Method for increasing yield of cultured algae in cultivation under strong light conditions, and method for culturing algae

Abstract

The present disclosure provides a method for increasing the yield of cultured algae in cultivation under strong light conditions, the method comprising: a step for preparing an algae culture medium containing an excess amount of iron; and a step for culturing algae under strong light conditions by using the algae culture medium.

Description

Method for increasing the yield of cultured algae in cultures involving high light conditions and method for culturing algae

[0001] The present disclosure relates to methods for increasing the yield of cultured algae and methods for culturing algae in cultures that include high light conditions.

[0002] Algae are widely used in various industrial fields, including aquaculture feed, energy sources, and dietary supplements, and are an important industrial resource. Light energy for photosynthesis is essential for algae growth. However, excessively strong light energy (strong light) can damage photosynthesis-related proteins, inhibiting algae growth (Non-Patent Document 1). This phenomenon, known as photoinhibition, is a major problem when culturing algae under strong light, including natural light. Therefore, there is a need for technologies that can overcome photoinhibition and enable algae to be cultivated under strong light.

[0003] One known method for culturing algae under strong light is to obtain and cultivate light-resistant algae by genetically modifying them to improve their ability to repair damaged proteins (Non-Patent Document 1). At the industrial level, examples include culturing using a shading net to reduce the amount of light (Non-Patent Document 2), and electrochemical cultivation using a medium containing a cell membrane-permeable electron mediator to reduce growth inhibition.

[0004] Genetic modification requires analysis to identify the target gene and genetic manipulation for the modification, and it takes time to obtain algae with the desired traits. Furthermore, installing a shade net can block excessive light during times of low light, such as in the morning and evening, which can have a negative impact on growth during those times. Electrochemical cultivation often requires a dedicated culture tank equipped with electrodes, which requires capital investment. Therefore, there is a need for a technology that can easily cultivate algae under strong light.

[0005] Japanese Patent Application Laid-Open No. 2016-182063

[0006] Saitama University website, https: / / www.saitama-u.ac.jp / topics_archives / 2019-0927-1631-9.html Saga Algae Biomass Council website, https: / / www.saga-abc.jp / main / 375.html

[0007] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for increasing the yield of cultured algae in cultures including high light conditions, and a method for culturing algae.

[0008] One aspect of the present disclosure is a method for increasing the yield of cultured algae in cultures involving high light conditions, the method comprising the steps of preparing an algal culture medium containing an excess amount of iron, and culturing algae under high light conditions using the algal culture medium.

[0009] Another aspect of the present disclosure is a method for culturing algae, the method comprising culturing algae under high light conditions using a medium containing an excess amount of iron.

[0010] According to the present disclosure, a method for increasing the yield of cultured algae in cultures involving high light conditions and a method for culturing algae can be provided.

[0011] The graph shows the change over time in cell density of algae cultured in the iron fertilization area (white circles) and the control area (black circles). The iron fertilization area received another iron fertilization on the third day.

[0012] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0013] <Method for increasing the yield of cultured algae in cultures involving high light conditions>

[0014] In one aspect, a method for increasing the yield of cultured algae in a culture that includes high-light conditions is provided, the method comprising the steps of preparing an algal culture medium containing an excess amount of iron and culturing algae in the algal culture medium under high-light conditions. By using such a method, it is possible to reduce the growth inhibition of algae caused by high-light conditions and increase the yield of cultured algae in a culture that includes high-light conditions without using genetic modification.

[0015] The present disclosure does not limit the mechanism by which an algal medium containing excess iron increases the yield of cultured algae in cultures under high light conditions. However, because some photosynthesis-related proteins (e.g., D1 protein) that are damaged by high light contain iron in their reaction centers, supplying iron as a component may promote the regeneration of damaged photosynthesis-related proteins.

[0016] The disclosed method includes preparing an algae culture medium containing an excess amount of iron. In this disclosure, "containing an excess amount of iron" can mean containing a concentration of iron that exceeds the concentration necessary and sufficient for algae growth. Here, "a concentration necessary and sufficient for algae growth" refers to a concentration that exceeds the concentration necessary and sufficient for algae growth under corresponding non-high light conditions (e.g., 50 μmol photons / m ２ / s), and the lowest iron concentration c above which an increase in the iron concentration does not result in an increase in the algae growth rate. Thus, "containing an excess amount of iron" can mean containing an iron concentration greater than c in a given medium. The excess iron concentration can be, for example, 2-fold or more or 4-fold or more, e.g., 2-10-fold or 4-7-fold the iron concentration c.

