KR102301268B1 - Composition for cryopreservation of filamentous cyanobacteria comprising alginate and DMSO and method for cryopreservation of filamentous cyanobacteria using the same - Google Patents

Abstract

The present invention relates to a composition for cryopreservation of filamentous cyanobacteria containing alginate and DMSO and a method for cryopreserving filamentous cyanobacteria using the composition, and the present invention relates to filamentous cyanobacteria managed by conventional subculture. As an improved method for preserving in vivo culture, the composition for cryopreservation of the present invention may be usefully utilized for maintenance and management of biological resources.

Description

Composition for cryopreservation of filamentous cyanobacteria comprising alginate and DMSO and method for cryopreservation of filamentous cyanobacteria using the same

The present invention relates to a composition for cryopreservation of filamentous cyanobacteria comprising alginate and DMSO, and a method for cryopreserving filamentous cyanobacteria using the composition.

As interest in biological resources in each country increases due to the entry into force of the Nagoya Protocol, biological resource banks in the United States, Germany, and Japan are securing, conserving, and managing biological resources of various taxa including animals, plants, and microorganisms. Various domestic biological resource banks are also managing microbial resources such as bacteria and fungi efficiently. Microbial resources such as bacteria and fungi are relatively easy to maintain as active cultures in solid and liquid medium, and cryogenic freezing is stored at -80°C or below or -160°C or below using a freezer or liquid nitrogen when necessary. Relatively easy to preserve.

Among various microorganisms, filamentous cyanobacteria (cyanobacteria or cyanobacteria) are a kind of bacteria that grow in a filamentous structure such as a thread, and are recently attracting attention in various research areas. Filamentous cyanobacteria play an important ecological role in fixing nitrogen in the air using specialized cells called heterocysts. However, it is also a causative species of harmful algal blooms called 'green algae' in rivers and lakes. Filamentous cyanobacteria, such as Anabaena , limit the use of drinking water sources by producing toxins or off-flavor substances that cause an earthy odor. On the other hand it produces toxins which filamentous cyanobacteria, such as the two-day Pirelli nematic (Geitlerinema) or via ringeu (Lyngbya) is also used as a biomaterial for drug development.

Therefore, it is necessary to efficiently preserve and manage filamentous cyanobacteria, which are important biological resources environmentally, ecologically, and biologically. However, in most culture collections, cyanobacteria are managed only as a living collection of active cultures by periodic subculture under photosynthetic conditions. The reason is that cryopreservation of filamentous cyanobacteria is difficult compared to cryopreservation of general bacteria and fungi.

After adding a preservative such as dimethyl sulfoxide (hereinafter 'DMSO'), methanol, or glycerol to the cells, the temperature is slowly lowered to cryogenic temperatures (-80°C to -160°C) for cryopreservation without damage to the cells. Methods have been limitedly studied in unicellular cyanobacteria such as Microcystis. However, filamentous cyanobacteria with specific structures such as heterogeneous cells and dormant cells may suffer irreparable damage during the freezing process due to the morphological complexity of the cells. Therefore, it is necessary to improve the viability while maintaining the morphological structure of filamentous cyanobacteria rather than simply applying the cryopreservation method developed in the prior art.

Meanwhile, Korea Patent No. 1767195 discloses 'a composition for cryopreservation of cells and a method for cryopreserving cells using the same', and Korean Patent Publication No. 2018-0070937 discloses 'a composition for cryopreserving microalgae and freezing microalgae using the same'. 'preservation method' is disclosed, but the composition for cryopreservation of filamentous cyanobacteria comprising alginate and DMSO of the present invention and a method for cryopreservation of filamentous cyanobacteria using the composition are not described.

The present invention was derived from the above needs, and the present inventors immobilized filamentous cyanobacteria into microcapsules using sodium alginate for cryopreservation of filamentous cyanobacteria, and then mixed with DMSO and frozen at -80°C. As a result of preservation, the present invention was completed by confirming that the survival rate of filamentous cyanobacteria after cryopreservation was significantly increased compared to the condition of using DMSO alone, a representative cryoprotectant.

In order to solve the above problems, the present invention provides a composition for cryopreservation of filamentous cyanobacteria comprising sodium alginate and calcium chloride as active ingredients.

