Method for producing polyhydroxyalkanoic acid using alginic acid as a carbon source

Abstract

To provide methods for producing polyhydroxyalkanoic acid (PHA) using alginic acid as carbon source, and microorganisms capable of synthesizing PHA using alginic acid as a carbon source.SOLUTION: Disclosed herein a method for producing PHA comprising culturing a microorganism having an alginic acid assimilation ability and a PHA synthesizing ability in a culture medium containing any one of selected from the group consisting of alginic acid and decomposition products thereof, and extracting PHA from the culture. Also disclosed is a bacterial strain of the genus Cobetia having 16S rRNA gene which comprises: (a) a base sequence represented by a specific sequence number; or (b) a base sequence having 90% or more identity to a base sequence represented by a certain sequence number.SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid using alginic acid as a carbon source. [Background technology]

[0002] Biodegradable plastics are polymeric materials that have the same properties and functions as ordinary plastics when not in contact with microorganisms, but when placed in the natural environment or a composting system, they are assimilated by microorganisms and ultimately decompose into carbon dioxide and water. There are two types of biodegradable plastics: biodegradable petroleum-derived plastics, which use fossil resources as raw materials, and biodegradable bioplastics, which are made from biomass. With the increase in carbon dioxide emissions and global warming becoming problems, there is a demand for materials like bioplastics, which are made from biomass and ultimately decompose into water and carbon dioxide, and do not accumulate in the environment.

[0003] The Japan Bioplastics Association states that a product must have a bioplastic content of 25% by mass or more in order to comply with the Biomass Plastic Identification Standards. Products with a higher bioplastic content have a lower environmental impact. Examples of bioplastics made from 100% biomass raw materials include polylactic acid and polyhydroxyalkanoic acid (PHA).

[0004] PHAs are aliphatic polyesters synthesized by microorganisms. They play a physiological role as carbon sources and energy compounds. They accumulate intracellularly when a carbon source is present under nutrient-depleted conditions, such as nitrogen and phosphorus shortages. Polyhydroxybutanoic acid (P(3HB)), one of the most studied PHAs, was discovered in Bacillus megaterium by Dr. Lemoigne in 1925. Since then, its synthesis by over 300 species of bacteria, including hydrogen-producing bacteria and cyanobacteria, has been reported. Due to its brittle nature (elongation at break of 5%), P(3HB) was considered inferior to polypropylene for commercial use. However, since Wallen and Rohwedder discovered a hydroxyalkanoic acid (HA) unit different from the 3-hydroxybutanoic acid unit (3HB) in 1974, various HAs have been reported, with over 150 types of monomer units reported to date. Furthermore, various studies have shown that the physical properties of PHAs can be improved by incorporating monomer units other than 3HB to form copolymers (Non-Patent Documents 1-6).

[0005] Microbial biosynthesis of PHA using various organic substances as carbon sources has been reported. For example, Patent Document 1 discloses a method for producing polyhydroxyalkanoic acid by a microorganism, comprising culturing a microorganism belonging to the genus Cupriavidus, Ralstonia, or Alcaligenes that can assimilate lignin derivatives and / or their polyhydroxyalkanoic acid biosynthesis intermediates in a medium containing, as a carbon source, at least one substance selected from the group consisting of lignin derivatives and their polyhydroxyalkanoic acid biosynthesis intermediates, and recovering polyhydroxyalkanoic acid from the microbial cells. Patent Document 2 discloses a method for producing a polyhydroxyalkanoic acid copolymer, which comprises (i) culturing a transformant in which a polyhydroxyalkanoic acid polymerase mutant gene and a leucine metabolic gene derived from a Clostridium microorganism have been introduced into a microorganism in the presence of sugars, and (ii) collecting a polyhydroxyalkanoic acid copolymer containing 3-hydroxybutanoic acid and 3-hydroxy-4-methylvaleric acid as monomer units from the resulting culture, wherein the sugars are monosaccharides (glucose).

[0006] Patent Document 3 discloses a method for producing polyhydroxyalkanoic acid by microorganisms by polymerizing monomer units containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, characterized in that the method uses fats and oils containing 41% by weight or more of lauric acid as a constituent fatty acid and / or fatty acids as a carbon source, and cultures the microorganisms while adjusting the oxygen transfer rate so that the average hourly productivity of polyhydroxyalkanoic acid from the start of culture is 1.5 g / L / h or more. Other examples include a method for producing polyhydroxyalkanoic acid or a polyhydroxyalkanoic acid copolymer using a microorganism that uses sugars (glucose, fructose, sucrose), fats or oils or fatty acids as a carbon source (Patent Documents 4 to 7); a method for producing polyhydroxyalkanoic acid by fermentation using a microorganism, in which the carbon source includes a carboxylic acid having four carbon atoms and / or an alcohol having four carbon atoms and a derivative thereof (Patent Document 8); a method for producing a microbial polyester, in which a microorganism capable of producing a polyester is cultured in a medium containing 1-hexene as the sole carbon source (Patent Document 9); and a method for producing polyhydroxyalkanoic acid, in which a microorganism capable of growing by assimilating acetic acid or a salt thereof and producing and accumulating polyhydroxyalkanoic acid is cultured in a medium containing acetic acid or a salt thereof as the sole carbon source (Patent Document 10).

[0007] PHA production using sugars and alcohol extracted from edible raw materials such as corn and sugarcane competes with food supplies, driving up prices. Therefore, research into utilizing unused biomass is expected to be a technology with minimal impact on the global environment. In recent years, seaweed has attracted global attention as one of the unused biomass resources. In Japan, a large amount of seaweed is discarded during seaweed production and processing, with approximately 60% of the harvest in wakame seaweed processing being discarded (Non-Patent Document 7). This seaweed waste must be disposed of as industrial waste, placing a heavy burden on fishermen and companies, and illegal dumping of seaweed waste is also a problem. Seaweeds belonging to the brown algae family, such as kelp and wakame seaweed, have been reported to contain 20–40% alginic acid per dry weight (Non-Patent Document 8). Furthermore, the alginic acid content in seaweed remains nearly constant throughout the year (Non-Patent Document 9). This makes alginic acid a promising raw material from marine resources. However, although there have been reports of PHA biosynthesis using cellulose and mannitol, which are major components of seaweed other than alginic acid, as carbon sources (Non-Patent Documents 10-12), there have been no reports of PHA biosynthesis using alginic acid as a carbon source. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-077103 [Patent Document 2] Japanese Patent Application Publication No. 2015-33 [Patent Document 3] Japanese Patent Application Laid-Open No. 2013-9627 [Patent Document 4] WO2012 / 102371 [Patent Document 5] WO2009 / 147918 [Patent Document 6] Japanese Patent Application Laid-Open No. 2009-225662 [Patent Document 7] Patent Application No. 2006-238032 [Patent Document 8] Japanese Patent Application Laid-Open No. 2009-225775 [Patent Document 9] Japanese Patent Application Laid-Open No. 2001-178487 [Patent Document 10] Japanese Patent Application Laid-Open No. 2001-178484 [Non-patent literature]

