Method for selecting algae having nitrogen fixation ability

Abstract

To provide a method for selecting algae having nitrogen fixation ability.SOLUTION: Provided is a method for selecting algae having nitrogen fixation ability, the method comprising a step of preferentially proliferating algae having nitrogen fixation ability by culturing a plurality of kinds of algae in a medium containing dissolved inorganic nitrogen at 0.9 to 9.0 mmol / L.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for selecting algae capable of fixing nitrogen. [Background technology]

[0002] Many species of microalgae have the ability to fix nitrogen, and these algae are attracting attention as a new biological fertilizer (Non-Patent Document 1). A common method for selecting algae with nitrogen fixation is to culture them in a medium from which the nitrogen components have been removed, and then select species that can grow in such a medium (Non-Patent Document 2).

[0003] When the nitrogen source in the culture medium is removed, the algae obtain nitrogen through nitrogen fixation, slowing down their growth rate. Therefore, selection using a nitrogen-free culture medium generally takes several weeks to a month or more. Such long-term cultivation can lead to contamination with mold and other microorganisms, making efficient selection of algae difficult. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Ammar, Esraa E. et al. “Algae as Bio-fertilizers: Between current situation and future prospective.” Saudi journal of biological sciences vol. 29(5) (2022): 3083-3096. doi:10.1016 / j.sjbs.2022.03.020 [Non-patent document 2] Zarezadeh, S. et al. Effects of cyanobacterial suspensions as bio-fertilizers on growth factors and the essential oil composition of chamomile, Matricaria chamomilla L.. J Appl Phycol 32, 1231-1241 (2020). https: / / doi.org / 10.1007 / s10811-019-02028-9 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for selecting algae having nitrogen fixation ability. [Means for solving the problem]

[0006] One aspect of the present disclosure is a method for selecting algae capable of fixing nitrogen, the method comprising a step of culturing multiple types of algae in a medium having a dissolved inorganic nitrogen concentration of 0.9 to 9.0 mmol / L, thereby preferentially growing algae capable of fixing nitrogen. [Effects of the Invention]

[0007] According to the present disclosure, a method for selecting algae having nitrogen fixation ability can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] The appearance of BG11-1 / 10N agar medium and BG11-N agar medium (control) on day 7 after inoculation with enrichment cultures was shown. Enrichment cultures prepared from soil sediment isolates collected at 20 locations were used. The numbers in the upper left corner of each plate pair (1–20) indicate the identification numbers of the soil sediment isolates. DETAILED DESCRIPTION OF THE INVENTION

[0009] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0010] In one aspect, a method for selecting algae capable of fixing nitrogen is provided, which includes a step of preferentially growing algae capable of fixing nitrogen by culturing multiple types of algae in a medium having a dissolved inorganic nitrogen concentration of 0.9 to 9.0 mmol / L.

[0011] In this disclosure, algae capable of fixing nitrogen refer to algae that have the ability to convert molecular nitrogen (N) into forms that are readily available to living organisms, such as ammonia, nitrate, nitrite, etc. As will be understood by those skilled in the art, algae capable of fixing nitrogen can grow independently of nitrogen compounds other than molecular nitrogen in the environment.

[0012] The selection method of the present disclosure includes a step of culturing multiple types of algae in a medium with a dissolved inorganic nitrogen concentration of 0.9 to 9.0 mmol / L, thereby preferentially growing algae with nitrogen fixation ability. It has been found that a dissolved inorganic nitrogen concentration within this range limits the growth of algae without nitrogen fixation ability because the concentration of efficiently available nitrogen is sufficiently low, while moderately increasing the growth efficiency of algae with nitrogen fixation ability. The culture for selection can be carried out for, for example, 3 days to 2 weeks.

[0013] The multiple types of algae disclosed herein may be multiple types of algae contained in a sample collected from a field. The sample collected from a field may be a sample containing soil and / or environmental water. Multiple types of algae can be obtained, for example, by adding sterile water to soil obtained from a field to suspend it, isolating the organisms dispersed in the supernatant, and seeding them in an algae culture medium for enrichment culture. The field in this disclosure is not limited to any natural environment in which algae can live, and may include terrestrial, marine, or freshwater fields.

[0014] 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 include Aurantiochytrium, Chlamydomonas, Chlorella, Schizon algae, Spirulina, Botryococcus, Euglena, Haptophyte, Prasinophyte, Chlorophyte, Brown algae, Red algae, Cyanobacteria, Diatoms, Xanthophyte algae, Chrysophyte algae, Dinoflagellates, and Seaweed. The multiple types of algae in the present disclosure may be a population containing multiple species of algae, or a population of algae with genetic variation within the same species.

