JP6763123B2 - Microalgae culture method - Google Patents

Description

The present invention relates to a method for culturing microalgae, which cultivates microalgae using wastewater such as factory wastewater and circulating water.

In recent years, there have been reports of Patent Document 3, Non-Patent Document 1 and Non-Patent Document 2 as algae culture techniques using sewage. The growth of algae requires carbon dioxide, water, nitrogen, phosphorus, and light, which are necessary for photosynthesis, but sewage contains water, nitrogen, phosphorus, and carbonate ions, which supply light. Furthermore, it is said that algae can be cultivated by using carbon dioxide in the air. In addition, since nitrogen is consumed during algae culture, it is considered that nitrogen in sewage is used for algae growth, and nitrogen can be removed by separating the algae and treated water by solid-liquid separation.

Further, Patent Document 4 proposes a method for culturing algae using permeated water obtained by membrane-filtering wastewater containing at least one of nitrogen and phosphorus. The problem is to suppress so-called contamination, in which the growth of microalgae is inhibited by microorganisms such as bacteria, protozoa, and metazoans contained in the wastewater during the growth of microalgae. The purpose is to filter to remove microorganisms. In addition, since nitrogen is consumed during algae culture, it is considered that nitrogen in wastewater is used for algae growth, and nitrogen can be removed by separating the algae and treated water by solid-liquid separation.

JP-A-2009-142787 Japanese Unexamined Patent Publication No. 2004-230338 Japanese Unexamined Patent Publication No. 8-107782 Japanese Unexamined Patent Publication No. 2014-60967 International Publication No. 2014/1093930 JP-A-5-285491 Japanese Unexamined Patent Publication No. 2012-175964

Y. S. Yun, S.M. B. Lee, J.M. M. Park, CI. Lee, J.M. W. Yang (1997), Carbon Dioxide Fixation by Algae Cultivation Using Wastewater Nutrients, J. Mol. Chem Tech. Biotechnol. , Vol. 69, pp. 451-455 Ryo Honda, J.M. Boonnorat, C.I. Chiemchaisri, Kazuo Yamamoto (2011), Development of biomass production and carbon dioxide fixation process from treated sewage water by concentrated culture of algae using a membrane separation photobioreactor, Proceedings of the 45th Annual Meeting of the Japan Society on Water Environment, p. 233 Takatake Hirata, Osamu Miki, Mako Okumura, Hirotaka Nakayama, Marine microalgae Nannochloropsis sp. Examination of culture conditions, 48th Annual Meeting of the Japan Society for Water Environment, p. 148 Haruko Takeyama, Energy Production and Business Outlook by Microalgae, p. 1 (2012) Makoto Watanabe, Algae Handbook, pp. 160-162 (2012) Collos, Y. , & Harrison, P.M. J. (2014), Acclimation and toxicity of high ammonium concentrations to unicellular algae. Marine pollution bulletin, 80 (1), pp. 8-23. Yu Nakayama et al., Observational research on water and heat transport in urban artificial drainage systems, Proceedings of Hydroengineering, Vol. 50 (2006) Makoto Watanabe, Algae Handbook, pp. 351 (2012) Makoto Watanabe, Algae Handbook, pp. 345-347 (2012) A Study on Total Nitrogen Inflow Load at Shibaura Water Reclamation Center, Tokyo Metropolitan Government Bureau of Sewerage (2008), [online], [Search on October 23, 2014], Internet <URL: http: // www. gesui. metro. tokyo. jp / gijiutou / gn20 / nenpou 2008 / 5-1-8. pdf> Mitsui Bussan Strategic Research Institute, Microalgae as Biomass Resources (2011), [online], [Searched on October 29, 2014], Internet <URL: http: // mitsui. mgssi. com / issues / reports / r1112j_uno. pdf> Szabo Milan et al. (2014), Photosynthetic acceleration of Nannochloropsis oculata investified by multi-wavelength fluorescence analysis analysis, Bioresours. 521-529. Makoto Watanabe, New Energy Algae Biomass, p. 150 (2010)

In the conventional method for growing microalgae using sewage, the concentration of nitrogen contained in sewage is about 30 mg-N / L in total nitrogen, of which ammonia nitrogen is about 20 mg-N / L, according to Non-Patent Document 10. is there. On the other hand, for example, the nitrogen concentration of wastewater discharged in the coke manufacturing process using a coke oven is 100 mg-N / L or more, and most of them are ammonia nitrogen (nitrogen atoms constituting ammonia molecules).