[0009] [Non-Patent Document 1] Kenichiro Matsumoto, Yoshiharu Doi, Biosynthesis of Biodegradable Polyesters: Current Status and Prospects, Journal of the Society of Synthetic Organic Chemistry, Vol. 61, No. 5, pp. 489-495, 2003 [Non-patent document 2] K. Sudesh, T Iwata, Sustainability of Biobased and Biodegradable Plastics, Clean 2008, 36(5-6), 433-442 [Non-patent document 3] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Prog. Polym. Sci., 25(10), 1503-1555, (2000) [Non-patent document 4] Tadahisa Iwata, Structure, Material and Biodegradability of Microbial Polyesters, Journal of the Crystallographic Society of Japan, 55, 188-196(2013) [Non-Patent Document 5] Wallen LL, Rohwedder WK, Poly-β-hydroxyalkanoate from activated sludge, Environ Sci Technol 8: 576-579 (1974) [Non-patent document 6] JML Dias, PC Lemos, LS Serafim, C. Oliveria, M. Eiroa, MGE Albuquerque, AM Ramos, R. Oliveira, M. A M. Reis, Recent Advances in Polyhydroxyalkanoate Production by Mixed Aerobic Culture: From the Substrate to the Final Product, Macromol. Biosci., 6(11), 885-906 (2006) [Non-Patent Document 7] Shinichiro Fujii, Takashi Korenaga, Material flow analysis and zero-emission technology in the wakame seaweed processing industry, Journal of Environmental Science 13(5), 586-592, (2000) [Non-patent document 8] Hiroyuki Takeda, Fuminori Yoneyama, Shigeyuki Kawai, Wataru Hashimoto and Kousaku Murata, Bioethanol production from marine biomass alginate by metabolically engineered bacteria, Energy Environ. Sci., 4, 2575-2581, (2011) [Non-Patent Document 9] T. Kimura, K. Ueda, R. Kuroda, T. Akao, N. Shinohara, T. Ushirokawa, A. Fukagawa and T. Akimoto, Nippon Suisan Gakkaishi, The seasonal variation in polysaccharide content of brown alga akamoku Sargassum horneri collected off Oshima Island (Fukuoka Prefecture), Nippon Suisan Gakkaishi, 73(4), 739-744, (2007) [Non-Patent Document 10] LE Alva Munoz, MR Riley, Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans, Biotechnol. Bioeng., 100(5), 882-888, (2008) [Non-Patent Document 11] F. Cerrone, R. Davis, ST Kenny, T. Woods, A. O'Donovan, VK Gupta, M. Tuohy, RP Babu, P. O'Kiely, K. O'Connor, Use of a mannitol rich ensiled grass press juice (EGPJ) as a sole carbon source for polyhydroxyalkanoates (PHAs) production through high cell density cultivation, Bioresour. Technol., 191, 45-52, (2015) [Non-Patent Document 12] M. Yamada, A. Yukita, Y. Hanazumi, Y. Yamahata, H. Moriya, M. Miyazaki, T. Yamashita, H. Shimoi, Poly(3-hydroxybutyrate) Production using mannitol as a sole carbon source by Burkholderia sp. AIU M5M2 isolated from a marine environment, Fisheries Science, 84(2), 405-412, (2018) Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, PHA biosynthesis using sugars, alcohols, and other carbon sources has been reported in the past. However, because these carbon sources compete with food, there has been a need to develop a method for producing bioplastics using unused biomass as a raw material.

[0011] Alginic acid is abundant in seaweed and is a promising marine resource. However, there have been no reports on PHA biosynthesis using alginic acid as a carbon source. Therefore, the present invention aims to provide a method for producing PHA using alginic acid as a carbon source and to provide a microorganism capable of synthesizing PHA using alginic acid as a carbon source. [Means for solving the problem]

[0012] As a result of extensive research to solve the above problems, the inventors discovered a microorganism capable of synthesizing PHA using alginic acid as a carbon source, and a method for producing PHA using this microorganism, thereby completing the present invention. Specifically, the present invention provides the following [1] to

[13] . [1] A method for producing polyhydroxyalkanoic acid (PHA), comprising culturing a microorganism capable of assimilating alginic acid and synthesizing PHA in a culture medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture. [2] The manufacturing method described in [1], which includes a preliminary step of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or acid. [3] The method according to [1] or [2], wherein the microorganism is selected from marine bacteria belonging to the Gammaproteobacteria class. [4] The method according to any one of [1] to [3], wherein the microorganism is any one selected from bacteria belonging to the genus Cobetia. [5] The method according to any one of [1] to [4], wherein the microorganism is selected from the following 1) or 2): 1) Microorganisms deposited under accession number NITE P-02758 or NITE P-02759. 2) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b): (a) The base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2. [6] The method according to any one of [1] to [5], wherein the microorganism is an isolated microorganism, a deposited microorganism, or a genetically modified microorganism. [7] The method according to any one of [1] to [6], wherein the concentration of alginic acid or alginic acid decomposition product in the medium is in the range of 0.5% by mass to 20% by mass. [8] The method according to any one of [1] to [7], wherein the concentration of sodium chloride in the medium is in the range of 0.5% by mass to 20% by mass. [9] The method according to any one of [1] to [8], wherein the PHA contains a 3-hydroxybutanoic acid monomer unit.

[10] The method according to [9], wherein the PHA is poly(3-hydroxybutanoic acid).