[0015] As will be understood by those skilled in the art, dissolved inorganic nitrogen in the present disclosure can refer to nitrogen in a form that can be readily utilized by plants as a nitrogen source, and includes dissolved ammonia nitrogen (NH4 + ), nitrite nitrogen (NO2 - ) and nitrate nitrogen (NO3 - Therefore, when the dissolved inorganic nitrogen is in a specific concentration range (for example, 0.9 to 9.0 mmol / L), it is possible to combine the nitrogen contained in ammonia nitrogen (NH4 + ), nitrite nitrogen (NO2 - ), nitrate nitrogen (NO3 - ) may mean that the sum of the molar concentrations of each falls within the concentration range.

[0016] In an embodiment, the concentration of dissolved inorganic nitrogen in the medium may be 0.5 mmol / L or more or 0.9 mol / L or more, preferably 1.0 mmol / L or more, and more preferably 1.5 mmol / L or more. In an embodiment, the concentration of dissolved inorganic nitrogen may be 9 mmol / L or less or 5 mmol / L or less, preferably 3.0 mmol / L or less, and more preferably 2.0 mmol / L or less. In an embodiment, the concentration of inorganic nitrogen may be 0.5 to 5 mmol / L or 0.9 to 9 mmol / L, preferably 1.0 to 3.0 mmol / L, and more preferably 1.5 to 2.0 mmol / L.

[0017] In the embodiment, the dissolved inorganic nitrogen is nitrate ions (NO3- ) in the embodiment. - The concentration of nitrate ions (NO3) in the embodiment may be 0.5 mmol / L or more, or 0.9 mol / L or more, preferably 1.0 mmol / L or more, and more preferably 1.5 mmol / L or more. - The concentration of nitrate ions (NO3) in the embodiment may be 9 mmol / L or less, or 5 mmol / L or less, preferably 3.0 mmol / L or less, and more preferably 2.0 mmol / L or less. - The concentration of the nitrogen-fixing algae in the culture medium may be 0.5 to 5 mmol / L, preferably 1.0 to 3.0 mmol / L, and more preferably 1.5 to 2.0 mmol / L. Even if the culture medium itself contains organic nitrogen (e.g., free amino acids) before the addition of the algal sample, this is not expected to have a short-term effect on the differential growth rates between nitrogen-fixing algae and non-nitrogen-fixing algae. However, the organic nitrogen compound content of the culture medium may typically be 1% by weight or less, for example, 0 to 1% by weight, 0 to 0.1% by weight, or 0 to 0.1% by weight.

[0018] The medium of the present disclosure is not limited as long as it can be used for culturing algae. The medium of the embodiment is preferably a solid medium. Such a solid medium can be prepared by a method known to those skilled in the art, including adding agar to a liquid medium and solidifying it. An example of a medium is a medium in which the dissolved inorganic nitrogen concentration of BG11 medium has been adjusted. Table 1 shows the composition of BG11-N agar medium, which does not contain a nitrogen source compared to standard BG11 agar medium known to those skilled in the art. Table 2 shows the composition of BG11-1 / 10N medium, which is used in the selection method of the embodiment and has a NaNO concentration of 150 mg / L compared to standard BG11 agar medium. Table 3 also shows the composition of BG11 medium with a standard NaNO concentration. As can be seen from Tables 2 and 3, BG11-1 / 10N agar medium has a dissolved inorganic nitrogen concentration approximately one-tenth that of BG11 medium.

[0019] [Table 1]

[0020] [Table 2]

[0021] [Table 3]

[0022] In the selection method of the present disclosure, multiple types of algae are cultured in a medium containing dissolved inorganic nitrogen at a concentration as described above, for example, 0.5 to 5 mmol / L, 0.9 to 9 mmol / L, 1.0 to 3.0 mmol / L, 1.5 to 2.0 mmol / L, or 0.9 to 9.0 mmol / L. This culture allows algae with nitrogen fixation to grow preferentially compared to a medium containing a dissolved inorganic nitrogen concentration outside the above range. More specifically, using a medium with a dissolved inorganic nitrogen concentration below the upper limit of the above range can slow the growth rate of algae without nitrogen fixation. Furthermore, using a medium with a dissolved inorganic nitrogen concentration above the lower limit of the above range can accelerate the onset of initial growth of algae with nitrogen fixation. In this case, it may be possible to confirm colony formation of algae with nitrogen fixation within about one week or less. This makes the selection method less susceptible to interference from mold and other microbial contamination. In this disclosure, "preferentially growing algae capable of fixing nitrogen" may mean growing algae capable of fixing nitrogen relatively faster by decreasing the growth rate of algae that do not have the ability to fix nitrogen compared to under other conditions and / or increasing the growth rate of algae that have the ability to fix nitrogen.