Here, in the coke manufacturing process, wastewater is generated and discharged by the following process. That is, the COG (coke oven gas) generated from the coke oven is washed and cooled with cheap water. Ammonia water that has been washed and cooled by COG contains ammonia nitrogen, tar, sludge, COD (Chemical Oxygen Demand) components, and the like. After washing and cooling the COG, the ammonia water is sequentially introduced into the tar decanter and the ammonia stripping device. The tar decanter removes tar and sludge from the aqueous solution, and the ammonia stripping device removes most of the ammonia nitrogen from the aqueous solution. However, a large amount of ammonia nitrogen (that is, at least 100 mg-N / L or more) still remains in the ammonia discharged from the ammonia stripping device. The aqueous solution discharged from the ammonia stripping device is diluted with seawater and then subjected to activated sludge treatment. In activated sludge treatment, COD components are mainly removed from cheap water. The cheap water after the activated sludge treatment, that is, the activated sludge treated water is discharged as wastewater. As described above, the wastewater discharged in the coke manufacturing process contains a high concentration of ammonia nitrogen.

Non-Patent Document 6 summarizes the EC50 value of algae growth inhibition by ammonia. Non-Patent Document 6 states that when the total ammonia concentration is several thousand μM, that is, the ammonia nitrogen concentration is several tens of mg / L, many green algae, blue algae, algae, dinophyceae, and raphidophytes are used. It has been shown that growth inhibition is observed. Therefore, conventionally, the ammonia nitrogen concentration as a culture condition for algae is generally set to about several tens of mg / L or less. That is, it was considered difficult to grow microalgae using activated sludge treated water.

In addition, the microalgae used for sewage are freshwater, but since cheap water is diluted with seawater before activated sludge treatment, it is limited to marine algae, not freshwater algae, so it is used for sewage. It was difficult to grow fine algae in activated sludge treated water.

Further, the water temperature of sewage is easily affected by the outside air temperature, and according to Non-Patent Document 7, it can be read that there is a fluctuation of about 16 to 28 ° C. throughout the year. On the other hand, according to Patent Document 6, the water temperature is set to about 30 ° C. in the activated sludge treatment of cheap water. Microorganisms generally have an optimum temperature, and the temperature range in which they can be cultivated is different. Therefore, it may be difficult to cultivate microalgae that can be cultivated in sewage as they are in activated sludge-treated water.

Furthermore, phenolic persistent COD components remain in the activated sludge-treated water, and their effects on microalgae are unknown.

To summarize the above, it is impossible to divert the prior art as it is in the following points.
1) Since freshwater is diluted with seawater before being treated with activated sludge, it is difficult for freshwater microalgae to treat freshwater containing seawater.
2) Most of the nitrogen components contained in activated sludge treated water are ammoniacal nitrogen compared to sewage, and the concentration level of activated sludge treated water is much higher in activated sludge treated water than in fresh water.
3) Compared to sewage, activated sludge treated water is generated from an activated sludge treatment tank with a water temperature of about 30 ° C, so the temperature conditions are not suitable for microalgae applied to sewage.
4) Activated sludge treated water has a higher pH than fresh water. (A lot of free ammonia)
5) Phenolic persistent COD components remain in the activated sludge-treated water, and their effects on microalgae are unknown.
Further, although Patent Document 4 proposes the growth of microalgae by permeated water obtained by membrane-filtering wastewater, there is no description regarding the nitrogen concentration of wastewater and the like, and although a plurality of algae types are exemplified, they are carried out. Nothing has been shown to be possible.

From the above, it was difficult to culture microalgae using activated sludge-treated water containing a high concentration of ammonia nitrogen.

Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to cultivate microalgae using wastewater containing a high concentration of ammonia nitrogen, for example, activated sludge treated water. It is an object of the present invention to provide a new and improved method for culturing microalgae and an apparatus for culturing microalgae.

In order to solve the above problems, according to a certain viewpoint of the present invention , activated sludge is separated from activated sludge treated water obtained by treating cheap water including wastewater discharged in the coke manufacturing process with activated sludge. Chlorella sp. Is a microalga grown using treated water as a medium . A method for culturing microalgae, which comprises putting activated sludge-treated water containing 100 mg-N / L or more of ammonia nitrogen into an algae culture tank and irradiating the microalgae with light .

Further, the N / P mass ratio of the algae culture tank may be 16 to 300.

In addition, a dissolved inorganic carbon source may be added to the algae culture tank.

Further, the photon density of light may be 60 μmol / m ² / sec or more.

In addition, phosphate may be added to the algae culture tank.

As described above, according to the present invention, it is possible to cultivate microalgae using wastewater containing a high concentration of ammonia nitrogen. Since nitrogen is required for the growth of microalgae, nitrogen resources can be effectively utilized by culturing microalgae that can utilize wastewater containing a large amount of nitrogen, which is a nutrient source. Further, when the wastewater becomes activated sludge treated water, the activated sludge treated water has a higher water temperature than sewage, so that it can be expected that the growth rate of microalgae will be higher.

