JP2976027B1 - Marine bacteria isolation method - Google Patents

Abstract

Abstract: A method for selectively separating marine bacteria, comprising separating marine bacteria using a medium to which bacteria belonging to the genus Moraxella which activates the growth of marine bacteria are applied in advance. [Effect] It is possible to selectively and efficiently collect marine bacteria of the genus Labyrinthula from the marine environment, for which there has been no effective separation method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for selectively separating marine bacteria, and more particularly, to a method for selectively separating marine bacteria belonging to the genus Labyrinthura using a specific medium. It is about.

[0002]

2. Description of the Related Art Labyrinthula is a marine fungus inherent in the marine environment, and is known as spoilage fungi such as seagrass such as eelgrass and mangrove leaf. And
Since this bacterium is closely related to the decomposition of organic substances in the natural ecosystem of the marine environment, it can be used as a new microbial resource, such as for the production of cellulolytic enzymes.

Generally, in order to separate marine bacteria from the natural world, diluents and immersion liquids are collected from the surface water of the habitat or a collected material such as seaweed or defoliation, and the diluent or immersion liquid is used as a separation source. A method of applying an appropriate culture medium to an agar medium prepared with seawater or artificial seawater, or 1 μm
A method of immersing the sample seawater or the collected matter with a filter having a pore size of about 10 μm to release the marine bacteria by filtering the sample water, filtering out the marine bacteria, and attaching the filter to the agar medium for culturing. After that, there is a method of separating the emerged bacteria. In the case where the growth of microorganisms is observed on the surface of the seaweed or the like as a sample, a method may be employed in which the cells are directly cut off using fine-tipped tweezers or needles and transplanted to the agar medium. However, in these methods, the medium is often covered by the growth of a large amount of bacteria and mold, and the growth of the target Labyrinthula is suppressed,
It is difficult to select and isolate Labyrinthula effectively.

[0004] Thraustochytrium and Schizochytrium belonging to the family Thraustochytrium belonging to marine fungi are selectively applied to pine pollen by a pine pollen fishing method and then applied to an agar medium. In this way, it is possible to increase the selectivity to a certain degree and to perform the separation. However, in this pine pollen fishing method, Labyrinthula genus Labyrinthula is not isolated. This is in contrast to the zoospore cells released by marine fungi of the Thraustochytrium family, which are easily attracted to the pine pollen used as fishing bait.
It is considered that Labyrinthula does not show the same behavior because it has a different life cycle and growth mode from the bacteria of the family Thraustochytrium. In addition, Labyrinthula spp.
It has a characteristic spindle-shaped cell morphology and can be easily distinguished from spherical Thraustochytrium. From the above, development of an effective and selective separation method for marine bacteria belonging to the genus Labyrinthura has become an urgent issue.

[0005]

SUMMARY OF THE INVENTION The present invention provides a method for effectively selecting and separating Labyrinthula from seawater or the marine environment.

[0006]

Means for Solving the Problems The present inventors have studied the marine fungi of the genus Labyrinthula expected to be used as a new marine microbial resource, and studied the ecological characteristics and the like.
As a result of intensive studies on a separation method using its ecological characteristics, marine bacteria belonging to the genus Moraxella were applied to the agar plate medium used for separation in advance, and after cultivation, the marine By inoculating a collection from the environment onto the agar plate medium, it was found that it was possible to selectively grow Labyrinthula spp.From this finding, Labyrinthula spp. A method that can be selectively separated has been developed, and the present invention has been completed.

[0007] That is, the present invention is a method for selectively separating marine bacteria, which comprises separating marine bacteria using a medium to which bacteria that activate the growth of marine bacteria are applied in advance. Labyrinthula bacteria are mentioned as the marine bacteria, and bacteria belonging to the genus Moraxella are bacteria activating the growth of the marine bacteria.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. 1. Preparation of agar medium The agar plate medium used for the isolation of Labyrinthula is prepared as follows. That is, a solution prepared by dissolving 2 g of glucose and an appropriate nitrogen source such as yeast extract and peptone in 15 g of agar in 1 liter of natural seawater or artificial seawater is steam-sterilized and used for ordinary microbial culture experiments. Put it in a Petri dish (9cm in diameter is often used). The concentration of natural seawater or artificial seawater used here is adjusted to 20 to 100% of that of ordinary seawater, and a concentration of 50 to 90% is desirable for culturing Labyrinthula. As the artificial seawater, commercially available tropic marine (manufactured by Aqua Alien Technik) can be used.

[0009] 2. Application of Bacteria to Agar Medium The agar plate medium prepared in the manner described above, which has been previously coated with bacteria and cultured, is used as a selective culture medium for marine bacteria. As the bacteria used here, marine bacteria are desirably used, and typical examples thereof include Moraxella bacteria. If the concentration of seawater used for preparing the agar plate medium is 50% or less, non-marine bacteria can also be used. As bacteria to be applied, the bacteria are prepared in advance using glucose (1%) and yeast extract (1%) in artificial seawater having a concentration of 50% (in the case of using non-marine bacteria, tap water or distilled water, etc.). The obtained culture solution is inoculated, and cultured with shaking at a culture temperature of 25 to 30 ° C. for 2 to 5 days. Culture conditions are performed under culture conditions suitable for culturing the bacteria to be used. The bacterial culture solution thus prepared was aseptically inoculated in 100 microliter portions onto an agar plate medium prepared in a petri dish, spread on the surface of the agar plate medium with a conical bar or the like, and further spread at 25 to 30 ° C for 2 to 2 hours. The bacteria are allowed to grow by static culture for 5 days.

