JP5559690B2 - Parachlorella new microalgae - Google Patents

Description

  The present invention relates to a novel microalgae capable of producing alginic acid, a method for using the same, and the like.

Previously, unicellular algae that had been classified as genus Chlorella have been shown to be a multi-lineage group spanning Chlorophyceae and Treboxyphyceae as a result of recent molecular phylogenetic analysis. (Non-patent document 1: Friedl, 1995 and Non-patent document 2: Huss et al., 1999). Furthermore, Ustinova et al. (3) by performing a molecular phylogenetic analysis using 18S rDNA and 16S rDNA, Chlorella Kesureri (Chlorella kessleri) another from other Chlorella among Torebokishia Motsuna Shown to form a group. Therefore, the establishment of the genus Parachlorella was advocated, and the scientific name of Chlorella kesleri was changed to Parachlorella kessleri accordingly . As other algae belonging to the genus Parachlorella, there is Closteriopsis acicularis , which is said to be morphologically not similar to Parachlorella quercerii. For the classification system of green algae in general, refer to Non-Patent Document 4 (Proeshold and Leliaert).
Alginic acid is a viscous polysaccharide contained between cells and cell walls of brown algae and some red algae. Alginic acid or a salt thereof is widely used as a thickening stabilizer or gelling agent in food additives, pharmaceuticals, cosmetics, dental materials and the like. Alginic acid is a kind of dietary fiber and has attracted attention in recent years. Furthermore, alginic acid, in particular, alginic acid degradation products (oligomers), also called alginic acid oligosaccharides, or low molecular weight alginic acid have been reported to have various physiological activities.
As for the algae belonging to the treboxya algae class, no examples in which sodium alginate was contained or metabolized have been reported.
Friedl, 1995 (J. Physol. 31: 632-639). Huss et al. 1999 (J. Physol. 35: 587-598). Ustinova et al. , 2001 (Eur. J. Physol. 36: 341-351) Thomas Proeshold and Frederik Leliaert, "Chapter 7 Systems of the green algae: conflict of the class and the ed Jeul et al." , "Unraveling the algae: the past, present and future of algal systems", (Taylor and Francis, 2007) Regional research and development promotion base support project (RSP project) feasibility study research report “Search for the potential application of alginic acid degradation products to foods and pharmaceutical materials”, Masami Watanabe et al., 2000 Uno et al. , 2006 (Biosci. Biotechnol. Biochem. 70 (12): 3054-3057)

  An object of the present invention is to provide a useful novel Parachlorella microalgae and a method for using the same.

The present inventors have found that Parachlorella microalgae isolated from nature are novel and have an ability to produce alginic acid, in particular, an alginic acid having a relatively low molecular weight, thereby completing the present invention. That is, the present invention
[1] para genus Chlorella microalgae with alginate-producing ability, para Chlorella Bainosu (. Parachlorella sp binos);
[2] The microalgae according to [1], having the following characteristics;
Form: Single cell, spherical, size of about 10 μm to about 15 μm
Reproductive form: Endospore-forming quadrant or endospore-forming octa-splitting ruthenium red staining: Positive;
[3] The microalgae according to the above [2], which has a size of about 12 μm to about 15 μm when cultured in an inorganic III medium to be described later, and has a reproductive pattern of endospore-forming quadrisection type;
[4] The microalgae according to [2], which has a size of about 10 μm to about 12 μm when cultured in an Ogbonna medium described below;
[5] The microalgae according to any one of [1] to [4] above, wherein the content of chlorophyll a and chlorophyll b in the cell is about 1: 1 to about 2.5: 1;
[6] The microalgae according to any one of [1] to [5], which has an 18S rRNA gene represented by SEQ ID NO: 1.
[7] The microalgae according to any one of [1] to [6], which has a deposit number of FERM P-21513 (FERM BP-10969);
[8] A method for producing alginic acid or alginate, comprising culturing the microalgae according to any one of [1] to [7], and recovering alginic acid or alginate from the culture or culture medium;
[9] The method for producing alginic acid or alginate according to [8], wherein the culturing step is performed using a neutral medium;
[10] The method for producing alginic acid or alginate according to [9], wherein the culturing step includes a step of culturing the microalgae using an alkaline medium after using a neutral medium;
[11] The method for producing alginic acid or alginate according to any one of [8] to [10], wherein the culturing step includes a step of culturing the microalgae in an open system;
[12] Alginic acid or alginate produced by the method according to any one of [8] to [11] is provided.