It is explanatory drawing which shows the outline of the culture apparatus of microalgae which concerns on embodiment of this invention. It is a graph which shows the measurement result of Example 1. FIG. It is a graph which shows the measurement result of Example 2. It is a graph which shows the measurement result of Example 3. It is a graph which shows the measurement result (nannochloropsis) of Example 4. It is a graph which shows the measurement result (Chlorococcales) of Example 4. It is a graph which shows the correspondence relationship between the N / P mass ratio in the algae culture tank, the algae concentration and the specific growth rate for each type of algae.

A preferred embodiment of the present invention will be described in detail below with reference to the accompanying drawings. In the present specification and the drawings, components having substantially the same functional configuration are designated by the same reference numerals, so that duplicate description will be omitted.

<1. Configuration of microalgae culture device>
First, the configuration of the microalgae culture apparatus according to the present embodiment will be described with reference to FIG. The microalgae culture apparatus includes an algae culture tank 3, a light source 4, and a solid-liquid separation layer 5. The algae culture tank 3 is a culture tank for culturing microalgae 2. At least drainage 1 and microalgae 2 are charged into the algae culture tank 3.

Wastewater 1 is wastewater containing at least 100 mg-N / L of ammonia nitrogen. Examples of such wastewater 1 include the above-mentioned activated sludge treated water and the like. Here, the activated sludge treatment method for treating the activated sludge of the cheap water is not particularly limited, and any method may be used as long as the cheap water generated from the coke oven can be treated. Examples of the type of activated sludge treatment method include a standard activated sludge method and a nitrification denitrification method. In the standard activated sludge method, after the cheap water flows into the sludge tank, the activated sludge is aerated with air. As a result, the sludge in the sludge tank oxidatively decomposes the COD component. In the nitrification denitrification method, a sludge tank consisting of a nitrification tank and an oxygen-free tank is used to reduce COD components and ammonia nitrogen in cheap water. Details of the nitrification denitrification method are disclosed in Patent Documents 1 and 2.

The microalgae 2 are microalgae that can be cultivated even in waste water 1 containing 100 mg-N / L or more of ammonia nitrogen. The microalgae 2 are, for example, microalgae of the genus Nannochloropsis and microalgae that can live in activated sludge-treated water.

The microalgae of the genus Nannochloropsis are described in, for example, Nannochloropsis sp. (Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis oceanica, Nannochloropsis atomus, Nannochloropsis atomus, Nannochloropsis masuna, Nanochloropsis macrona, Nanochloropsis As commercially available products, Marine Fresh (including Nannochloropsis maritima) manufactured by Marine Tech Co., Ltd. is known. Such marine fresh can also be used in this embodiment. Microalgae that can live in activated sludge-treated water are, for example, Chlorococcales. These microalgae may be cultured alone or mixed. Furthermore, as a result of the inventor's study on microalgae that can live in activated sludge-treated water, in particular, Chlorella sp. Was found to be excellent in proliferation. Chlorella sp. According to Non-Patent Document 13, it is described that conventionally, it was classified into Chlorococcales based mainly on morphological observation. Therefore, the above-mentioned Chlorella cales includes Chlorella sp. Is included.

As a result of diligent research on microalgae, the present inventor has found that microalgae that can be cultivated exist even in waste water 1 containing 100 mg-N / L or more of ammonia nitrogen. Hereinafter, the studies conducted by the present inventor will be described.

Microalgae are non-patents among the algae described in Non-Patent Document 4 (organisms that generate oxygen, excluding so-called land plants (moss, fern, seed plants)). It refers to the microorganisms of several tens of microns that live in the ocean and fresh water described in Document 11. Microalgae grow by ingesting nitrogen and phosphorus.

Here, when activated sludge treated water is used as the wastewater 1, the wastewater 1 is diluted with seawater. Therefore, the present inventor focused on marine microalgae. As marine microalgae, Nannochloropsis, Quitoceras calcitrans, Quitoceras glacilis, Tetracermis, Pablo balseri and the like are known.

Then, the inventors focused on Nannochloropsis and attempted to culture microalgae of the genus Nannochloropsis in activated sludge-treated water containing a high concentration of ammonia nitrogen. As a result, the present inventor has found that microalgae of the genus Nannochloropsis can be cultivated even in activated sludge-treated water having an ammonia nitrogen concentration of 300 mg-N / L. Although Non-Patent Document 3 describes the culture of Nannochloropsis, the ammonia nitrogen concentration in the culture solution was not specified. That is, Non-Patent Document 3 does not disclose any knowledge that microalgae of the genus Nannochloropsis can be cultivated even in activated sludge-treated water having an ammonia nitrogen concentration of 300 mg-N / L. No suggestion was given.

