Methods for isolating algae

Abstract

This provides a method for isolating algae. [Solution] A method for isolating algae, comprising the step of culturing algae attached to a water intake filter that has filtered water containing algae.

Description

Technical Field

[0001] The present disclosure relates to a method for isolating algae.

Background Art

[0002] Microalgae are important as a direct or indirect feed for aquatic products, but the types of algae that are generally cultured and commercially available are limited. In order to achieve stable production of fish and shellfish, it is required that producers can easily obtain the desired microalgae and use them for production.

[0003] In the conventional isolation of phytoplankton, first, concentration or pre-culture is performed using a plankton net or a membrane filter according to the size or characteristics of the algae to be cultured. Thereafter, the algae that grow on the agar medium are isolated from the colonies, and the algae that do not grow on the agar medium are obtained one by one under a microscope. Then, a culture strain of single algae is obtained by culturing in a medium containing antibiotics or the like.

[0004] Since the isolation method as described above requires specialized knowledge such as handling of equipment, the number of operators who can obtain the target microalgae is limited. There is a need to develop a method that can be easily obtained by aquaculture operators who need microalgae feed.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] This disclosure is made to solve the problems described above and aims to provide a method for isolating algae. [Means for solving the problem]

[0007] One aspect of the present disclosure is a method for isolating algae, which includes the step of culturing algae attached to a water intake filter that has filtered water containing algae. [Effects of the Invention]

[0008] This disclosure provides a method for isolating algae. [Brief explanation of the drawing]

[0009] [Figure 1] The image shows the appearance of these cartridge filters, which have algae attached to them, after filtering raw water through a spool cartridge filter with a filter accuracy of 5 μm (left) and then filtering the filtrate through a spool cartridge filter with a filter accuracy of 1 μm (right). [Figure 2] This shows the filter fibers of a 1 μm precision filter immersed in culture medium in a flask. [Figure 3] Microscopic images of a small diatom isolated in the example (left) and a commercially available diatom (right) are shown. [Modes for carrying out the invention]

[0010] The following describes non-limiting embodiments of this disclosure. This disclosure is not limited to the embodiments described below.

[0011] The types of algae described herein are not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae may be any of the following classifications: Aurantiochytrium, Chlamydomonas, Chlorella, red algae Schizoan, Spirulina, Botryococcus, Euglena, haptophytes, Prasinophytes, green algae, brown algae, red algae, cyanobacteria, diatoms, yellow-green algae, golden algae, dinoflagellates, and seaweed. More specific examples of algae include those of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis.

[0012] In this disclosure, the water containing algae is not limited and may be environmental water such as seawater, freshwater, or brackish water. Filtration devices are installed in water intake facilities such as aquaculture farms to remove suspended solids and organisms from the environmental water. The filters in the filtration devices are usually discarded after filtration, but they often have a discoloration thought to be derived from microalgae. The inventors considered that this could be used as a source for a simple method of obtaining microalgae. In fact, by simply immersing a filter that has passed through natural seawater (typically used as an intake filter in aquaculture farm water intake facilities) in a seawater culture medium and cultivating it, it was possible to obtain algae of a different size than commercially available products that are suitable for aquaculture feed.

[0013] The intake filter in the embodiment is not limited to any filter used to filter the environmental water being taken in. The intake filter may be an intake filter for an aquaculture farm. The intake filter may be a membrane filter or a depth filter, but a depth filter is preferred. An example of a depth filter used as an intake filter is a spool filter, which may be used as a cartridge filter. The material of the filter is not limited, but polypropylene or cotton may be used for a spool filter.