[11] A step of obtaining polyhydroxyalkanoic acid (PHA) by carrying out the method according to any one of [1] to

[10] ; A method for producing a bioplastic product, comprising the step of obtaining a plastic product containing 25% by mass or more of the PHA obtained in the above step.

[12] Microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759.

[13] A bacterium of the genus Covetia having a 16S rRNA gene containing the following nucleotide sequence (a) or (b): (a) The base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2. [Effects of the Invention]

[0013] According to the present invention, a method for producing a bioplastic using alginic acid as a carbon source can be provided. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the biosynthetic pathway of PHA. [Figure 2] Figure 2A shows an example of the state of a natural sample cultured in seawater, and Figure 2B shows an example of the state of a natural sample cultured in agar medium (ZoBell 2216E seawater medium). [Figure 3-1] Figure 3-1 shows some of the results of culturing 121 samples out of the 171 strains obtained in the primary screening. [Figure 3-2] Figure 3-2 shows some of the results of culturing 121 samples out of the 171 strains obtained in the primary screening. [Figure 4A] FIG. 4A shows the results obtained by GC-MS for the 5-28-6-1 strain. [Figure 4B] FIG. 4B shows the results obtained by GC-MS for the 5-11-6-3 strain. [Figure 5A] FIG. 5A shows the results of 1H-NMR analysis of the 5-28-6-1 strain. [Figure 5B] FIG. 5B shows the results of 1H-NMR analysis of the 5-11-6-3 strain. [Figure 6] Figure 6 shows the nucleotide sequences of the 16S rRNA gene. A: strain 5-28-6-1, B: strain 5-11-6-3. [Figure 7-1] Figure 7-1 shows an alignment of the nucleotide sequences of the 16S rRNA gene. [Figure 7-2] Figure 7-1 shows the continuation of the alignment of the 16S rRNA gene nucleotide sequences. [Figure 8] FIG. 8 shows the Gram staining images of the microorganisms strains 5-28-6-1 and 5-11-6-3. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0016] The present invention provides a method for producing polyhydroxyalkanoic acid (PHA), which comprises culturing a microorganism capable of assimilating alginic acid and synthesizing PHA in a culture medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture.

[0017] The production method of the present invention can be carried out using microorganisms capable of alginate assimilation and PHA synthesis. Alginate assimilation is the ability to grow using alginate as a carbon source. PHA synthesis is the ability to biosynthesize PHA. Microorganisms capable of alginate assimilation and PHA synthesis can be said to be microorganisms capable of synthesizing PHA using alginate as a carbon source. Microorganisms capable of alginate assimilation and PHA synthesis may be microorganisms that can assimilate alginate decomposition products in addition to alginic acid and synthesize PHA.

[0018] Whether a microorganism has the ability to utilize alginate can be determined by culturing it in a medium containing alginate as the sole carbon source and determining whether the microorganism grows. For example, a test microorganism is cultured in a medium containing 1% by mass of sodium alginate as the sole carbon source under conditions suitable for the growth of the test microorganism (temperature, pH, oxygen, sodium salt concentration, etc.). If the test microorganism grows, it can be determined that the test microorganism has the ability to utilize alginate. Growth of the test microorganism can be measured by the turbidity method, the plate culture method, the dry cell weight method, etc.

[0019] Whether a microorganism has the ability to synthesize PHA can be determined by culturing it in a nitrogen- or phosphorus-limited medium and determining whether it synthesizes PHA. For example, a test microorganism is cultured in a nitrogen-limited inorganic salt medium containing 1-2% glucose as the sole carbon source under conditions suitable for its growth. If the test microorganism accumulates PHA within its body, it can be determined that the test microorganism has the ability to synthesize PHA. PHA accumulation can be confirmed by Nile Red or Nile Blue A staining, as well as by observing intracellular PHA granules with a transmission electron microscope, NMR analysis of chloroform extracts obtained from dried cells, and gas chromatography or gas chromatography-mass spectrometry of ethanolyzed chloroform extracts. Furthermore, if the accumulated PHA is PHB, it can be converted to crotonic acid with sulfuric acid and then analyzed by high-performance liquid chromatography to determine PHB accumulation.

[0020] The microorganisms having the ability to utilize alginate and synthesize PHA may be halophilic marine bacteria belonging to the class Gammaproteobacteria. The class Gammaproteobacteria belongs to the phylum Proteobacteria, which is a type of eubacteria. Bacteria belonging to the class Gammaproteobacteria are gram-negative bacteria. Marine bacteria are bacteria that live in the ocean and can grow well in seawater. Halophilic bacteria are bacteria that can grow well at a sodium salt concentration of about 0.5 to 20%.

[0021] The microorganism capable of alginate utilization and PHA synthesis may be a bacterium belonging to the genus Cobetia, such as Cobetia marina, Cobetia pacifica, Cobetia amphilecti, and Cobetia litoralis, as well as undescribed species inferred to belong to the genus Cobetia from 16S rDNA sequences. The microorganism capable of alginate utilization and PHA synthesis of the present invention can be selected from the genus Cobetia, preferably Cobetia marina and its related species (including undescribed species), more preferably from the species to which the 5-28-6-1 and 5-11-6-3 strains described in the Examples below belong, with the 5-28-6-1 and 5-11-6-3 strains being particularly preferred. Cobetia marina was previously classified as Arthrobacter marinus, Pseudomonas marina, Dalya marina, and Halomonas marina (Arahal DR, et al., (December 2002) Systematic and Applied Microbiology. 25 (2): 207-11).

[0022] In one embodiment of the present invention, the microorganism capable of assimilating alginic acid and synthesizing PHA can be any one selected from the following 1) or 2). 1) Microorganisms deposited under accession number NITE P-02758 or NITE P-02759. 2) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b): (a) The base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0023] The microorganism can be a microorganism that has a 16S rRNA gene containing a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and that has the ability to assimilate alginate and synthesize PHA. Furthermore, the microorganism can be a microorganism belonging to the genus Covetia that has a 16S rRNA gene containing a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and that has the ability to assimilate alginate and synthesize PHA. The microorganism may also be a microorganism belonging to the genus Covetia, which has a 16S rRNA gene containing a base sequence in which one or several bases have been substituted, inserted, deleted and / or added in the base sequence set forth in SEQ ID NO: 1 or 2, and which has the ability to utilize alginate and synthesize PHA.