Furthermore, it has been reported that high concentrations of ammonia nitrogen have growth inhibition for algae. For example, Non-Patent Document 6 lists EC50 values for algae growth inhibition. In Non-Patent Document 6, many green algae, blue algae, dinophyceae, dinophyceae, and raphidophytes grow when the total ammonia concentration is several thousand μM, that is, the ammonia nitrogen concentration is about several tens of mg / L. It has been described to show inhibition. Therefore, conventionally, it has been recognized that it is necessary to keep the ammonia nitrogen concentration to several tens of mg / L or less in order to culture algae. Therefore, it was not known that microalgae of the genus Nannochloropsis could be cultivated in activated sludge-treated water having an ammonia nitrogen concentration of 100 mg-N / L or more.

On the other hand, the present inventor has found that microalgae of the genus Nannochloropsis can be cultivated even in activated sludge-treated water having a concentration of ammonia nitrogen one order higher than before. Furthermore, the present inventor observed the activated sludge-treated water in the solid-liquid separation layer (the tank that separates the solid content (sludge) in the activated sludge-treated water from the activated sludge-treated water), and found that it was fine in the solid-liquid separation layer. We found that algae are growing. Then, the present inventor collected these microalgae and tried to culture them in activated sludge-treated water containing a high concentration of ammonia nitrogen. As a result, the present inventor has found that the microalgae can be cultivated even in activated sludge-treated water having an ammonia nitrogen concentration of 1,000 mg-N / L. Then, the present inventor separates the cultured microalgae with a digital pipette, drops them on a lined slide glass, covers the glass, and uses a biological microscope (BX43 manufactured by Olympus Corporation) to perform a microscope at a magnification of 200 to 400 times. Upon observation, it was found that the algae were Chlorococcales. Furthermore, as a result of genetic analysis using a DNA sequencer of 3130 Genetic Analyzer manufactured by Applied Biosystems, the main species of the algae was Chlorella sp. I found that.

Chlorella sp. From microalgae growing in the solid-liquid separation layer. In order to isolate the algae, a micropipette method or the like in which only the target algae are sucked up with a pipette while observing with a microscope can be used. The isolated microalgae are often used in the desired growth method after undergoing pre-culture in which the algae are grown to a certain concentration in the medium. Alternatively, among the microalgae that can live in activated sludge-treated water, Chlorella sp. When there is no microorganism having the same proliferative property as the above and the same ammonia resistance, the microalgae growing in the solid-liquid separation layer may be cultured using activated sludge-treated water as a medium. In this case, Chlorella sp. Can be dominant. In this case, it is desirable to carry out a plurality of subcultures (culture in which a part of the target algae is transferred to a new medium and proliferates) in order to promote dominance. By these methods, Chlorella sp. By isolating or dominating the algae, high-purity algae containing no diatoms, filamentous fungi, protozoa, etc. can be obtained, and contamination can be eliminated. That is, a higher growth rate and growth rate can be obtained.

As described above, the present inventor has found that there are microalgae that can be cultivated in activated sludge-treated water containing 100 mg-N / L or more of ammonia nitrogen, and based on such knowledge, the present embodiment relates to the present embodiment. I came up with a culture device and a culture method.

On the other hand, dissolved inorganic carbon is also required for culturing microalgae 2. Dissolved inorganic carbon, for example carbon dioxide _(CO 2), and carbonate _(H 2 _CO 3), bicarbonate ions (HCO ₃ ^-), a carbonate ion _(CO ^{3 2-),} etc., is usually included in the waste water 1 There is. However, in order to grow the microalgae 2 more, a dissolved inorganic carbon source, that is, a material that can become dissolved inorganic carbon in the wastewater 1, may be added to the wastewater 1. Examples of the dissolved inorganic carbon source include carbon dioxide gas and various carbonates. The carbonate may be, for example, a sodium salt, a potassium salt, a calcium salt, or the like. In addition, Non-Patent Document 5 discloses that carbonate can be used for culturing algae. Therefore, the present inventor verified whether the microalgae 2 could be cultivated with carbonate, and was able to cultivate without any particular problem. Therefore, carbonate can be used as a dissolved inorganic carbon source.

The introduced amount and method of adding the dissolved inorganic carbon source are not particularly limited, and the added amount and method adopted in the known algae culture method can also be applied to this embodiment. For example, when carbon dioxide gas is introduced into the drainage 1 as a dissolved inorganic carbon source, an air diffuser may be arranged at the bottom of the algae culture tank 3 and carbon dioxide gas may be introduced (supplied) into the drainage 1 from the air diffuser. .. Further, the gas flow rate of carbon dioxide gas may be, for example, about 0.25 vvm (volume gas per volume birth per min) (see Non-Patent Document 8).