[0014] The intake filter in the embodiment is an intake filter that has filtered water containing algae. Filtration is performed by passing water containing algae through the intake filter. The intake filter in the embodiment may be a filter used in an intake facility for filtering raw water or for further filtering of filtered water obtained by filtering raw water once or more times. The raw water may be water obtained by acquiring environmental water consisting of seawater, freshwater, or brackish water. For filtration using the intake filter, the raw water may be used directly, or filtered water obtained by filtering the raw water beforehand using a filter other than the intake filter may be used. When using raw water that has been filtered once or more times, the filtration is performed with a filter accuracy that allows at least a portion of the algae contained in the raw water to pass through. The filter accuracy of the filter used for such pre-filtration may be 1 μm to 50 μm, 2 μm to 25 μm, 3 μm to 10 μm, or 3 μm to 5 μm. By using a filter in an intake facility, intake filtration filters that would normally be discarded can be effectively utilized as a source of algae for aquaculture feed. Furthermore, since fisheries operators without specialized knowledge can easily obtain new microalgae at their aquaculture sites, it becomes possible to obtain algae that are suited to the dietary needs of the organisms being raised and the water quality of the area where the algae are grown. This will lead to improved breeding performance for aquatic products that are difficult to cultivate with currently available commercially.

[0015] The intake facility in the embodiment may be an intake facility for taking in environmental water that may contain algae, such as seawater, freshwater, or brackish water. The intake facility in the embodiment may be an intake facility for tap water, agricultural water, industrial water, and aquaculture water, and may be an intake facility for a water treatment plant, irrigation facility, factory, or aquaculture farm. The intake facility in the embodiment is preferably an intake facility for aquaculture water or an intake facility for an aquaculture farm, and is particularly preferably an intake facility for an aquaculture farm. An intake facility for an aquaculture farm has the advantage of being easily accessible to fisheries operators and being usable to easily obtain new microalgae at the aquaculture site. In one embodiment, the method includes taking in water from environmental water through a conduit to an aquaculture tank, and cultivating aquatic products in the aquaculture tank, wherein the intake filter is located in the middle or at the end of the conduit to filter the water passing through the conduit. Aquatic products mean aquatic organisms cultivated for commercial purposes, as is usually understood by those skilled in the art.

[0016] In this disclosure, filter accuracy is defined based on the particle size of the particles that the filter can capture, and means the smallest particle size that the particles that the filter can capture may have. Here, "capable of capturing" may mean that the proportion of particles captured by the filter is above a certain value. "Capable of capturing" may mean, for example, that the proportion of particles captured by the filter is 1% or more, 10% or more, 20% or more, 30% or more, 50% or more, 90% or more, or 99% or more, and in this case, the smallest particle size that can capture any of these proportions may be used as the filter accuracy. In the embodiments, the filter accuracy preferably means the smallest particle size that the particles that the filter captures in proportion to 30% or more may have. The filter accuracy of the water intake filter in the embodiment may be 0.5 μm to 200 μm, 0.5 μm to 50 μm, 0.5 μm to 20 μm, 0.5 μm to 10 μm, 0.5 μm to 5 μm, 0.5 μm to 3 μm, or 1 μm to 2 μm.

[0017] The method for isolating algae in the embodiment includes a step of culturing algae attached to an intake filter that has filtered water containing algae. The algae attached to the intake filter that has filtered water containing algae in the embodiment may be algae that were present in the raw water and attached to the filter during the filtration process. The cultivation in the embodiment can be carried out using a culture medium for algae cultivation known to those skilled in the art, sterile water such as sterile seawater, or a culture medium containing a mixture thereof. Culture media known to those skilled in the art include KW21 medium (available from Daiichi Seimo Co., Ltd.), IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, Koren-Hutner (KH) medium, Cramer-Myers (CM) medium, and BG11 medium. Culture media containing these media, modified media of these media, or a culture medium containing a mixture of several of these media or modified media can be used for cultivation in the embodiment. The culture medium may contain one or more agents from among antibiotics, antibacterial agents, antifungal agents, and antiprotozoal agents. These agents can be added to the culture medium at concentrations suitable for algal cultivation, as known to those skilled in the art. Examples of such agents and their concentrations include 0.05 mg / mL to 0.2 mg / mL penicillin, 0.02 mg / mL to 0.1 mg / mL streptomycin, 0.05 mg / mL to 0.2 mg / mL chloramphenicol, 0.02 mg / mL to 0.2 mg / mL kanamycin, 0.01 mg / mL to 0.05 mg / mL nystatin, and 0.001 mg / mL to 0.2 mg / mL trimethoprim.