[0024] The nucleotide sequence can be analyzed by a conventional method. For example, a method for amplifying the 16S rRNA gene from the DNA of a microorganism includes, but is not limited to, PCR using primers. The amplified product obtained by PCR can be subjected to a DNA sequencer or the like to analyze the nucleotide sequence.

[0025] Sequence identity is defined as the percentage of identical bases between two sequences after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Sequence identity can be determined using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ClustalX, or Megalign (DNASTAR) software.

[0026] As used herein, the range of "one or several" in the "base sequence in which one or several bases have been substituted, inserted, deleted, and / or added" is not particularly limited, but means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0027] The microorganism can be an isolated microorganism, a deposited microorganism, or a genetically modified microorganism. Isolated microorganisms are microorganisms isolated from natural samples. Examples of natural samples include seawater, marine organisms, seaweed, brackish water, brackish water organisms, rocks, soil, and sand. Natural samples can be cultured in any medium, such as ZoBell 2216E seawater medium, MS medium, or Daigo IMK medium, at 10°C to 42°C for 24 to 72 hours at a pH of 5 to 10, and colonies can be isolated. Prior to culturing in ZoBell 2216E seawater medium or MS medium, the microorganisms can be precultured in filtered seawater. Isolated colonies can be cultured in an alginate-containing medium, and colonies that show growth in the alginate-containing medium and are judged to be synthesizing PHA can be selected. Nile Red is a hydrophobic dye that reacts with hydrophobic substances such as neutral lipids and PHAs in living organisms to produce a red color, making it useful for screening microorganisms capable of synthesizing PHA.

[0028] Furthermore, for PHA synthesis, PHA can be extracted from dried microbial cells with an organic solvent such as chloroform, and then precipitated by adding an organic solvent such as methanol or hexane, followed by screening using analytical techniques such as gas chromatography, liquid chromatography, NMR, IR, etc. Furthermore, in some cases, the strains of isolated colonies can be identified based on their 16S rDNA sequences or bacteriological properties.

[0029] The deposited microorganisms can be obtained by selecting microorganisms capable of alginate assimilation and PHA synthesis from the deposited microbial material using the same techniques as those described for the isolated microorganisms. For example, microorganisms capable of alginate assimilation and PHA synthesis can be selected from the deposited marine bacteria belonging to the class Gammaproteobacteria. Furthermore, microorganisms capable of alginate assimilation and PHA synthesis can be selected from the deposited microorganisms belonging to the genus Covetia. The genetically modified microorganism may be a microorganism in which, for example, an alginate decomposing enzyme gene, a PHA synthase gene, and / or genes related thereto have been modified.

[0030] The polyhydroxyalkanoic acid (PHA) for which the production method is provided by the present invention is a polyester known to accumulate in the bodies of certain microorganisms, and has the following chemical formula: [ka] [wherein R may be the same or different and is a linear or branched alkyl group having 1 to 14 carbon atoms, and n is the number of repetitions and is an integer of 2 or more, preferably an integer of 100 or more, and preferably an integer of 100,000 or less]. Specific examples of PHAs include those having the following chemical formula: [ka] Examples of the polyhydroxybutanoic acid include polyhydroxybutanoic acid (P(3HB) or PHB) having the formula: Representative copolymers include poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)], poly(3-hydroxybutanoic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)], poly(3-hydroxypropionic acid) [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)], and poly(3-hydroxybutanoic acid-co-glycolic acid) [P(3HB-co-GL)].

[0031] The biosynthetic pathway of P(3HB) has been well studied in Ralstonia eutropha. It has been reported that two acetyl-CoA molecules are condensed to acetoacetyl-CoA by β-ketothiolase (PhaA), which is then reduced to (R)-3-hydroxybutyryl-CoA by NADPH-dependent acetoacetyl-CoA reductase (PhaB), followed by polymerization by PHA synthase (PhaC) (Fig. 1) (Yamada M. et al., Oleoscience, 5 (11), 523-532 (2005); K. Sudesh et al., Prog. Polym. Sci. 25 (2000) 1503-1555). Furthermore, the biosynthesis of medium-chain 3HA (6-14 carbon atoms) has been studied in Pseudomonas bacteria and Aeromonas caviae, and it has been reported that the β-oxidation of fatty acids and the biosynthetic pathway are involved (Fig. 1) (Hiromi Matsuzaki et al., Journal of the Japan Oil Chemists' Society, 48(12), 1353-1363, 1999; T. Fukui et al., J. Bacteriol, 180(3), 667-673, 1998; T. Fukui et al., J. Bacteriol, 179(15), 4821-4830, 1997).

[0032] The polyhydroxyalkanoic acid (PHA) produced by the method of the present invention can have a molecular weight of 200,000 to 2,000,000. The polyhydroxyalkanoic acid (PHA) produced by the present invention may contain a 3-hydroxybutanoic acid monomer unit, such as poly(3-hydroxybutanoic acid-co-3-hydroxypropionic acid) [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)], poly(3-hydroxybutanoic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)], poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)], or poly(3-hydroxybutanoic acid-co-glycolic acid) [P(3HB-co-GL)]. The polyhydroxyalkanoic acid (PHA) for which the production method is provided by the present invention can be poly(3-hydroxybutanoic acid).

[0033] Culture media and conditions for PHA biosynthesis In the production method of the present invention, the microorganism is cultured in a medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and the microorganism can synthesize PHA using any one selected from the group consisting of alginic acid and alginic acid decomposition products as a carbon source. Alginic acid is a polysaccharide found in brown algae and the like, and is a polymer formed by linear polymerization of two types of uronic acid (β-D-mannuronic acid (M) and its C-5 epimer, α-L-guluronic acid (G)). The alginic acid used in the present invention is not particularly limited and may have any degree of polymerization or any ratio of M to G (M / G ratio). The alginic acid of the present invention also includes alginates, alginate esters, etc. Examples of alginates include sodium alginate, potassium alginate, ammonium alginate, and calcium alginate. Examples of alginic acid decomposition products include partial enzymatic decomposition products and partial hydrolysis products, such as uronic acid.