Further, oxygen necessary for respiration of microalgae is input to the algae culture tank 3. The method of adding oxygen is not particularly limited. For example, oxygen in the air may be introduced into the algae culture tank 3 by introducing air into the drainage 1 from the above-mentioned air diffuser pipe. Here, when carbon dioxide gas is used as the dissolved inorganic carbon source, the volume ratio of carbon dioxide gas to the mixed gas of carbon dioxide gas and air may be, for example, 0.2 to 5% by volume (Non-Patent Document). 8).

Phosphorus is also required for culturing microalgae 2. When activated sludge treated water is used as wastewater 1, the activated sludge treated water usually contains about 0 to 1 mg-P / L of phosphorus, but most of the phosphorus remains depending on the state of the activated sludge in the activated sludge tank. It may not be. Therefore, when the phosphorus in the wastewater 1 is insufficient, a phosphorus source (for example, phosphoric acid) may be added to the wastewater 1. As shown in Example 4 described later, the phosphorus concentration in the algae culture tank 3 is preferably 1 mg-P / L or more. The phosphorus concentration in the algae culture tank 3 can be measured by JIS K1020 46.1.1 molybdenum blue absorptiometry or the like.

Further, as shown in Example 6 described later, the mass ratio of the ammonia nitrogen concentration to the phosphorus concentration (hereinafter, also referred to as “N / P mass ratio”) in the algae culture tank 3 is 16.7 to 300. Is desirable. Generally, the N / P mass ratio is 16 in the Redfield ratio. If the phosphorus concentration is too high, that is, the N / P mass ratio is too low, the calcium phosphate salt is used when culturing the microalgae 2 using treated water containing a large amount of calcium salt such as cheap water treated water. White turbidity may occur. When white turbidity occurs in the algae culture tank 3, the light is blocked by these white turbidity, so that the growth of microalgae is suppressed. Therefore, the N / P mass ratio is preferably a certain value or more. Further, the phosphorus concentration may be adjusted by adding a minimum required amount, and if too much phosphorus is added, the cost increases. Therefore, it is preferable in terms of cost that the phosphorus concentration is low, that is, the N / P mass ratio is high. Further, as shown in Example 6 described later, even if the phosphorus concentration is 1 mg-P / L or more, if the N / P ratio is higher than 300, the algae concentration or the specific growth rate is affected. There is a certain preferred upper limit for the / P mass ratio. Therefore, it is desirable that the N / P mass ratio be adjusted within the above-mentioned constant range. Specifically, as described above, the N / P mass ratio is preferably 16.7 to 300. In addition, wastewater 1 (for example, activated sludge treated water) having a very high ammonia nitrogen concentration is put into the algae culture tank 3. Therefore, since the concentration of ammonia nitrogen in the algae culture tank 3 is very high, the N / P mass ratio may be adjusted by charging a phosphorus source (for example, phosphoric acid) into the algae culture tank 3. In addition, there are cases where it is desired to increase the N / P mass ratio, for example, when a phosphorus source is added too much. In this case, an ammonia source (for example, a solid or solution of ammonium chloride, ammonium sulfate, etc.) may be charged into the algae culture tank 3.

The shape of the algae culture tank 3 itself is not particularly limited. For example, the algae culture tank 3 is preferably, but is not limited to, a rectangular parallelepiped type or a cylindrical one designed in a sewage treatment plant or a factory wastewater treatment plant. Further, as the number of microalgae in the algae culture tank 3 increases, it becomes difficult for dissolved inorganic carbon to spread throughout the microalgae. Therefore, the algae culture tank 3 may be provided with a stirring device. Further, even if the algae culture tank 3 has a large capacity, the microalgae 2 can be cultured if water, nitrogen, light, phosphorus, and a dissolved inorganic carbon source are appropriately supplied to the microalgae 2. However, it is considered that it is difficult for light to reach the bottom of the algae culture tank 3. In that case, among the open pond systems described in Non-Patent Document 9, the raceway type culture pond provided with the loop circulation type water channel having a water depth of about 0.3 m is used as the algae culture tank 3 to generate sunlight. Can reach the bottom sufficiently.

The light source 4 supplies light to the microalgae in the algae culture tank 3. Here, it is desirable that the wavelength of the light emitted from the light source 4 includes the wavelength required for photosynthesis by microalgae. As the light source 4, for example, an incandescent lamp, a fluorescent lamp, an LED (light emitting diode), or the like is desirable, but the light source 4 is not limited thereto. Instead of irradiating the light from the light source 4, the fine algae 2 may be irradiated with sunlight. In this case, the light source 4 does not necessarily have to be provided in the culture apparatus.

