was measured to investigate whether microalgae were being cultured using nutrients in the digestive fluid. The microalgae concentration was calculated from the weight obtained by collecting microalgae from the culture medium using a 0.45 μm microfiltration membrane, drying at 105°C for 24 hours, and weighing the sample. 3- and NH4 + The concentration was measured using ion chromatography (DIONEX DX-120, Thermo Fisher Scientific KK) or ion chromatograph (IC-2010, Tokyo Kaken Co., Ltd.). Additionally, distilled water was added on days 3, 6, 10, 12, 14, 17, 21, and 25, the digestate in the apparatus was replaced on days 8 and 21, and KH3PO4 was added on day 14. [Table 6]

[0065] Figure 9 shows the amount of microalgae in the culture tank from day 1 to day 28. Note that the concentration was not measured from day 8 to day 10. No microalgae growth was observed until around day 9 after inoculation, but from around day 10 the culture medium turned green and microalgae growth could be visually confirmed. On day 12, 1 liter of culture medium (containing 0.16 g of microalgae) was collected, and thereafter the amount of microalgae gradually decreased, and the color of the culture medium became lighter, which could be visually confirmed. After measuring the amount of microalgae on day 28, the membrane separation device was disassembled and it was found that microalgae had entered the gaps in the device's flange and were growing there. The amount of microalgae that had entered the inside of the device was brushed into the culture medium and calculated to be 0.5 g.

[0066] PO4 in digestive fluid and culture medium from day 1 to day 28 3- The quantities are shown in Table 7 and Figure 10. Note that the PO4 on days 8 and 9 3- The quantity was not measured. [Table 7]

[0067] As shown in Table 7, the digestive fluid contains PO4 3- It contains almost none. Therefore, PO4 in the culture medium 3- It is derived from the added KH3PO4, and PO4 3- It was shown that there was almost no movement between the digestant and the culture medium. Furthermore, PO4 was present in the culture medium at a nearly constant rate. 3- Since PO4 is being consumed, 3- It has been shown that microalgae can be cultured using this method.

[0068] Furthermore, the growth rate of microalgae was calculated at regular intervals (days 1-6, 10-14, 15-19, and 19-28). The calculated growth rates of microalgae ranged from 78 to 234 g / m². 3 The growth rate of microalgae during each period was (PO4 in the culture medium on the first day). 3- Quantity (mg) - PO4 in culture medium on the final day 3- The growth rate of microalgae from day 1 to day 6 was calculated as (amount (mg)) x phosphate consumption of microalgae (0.044 mg) / volume of solution (L) / number of days (day). For example, the growth rate of microalgae from day 1 to day 6 was calculated as (PO4 in the culture solution on day 1). 3- Amount (547.63 mg) - PO4 in culture medium on day 6 3- amount (440.74 mg)) x phosphoric acid consumption by microalgae (0.044 mg) / liquid volume (3.8648 L) / number of days (5 day) = 125.71 mg / L / day (g / m 3 It becomes / day).

[0069] These results suggest that supplying a high concentration of phosphate ions to the culture medium activates the cultivation of algae.

[0070] NH4 in digestive fluid and culture medium from day 1 to day 28 + The quantities are shown in Table 8 and Figure 11. Note that the NH4 on days 8 and 9 + The quantity was not measured. [Table 8]

[0071] Up to 7 days after microalgae sowing, both the digestive fluid and culture medium contain NH4. + A decrease in the amount was observed, NH4 + The fact that NH4 is moving from the digestive fluid to the culture medium via the membrane, and that microalgae are causing NH4 in the culture medium + This suggested that it was being consumed. After changing the digestive fluid on the 8th day, NH4 was administered from the 10th to the 13th day. + Although the total amount has decreased, NH4 in the culture medium+ This could not be confirmed. This is because NH4 is produced by the proliferating microalgae. + This is thought to be because the consumption of NH4 exceeded the supply. Also, from day 13 onwards, NH4 + This can now be confirmed, and it is due to a decrease in the growth rate of microalgae in the culture medium, resulting in NH4 production by microalgae. + Consumption of NH4 is reduced, + This is thought to be because the supply exceeded the consumption. [Industrial applicability]

[0072] According to the present invention, nutrients can be supplied from digestate at a supply rate and in the required amount suitable for algal cultivation. Furthermore, since the present invention can supply nutrients without requiring pretreatment such as removal of turbidity components, it is possible to cultivate and harvest algae at low cost. Furthermore, it will become possible to cultivate algae on a large scale, which is expected to lead to the practical application of commercial-scale production of biofuels and bioenergy.

Claims

1. A method for culturing algae using a reaction vessel equipped with a digestion tank containing a digestion fluid with a high concentration of nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae, A method for culturing algae, comprising the step of supplying nutrients contained in the digestive fluid to the culture medium via a membrane by diffusion utilizing the concentration difference of nutrients in the digestive fluid tank and the algae culture tank.

2. The rate of nutrient supply is 177–188 g-N / m ２ The method according to claim 1, wherein / d.

3. The film is 0.0193 m ２ The method according to claim 1 or 2, having the above area.

4. The cultivation rate of algae is 49–234 g / m ３ The method according to any one of claims 1 to 3, wherein / d.

5. The method according to any one of claims 1 to 4, further comprising the step of circulating each liquid within the same tank using the pumps, wherein the digestate tank and the algae culture tank are further equipped with pumps, and the volume of the digestate and culture liquids is kept the same.

6. CO2 in the culture tank ２ The method according to any one of claims 1 to 5, further comprising the step of supplying.

7. In the culture tank, phosphate ions (PO ４ ３－ The method according to any one of claims 1 to 6, further comprising the step of supplying ).

8. The method according to any one of claims 1 to 7, wherein the digestate is a methane fermentation digestate.

9. The method according to any one of claims 1 to 8, wherein the culture medium is dechlorinated tap water, groundwater, or river / lake water.

10. The method according to any one of claims 1 to 9, wherein the algae are microalgae.

11. The method according to any one of claims 1 to 10, wherein the membrane is a microfiltration membrane (MF membrane).

12. The method according to any one of claims 1 to 11, wherein the nutrients include at least one selected from the group consisting of ammonia nitrogen, nitrate nitrogen, phosphate phosphorus, orthosilicic acid, potassium, calcium, magnesium, and sulfur.

13. An algae cultivation system comprising a digestion tank, a membrane with a pore size of 0.45 μm or less, and a culture tank, wherein the digestion tank contains a digestion fluid containing high concentrations of nutrients, the culture tank contains a culture medium and algae, and the membrane is installed as a partition between the digestion tank and the culture tank.

14. The algae cultivation system according to claim 13, characterized in that the algae in the culture tank consume nutrients, thereby maintaining a concentration difference between the digestive fluid tank and the culture tank, and supplying nutrients contained in the digestive fluid to the culture medium by concentration diffusion.

15. The algae cultivation system according to claim 13 or 14, wherein the digestion tank, the membrane, and the culture tank are arranged in a horizontal line in that order.

16. The algae cultivation system according to claim 13 or 14, wherein the digestion tank, the membrane, and the culture tank are arranged in a vertical line in that order.

17. A method for supplying nutrients using a reaction vessel equipped with a digestion tank containing a digestion fluid containing high-concentration nutrients, a membrane with a pore size of 0.45 μm or less, and a culture tank containing a culture medium and algae, A method for supplying nutrients, characterized in that algae in the culture tank consume nutrients, thereby maintaining a concentration difference of nutrients between the digestive fluid tank and the culture tank, and supplying nutrients contained in the digestive fluid to the culture medium through a membrane by concentration diffusion.

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