[0034] The concentration of alginic acid or alginic acid decomposition product in the culture medium for PHA biosynthesis can be appropriately set depending on the type of alginic acid or alginic acid decomposition product and the type of microorganism, but is preferably in the range of 0.5% to 20% by mass, more preferably 1% to 10% by mass, and particularly preferably 1% to 5% by mass. For example, in the case of sodium alginate, the concentration is preferably in the range of 0.5% to 20% by mass, more preferably 1% to 10% by mass, even more preferably 1% to 5% by mass, and particularly preferably 2% to 4% by mass.

[0035] A nitrogen-limited inorganic salt medium can be used as a microbial culture medium for PHA biosynthesis. In addition to the carbon sources mentioned above, the medium can contain an inorganic nitrogen source (e.g., ammonium sulfate, ammonium nitrate, urea). The medium can also contain minerals and trace elements, such as salts of phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), cobalt (Co), copper (Cu), zinc (Zn), and nickel (Ni). Specific examples of substances containing minerals and trace elements include calcium chloride, magnesium sulfate, potassium nitrate, sodium chloride, iron (III) chloride, calcium chloride, cobalt chloride, copper (II) sulfate, nickel (II) chloride, and chromium (III) chloride.

[0036] In the production method of the present invention, cultivation for PHA biosynthesis can be carried out at a temperature in the range of about 20°C to 40°C and a pH in the range of about 5 to 10. The cultivation temperature and pH can be adjusted appropriately depending on the microorganism being cultivated. For example, among the above-mentioned microorganisms, 1) the microorganism deposited under accession number NITE P-02758 (strain 5-11-6-3 in the Examples) and 2) the microorganism deposited under accession number NITE P-02759 (strain 5-28-6-1 in the Examples) are microorganisms isolated by screening under acidic conditions. Therefore, cultivation for PHA synthesis using the microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759 can be carried out in an acidic medium.

[0037] When marine bacteria are used as microorganisms capable of alginate utilization and PHA synthesis, the concentration of sodium chloride in the culture medium can be set appropriately depending on the type of marine bacteria, etc., but it is preferable to adjust it to a range of 0.5% to 20% by mass, more preferably to a range of 1% to 10% by mass, and particularly preferably to a range of 1% to 8% by mass.

[0038] In the production method of the present invention, the culture for PHA synthesis is carried out under aerobic conditions, and an aeration stirring type culture tank or a bubble column type culture tank can be used. The size of the culture tank can be, for example, 150 milliliters or more. When culturing is carried out on an industrial scale, the size of the culture tank can be, for example, 100 liters or more. The culture can be carried out while continuously or intermittently supplying a carbon source and other nutrient sources. The carbon source concentration in the medium can be set in the range of about 0.5% by mass to about 5.0% by mass. The stirring speed can be, for example, 100 to 120 rpm. The number of days for culture can be set in the range of 1 to 7 days, but is not limited thereto. The culture can be carried out while controlling conditions such as temperature, pH, carbon source concentration, aeration rate, stirring speed, and culture time.

[0039] PHA recovery PHA accumulated within the microbial cells can be recovered by known methods (e.g., Patent Document 3). For example, the cells are separated from the culture using a separation means such as a centrifuge, washed with distilled water, methanol, or the like, and dried. PHA is extracted from these dried cells using an organic solvent such as chloroform. Cell components are removed from this organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. The supernatant is then removed by filtration or centrifugation, and the PHA can be recovered by drying.

[0040] The production method of the present invention can include a previous step of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or acid. Extraction of alginic acid from seaweed can be performed by known methods (for example, JP 2017-21034 A). For example, alginic acid can be extracted from seaweed by the following method. In seaweed, alginic acid (AL-) is trapped as an inorganic ion salt with calcium ions, etc., so sodium carbonate is added to the seaweed and heated to extract sodium ions (Na + ) and calcium ions (Ca 2+ ) is subjected to ion exchange to extract alginic acid as water-soluble sodium alginate (alginic acid extraction). Next, calcium carbonate is removed from the alginic acid extract. The alginic acid extract from which calcium carbonate has been removed is acidified to precipitate alginic acid, or calcium ions are added to precipitate calcium alginate, which is then filtered to obtain alginic acid or calcium alginate.

[0041] The seaweed from which alginic acid is extracted is a macroalgae selected from brown algae, red algae, green algae, etc., with brown algae being particularly preferred because brown algae are rich in alginic acid. Examples of brown algae include macroalgae belonging to the Laminariales and Fucales orders, such as kelp, wakame, and giant duckweed, but are not limited to these, and brown algae growing in the oceans of various parts of the world can be used.

[0042] The extracted alginic acid may be hydrolyzed with an enzyme or acid and then added to the medium as a carbon source. For enzymatic hydrolysis, alginate lyase (EC.4.2.2.3) or the like can be used. For acid hydrolysis, inorganic acids such as hydrochloric acid and phosphoric acid, sulfonic acids such as sulfuric acid, acetic acid, and carboxylic acids such as formic acid can be used.

[0043] The present invention provides a method for producing a bioplastic product, which includes the steps of carrying out the above-described production method to obtain polyhydroxyalkanoic acid (PHA) and obtaining a plastic product containing 25% by mass or more of the PHA obtained in the previous step. The Japan Bioplastics Association states that a product must have a bioplastic content of 25% by weight or more in order to meet the biomass plastic identification standards. The bioplastic content is the ratio of the mass of biomass plastic components in a biomass plastic product to the total mass.

[0044] The PHA produced by the production method of the present invention can be used as a raw material for various plastic products. Examples of plastic products containing PHA include films and sheets that can be used as packaging for frozen foods or cushioning materials, stationery such as ballpoint pens, disposable tableware such as straws, forks, spoons, and plates, civil engineering and construction materials, multipurpose films for agricultural and fishery materials, seedling pots, fishing lines, fishing nets, etc.