In addition, the present invention provides a method for cryopreservation of filamentous cyanobacteria, comprising the step of mixing the composition for cryopreservation with filamentous cyanobacteria.

The present invention is an improvement of the method for preserving the in vivo culture of filamentous cyanobacteria managed by periodic subculture, in which filamentous cyanobacteria are immobilized using alginic acid microcapsules and mixed with DMSO to improve viability. Since the cryopreservation method of cyanobacteria is provided, it will be usefully utilized for the maintenance and management of biological resources such as filamentous cyanobacteria.

1 is an optical micrograph of three types of filamentous cyanobacteria used in the present invention. (A) Anabaena variabilis FBCC10004, (B) Geitlerinema sp. FBCC020002, (C) Oscillatoria sp. FBCC02001. Scale bar: 20 μm.
2 is a view of alginic acid microcapsules formed by 2.0% sodium alginate (sodium alginate) and calcium chloride (calcium chloride) concentration. (A) 2% calcium chloride, (B) 2.5% calcium chloride, (C) 3% calcium chloride. Scale bar: 1mm.
3 is a view of alginate microcapsules formed by 2.5% sodium alginate and calcium chloride concentrations. (A) 2% calcium chloride, (B) 2.5% calcium chloride, (C) 3% calcium chloride. Scale bar: 1mm.
4 shows a state in which three types of cyanobacteria are immobilized in alginic acid microcapsules and cells are captured inside the microcapsules. (A) Anabaena variabilis FBCC10004, (B) Geitlerinema sp. FBCC020002, (C) Oscillatoria sp. FBCC02001. Scale bar: 1mm.
FIG. 5 is a diagram showing the survival rate after cryopreservation of three filamentous cyanobacteria using the cryoprotectant DMSO alone ('general cryopreservation) or alginic acid microcapsules and DMSO together ('microcapsule use').
6 is a diagram showing the difference immediately after the start of cryopreservation of cyanobacteria A. variabilis using alginic acid microcapsules and the cryoprotectant DMSO and 2 weeks after thawing and recultivation. (A) Left: right after cryopreservation, right: after thawing and 2 weeks of culturing, (B) inside the microcapsule immediately after cryopreservation, (C) inside the microcapsule after thawing and 2 weeks after culturing.
7 shows the structural differences in cryopreservation of filamentous cyanobacteria using microcapsules using a transmission electron microscope (TEM). A is a cross-section of filamentous cyanobacteria A. variabilis cultured without using any medium; B is one of A. variabilis cryopreserved using only a freezing protective agent DMSO section, C is the cross-section of the A. variabilis case of immobilizing a filamentous cyanobacteria inside alginate microcapsules and cryopreserved using a freezing protective agent DMSO at the same time, D is a cross-sectional view of A. variabilis cells immobilized inside microcapsules after thawing and 2 weeks of culturing.

In order to achieve the object of the present invention, the present invention provides a composition for cryopreservation of filamentous cyanobacteria comprising sodium alginate and calcium chloride as active ingredients.

In the process of freezing for long-term preservation of cells and thawing for reuse, cells are damaged by temperature change. When freezing proceeds, the moisture outside the cell is first frozen, and as a result, the environment outside the cell is more salty than the inside of the cell. (plasmolysis) occurs. To prevent this, a cryoprotectant is used, and the cryoprotectant is divided into a cytoprotective type colligative cryoprotectant and a cell penetrating type cryoprotectant. Cytoprotective short-acting cryoprotectants inhibit the formation of extracellular ice crystals, and cell penetrating cryoprotectants penetrate the cell membrane to replace intracellular moisture and inhibit the formation of ice crystals upon freezing. In particular, for cryopreservation of microalgal cells, a cell-penetrating cryoprotectant is not used due to its nature, and a cell-penetrating cryoprotectant, specifically, for example, DMSO or glycerol, etc. has been conventionally used.