[0017] The iron in the present disclosure is preferably dissolved in the medium in the form of iron ions, and such iron ions are divalent (Fe 2+ or Fe(II)) or trivalent (Fe 3+ The iron ion may be an iron ion of the formula (Fe(III) or Fe(III)). In an embodiment, the iron ion may be chelated with a chelating agent such as EDTA. An example of a chelated iron ion is Fe(III)-EDTA.

[0018] In embodiments, the concentration of excess iron may be, for example, 20 μmol / L or more, preferably 30 μmol / L or more, more preferably 40 μmol / L, and even more preferably 50 μmol / L or more, and may be, for example, 300 μmol / L or less, preferably 200 μmol / L or less, more preferably 160 μmol / L or less, and even more preferably 140 μmol / L or less (as iron ions). In embodiments, the concentration of excess iron may be 20 to 300 μmol / L, preferably 30 to 200 μmol / L, more preferably 40 to 160 μmol / L, and even more preferably 50 to 140 μmol / L. These iron concentrations may be the total iron concentration, including the iron originally contained in the algal culture medium or other solution used for algae cultivation, as described below, plus any additional iron added. The form of the added iron is not limited, but is preferably in the form of a metal salt or chelated iron, and more preferably in the form of Fe(III)-EDTA. The timing of adding iron is not limited, but may be one or more time points selected from the first day of culture or one, two, three, or four or more days after the start of culture.

[0019] The type of algae in the present disclosure is not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae that may be used include Aurantiochytrium, Chlamydomonas, Chlorella, Schizon algae, Spirulina, Botryococcus, Euglena, Haptophyte, Prasinophyte, Chlorophyte, Phaeophyte, Rhodophyte, Cyanobacteria, Diatoms, Xanthophyte, Chrysophyte, Dinoflagellates, and Seaweed. More specific examples of algae include algae of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis. Among these, algae of the Chaetoceros genus, such as Chaetoceros gracilis, are preferred. The algae in the present disclosure may be a single species of algae or a population containing multiple species of algae. A genetically homogeneous population of algae (pure lines) of the same species may be used, or a population of algae having genetic variation may be used. The algae of the present disclosure may be derived from an isolated alga.

[0020] The cultured algae in the present disclosure may be cultured in a closed culture facility, such as a bioreactor, flask, culture tank, or other closed culture vessel, or may be cultured in an open culture facility, such as an open pond, but even in this case, it is preferable that the contents of the culture facility, such as the medium, are not freely exchanged with environmental water and are limited.

[0021] Algae can be cultured using algae culture media known to those skilled in the art, seawater, diluted seawater, artificial seawater, mixtures of these, or solutions with a partial composition similar to these. The algae culture media can be any aqueous liquid capable of growing natural algae, or an aqueous algae culture medium with a defined composition can be used. The medium can be any medium for algae culture known to those skilled in the art, including, for example, a medium containing nutrients, a carbon source, rare metals, etc. Specific examples of the aqueous medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified versions of these media, and mixtures of these media or modified media. IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L Thiamin-HCl, 0.0015mg / L Biotin, 0.0015mg / L Vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2 to 1 mM Na2SiO3 may be added to the IMK medium in addition to the above components.mSWM-3 medium contained 17 mg NaNO3, 1.56 mg NaH2PO4・2H2O, 5.68 mg Na2SiO3・9H2O, 1.12 mg Na2EDTA・2H2O, 0.084 mg Fe-EDTA, 0.0346 μg Na2SeO3, 1 ml P-1 metal solution (618.3 mg H3BO4, 69.25 mg MnCl2・4H2O, 5.45 mg ZnCl2, 238 μg CoCl2・6H2O, 100 ml distilled water), 0.2 μg Vitamin B12, 1 mL Vitamin mixed solution S3 (5 mg Thiamine HCl, 1 mg Nicotinic acid, 1 mg Calcium pantothenate, 0.1 mg p-Aminobenzoic acid, 0.01 mg Biotin, 50 mg Inositol, 0.02 mg Folic acid, 30 mg Thymine, 100 mL The medium (pH 7.7-7.8) is prepared by mixing 50 mg Tris (hydroxymethyl) aminomethane and 98 mL seawater.

[0022] The algae of the present disclosure may be cultured under natural light and / or artificial light. When cultured under natural light, a shading sheet may be used to block light. Shading with such a shading sheet may be used only in the early stages of culture, or at other times. Algae may be cultured indoors or outdoors. Algae may be cultured in an atmosphere of 0.5% or more, 1% or more, or 2% or more CO . ２ This may involve ventilation.