[0010] 3. Moraxella bacterium As a bacterium suitable for separating Labyrinthula, for example, Moraxella phenylpyruvica LB004 is used.
This fungus was isolated from the surface seawater near Iriomote Island by the present inventors, and the mycological properties of this fungus are as follows. Bacteriological properties of Moraxella phenylpyruvica LB004 It takes many forms such as linkage, elongation, filament and sphere on agar medium. Juvenile cells are 0.6 × 1.0 (μ
m) and aging cells are often 1.2 × 2.0 (μm) spherical bacilli. Gram negative, does not form spores,
No motility is seen. Optimal growth temperature is around 25 ℃, 5 ℃
However, it grows but does not grow at 37 ° C. Colonies are round, smooth at all edges, convex, shiny, translucent, off-white. It has catalase activity and oxidase activity. Also, the OF test is negative. NaCl is 4
% Or 9.5%. This Moraxella phenylpyruvica LB004 was identified by FERM P-16 on August 24, 1998 by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
Deposited as 954.

4. Sources of Labyrinthula spp.The distribution range of Labyrinthula spp. Is known to cover a wide range of marine environments from the tropics to the polar regions. The sources of such separation are seawater, various seaweeds, seagrass such as eelgrass, and hilt. For example, plants that grow along the coast, defoliation, wood chips, mud sand, and various organic substances collected in seawater or along the coast can be used. However, in general, when considering the distribution density of Labyrinthula and the growth activity of isolates, the tropical and subtropical waters are taken as the harvesting area, and the leaves of eelgrass and deciduous leaves of the eelgrass collected along the coast are considered as sources. It is desirable to do.
It is considered that these collections have been immersed in seawater for a certain period of time and have Labyrinthura sp. Attached thereto. In order to separate Labyrinthula, it is also possible to intentionally immerse these leaf pieces, wood pieces, and the like in seawater for a certain period of time and then use them as a separation source.

5. Sample preparation method These specimens, which are the separation source of Labyrinthula, are cut into fragments of about 1 cm x 1 cm, the surface is washed several times with sterilized water, etc., and brought into the water adhering to the surface. After removing as much of the bacteria or mold spores as possible, the cells are attached to the center of an agar plate medium coated and cultured with the aforementioned Moraxella bacteria. Use this agar plate medium for 20 ~
Incubate at 30 ° C. for 2-7 days.

6. Detection method During the culture period, observe the agar plate medium with a microscope every day,
Investigate the appearance of Labyrinthula. The microscope magnification is
At 80-400x, an inverted microscope is suitable for observation,
A stereomicroscope or the like can also be used. Labyrinthula is composed of spindle-shaped cells with a length of about 10 μm, which spreads on an agar plate medium while sliding in a daisy chain or a lump. Due to this motility, reticulated or fungal hyphae appear to spread and show a radial spread from around the sample.
In about 5 days, the sample reaches the periphery of the petri dish from the center of the petri dish on which the sample is attached. At this time, various bacteria other than Labyrinthula bacteria, molds, yeasts, and protozoa also appear around the sample, but they are prevented from growing and spreading around by the applied Moraxella bacteria. . Labyrinthula invades colonies of Moraxella spp., Repeats vigorous growth, and spreads like a mesh. When a similar separation method is attempted using an agar plate medium not coated with Moraxella bacteria, bacteria, molds, yeasts, etc. predominantly appear to suppress the appearance of Labyrinthula sp. It was observed that the amount of growth and the speed of spreading in a network were small. From this, it was observed that Moraxella bacteria not only inhibit the growth of other bacteria other than Labyrinthula, but also fungi and yeasts, and also positively promote the growth of Labyrinthula. Was done.

7. Characteristics of Labyrinthula Labyrinthula has the following mycological properties, and is easily distinguished from other microorganisms by its morphology and motion microscopy. Mycological properties of Labyrinthula The vegetative cells of Labyrinthula are characteristically spindle-shaped and gliding through a protoplasmic network. The taxonomy ranks the Labyrinthura family, which consists of one genus Labyrinthula, of which eight species are known ("Handbook of Protoctista", David Porter
Authors, 1990). The order Labyrinthura is composed of Labyrinthurae and Thraustochytrium, and the family Thraustochytrium is composed of thirty species of seven genera such as Thraustochytrium and Schizochytrium. The fungi of the Thraustochytrium family have spherical or egg-shaped vegetative cells, and are easily distinguished from Labyrinthula. Labyrinthula is known to break the cell walls of seaweeds and seaweeds and degrade the inside. For example, Labyrinthura zostellae kills eelgrass. Labyrinthula sp. Has strong cellulase activity due to such properties, but it is difficult to separate it, so there are many unclear points.