[0023] The selection method of the embodiment further includes a step of isolating the algae grown in the culturing step. Isolation of the algae can be performed by collecting algal cells from colonies formed on the solid medium using sterilization procedures known to those skilled in the art and seeding them on another medium. In the selection method of the embodiment, the isolated algae can be further selected by one or more additional steps of culturing them in a medium with a dissolved inorganic nitrogen concentration as described above, for example, 0.5-5 mmol / L, 0.9-9 mmol / L, 1.0-3.0 mmol / L, 1.5-2.0 mmol / L, or 0.9-9.0 mmol / L, thereby preferentially growing algae with nitrogen fixation ability. The nitrogen fixation ability of the isolated algae can be confirmed, for example, by culturing them in a medium without added inorganic nitrogen or with a dissolved inorganic nitrogen concentration of less than 0.5 mmol / L, such as BG11-N, for a relatively long period of time, optionally in comparison with a non-nitrogen-fixing control algae.

[0024] In the selection method of the embodiment, multiple types of algae can be contained in the same mixture and cultured together in contact with a medium. In a selection method including such a culture format, a solid medium contained in a petri dish can be used. The mixture of the embodiment can be a mixture containing a population of algae collected from the field. An example of a mixture collected from the field is an enrichment culture of algae obtained from soil. Alternatively, multiple types of algae can be isolated into single individuals or single cells and cultured in a solid or liquid medium contained in a microwell plate. As the microplate for such a culture format, 96-well plates and 384-well plates known to those skilled in the art can be used. [Example]

[0025] Examples of the present disclosure will be described below, but the present disclosure is not limited to the examples described below.

[0026] An appropriate amount of sterile water was added to 20 samples (identification numbers: 1-20) of soil sediment isolation sources from Kumejima, which were then stirred and suspended (with occasional stirring) and left to stand for several days. The organisms dispersed in the supernatant suspension were used for isolation. The sterile water suspension of the isolation source was decanted and filtered through a 300 μm metal filter to remove debris, and the filtrate was collected. An appropriate amount of the collected filtrate was inoculated onto previously prepared sterile "BG11 medium" and subjected to enrichment culture under weak light.

[0027] The precipitate collected from the enrichment cultured algae was plated on BG11-1 / 10N agar medium and BG11-N agar medium and spread using a spreader. The agar medium plated with the enrichment culture was cultured under the conditions shown in Table 4, and the culture was continued until algal colonies began to grow on the plate. Figure 1 shows the appearance of the BG11-1 / 10N agar medium and BG11-N agar medium plated with each sample (identification number: 1 to 20) on the 7th day of culture.

[0028] [Table 4]

[0029] In the culture using BG11-N agar medium, almost no algal colonies were observed on the seventh day of culture, whereas in the culture using BG11-1 / 10N agar medium, algal colonies were formed in multiple samples. When the algae contained in some of the colonies on BG11-1 / 10N agar medium were transferred to nitrogen-deficient medium (BG11-N) and cultured, growth was observed, confirming that algae with nitrogen-fixing ability can be isolated by selective culture using BG11-1 / 10N medium.

[0030] The above examples demonstrate that algae with nitrogen fixation ability can be efficiently selected by culturing algal samples in a medium to which dissolved inorganic nitrogen has been added at a concentration lower than that in normal BG11 medium but higher than that in BG11-N.

Claims

1. A method for selecting algae having nitrogen fixation ability, comprising: and a step of culturing multiple types of algae in a medium with a dissolved inorganic nitrogen concentration of 0.9 to 9.0 mmol / L, thereby preferentially growing algae having nitrogen fixation ability. method.

2. The method of claim 1 , further comprising the step of isolating the algae grown in the culturing step.

3. The method according to claim 1 or 2, wherein the plurality of types of algae are contained in the same mixture and are cultured together in contact with the medium.

4. The method of claim 3 , wherein the mixture is a mixture comprising a field-harvested algae population.

5. The dissolved inorganic nitrogen is nitrate ions (NO 3 - 3. The method of claim 1 or 2, comprising:

6. Nitrate ions (NO 3 - 6. The method according to claim 5, wherein the concentration of the soluble ...

7. The method according to claim 6, wherein the concentration of the dissolved inorganic nitrogen is 1.0 to 3.0 mmol / L.

8. The method of claim 1 or 2, wherein the medium is a solid medium.

Images