In the cryopreservation composition of the present invention, the sodium alginate and calcium chloride may be mixed with each other at a concentration of 2.3 to 2.7% (w/v) to form alginic acid microcapsules, and specifically, 2.3 to 2.7% (w/v) v) may be to form alginic acid microcapsules by dripping a sodium alginate solution at a concentration of 2.3 to 2.7% (w/v) into a calcium chloride solution, more specifically, at a concentration of 2.5% (w/v). It may be to form alginate microcapsules by dropping a sodium alginate solution into a calcium chloride solution having a concentration of 2.5% (w/v), but is not limited thereto. In addition, the sodium alginate solution having a concentration of 2.3 to 2.7% (w/v) is preferably prepared in a mixed state with the culture solution of filamentous cyanobacteria before being instilled into the calcium chloride solution.

In addition, the cryopreservation composition of the present invention may further include DMSO (dimethyl sulfoxide), and the DMSO may preferably be included in an amount of 5 to 20% (v/v) of the cryopreservation composition, more preferably Preferably, it may be included in an amount of 7 to 15% (v/v), and more preferably, it may be included in an amount of 10% (v/v), but is not limited thereto.

In the cryopreservation composition of the present invention, the filamentous cyanobacteria are not limited thereto, but Anabaena sp. (preferably Anabaena variabilis ), Gaitlerinema species ( Geitlerinema sp.) or Oscillatoria sp.).

The composition for cryopreservation of filamentous cyanobacteria according to the present invention is characterized in that the survival rate of filamentous cyanobacteria is significantly increased after cryopreservation at -80°C compared to DMSO, a cryoprotectant that has been used in the past.

The present invention also provides a method for cryopreserving filamentous cyanobacteria, comprising mixing the cryopreservation composition with filamentous cyanobacteria. The cryopreservation method according to the present invention is more specifically,

(a) Preparing a mixture of sodium alginate and filamentous cyanobacteria by adding the culture medium of filamentous cyanobacteria to a sodium alginate solution of 2.3-2.7% (w/v) ^{concentration and adjusting the concentration of 10 4} to 10 ^{6 cells per ml} ;

(b) dropping the mixed solution of sodium alginate and filamentous cyanobacteria into a calcium chloride solution having a concentration of 2.3 to 2.7% (w/v) to form alginate microcapsules in which filamentous cyanobacteria are immobilized; and

(c) adding the alginic acid microcapsules in which the filamentous cyanobacteria are immobilized to a liquid medium containing DMSO and cooling to -80°C; may include, but is not limited to.

Filamentous cyanobacteria cryopreserved by the method according to the present invention can be cultured as alginic acid microcapsules after thawing (or thawing). Filamentous cyanobacteria fixed (captured) in alginate microcapsules can proliferate during the culture process and cause cracks in the microcapsules to spread out of the microcapsules. Alternatively, after thawing, the microcapsules may be pulverized and cultured.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Identification and culture of filamentous cyanobacteria

Three species of filamentous cyanobacteria in a pure state were isolated from the Nakdong River freshwater system. For pure separation of filamentous cyanobacteria, a microglass tube separation method using an inverted microscope and a colony separation method using a solid medium were performed in parallel. First, while observing the sample under an inverted microscope, filamentous cyanobacteria were isolated with a heated Pasteur glass pipette. The primary isolated filamentous cyanobacteria were cultured in BG11 liquid medium for 2 weeks at a light intensity of 200 μmol/m2/s and a temperature of 25°C. Secondary separation was performed to purely isolate the cultured cyanobacteria. To this end, a BG11 solid medium supplemented with 1.5% agar was made, and the primary cultured filamentous cyanobacteria were plated 2-3 times on the solid medium and stationary cultured under the same conditions for 14 days. Filamentous cyanobacteria were grown on a solid medium and pure colonies were isolated. Filamentous cyanobacteria isolated purely through microglass tube separation and colony separation on solid medium were then sequenced with 16S rRNA gene for species identification. The phylogenetic relationship with other cyanobacteria was comparatively analyzed as a result of the analyzed base sequence, and the species was identified by comparing the image data of the cells observed with an optical microscope with the illustrated data. In addition, as a result of sequencing, it was possible to simultaneously confirm the state of pure separation without contamination. As a result, filamentous cyanobacteria isolated from the Nakdong River freshwater system were identified as Anabaena variabilis FBCC10004, Geitlerinema sp. FBCC020002) and Oscillatoria sp. FBCC020001 (Oscillatoria sp. FBCC020001) ( 1). The FBCC number means the biological resource number of the resource bank operated by the National Nakdong River Biological Resources Center.