According to the present invention, a novel useful microalgae and its application are provided. The Parachlorella microalgae of the present invention can be easily grown under a general environment and secretes sodium alginate, so that it can be used for the production of alginic acid and its salts.
The microalgae of the present invention has a high chlorophyll content, particularly a high content of chlorophyll b that absorbs blue light reaching deep water. Therefore, the microalgae of the present invention can efficiently perform photosynthesis even in an overcrowded culture condition with a large number of cells in a deep culture vessel.
In addition, the microalgae of the present invention performs an endospore-forming tetrasplitting type or an endospore-forming eight-splitting division mode. That is, the mother cell wall is removed immediately after division, and the cell wall is formed after division is completed through a state close to a protoplast consisting of only the cell walls of immature daughter cells. Therefore, it is also convenient when extracting cell contents, further performing transformation using gene transfer, gene recombination, or breed improvement using RNAi.

FIG. 1-1 is a diagram showing micrographs of the Parachlorella querseri strain and the strain of the present invention. Panel A is Parachlorella kueseri, 2152 strains; Panels B-E are Parachlorella kueseri, 2153 strains; Panels F and G are Parachlorella kueseri, 2154 strains; Panel H is a strain of the present invention. For panels D and H, a fluorescence microscope was used, and for other cases, an optical microscope was used.
FIG. 1-2 is a diagram showing micrographs of the Parachlorella kucereri strain and the strain of the present invention. Panel A is Parachlorella kueseri, 2152 strains; Panels B-E are Parachlorella kueseri, 2153 strains; Panels F and G are Parachlorella kueseri, 2154 strains; Panel H is a strain of the present invention. For panels D and H, a fluorescence microscope was used, and for other cases, an optical microscope was used.
1-3 is a figure which shows the microphotograph of the parachlorella kesrel strain and the strain | stump | stock of this invention. Panel A is Parachlorella kueseri, 2152 strains; Panels B-E are Parachlorella kueseri, 2153 strains; Panels F and G are Parachlorella kueseri, 2154 strains; Panel H is a strain of the present invention. For panels D and H, a fluorescence microscope was used, and for other cases, an optical microscope was used.
1-4 is a figure which shows the microphotograph of the parachlorella kesrel strain and the strain | stump | stock of this invention. Panel A is Parachlorella kueseri, 2152 strains; Panels B-E are Parachlorella kueseri, 2153 strains; Panels F and G are Parachlorella kueseri, 2154 strains; Panel H is a strain of the present invention. For panels D and H, a fluorescence microscope was used, and for other cases, an optical microscope was used.
FIG. 2 is a transmission electron micrograph image (10,000 times) of the strain of the present invention. In the figure, “C” represents a chloroplast and “N” represents a nucleus.
FIG. 3 is a diagram showing a differential interference microscopic image (400 times) of the strain of the present invention. The part surrounded by a circle represents the cell of the strain of the present invention, and the part surrounded by a dotted line represents a sodium alginate layer and commensal bacteria.
FIG. 4 is a diagram showing the results (spectrum) of the FT-IR (Fourier transform infrared spectrophotometer) measurement of the cell lysate of the present invention and the standard sodium alginate.
FIG. 5 is a view showing a molecular phylogenetic tree containing the strain of the present invention and related species, which was prepared based on the 18S rRNA sequence. The lower left scale represents 0.01 genetic distance.
FIG. 6 is a diagram showing alginate production by the strains of the present invention cultured in media having different pHs.