As shown in Example 2 described later, the present inventor found that the photon density of light was 60 μmol / m ² / sec or more and the algae increased significantly. Therefore, the photon density of light is preferably 60 μmol / m ² / sec or more. Further, although the upper limit of the photon density is not particularly specified, Non-Patent Document 12 describes that the daytime peak of sunlight is 2,000 μmol / m ² / sec, and Patent Document 7 is exposed to excessive sunlight. Since it is described that the site is stagnant due to strong light inhibition, it can be said that about 2,000 μmol / m ² / sec is an upper limit that does not cause forced inhibition. However, this photon density is the latest in algae. The photon density can be measured using, for example, a photon meter MQ-200 manufactured by Fujiwara Seisakusho Co., Ltd.

After the microalgae 2 are cultivated in the algae culture tank 3, the waste water 1 is charged into the solid-liquid separation layer 5 together with the microalgae 2. In the solid-liquid separation layer 5, the mixture of wastewater 1 and microalgae 2 is separated into wastewater 1 and microalgae 2. The wastewater 1 after separation is discharged to the outside of the apparatus as the treatment completed water 6. The solid-liquid separation layer 5 is provided in the culture device when the culture device performs continuous treatment (treatment for continuously culturing the microalgae 2). Therefore, when the culture device performs the badge treatment, the solid-liquid separation layer 5 does not have to be provided in the culture device.

<2. Microalgae culture method>
Next, a method for culturing microalgae using the culturing apparatus shown in FIG. 1 will be described. First, the drainage 1 and the microalgae 2 are put into the algae culture tank 3. Then, the microalgae 2 are irradiated with light from the light source 4. As a result, the microalgae 2 are cultured in the algae culture tank 3. The amount of light from the light source 4 may be appropriately adjusted according to the growth condition of the microalgae 2. Then, the waste water 1 is charged into the solid-liquid separation layer 5 together with the fine algae 2. In the solid-liquid separation layer 5, the mixture of wastewater 1 and microalgae 2 is separated into wastewater 1 and microalgae 2. The wastewater 1 after separation is discharged to the outside of the apparatus as the treatment completed water 6.

As described above, according to the present embodiment, the microalgae 2 can be cultured using the waste water 1 containing ammonia nitrogen at 100 mg-N / L or more.

(Example 1: Propagation of nannochloropsis using activated sludge treated water)
Next, examples of the present invention will be described. In Example 1, microalgae of the genus Nannochloropsis (Marine Fresh of Marine Tech Co., Ltd.) were cultivated as microalgae 2. Specifically, after adding microalgae 2 to the culture tube container so as to have a concentration of about 1.0 × 10 ⁴ cell / mL, 10 mL of activated sludge-treated water was placed in the culture tube container. Here, the concentration (cell / mL) of the microalgae 2 was measured by directly counting the diluted culture solution with a biological microscope (BX43 manufactured by Olympus Corporation) using a hemocytometer. Moreover, when the ammonia nitrogen concentration in the activated sludge treated water was measured by the indophenol blue absorptiometry, the ammonia nitrogen concentration was 188 mg-N / L. Moreover, when the phosphorus concentration in the activated sludge-treated water was measured by the molybdenum blue absorptiometry, the phosphorus concentration was 0.18 mg / L. Then, the microalgae 2 was cultured with the photon density in the culture tube set to 60 μmol / m ² / sec, the light irradiation cycle set to 12 hours light period / 12 hours dark period, and the water temperature set to 30 ° C. These culture conditions were constant during the culture. The photon density was measured using a photon meter MQ-200 manufactured by Fujiwara Seisakusho. As a result, the algae concentration increased from 1.0 × 10 ⁴ cell / mL to 7.0 × 10 ⁴ cell / mL within 2 weeks from the start of the culture. The measurement results are shown in FIG. From this, it was found that Nannochloropsis can be cultured in activated sludge-treated water.

(Example 2: Propagation of microalgae living in activated sludge treated water using activated sludge treated water)
In Example 2, Chlorococcales were cultured as microalgae 2. Specifically, after adding microalgae 2 to the culture tube container so as to have a concentration of about 2.2 × 10 ⁴ cells / mL, 10 mL of activated sludge-treated water was placed in the culture tube container. The ammonia nitrogen concentration of the activated sludge treated water was 188 mg-N / L, and the phosphorus concentration was 0.18 mg / L. Then, the culture was carried out with the photon density in the culture tube set to 60 μmol / m ² / sec, the light irradiation cycle set to 12 hours light period / 12 hours dark period, and the water temperature set to 30 ° C. These culture conditions were constant during the culture. As a result, the algae concentration increased from 2.2 × 10 ⁴ cell / mL to about 4.4 × 10 ⁴ cell / mL within 2 weeks from the start of the culture. The measurement results are shown in FIG. From this, it was found that Chlorococcales can be cultured in activated sludge-treated water.