The three types of filamentous cyanobacteria were cultured using a 50 ml T-flask. In the same manner as in the separation process described above, incubation was performed at 200 μmol/m2/s under light intensity and at 25° C., and sequentially diluted samples were placed in a haematocytometer to check the concentration of the cultured cells, followed by an inverted microscope. was used, and the concentration of cells present in the culture medium was determined by reflecting the diluted fold. After confirming the cell concentration of the culture medium, the cells were suspended ^{and prepared at the same concentration (10 5} cells/ml), and this was used as the initial cell concentration for cryopreservation.

2. Microcapsule immobilization method of filamentous cyanobacteria using alginic acid microcapsules

In order to immobilize the cultured filamentous cyanobacteria into the alginic acid microcapsules, the optimal conditions for preparing the alginic acid polymer were first determined. First, 2% and 2.5% sodium alginate solutions and 2%, 2.5% and 3% calcium chloride solutions were prepared. Thereafter, using a Pasteur pipette, 20 μl of sodium alginate solution was dropped into a beaker containing calcium chloride solution to prepare calcium-alginic acid polymer. The size of the calcium-alginic acid microcapsules prepared in 6 combinations of sodium alginate solution 2 concentration conditions and calcium chloride 3 concentration conditions was confirmed. As a result, microcapsules were formed to be large and soft when the concentration of sodium alginate was high and the concentration of calcium chloride was low. Conversely, when the concentration of sodium alginate was low and the concentration of calcium chloride was high, it was observed that small but rigid formation was observed ( FIGS. 2 and 3 ).

Microcapsules made of 2.5% calcium chloride and 2.5% sodium alginate among the six conditions were used for subsequent experiments. In order to immobilize filamentous cyanobacteria in the microcapsule, the cell culture medium was added to 2.5% sodium alginate solution to prepare a concentration of ^{10 5 cells/ml.} Thereafter, alginic acid microcapsules were formed by dropping a mixture of alginic acid and filamentous cyanobacteria into a 2.5% calcium chloride solution one drop at a time using a Pasteur pipette. Filamentous cyanobacteria cells were fixed inside the spherical alginate microcapsules with a diameter of about 2 mm, and this was confirmed under a microscope (FIG. 4).

3. Method for confirming the viability of cryopreservation using alginic acid microcapsules

In the present invention, (1) DMSO was used as a cryopreservative composition and (2) Alginic acid microcapsules and DMSO were used as a cryopreservative composition to compare the cases in which alginic acid microcapsules were used and those in which they were not used.

For experimental condition (1), DMSO cryoprotectant was added to the BG11 liquid medium in which 3 types of filamentous cyanobacteria cells were cultured in a concentration gradient of 0%, 5%, 10%, 15% and 20% (v/v). added. After acclimatization of the mixed solution at 4° C. for 12 hours, 1 ml each was dispensed into cryogenic vial tubes. The dispensed ultra-low temperature cryopreservation tube was cooled using a controlled rate freezer. The cooling conditions were frozen at -1°C/min to -40°C, and after maintaining the temperature at -40°C for 15 minutes, two-step cooling was performed again to -80°C at -1°C/min. After freezing, it was stored in a -80°C deep freezer for 1 month.

For the experimental condition (2), 1 ml of BG11 liquid medium containing DMSO cryoprotectant was first dispensed into a tube for cryopreservation at ultra-low temperature, and about 20 microcapsules of alginic acid in which cells were immobilized were added to the tube. Thereafter, it was cooled and stored in the same manner.

In order to analyze the viability of the filamentous cyanobacteria regenerated cells, the cryopreserved filamentous cyanobacteria vial was taken out of the cryogenic freezer and thawed in a 37°C water bath for 5 minutes. After centrifuging the tube at 6,500x g for 5 minutes, the supernatant was discarded, the alginic acid microcapsules having filamentous cyanobacteria fixed therein were recovered and added to fresh BG11 medium without cryopreservative. After thawing, the cells were stored in a dark room at room temperature for 24 hours, then 20 μl of each was dispensed into a 96-well plate, and 180 μl of BG11 medium was dispensed into each well and cultured for 2 weeks. to evaluate the survival rate. For cell counting, microcapsules were crushed and the cells inside were suspended.