1. Method for Isolating Microalgae of the Present Invention The microalgae of the present invention can be isolated from a freshwater sample collected outdoors by subculture. Specifically, for example, a small amount of sample is added to a liquid medium (2.5 g / L KNO ₃ , 7.5 g / L MgSO ₄ .7H ₂ O, 17.5 g / L KH ₂ PO ₄ , 2.5 g / L CaCl ₂ , 2.5 g / L NaCl, 10 g of each solution of 20 g / L NH ₄ H ₂ PO ₄ and 940 mL of distilled water, 1 drop of 1% FeCl ₃ and 2 mL of Arnons A5 solution (composition: 2.86 g in distilled water) / L H ₃ BO ₄ , 1.81 g / L MnCl ₂ .4H ₂ O, 0.222 g / L ZnSO ₄ .7H ₂ O, 0.079 g / L CuSO ₄ .5H ₂ O, 0.015 g / L (NH ₄ ) 5Mo ₇ O ₂₄ · 4H ₂ O) added, pH 6.5; hereinafter referred to as “inorganic III medium”), 100 μL of this culture medium is collected, and 1. 5% W / V) was cultured similarly spread on plate medium plus agar (hereinafter referred to as "plating medium"), to select the green colonies. After this operation was repeated until the colonies became uniform, LB plate medium (5 g peptone, 2.5 g yeast extract, 0.5 g NaCl was dissolved in 1,000 mL of distilled water and 1.5% (W / V). Confirm that there is no bacterial contamination at pH 6.5) with agar added.
The microalgae of the present invention can grow under aerobic or anaerobic conditions in a general medium such as fresh water or LB medium, but in the above liquid medium, room temperature to 30 ° C, It grows and grows particularly well under light conditions and aerobic (shaking culture) conditions. The microalgae of the present invention is photorequiring (minimum 700 Lux, preferably about 2,000 to about 20,000 Lux, most preferably about 3,000 to about 8,000 Lux).
As for the subculture conditions, it is desirable to subculture at intervals of about 2 weeks to about 1 month.
The isolated strain described in Example 1 described later was deposited on February 28, 2008 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, 6th), It was assigned the accession number FERM P-21513 and transferred to the international deposit under the "Budapest Convention on the International Approval of Deposit of Microorganisms in Patent Procedures" as FERM BP-10969 on May 23, 2008.
2. Algalological properties of the microalgae of the present invention Morphological features The microalgae of the present invention is a single cell, is spherical, and has a size of about 12 μm to about 15 μm. The amount of chlorophyll is high, it is green, and it may have multiple mitochondria. When measured by absorptiometry, the total amount of chlorophyll is about twice that of chlorella ( Chlorella vulgaris ), and reaches about 800 to 1,000 mg per 100 g of dry weight of algae. In chlorella, chlorophyll a accounts for about 75 to 90% of the total amount of chlorophyll, whereas in the microalgae of the present invention, the ratio of chlorophyll b is large, and the ratio of chlorophyll a: chlorophyll b content is about 1: 1. ˜about 2.5: 1 (about 50% vs. about 50% to about 71% vs. about 29%).
The cell size may vary depending on the culture conditions. Ogbonna medium (500 mg / L KNO ₃ , 1800 mg / L MgSO ₄ .7H ₂ O, 1250 mg / L KH ₂ PO ₄ , 100 mg / L K ₂ HPO ₄ , When cultured in 30 mg / L FeSO ₄ · 7H ₂ O, 40 mg / L EDTA, Arnons A5 solution 1 mL / L and glucose 5 g / L; pH 7.5), it can be smaller than when cultured in inorganic III medium (about 10 μm to about 12 μm).
B. Reproductive patterns There are many types of treboxya algae called endospore-forming types. The mode of division of the microalgae of the present invention was determined to be an endospore-forming quartile type based on microscopic observation.
In addition, when it culture | cultivated in Ogbonna culture medium, it became clear that it might take the endospore formation octosis type division | segmentation mode.
C. Physiological and biochemical properties Microalgae have a very high ability to fix CO _{2 and} are known to synthesize starch as a photosynthetic product. However, the microalgae of the present invention was found to be negative for the iodine starch reaction.
In the microalgae of the present invention, some individuals are stained red by ruthenium red staining. Therefore, it is considered that the microalgae of the present invention itself secretes a substance containing uronic acid. In particular, the microalgae of the present invention can secrete alginic acid as a viscous polysaccharide-like substance. Depending on the growth environment, this alginic acid may create a biofilm-like commensal microbial community in which microorganisms are trapped (FIG. 3).
3. Molecular phylogeny based on the positional 18S rRNA sequence taxonomic of this strain, microalgae of the present invention belongs to the para Chlorella (Parachlorella) genus, and closely known species para Chlorella Kesureri (Parachlorella kessleri) It became clear that there was. FIG. 5 shows a molecular phylogenetic tree containing the strain of the present invention and related species.