[0045] microorganisms Covetia The present inventors have discovered the ability to utilize alginate and synthesize PHA in several microbial strains isolated from seaweed washed up on Goishi Beach in Ofunato Bay, Ofunato City, Iwate Prefecture. In particular, the microbial strains 5-28-6-1 and 5-11-6-3 described in the Examples are Gram-negative bacilli (Figure 8) and grow in media with an NaCl concentration of approximately 5%. The microbial strains 5-28-6-1 and 5-11-6-3 each have a 16S rRNA gene containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2. Analysis of a sequence database based on the 16S rRNA gene sequence using the homology search program BLAST (Altschul SF et al., 1990 Basic local alignment search tool. J. Mol. Biol. Vol. 215, pp. 403-410) suggests that they may belong to the genus Covetia.

[0046] Covetia bacteria are aerobic, slightly halophilic (0.5-20%), and reported to grow at temperatures between 4 and 42°C and pH between 4 and 11 (Romanenko LA et al., International Journal of Systematic and Evolutionary Microbiology, 63, 288-297, (2013)). They have also been reported to accumulate P(3HB) intracellularly using glucose as a carbon source (Romanenko LA et al. 2013; Arahal DR et al., Appl. Microbiol. 25, 207-211, 2002). However, there have been no reports of PHA accumulation using alginate as a carbon source prior to this application. Covetia bacteria are also reported to have round, shiny, smooth, and cream-colored colonies (Romanenko LA et al. 2013). The colonies of strains 5-28-6-1 and 5-11-6-3 observed during single isolation were also round, shiny, smooth, and cream-colored, consistent with the colony morphology reported for the above-mentioned Covetia bacteria. Furthermore, the conditions under which strains 5-28-6-1 and 5-11-6-3 can grow also matched those reported for the above-mentioned Covetia bacteria. Therefore, it was suggested that strains 5-28-6-1 and 5-11-6-3 may belong to the Covetia genus.

[0047] A comparison of the phenotypes of strain 5-11-6-3 with strain DSM4741 (Arahal DR et al., 2002, supra), the type strain of Cobetia marina, revealed that Cobetia marina can grow in a medium with a 0% NaCl concentration, whereas strain 5-11-6-3 cannot grow in a medium with a 0% NaCl concentration. Furthermore, the strains differ in their ability to assimilate L-arabinose and D-mannose (Table 22 in Example 6). Therefore, strain 5-11-6-3 is a Cobetia bacterium, and although closely related to Cobetia marina, it may be a different species.

[0048] The present invention provides a bacterium of the genus Covetia that has the ability to utilize alginate and synthesize PHA. The present invention provides a bacterium of the genus Covetia having a 16S rRNA gene comprising the following base sequence (a) or (b): (a) the base sequence set forth in SEQ ID NO: 1 or 2; (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0049] The present invention provides a bacterium of the genus Covetia having a 16S rRNA gene comprising the following nucleotide sequence (a) or (b), and having the ability to utilize alginate and synthesize PHA: (a) the base sequence set forth in SEQ ID NO: 1 or 2; (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0050] The nucleotide sequence and nucleotide sequence identity can be analyzed by the methods described above. Whether a bacterium has the ability to utilize alginate and / or synthesize PHA can be determined by the methods described above. The Covetia bacterium of the present invention has the ability to utilize alginic acid and synthesize PHA, and therefore can be used in a method for producing bioplastics using seaweed as a raw material.

[0051] microorganism deposit The 5-28-6-1 strain has been deposited with the following depository by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture, Japan). The 16S rRNA gene of the 5-28-6-1 strain has the base sequence shown in column number 1. (i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NITE-NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (ii) Trust date (deposit date): August 8, 2018 (iii) Accession number NITE P-02759 (Cobetia sp. IU180733JP01 (5-28-6-1) strain)

[0052] The 5-11-6-3 strain has been deposited with the depository institution by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture) as follows: The 16S rRNA gene of the 5-11-6-3 strain has the nucleotide sequence set forth in SEQ ID NO:2. (i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NITE-NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (ii) Trust date (deposit date): August 8, 2018 (iii) Accession number NITE P-02758 (Cobetia sp. IU180733JP01 (5-11-6-3) strain)

[0053] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" is based on mass, and numerical ranges are stated as including their endpoints.

[0054] Reagents and equipment The reagents and equipment used in the examples are as follows. [Table 1]

[0055] [Table 2]

[0056] [Table 3]

[0057] [Table 4]

[0058] [Table 5]

[0059] [Table 6] [Example]

[0060] Example 1 Isolation of bacteria from natural samples A total of 31 natural samples were collected from the vicinity of Goishi Beach in Ofunato City, Iwate Prefecture on June 20, 2017. After collection, they were stored at 4°C and used in the experiment. The natural samples included seawater, seaweed, shells, and sea slugs.

[0061] The collected natural samples were cultured for 2-3 days (scale: 5 mL test tube, temperature: 30°C, stirring speed: 111 rpm, medium: ZoBell 2216E seawater medium, pH: 5, 7, or 9). By using seawater from Ofunato City, Iwate Prefecture as the medium, the initial culture was performed under conditions similar to those in the environment where the samples were collected. HCl and NaOH were used to adjust the pH. The seawater for the medium was filtered through filter paper (No. 2, 110 mm) before use. 5 mL of the prepared medium was dispensed into each test tube and autoclaved (121°C, 15 min).

[0062] The seaweed samples were cut into pieces approximately 1cm x 1cm in size and added directly to the medium. Solid samples (shells, sea slugs) were suspended in 5mL of 0.1M potassium phosphate buffer (KPB, pH 7.0), and 200μL of the suspension was added to the medium. Shells were crushed and the contents ground in a mortar and pestle and then suspended in KPB, while sea slugs were ground in a mortar and the resulting liquid was suspended in KPB. 200μL of liquid samples (seawater) were added directly to the medium.

[0063] On the first day of culture, regardless of pH, the medium, which was clear immediately after inoculation, turned white or yellow and cloudy in all samples. Single bacteria were isolated from the culture medium on the second or third day. Figure 2A shows the culture state of some samples.

[0064] To obtain a single colony from the culture medium containing the natural sample, single bacterial isolation was performed using the medium shown in Table 7 at a culture temperature of 30°C for 3 days. The culture medium containing the natural sample was diluted and inoculated onto the medium shown in Table 7. The natural sample was diluted by taking 10 μL of the culture medium and mixing it with 990 μL of sterilized RO water. This procedure was repeated for 10 4 ,10 6 ,10 8 The plates were dilution by 2:1 and 200 μL of each was inoculated. Observation was carried out daily, and plates with sufficient growth were stored at 4°C until the next experiment.