Example 1. Analysis of the survival rate improvement effect of cryopreservation using alginic acid microcapsules

As a result of analyzing the survival rate under the conditions of 5%, 10%, 15%, and 20% of DMSO cryoprotectant, the optimal concentration of cryopreservative was 10% (v/v), and under this condition, 3 filamentous cyanobacteria The average survival rate was observed to be 15%. In addition, when the cryoprotectant of DMSO was not added, the survival rate was confirmed to be less than 5% (FIG. 5). On the other hand, when filamentous cyanobacteria were immobilized in alginic acid microcapsules and cryopreserved using DMSO as a cryoprotectant, the survival rate was excellent compared to the survival rate using DMSO alone under the same conditions. In particular, as a result of calculating the relative survival rate based on the survival rate of DMSO alone, it was found that after immobilization with alginic acid microcapsules, the survival rate was improved by more than 200% under the condition of using DMSO together. In particular, when the concentration of the cryoprotectant DMSO was 0 or 20%, it was confirmed that the survival rate was increased by 300% or more in the condition in which filamentous cyanobacteria were immobilized and cryopreserved using alginic acid microcapsules compared to the cryopreservation condition using only DMSO. From the above results, it was found that the survival rate after cryopreservation was increased by immobilizing filamentous cyanobacteria using alginic acid microcapsules.

As a result of thawing (thawing) the frozen cyanobacteria Anabaena variabilis inside alginate microcapsules and recultivating them as they were captured in the microcapsules, the concentration of filamentous cyanobacteria inside the microcapsules increased after 2 weeks of culturing. was observed to change to dark green (FIG. 6).

In addition, structural differences between cryopreservation using microcapsules were confirmed using transmission electron microscopy (TEM). In general, the cross-section of filamentous cyanobacteria A. variabilis cultured without using any medium was circular or oval depending on the slope of the cross-section from which the cells were cut. The inside of the cell shows a thylakoid membrane with a wrinkled structure and a lipid body and carboxysome inside in the form of a circle, and the cytoplasm is filled between these organelles. can be confirmed (FIG. 7A). A general cryopreservation method using only the cryoprotectant DMSO confirmed that the A. variabilis cells were destroyed or became atypical cells (FIG. 7B). On the other hand, when filamentous cyanobacteria were immobilized inside alginate microcapsules and cryopreserved using the cryoprotectant DMSO at the same time , it could be observed that A. variabilis cells were collected in the alginate medium in various shapes such as round, ovoid, oval, and irregular shapes. there was. In addition, as the cytoplasm was reduced, the cells contracted, and a dark line with deep wrinkles was evident on the surface (FIG. 7C). As such, the A. variabilis cells inside the microcapsule recovered from an oval to a spherical shape through culturing for 2 weeks after thawing. .

Claims (6)

delete delete delete delete delete (a) Anabaena species ( Anabaena sp.), Geitlerinema species ( Geitlerinema sp.) or Oscillatoria species ( Oscillatoria sp.) in a sodium alginate solution of 2.3 ~ 2.7% (w / v) concentration preparing a mixed solution of sodium alginate and Anabena species, Gaitlerinema species or Oscilatoria species by adding the mixture to a ^{concentration of 10 4 to 10 6 cells per ml;}
(b) by dropping the sodium alginate and the mixture of Anabena species, Gaitlerinema species or Oscilatoria species into a calcium chloride solution of 2.3 to 2.7% (w/v) concentration, Anabena species, Gaitlerinema species or Forming alginic acid microcapsules in which Oscilatoria species are immobilized; and
(c) the Anabena species, Gatlerinema species or Oscilatoria species are fixed alginic acid microcapsules are added to a liquid medium containing 5-20% (w/v) DMSO, cooled to -80 ℃ and preserved Anabena species, Gaitlerinema species or Oscilatoria species cryopreservation method comprising the step of;

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