The base sequence of the 18S rRNA gene of the strain of the present invention is shown in SEQ ID NO: 1 in the sequence listing.
4). Alginic acid production method using the microalgae of the present invention As described above, since the microalgae of the present invention produces alginic acid, by culturing the microalgae of the present invention and recovering alginic acid from the culture or culture solution Alginic acid can be obtained.
Specifically, for example, the microalgae of the present invention can be cultured in a large amount in the above-mentioned liquid medium (inorganic III medium) or other medium such as Ogbonna medium, and alginic acid can be separated and recovered by filtration from the culture solution. it can. It is advantageous in terms of growth rate that the culture is performed in a neutral (eg, pH 6 to 8, preferably pH 6.5 to 7.5) culture solution. By giving a stimulus such as contamination of various bacteria during the culture, secretion of alginic acid is promoted. Therefore, for example, at the final stage of the culture in which the microalgae of the present invention is sufficiently grown, the culturing is further performed in an open system for several tens of minutes to several hours, so that the alginate can be easily separated and recovered. In addition, it was found that by making the pH of the culture medium alkaline, the amount of alginic acid increased, and in particular, a relatively large amount of alginic acid having a relatively low molecular weight (low polymerization degree) was produced. Therefore, the culture of the microalgae of the present invention can be performed using a liquid medium (inorganic III medium) or other medium such as Ogbonna medium as described above, but the separation of alginic acid secreted into the culture solution is possible. -It is advantageous to culture in an alkaline medium before recovery. Either one or both of the culture in an open system and the culture in an alkaline medium may be performed at the final stage of the culture.
In addition, the method used for extracting alginic acid from brown algae can be applied to the microalgae of the present invention. In this case, after drying and pulverizing the microalgae of the present invention, alkali (sodium hydroxide, sodium carbonate, etc.) can be added to elute it from the cells as sodium alginate (gel) and collected by filtration. .

Unless otherwise stated, all culture media and instruments used were sterilized with 121 ° C. (autoclave) or 80% (V / V) ethanol.
1. Isolation and selection of this strain Using an aqueous solution sample collected from a factory wastewater treatment plant in Gifu Prefecture, light irradiation liquid subculture was performed by dilution to isolate microalgae.
The “liquid medium” (inorganic III medium) used in this experiment was prepared as follows. 2.5 g / L KNO ₃ , 7.5 g / L MgSO ₄ .7H ₂ O, 17.5 g / L KH ₂ PO ₄ , 2.5 g / L CaCl ₂ , 2.5 g / L NaCl, and 20 g / L NH 10 mL of each aqueous solution of ₄ H ₂ PO ₄ was added to 940 mL of distilled water, 1 drop of 1% (W / V) FeCl ₃ and 2 mL of Arnons A5 solution were added, and the pH was adjusted to 6.5. And autoclaving at 121 ° C. for 15 minutes. The solid medium was prepared by adding agar to the liquid medium to a concentration of 1.5% (W / V) before adjusting the pH (plate medium).
“LB (liquid) medium” was prepared by dissolving 5 g peptone, 2.5 g yeast extract, 0.5 g NaCl in 1,000 mL of distilled water, adjusting the pH to 6.5, and then autoclaving at 121 ° C. for 15 minutes. . The solid medium was prepared by adding 1.5% (W / V) agar to the liquid solution before adjusting the pH (LB solid medium).
100 μL of the aqueous solution sample was weighed and cultured in a liquid medium. The culture conditions were basically room temperature or 30 ° C., light conditions, stirring with a stirrer, and cultured for 1 week. 100 μL of this culture solution was weighed, applied to a flat plate medium, and cultured basically under the same culture conditions as described above.
Among the colonies formed on the plate medium, colonies showing a green color were picked up, placed in a liquid medium, and cultured under basically the same culture conditions as described above.
The above liquid culture and plate culture cycle was repeated until no other colonies appeared on the plate medium.
As a result of liquid culture after repeating the above cycle twice (cycle 2) and three times (cycle 3) and microscopic observation of the plate medium, several types of bacteria other than green microorganisms were confirmed in these samples. . This included motility microorganisms.