[8] Purification of Labyrinthula In order to pick up Labyrinthula, a mesh-like cell mass (approximately 5 mm x 5 mm) that has reached near the edge of the Petri dish is sterilized using a platinum loop or the like that is used for transplanting the bacteria together with the agar medium. Cut and transfer to the center of agar plate medium not coated with Moraxella bacteria. Again, this agar plate medium was
Incubate at ３０30 ° C. for 2-7 days. Labyrinthula spp. Grows with a network spread in the center of the transplanted agar pieces. At this time, since Moraxella bacteria grow together with Labyrinthula, in order to obtain pure Labyrinthula, the cell mass grown near the edge of this petri dish was further transferred to an agar plate medium containing an antibiotic. Alternatively, it is transplanted into a liquid culture medium to isolate Labyrinthula spp. As the antibiotic to be used, chloramphenicol, penicillin, streptomycin, or the like, which is generally used, can be used. In the case of an agar plate medium using chloramphenicol, medium 1
The medium is added at a concentration of 50 to 400 mg per liter, and the medium is then prepared according to a conventional method. In the case of a liquid medium, chloramphenicol is added to a 50% glucose yeast extract artificial seawater medium at a concentration of 20 to 200 mg per liter of the medium. By such a separation method, Labyrinthula can be isolated.However, in order to preserve Labyrinthula, the Labyrinthula in a state before isolation from Moraxella is not added to an agar slant medium without an antibiotic. And transplant it to 1
Store at 0-15 ° C at low temperature.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. However, the present invention is not limited to these examples. [Example 1] Isolation of Labyrinthula was carried out using fallen leaves of hirugi or eelgrass collected on Iriomote Island. The composition of the agar plate medium used for the separation was 2 g of glucose, 1 g of yeast extract, 15 g of agar, and 1 L of 90% natural seawater.
A medium (MA) coated with Moraxella phenylpyruvica LB004 on this medium, a medium (MB) not coated as a control, and a medium (MC) containing 200 mg of chloramphenicol at the time of preparation were used. . The sampling points and the number of samples (in parentheses) are the Iriomote-jima Tomogawa River estuary (Hiragi 12 points), the Urauchi River estuary (Hiragi 16 points) and Funaura Bay (Hiragi 12 points, eelgrass 4 points). Table 1 summarizes the results of the isolation of Labyrinthula using three types of media.

[0017]

[Table 1]

Although there was not much difference when hirugi of Funaura Bay was used as a separation source, in other samples, a medium (MA) coated with Moraxella bacteria was used two to three times more frequently. Labyrinthula was detected. In the case of the culture medium (MB) to which Moraxella spp. Is not applied, although there are samples in which the appearance of Labyrinthula spp. Is observed, in many cases, contamination of bacteria and mold is large, and isolation of Labyrinthula spp. It was difficult. In the medium (MC) to which chloramphenicol was added, although the growth of bacteria was suppressed, the appearance of mold was large, and the appearance of Labyrinthula was not observed.

Example 2 In order to isolate Labyrinthula sp. Appearing on the MA medium, a liquid medium containing 50 mg / L of chloramphenicol was used at room temperature for the purpose of removing Moraxella spp. The cells were cultured. The liquid medium is prepared by adding 2 g / L of glucose and 1 g / L of yeast extract to 50% artificial seawater. The sample in which Labyrinthula spp. Of Example 1 has appeared is transplanted to an agar plate medium in a petri dish not coated with Moraxella spp., And cultured at room temperature. The cell mass extended to the edge of the petri dish was transplanted into the above-mentioned liquid medium, and cultured at 25 ° C. for 3 days. Microscopic photographs of the cultured Labyrinthula are shown in FIGS. 1 and 2. From this photograph, spindle-shaped flowing Labyrinthula cells are observed. There was no contamination with Moraxella bacteria, indicating that Labyrinthula was isolated.

[0020]

Industrial Applicability According to the present invention, marine fungi of the genus Labyrinthula can be selectively and efficiently collected from the marine environment. This isolation of Labyrinthula spp. Provides a new microbial resource according to the present invention because the fungus has the ability to produce cellulolytic enzymes.

[Brief description of the drawings]

FIG. 1 shows a micrograph of Labyrinthula spp. Invading a Moraxella spp. Colony before isolation.

FIG. 2 shows a micrograph of Labyrinthula isolated in liquid culture.

Continuing from the front page (72) Inventor Takanori Higashihara 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. Institute of Biotechnology and Industrial Technology (72) Inventor Ryuichiro Kurane 1-1-3 Higashi 1-3-1 Tsukuba City, Ibaraki Pref. Institute of Biotechnology, Industrial Technology Research Institute (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 1/00-7/08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for selective isolation of Labyrinthula sp., Comprising isolating Labyrinthula sp. Using a medium pre-coated with Moraxella sp. Which activates the growth of Labyrinthula sp.