(Example 3: Examination of photon density in growth of nannochloropsis using activated sludge treated water)
In Example 3, the same test as in Example 1 was carried out except that only the photon density in the culture tube container was changed stepwise. That is, in Example 3, the effect of photon density on the growth of nannochloropsis was examined. The photon densities were 42, 52, 60, 74 and 85 μmol / m ² / sec, and the cells were cultured for 2 weeks under the conditions of each photon density. As a result, the algae concentration increased from 2.5 × 10 ⁴ cell / mL to a maximum of 4.8 × 10 ⁵ cell / mL, and the algae increased according to the photon density. The measurement results are shown in FIG. In addition, algae increased significantly between the photon densities of 52 μmol / m ² / sec and 60 μmol / m ² / sec. From this, it was found that the photon density for culturing Nannochloropsis is preferably 60 μmol / m ² / sec or more.

(Example 4: Examination of addition of phosphoric acid in the growth of nannochloropsis and Chlorococcales using activated sludge treated water)
Next, the correlation between the culture amount of nannochloropsis and Chlorococcales and the phosphorus concentration was examined. Specifically, Nannochloropsis and Chlorococcales were placed in separate culture tube containers. The input amount of nannochloropsis was about 1.0 × 10 ⁴ cells / mL, and the input amount of Chlorococcales was about 2.1 × 10 ⁴ cells / mL. Next, 10 mL of activated sludge-treated water was placed in each culture tube container. The ammonia nitrogen concentration of the activated sludge treated water was 188 mg-N / L, and the phosphorus concentration was 0.18 mg / L. Further, 1 mg-P / L equivalent of phosphoric acid was added to each culture tube container. Then, the culture was carried out for 2 weeks with the photon density in the culture tube being 60 μmol / m ² / sec, the light irradiation cycle being 12 hours light period / 12 hours dark period, and the water temperature being 30 ° C. These culture conditions were constant during the culture. Further, the same test was conducted by changing the addition amount of phosphoric acid to 0 (without addition), 2, 5, and 10 mg-P / L. The measurement result for nannochloropsis is shown in FIG. 5, and the measurement result for Chlorococcales is shown in FIG.

As a result, under the culture conditions where the phosphoric acid concentration was 1 mg-P / L or more, the nannochloropsis concentration 2 weeks after the start of the culture was 1.0 × 10 ⁴ cell / mL regardless of the amount of phosphoric acid added. It increased to 3.4 × 10 ⁵ cell / mL or more. In addition, the Chlorococcales concentration increased from 2.1 × 10 ⁴ cell / mL to about 2.4 × 10 ⁵ cell / mL. Even when the amount of phosphoric acid added is zero, microalgae are cultivated. In this case, it is presumed that the microalgae were cultivated by phosphorus contained in the activated sludge-treated water. From these facts, it was found that the phosphorus concentration in the culture medium (corresponding to the waste water 1 in the embodiment) for culturing Nannochloropsis and Chlorococcales is preferably 1 mg-P / L or more.

(Example 5: Propagation of Chlorella sp. Using activated sludge treated water)
In Example 5, microalgae living in the activated sludge-treated water were separated as microalgae 2, and the separated microalgae were subcultured a plurality of times using the activated sludge-treated water as a medium. As a result, Chlorella sp. Was made dominant. Specifically, several mL of microalgae living in activated sludge-treated water were collected. Then, 800 ml of activated sludge-treated water as a medium and the separated microalgae were put into a 1 L culture container. Then, the microalgae 2 was cultured under the culture conditions of 800 mL of activated sludge-treated water, a photon density of 60 μmol / m ² / sec, a light irradiation cycle of 12 hours in the light period / 12 hours in the dark period, and a water temperature of 30 ° C. The culture of microalgae 2 was carried out until the concentration of microalgae 2 became a sufficient concentration of algae. Furthermore, several mL of microalgae were taken from the culture vessel and cultured under the same culture conditions. Such a culture treatment was carried out a plurality of times, and subculture was performed. Subcultured microalgae 2, ie Chlorella sp. Was put into a 1 L culture vessel. Then, the activated sludge-treated water was put into the culture vessel, and Chlorella sp. The concentration of was about 1.0 × 10 ⁴ cell / mL. The ammonia nitrogen concentration of the activated sludge treated water was 188 mg-N / L, and the phosphorus concentration was 0.18 mg-P / L. Further, 1 mg-P / L equivalent of phosphoric acid was added to the culture vessel. Then, under the culture conditions that the photon density in the culture vessel was 60 μmol / m ² / sec, the light irradiation cycle was 12 hours light period / 12 hours dark period, and the water temperature was 30 ° C., Chlorella sp. Was cultured for 2 weeks. These culture conditions were constant during the culture. As a result, the algae concentration increased from 1.0 × 10 ⁴ cell / mL to about 4.3 × 10 ⁶ cell / mL within 2 weeks from the start of the culture. From this, the dominant Chlorella sp. It was found that high-purity algae containing no diatoms, filamentous fungi, protozoa, etc. can be obtained and contamination can be eliminated by culturing in activated sludge-treated water. That is, a higher growth rate and growth rate were obtained.