Therefore, colonies obtained after repeating the above cycle three times or more were cultured in a liquid medium at 30 ° C. under bright conditions for 10 days. 10 μL of isodine (trade name, 700 mg / L solution of poppyon iodine) was added to this culture solution (5 mL), stirred well, and then allowed to stand at 30 ° C. under anaerobic and bright conditions for 24 hours. 100 μL of this liquid medium was added to 5 mL of liquid medium and cultured for 14 days at 30 ° C. under aerobic and light conditions.
Next, 0.01% (V / V) sodium hypochlorite was added to the culture solution and allowed to stand for 3 hours, and then the culture solution was applied to a plate medium and an LB plate medium at 30 ° C. Cultivation was carried out for 10 to 12 days under light and bright conditions.
As a result, it was possible to isolate a single colony containing no bacteria. This colony was grown under aerobic conditions in a liquid medium by culturing at 30 ° C. for 3 days.
This strain (Parachlorella binos; hereinafter may be abbreviated as “Binos”) was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on February 28, 2008, and the deposit number FERM P- No. 21513 was granted and transferred and accepted as an FERM BP-10969 on May 23, 2008, under the “Budapest Convention on the International Approval of Deposits of Microorganisms in Patent Procedures”.
2. Microscopic observation, dyeability, etc. i) Iodine starch reaction Starch was quantified by an enzymatic method using the strain obtained above and Chlorella ( Chlorella vulgaris ) manufactured by Chlorella Kogyo Co., Ltd. as a sample. Specifically, 50% ethanol is used to extract and wash low molecules, etc., insoluble matter is heated and gelatinized, treated with glucoamylase, and filtered to obtain the glucose of the filtrate, “CII Test Wako” (Trade name) was used for quantification. From this result, the amount of starch was calculated as glucose (%) × 0.9 = starch (%). As the amount of starch, 0.2% was obtained for the strain of the present invention, and 0.9% was obtained for chlorella. Obtained. However, in this method, since glucose is a constituent component and there is a possibility that a polysaccharide having a binding different from starch may be used as a measurement control, each sample (for the strain of the present invention, the color is dark and remains as it is). In this case, the iodine starch reaction was performed with respect to (using the liquid after filtration through a membrane filter having a pore diameter of 0.45 μm), and all of them were negative for the iodine starch reaction.
ii) Ruthenium red staining The strain obtained above was directly or after ruthenium red staining, and then observed under a microscope ("Leica DM1 6000B", manufactured by Leica). In ruthenium red staining, a microorganism sample is collected by centrifugation (3,000 rpm, 5 minutes), a ruthenium red aqueous solution (manufactured by Nacalai Tesque) is added, and the mixture is allowed to stand at room temperature for about 1 hour. This was done by washing.
iii) Microscopic observation Three strains (2152, 2153, and 2154) of nine types of Parachlorella querserili strains stored in the National Institute for Environmental Studies Microbial System Conservation Center were obtained, and the same observation was performed. Comparison with the strain of the present invention. In addition, there is no information on how far these Kessler strains are genetically separated.
The results of microscopic observation are shown in FIGS. As a result of observation, it was found that all of these three strains secreted some sort of viscous polysaccharide. Of these three strains, the 2152 strain is most similar to the parachlorella binos of the present invention in appearance, but has a smaller cell body than the binos (FIG. 1-1, panel A), Ruthenium red staining was negative. Since the 2153 strain was positive for ruthenium red staining in the same manner as the strain of the present invention (FIG. 1-4, panel H), the secretion was considered to be a compound containing uronic acid such as alginic acid (FIG. 1-1). Panel B to FIGS. 1-3, Panel E). However, the strain of the present invention was endospore-forming quadrilateral, whereas strain 2153 was endospore-forming eight-splitting. The 2154 strain could be seen as an individual having a larger cell body than that of the strain of the present invention, but the cell contained clearly less chloroplasts, so it seemed clear. It was clear from observation using a fluorescence microscope that there were few chloroplasts (FIGS. 1-3, Panel F and FIGS. 1-4, Panel G). It was also different from the strain of the present invention in that it was negative for ruthenium red staining.