[0023] The method of the present disclosure includes culturing algae under high light conditions using the prepared algal culture medium containing excess iron. In the present disclosure, "high light conditions" may refer to conditions including a light intensity at which light saturation occurs and at which increasing the light intensity above that intensity does not increase photosynthesis.

[0024] In this embodiment, the high light condition is 250 μmol photons / m 2 / s or more, 500 μmol photons / m 2 / s or more, or 1000 μmol photons / m 2 / s or more, and may be 3000 μmol photons / m 2 / s or less, 2500μmol photon / m 2 / s or less, or 2000 μmol photons / m 2 The high light condition of the embodiment may be 250 to 3000 μmol photons / m ２ / s, and may include a light amount of 500 to 2500 μmol photons / m ２ / s, and may include a light amount of 1000 to 2000 μmol photons / m ２ The light intensity may include a light intensity of 1 / s. These light intensities may be the maximum light intensity or the average light intensity during the culture step. Furthermore, these light intensities may be the light intensity temporarily irradiated during the culture step or the light intensity constantly irradiated.

[0025] <Method for cultivating algae>

[0026] In another embodiment of the present disclosure, there is provided a method for culturing algae, the method comprising culturing algae under high light conditions using a medium containing an excess amount of iron. The explanations given in the section <Method for increasing the yield of cultured algae in a culture including high light conditions> can be similarly applied to the various elements of this embodiment (including the excess amount of iron, the medium, the high light conditions, and the culture of algae).

[0027] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below. In particular, similar effects can be observed when using algae species and iron ion sources other than those exemplified below.

[0028] An outdoor aquarium was filled with 160 L of tap water and supplemented with 4.8 kg of artificial seawater Marine Standard, 40.32 g of Daigo IMK medium (manufactured by Shioya MS Co., Ltd.), and 9.09 g of sodium metasilicate nonahydrate. Daigo IMK medium is a commercially available medium designed to reliably and efficiently cultivate as many microalgae as possible. The manufacturer instructed the aquarium to dissolve 25.2 g of Fe-EDTA in 100 L of seawater, providing 5.2 mg / L (approximately 0.8 g / 160 L) of Fe-EDTA. Six such aquariums were prepared, with three serving as iron fertilizer test tanks, each supplemented with 3.2 g of Fe(III)-EDTA. The remaining three served as controls, containing Fe(III)-EDTA-free medium other than the Fe(III)-EDTA component of Daigo IMK medium. The media used in the control group is one commonly used by those skilled in the art for culturing certain species of algae, including Chaetoceros gracilis.

[0029] Plant 5 x 10 Chaetoceros gracilis in each tank. 5 Algae were added to the iron fertilizer group so that the concentration of cells / mL was reached, and cultivation was initiated under natural light. At the start of cultivation, a sheet with a shading rate of approximately 85% was placed over the entire tank, which was then removed after three hours. The algae cell density in the culture solution was measured daily. On the third day, an additional 4.0 g of Fe(III)-EDTA was added to the iron fertilizer group.

[0030] The maximum light intensity of natural light irradiated during cultivation was 1700 μmol photons / m 2 While almost no algae growth was observed in the control area, the algae growth rate in the iron fertilization test area was higher than that of the control area from the first day, reaching a maximum growth rate of 7.5 times that of the control area (Figure 1). These results confirm the effect of iron fertilization on promoting algae growth under strong light.

[0031] The above examples demonstrate that an algal medium containing excess iron increases the yield of cultivated algae in cultures containing high light conditions.

[0032] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various changes can be made to the configuration and details of the present invention within the scope of the present disclosure.

[0033] The present disclosure includes the following embodiments: (Item 1) A method for increasing the yield of cultured algae in a culture including high light conditions, the method comprising: preparing an algal culture medium containing an excess amount of iron; and culturing algae under high light conditions using the algal culture medium. (Item 2) The method according to item 1, wherein the concentration of the excess amount of iron is 20 to 300 μmol / L. (Item 3) The high light conditions are 250 to 3000 μmol photons / m ２ Item 4. A method for culturing algae, comprising culturing algae under strong light conditions using a medium containing an excess amount of iron.

Claims

1. A method for increasing the yield of cultured algae in a culture involving high light conditions, comprising: preparing an algal culture medium containing an excess amount of iron; and culturing algae under high light conditions using the algal culture medium.

2. The method of claim 1, wherein the concentration of the excess iron is 20 to 300 μmol / L.

3. The above strong light condition is 250 to 3000 μmol photons / m ２ 3. The method according to claim 1 or 2, comprising a light amount of 1 / s.

4. A method for culturing algae, comprising culturing algae under strong light conditions using a medium containing an excess amount of iron.

Images