[0065] [Table 7]

[0066] result Multiple colonies of different colors, morphologies, and sizes were observed on a single agar plate (Figure 2B). These were visually selected, and a total of 311 strains were obtained (Table 8). No notable trends in colony counts were observed at each pH.

[0067] [Table 8]

[0068] Example 2 Selection of PHA-synthesizing candidate strains using Nile Red (primary screening) In this example, as a primary screening, the strains obtained by the single cell isolation in Example 1 were cultured in a medium containing Nile Red to select candidate strains for PHA synthesis. Nile Red is a hydrophobic dye that reacts with neutral lipids within cells to produce a red color, making it possible to visually identify candidate strains. Two types of medium, one containing Nile Red and one not, were prepared, and the color change was observed with the naked eye by inoculating bacteria onto both plates. Red-colored colonies were stored at 4°C until the next experiment.

[0069] The composition and culture conditions of the Nile Red-containing medium are listed in Tables 9 and 10. The medium was autoclaved (121°C, 15 min). Trace Element was filter-sterilized (Minisart®-RC25, sartorius, RC, pore size 0.45 μm). Sodium alginate was autoclaved (121°C, 15 min) separately from the medium. Nile Red was dissolved in dimethyl sulfoxide and used without sterilization. The final concentration of sodium alginate in the medium was 1%. Colonies with different colors and morphologies were selected from ZoBell 2216E seawater plates, picked with a toothpick, and inoculated onto two plates. The plates were incubated at 30°C for 5 days, and the Nile Red-containing plates were incubated in the dark.

[0070] [Table 9]

[0071] [Table 10]

[0072] result The plates were observed with the naked eye every day, and red-colored strains were selected, yielding a total of 171 strains (Table 11). After the culture was completed, the strains were stored at 4°C. [Table 11]

[0073] Example 3 Selection by gas chromatography-mass spectrometry (GC-MS) method (secondary screening) In this example, selection was performed by GC-MS method as a secondary screening. Since Nile Red reacts with substances other than PHA and turns red, the strains selected in the primary screening were cultured, and then PHA polymers were extracted. The extracts were ethanolyzed, and PHA monomers were detected by GC-MS method for further selection. [ka]

[0074] (Culturing) The PHA-synthesizing candidate strains obtained in the primary screening were cultured in test tubes (10 mL). The medium listed in Table 12 was autoclaved (121°C, 15 min), and the Trace Element (Table 11) and sodium alginate were sterilized separately using the same method as above and then added to the medium. Colonies of the PHA-synthesizing candidate strains obtained in the primary screening were isolated with toothpicks and placed in the medium, along with the toothpicks, for shaking culture. Sodium alginate was used as the carbon source, with a final concentration of 1%. Culture was carried out at 30°C with an agitation speed of 111 rpm for 72 hours, with the culture time being extended for samples with slow growth.

[0075] [Table 12]

[0076] Of the 171 strains obtained in the primary screening, 121 samples could be cultured. Some of the culture results are shown in Figure 3. PHA was extracted from these samples and subjected to GC-MS analysis.

[0077] (PHA extraction) After the cultivation, the pH and OD of the culture solution were 660 The bacterial mass was measured, and the cells were recovered by suspension in RO water twice and centrifugation three times. Centrifugation was performed at 8,490 × g or 7,670 × g. The recovered cells were pre-frozen at -30°C. The tubes containing the cells were then capped with parafilm, punctured, and freeze-dried for at least 24 hours in a freeze dryer. After freeze-drying, the dried cells were weighed and crushed. They were then transferred to a screw-cap test tube, and 5 mL of chloroform was added per 200–300 mg of cells. After vortexing, the mixture was heated at 70°C for two days. Vortexing was performed as needed. After heating, the cells were removed by passing through a filter, and the filtrate was heated to dryness in a fume hood. The weight of the extract was determined by weighing the screw-cap test tube before and after filtration. The dried sample was stored at room temperature.

[0078] (GC-MS sample preparation) 1 mL of chloroform was added to the extract and dissolved by heating. 3.4 mL of 99.5% ethanol and 400 μL of HCl (stock solution) were added, vortexed for 1 minute, and heated in a heat block at 100°C for 4 hours (ethanolysis treatment) (Y. Arai, et al., Plant Cell Physiol. 43(5), 555-562, (2002)). During heating, vortexing was performed for 1 minute every 30 minutes. After cooling on ice, 4 mL of a 0.65 M NaOH + 0.9 M NaCl mixture and 2 mL of 0.25 M Na2HPO4 were added, vortexed for 1 minute, and the chloroform layer was confirmed to be neutral using pH paper. The solution was then centrifuged at 800 × g for 5 minutes, and the chloroform layer was collected at the bottom. The chloroform layer was passed through a column made by packing glass wool and Na2SO4 in a Pasteur pipette, and then transferred to a screw-cap test tube containing molecular sieves that had been preheated at 180°C for 2 hours for dehydration. This was used as a sample and stored at -30°C until analysis.

[0079] To measure the amount of synthesized PHA, P(3HB), one of the most common PHAs, was subjected to ethanolysis, and a calibration curve was created using 3HB ethyl as a standard substance. To create the calibration curve, P(3HB) was diluted with chloroform to prepare concentrations of 10, 50, 100, 500, and 1000 μg / mL, and each ethanolyzed sample was used.

[0080] (GC-MS method conditions) An HP-5 column (length 30 m, film thickness 0.25 μm, inner diameter 0.25 mm) was used. The analytical conditions are as shown in Tables 13 and 14. [Table 13]

[0081] [Table 14]

[0082] A peak in the mass spectrum of the 3HB ethyl standard was confirmed at a retention time of approximately 4.7. Analysis of 121 samples revealed that a peak in the mass spectrum of 3HB ethyl, thought to be derived from P(3HB), was detected in multiple samples. Fourteen samples that showed peaks between retention times of 4.76 and 5.00 were selected as candidate strains for PHA synthesis (Table 15). Of the 14 samples that showed peaks between retention times of 4.76 and 5.00, the analytical results for strains 5-28-6-1 and 5-11-6-3 are shown in Figures 4A and 4B.