These characteristics are summarized as follows.
iv) Electron microscope observation The strain of the present invention was also observed with a transmission electron microscope (Hitachi, "HITACHI H-7000"). An electron micrograph image (10,000 times) is shown in FIG. In the figure, “C” is a chloroplast and “N” is a nucleus. About 70% of the visual field area is chloroplasts, indicating that the strain of the present invention stores a large amount of chloroplasts. Note that the light-colored layer surrounding the outer periphery of the cell is considered to be alginic acid.
v) Observation of Microbial Community Containing Microalgae of the Present Invention The strain of the present invention cultured in an open system was observed with a differential interference microscope (Leica, “LEICA DM1 6000B”, magnification 400 ×). The result is shown in FIG. It has been found that at least four types of microorganisms including nitrogen-fixing bacteria are trapped by the viscous polysaccharide secreted by the strain of the present invention, thereby creating a symbiotic microbial community such as a biofilm.
3. Identification of secretions To Parachlorella binos (2,000 mg / L) cultured in a liquid medium as described above, Na ₂ CO ₃ was added to a final concentration of 2% (W / V). Cells were lysed. The lysate was centrifuged at 3,000 rpm for 10 minutes to precipitate impurities, and the supernatant was filtered through a 0.4 μm filter. The residue remaining on the filter was washed with 1N hydrochloric acid and 1N NaOH, and subjected to FT-IR (Fourier transform infrared spectrophotometer) measurement.
The result of this analysis is shown in FIG. When compared with the spectrum of the standard sodium alginate standard, it was strongly suggested that the polysaccharide secreted from the parachlorella binos of the present invention was alginic acid.
4). Measurement of chlorophyll content Parachlorella binos and chlorella ( Chlorella vulgaris , manufactured by Chlorella Kogyo Co., Ltd.) cultured as described above were used as samples, and the amount of chlorophyll was measured by spectrophotometry at the Japan Food Analysis Center did.
The results are shown in Table 2.
Vinos has a significantly higher total chlorophyll amount, chlorophyll a amount and chlorophyll b amount than chlorella, and is about twice as much as the total chlorophyll amount, chlorophyll a amount and chlorella. It was twice. The ratio of chlorophyll a and chlorophyll b was about 9: 1 for chlorella, but about 2: 1 for the strain of the present invention.
5. Base sequence analysis of 18S rRNA gene Genomic DNA extracted from Parachlorella binos of the present invention was used as a template, and the target gene was amplified by PCR using universal 18S rRNA gene primers, followed by agarose gel electrophoresis The target gene was excised from the gel and ligated to a TA vector (Promega, trade name “P-GemT”). Thereafter, E. coli was transformed with this plasmid, and after screening by blue-white selection, the plasmid was extracted by the alkali-SDS method. Subsequently, it was commissioned to Biomatrix Laboratories Co., Ltd., and the nucleotide sequence was analyzed from the sp6 and t7 sites of this plasmid.
The nucleotide sequence data was aligned with the closely related 18S rRNA gene sequences registered in NCBI using a published multiple alignment program “Clustal W” to construct a phylogenetic tree (neighboring linkage method).
The results are shown in FIG. As a result, it was found that the strain of the present invention is a new species closely related to Parachlorella kueseri.
Further, the base sequence of the 18S rRNA gene of the strain of the present invention obtained by the above analysis is shown in SEQ ID NO: 1 in the sequence listing.
6). Production of alginic acid by parachlorella binos in medium of different pH Incubated in inorganic III medium at 25 ° C. for over 1 week, parachlorella binos with a cell concentration of 10 ⁶ cells / L were placed in three containers. Dispensed. The pH of the culture solution in each container was adjusted to pH 5, pH 7, and pH 10 using hydrochloric acid and sodium hydroxide. This was cultured at 25 ° C. in the dark, and sampled on the first, second, third, sixth, and eighth days, and the amount of nonvolatile organic carbon (NPOC) was measured as an index of alginic acid production. .
Specifically, the amount of NPOC was measured as follows. The culture solution containing Parachlorella binos collected at each time point was centrifuged at 16,500 × g for 1 minute to sediment the cells. The supernatant was collected, and an equal volume of isopropanol was added, followed by centrifugation at 16,500 × g for 10 minutes. A sample obtained by dissolving the obtained pellet in sterilized distilled water was used as a sample. Using TPOC-v manufactured by Shimadzu Corporation, the amount of NPOC in each sample was measured in accordance with a measurement method by acidification and aeration treatment (JIS-K0551, ASTM-D2579).