(Example 6: N / P mass ratio examination in the growth of nannochloropsis and Chlorella sp. Using activated sludge treated water)
In Example 6, as microalgae 2, Nannochloropsis and Chlorella sp. Was placed in separate 1 L culture containers. Chlorella sp. Was obtained by the same method as in Example 5. The input amount of nannochloropsis was about 1.0 × 10 ⁴ cell / mL, Chlorella sp. The input amount of was about 2.1 × 10 ⁴ cell / mL. Next, activated sludge-treated water was placed in each culture vessel, and the ammonia nitrogen concentration was 100 to 1000 mg-N / L, the phosphorus concentration was 1 to 6 mg-P / L, that is, the N / P mass ratio was 16.7 to 1000. Adjusted to. Here, the concentration of ammonia nitrogen was adjusted with an ammonium chloride solution. The phosphorus concentration was adjusted by adjusting the amount of phosphoric acid charged into the culture vessel.

Then, the microalgae 2 was cultured for 2 weeks at a photon density of 60 μmol / m ² / sec in each culture vessel, a light irradiation cycle of 12 hours in a light period / 12 hours in a dark period, and a water temperature of 30 ° C. These culture conditions were constant during the culture. Two weeks after the start of culturing, the algae concentration and specific growth rate were compared. The result is shown in FIG. As shown in FIG. 7A, the concentration of nannochloropsis increased from 1.0 × 10 ⁴ cell / mL to a maximum of about 1.2 × 10 ⁶ cell / mL. On the other hand, as shown in FIG. 7 (b), Chlorella sp. The concentration of was increased from 2.1 × 10 ⁴ cell / mL to a maximum of about 7.3 × 10 ⁶ cell / mL. Looking at the correspondence between the N / P mass ratio and the algae concentration and specific growth rate, when the N / P mass ratio is 16.7 to 300, the average concentration of nannochloropsis is 9 × 10 (arithmetic mean). ^The specific growth rate was about ⁵ cell / mL, and the average (arithmetic mean) was about 0.5 (1 / d). On the other hand, when the N / P mass ratio is 16.7 to 300, Chlorella sp. The concentration of is 3.3 × 10 ⁶ cell / mL to 7.3 × 10 ⁶ cell / mL, which is sufficient as a concentration, and the specific growth rate is about 1.0 (1 / d) on average. Met. On the other hand, under the condition that the N / P mass ratio was 500 to 1,000, the algae concentration and the specific growth rate tended to decrease in all the algae. From this, it was found that the N / P mass ratio at which a good algae concentration and / or specific growth rate was obtained was 16.7 to 300.

Although the preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, the present invention is not limited to such examples. It is clear that a person having ordinary knowledge in the field of technology to which the present invention belongs can come up with various modifications or modifications within the scope of the technical ideas described in the claims. , These are also naturally understood to belong to the technical scope of the present invention.

1 Drainage 2 Fine algae 3 Algae culture tank 4 Light source 5 Solid-liquid separation tank 6 Treatment completed water

Claims (5)

Chlorella sp. Is a microalga that is separated from activated sludge-treated water obtained by treating cheap water containing wastewater discharged in the coke manufacturing process with activated sludge and grown using the activated sludge-treated water as a medium . A method for culturing microalgae, which comprises putting the activated sludge-treated water containing 100 mg-N / L or more of ammonia nitrogen into an algae culture tank and irradiating the microalgae with light. The method for culturing microalgae according to claim 1 , wherein the N / P mass ratio of the algae culture tank is 16 to 300. The method for culturing microalgae according to claim 1 or 2 , wherein a dissolved inorganic carbon source is added to the algae culture tank. The method for culturing microalgae according to any one of claims 1 to 3 , wherein the photon density of the light is 60 μmol / m ² / sec or more. The method for culturing microalgae according to any one of claims 1 to 4 , wherein a phosphate is added to the algae culture tank.

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