[0018] The culture of the embodiment may be carried out under natural light and / or artificial light irradiation, and may be accompanied by stirring and / or CO2 aeration. The culture of the embodiment may include a step of collecting algae attached to the intake filter at its initiation. The collection of algae may be carried out, for example, by cutting off a portion of the intake filter and immersing it in the culture medium along with the algae attached thereto.

[0019] The method for isolating algae according to the embodiment may include a step of sorting the cultured algae or their offspring according to size. The step of sorting according to size may be performed using two filters having different filter accuracies, and may include a step of passing the algae through a filter having a larger filter accuracy, one of the two filters having different filter accuracies; a step of filtering the algae that have passed through the filter having a larger filter accuracy, one of the two filters having different filter accuracies, through a filter having a smaller filter accuracy, and a step of culturing the algae attached to the filter having a smaller filter accuracy. Here, larger or smaller filter accuracy means, as described above regarding the definition of filter accuracy, that the particle size of the particles that can be captured is relatively larger or smaller, respectively. The filter accuracy of the filter having a larger filter accuracy may be 4 μm to 50 μm, 5 μm to 25 μm, or 5 μm to 10 μm, and the filter accuracy of the filter having a smaller filter accuracy may be 0.5 μm to 3 μm, 1 μm to 3 μm, or 2 μm to 3 μm. The filters used in the sorting process according to size may be membrane filters or depth filters, but membrane filters are preferred. When using membrane filters, the pore size of the filter with high filter accuracy may be 4 μm to 50 μm, 5 μm to 25 μm, or 5 μm to 10 μm, and the pore size of the filter with low filter accuracy may be 0.5 μm or more and less than 4 μm, 1 μm to 3 μm, or 2 μm to 3 μm.

[0020] The method for isolating algae according to the embodiment may include, before the culturing step, a step of filtering raw water containing algae or filtered water obtained by filtering the raw water one or more times, and a step of further filtering the filtered water generated in the filtering step with a water intake filter to obtain a water intake filter that has filtered water containing algae. The water intake filter is used as the water intake filter in the step of culturing the algae attached to the water intake filter that has filtered water containing algae. The raw water in the filtering step may be raw water obtained by acquiring environmental water such as seawater, fresh water, or brackish water containing algae. For filtration using the water intake filter, this raw water may be directly used, or filtered water obtained by previously filtering the raw water using a filter other than the water intake filter may be used.

[0021] The method for isolating algae according to the embodiment may further include a step of subculturing the algae selected in the screening step. In subculturing, a part of the algal culture is added to a fresh medium, and performing a new culture is repeated one or more times. As the medium used for subculturing, the medium described in the explanation of the step of culturing the algae attached to the water intake filter that has filtered water containing algae can be used.

Examples

[0022] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0023] Seawater was pumped up from a water area where the presence of algae was confirmed, according to the water depth. First, the raw water was simply filtered (sand filtration and pressure filtration), and the filtered water of this simple filtration was re-filtered with a filter in which two wound cartridge filters having filtration accuracies of 5 μm and 1 μm (typically used in the water intake facilities of fish farms) were connected in series. Water flow was continued until the cartridge filter was colored and the presence of residues was confirmed (Fig. 1).

[0024] After sufficient coloration was confirmed on the cartridge filter, the filter surface was cut off, placed in the culture medium, stirred, and cultivation was started (Figure 2). As the culture medium, KW21 (manufactured by Daiichi Seimo Co., Ltd.) was diluted with sterile seawater. Streptomycin 0.06 mg / ml, penicillin 0.1 mg / ml, kanamycin 0.04 mg / ml, and trimethoprim 0.0012 mg / ml were added to the culture medium.