The results are shown in FIG. In FIG. 6, the diamond (♦) represents pH 5, the square (■) represents pH 7, and the triangle (▲) represents pH 10. As is clear from FIG. 6, it was found that the strain of the present invention secretes a larger amount of alginic acid when cultured under alkaline conditions than when under acidic or neutral conditions.
7). Characterization of Parachlorella binos secretion In this experiment , Parachlorella binos were cultured in an inorganic III medium for 1 week or more at 25 ° C., and the cell concentration was 10 ⁶ cells / L or more. 0.5 mL of this culture solution containing Parachlorella binos was centrifuged at 16,500 × g for 1 minute to precipitate the cells. An equal amount of isopropanol was added to the supernatant, followed by centrifugation at 16,500 × g for 10 minutes. A sample obtained by dissolving the obtained pellet in sterile distilled water was used as a sample, and fractionated using an ultrafiltration membrane (As One, trade name “Vivaspin”). The amount of alginic acid contained in each fraction is determined according to the above 6. The NPOC was measured by the method described in 1.
As a result, all the alginic acid in the sample passed through the ultrafiltration membrane having a fractional molecular weight of 100,000. On the other hand, when an ultrafiltration membrane with a molecular weight cut off of 10,000 was used, a part was trapped but the rest passed.
Therefore, the alginic acid of the secretion of Parachlorella binos has a molecular weight of approximately 100,000 or less, most of which has a molecular weight of 20,000 or less, and is considered to include those having a molecular weight of 10,000 or less.
8). Cultivation of Parachlorella binos in different media Parachlorella binos of the present invention was cultured using the following media. “Ogbonna medium” was prepared as follows. 500 mg / L KNO ₃ , 1800 mg / L MgSO ₄ .7H ₂ O, 1250 mg / L KH ₂ PO ₄ , 100 mg / L K ₂ HPO ₄ , 30 mg / L FeSO ₄ .7H ₂ O, 40 mg / L EDTA in distilled water After adding 1 mL of Arnons A5 solution and 5 g of glucose and adjusting the pH to 7.5 using sodium hydroxide, it was autoclaved at 121 ° C. for 20 minutes. This medium is described in Ogbonna et al. J. of Applied Physology, Vol. 9, no. 4, pp. 359-366 (1997). The above 1. except that this medium was used instead of the liquid medium. The cells were cultured for 7 days under the same conditions (30 ° C., aerobic, bright conditions).
2. Microscopic observation was performed in the same manner as described above. When cultured in this medium, Parachlorella binos
Form: Single cell, spherical, size of about 10-12 μm
Reproductive pattern: Endospore-forming octuploid type.
This application is based on Japanese Patent Application No. 2008-220213 filed on Aug. 28, 2008. All the contents described in the specification and claims of Japanese Patent Application No. 2008-220213 Included in the application specification.
[Sequence Listing]

Claims (11)

Para genus Chlorella microalgae with alginate-producing ability, para Chlorella Bainosu (Parachlorella sp. Binos). The microalgae according to claim 1, having the following characteristics.
Form: Single cell, spherical, size 10 μm to 15 μm
Reproductive pattern: Endospore-forming quadrant or endospore-forming octa-splitting ruthenium red staining: Positive The microalgae according to claim 2, wherein when cultured in an inorganic III medium, the microalgae have a size of 12 µm to 15 µm and a reproductive mode of endospore-forming quadrilateral. The microalgae according to claim 2, having a size of 10 µm to 12 µm when cultured in an Ogbonna medium. The content of chlorophyll a and chlorophyll b in the cells is 1 : 1 to 2 . The microalgae according to any one of claims 1 to 4, which is 5: 1.   The microalgae according to any one of claims 1 to 5, which has an 18S rRNA gene represented by SEQ ID NO: 1.   The microalgae according to any one of claims 1 to 6, which has a deposit number of FERM P-21513 (FERM BP-10969).   A method for producing alginic acid or alginate, comprising culturing the microalgae according to any one of claims 1 to 7 and recovering alginic acid or alginate from the culture or culture medium.   The method for producing alginic acid or alginate according to claim 8, wherein the culturing step is performed using a neutral medium.   The method for producing alginic acid or alginate according to claim 9, wherein the culturing step includes a step of culturing the microalgae using an alkaline medium after using a neutral medium. The method for producing alginic acid or alginate according to any one of claims 8 to 10, wherein the culturing step includes a step of culturing the microalgae in an open system.