[0025] Using the same culture medium as described above, several subculturings were performed. To select the required algal size, syringe filters with pore sizes of 5 μm and 3 μm were used. First, the mixture was filtered through a 5 μm filter, then filtered again through a 3 μm filter, and the algae captured by the 3 μm filter were collected and the culture was repeated.

[0026] After repeated size sorting and subculturing, the selected size of algae became the dominant species, resulting in a culture containing algae with a nearly uniform appearance (Figure 3).

[0027] The results above illustrate that useful, uniformly quality algae can be isolated by cultivating algae attached to water intake filters in aquaculture farms and other facilities that filter water containing algae.

[0028] While this disclosure has been described with reference to several embodiments described above, it is not limited to the examples given in these embodiments. Various modifications can be made to the configuration and details of the present invention within the scope of this disclosure.

[0029] This disclosure includes the following embodiments. (Section 1) This process includes cultivating algae attached to an intake filter that has filtered water containing algae. Methods for isolating algae. (Section 2) The method according to item 1, wherein the intake filter is a filter used in an intake facility for filtering raw water or for further filtering of filtrate obtained by filtering raw water once or more times. (Section 3) The method according to item 2, wherein the water intake facility is a water intake facility for an aquaculture farm. (Section 4) The method according to any one of items 1 to 3, wherein the water intake filter is a depth filter. (Section 5) The method according to any one of items 1 to 4, wherein the water intake filter is a spool filter. (Section 6) The method according to any one of items 1 to 5, wherein the filter accuracy of the water intake filter is 0.5 μm to 50 μm. (Section 7) The method according to any one of claims 1 to 6, further comprising a step of selecting algae or their offspring cultured in the aforementioned culture step according to size. (Section 8) Before the aforementioned culturing step, A process of filtering raw water containing algae or filtrate obtained by filtering raw water once or more times, and The process includes further filtering the filtrate produced in the above filtering step with an intake filter to obtain an intake filter that has filtered out the water containing the algae. The method described in any one of items 1 to 7. (Section 9) The method according to item 7, further comprising the step of subculturing the algae selected in the selection step. (Section 10) The method according to claim 9, comprising repeating the step of sorting according to the size and the step of subculturing two or more times. (Section 11) The method according to claim 7, comprising the steps of: sorting according to size using two filters having different filter accuracies; passing the algae through the filter having a higher filter accuracy among the two filters having different filter accuracies; filtering the algae that have passed through the filter having a higher filter accuracy in the passing step using the filter having a lower filter accuracy among the two filters having different filter accuracies; and culturing the algae attached to the filter having a lower filter accuracy.

Claims

1. This process includes cultivating algae attached to an intake filter that has filtered water containing algae. Methods for isolating algae.

2. The method according to claim 1, wherein the intake filter is a filter used in a water intake facility for filtering raw water or for further filtering of filtered water obtained by filtering raw water once or more times.

3. The method according to claim 2, wherein the water intake facility is a water intake facility for an aquaculture farm.

4. The method according to any one of claims 1 to 3, wherein the water intake filter is a depth filter.

5. The method according to any one of claims 1 to 3, wherein the water intake filter is a spool filter.

6. The method according to any one of claims 1 to 3, wherein the filter accuracy of the water intake filter is 0.5 μm to 50 μm.

7. The method according to any one of claims 1 to 3, further comprising the step of selecting algae or their offspring cultured in the aforementioned culture step according to size.

8. Before the aforementioned culturing step, A process of filtering raw water containing algae or filtrate obtained by filtering raw water once or more times, and The process includes further filtering the filtrate produced in the above filtering step with an intake filter to obtain an intake filter that has filtered out the water containing the algae. The method according to any one of claims 1 to 3.

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