[0083] The amount of P(3HB) was determined from a calibration curve using 3HB ethyl as a standard substance, and the P(3HB) accumulation rate was calculated by dividing the amount of P(3HB) by the dry cell weight (Table 15). [Table 15]

[0084] In Examples 1 and 2, 31 samples of seaweed, etc., collected from the natural world were cultured in seawater medium and subjected to single-cell isolation, yielding a total of 311 strains. Selection was then performed using a medium containing Nile Red, yielding 171 PHA synthesis candidate strains. These strains were cultured in PHA synthesis medium, polymers were extracted, and samples subjected to ethanolysis were analyzed by GC-MS to further narrow down the candidate strains. As a result, 3HB ethyl, an ethanolysis product of P(3HB), one of the most common PHAs, was detected in 14 strains.

[0085] In GC-MS measurements, the sample is first converted into a monomer, so it is not possible to determine whether a polymer has been synthesized. 1 H-NMR analysis is required, 1 H-NMR analysis requires a certain amount of analytical sample. Therefore, in the following Example 4, polymer synthesis was performed by culturing on a multiple test tube and Sakaguchi flask scale. 1 The synthesis of PHA was confirmed by H-NMR analysis.

[0086] Example 4 1 H-Nuclear Magnetic Resonance ( 1 H-NMR) analysis 1. Cultivation at Sakaguchi flask scale The 14 strains suggested to be capable of PHA synthesis by the GC-MS method in Example 2 were cultured in a scale-up using a Sakaguchi flask. Seed culture was performed in a 10 mL test tube as described in Example 2, and main culture was performed by subculturing 3 mL of the seed culture. Culture at the Sakaguchi flask scale was performed at a temperature of 30°C and an agitation rate of 111 rpm for 24 to 72 hours, and the culture time was further extended for samples with slow growth. In this case, the culture time was extended for the 9-15-0-1 strain because of its slow growth.

[0087] The results of the main culture are shown in the table below. [Table 16]

[0088] As shown in the table above, the relationship between dry cell weight and extract weight over time was clarified, and in most samples, dry cell weight and extract weight peaked after 1-2 days of culture, and then tended to decrease after 3 days of culture. This is likely due to the fast growth of most strains, which may have led to the depletion of nutrients around the first or second day of culture. The fast growth and short time required for PHA synthesis are thought to be advantages when considering industrial use.

[0089] 2. Polymer Purification and Sample Preparation A chloroform extract was obtained from the culture by the PHA extraction method described in Example 3. This was dissolved in a small amount of chloroform, and an excess amount (10 times or more) of hexane was added thereto to precipitate the polymer, which was then purified. The purified polymer was dissolved by adding 2 mL of chloroform-D (D, 99.8%) + 1 v / v% TMS and heating.

[0090] 3.1 H-NMR analysis As a result of the analysis, peaks specific to P(3HB) homopolymer (1.26-1.28, 2.44-2.64, 5.24-5.28 ppm), a type of PHA, were observed in 13 of the 14 strains, while no peaks for the monomer 3HB were detected. Figures 5A and 5B show some of the analytical results (strains 5-28-6-1 and 5-11-6-3). Note that 3HB peaks were observed at 1.20-1.21, 2.30-2.43, and 4.13-4.19 ppm, so P(3HB) homopolymer can be distinguished from 3HB. The peak detected near 1.5 ppm is thought to be residual water, and the peak near 7.26 ppm is thought to be deuterated chloroform. The only strain that did not detect peaks specific to P(3HB) homopolymer was 9-10-8-2.

[0091] After culture in test tubes 1 H-NMR analysis confirmed that 13 strains were P(3HB) homopolymers, suggesting that the strains may convert alginate to acetyl-CoA and synthesize P(3HB) through the previously reported three-step enzymatic reaction (Sudesh K. et al., Prog. Polym. Sci. 25 (2000) 1503-1555; Hiroyuki Takeda et al., Energy Environ. Sci., 2011, 4, 2575).

[0092] Example 5 Species identification: Determination of 16S rRNA base sequence Nucleic acids were extracted from the bacterial cells, precipitated with ethanol, and then the nucleic acid concentration was measured and subjected to agarose gel electrophoresis. DNA extraction was confirmed in all samples, so they were subjected to the next step. PCR amplification of 16S rDNA or 18S rDNA was performed on the DNA extracts. Because it was not clear whether this strain was a bacterium or a fungus, both 16S rRNA primers and 18S rRNA primers were used. [Table 17]

[0093] PCR was performed using TaKaRa Ex Taq with the following reaction mixture: 0.25 μL of TaKaRa Ex Taq (5 U / μL); 5 μL of 10× Ex Taq Buffer; 4 μL of dNTP mixture (2.5 mM each); 3 μL of primers (10 μM each); 3-5 μL of template (<500 ng); and up to 50 μL of sterile MQ water. The reaction conditions were 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, with 30 cycles. As a result, a band of approximately 1.5 kbp corresponding to 16S rRNA was confirmed in all samples except for the 5-10-6-1 sample.

[0094] Subsequent experiments were carried out using strains 5-28-6-1 and 5-11-6-3, which had stable and high P(3HB) accumulation rates. The 16S rRNA of strains 5-28-6-1 and 5-11-6-3 was amplified by PCR and inserted into the pTA2 vector, which was then transformed into Escherichia coli JM109. Recombinants were randomly selected, and after 16 hours of cultivation, the cells were harvested and the plasmids were extracted.

[0095] The resulting plasmid DNA was subjected to sequence analysis, and the full-length 1,496-bp nucleotide sequence of the region flanked by the above-mentioned 16S rRNA gene amplification primers was determined for both strains 5-28-6-1 and 5-11-6-3. A homology search was performed using the BLAST program. The results showed that strains 5-28-6-1 and 5-11-6-3 shared 99% homology with the 16S rRNA of Cobetia marina. The 16S rRNA nucleotide sequences of strains 5-28-6-1 and 5-11-6-3 strongly suggested that they were Cobetia bacteria (Table 18). There were four base differences between strains 5-28-6-1 and 5-11-6-3 (Fig. 7).

[0096] [Table 18]

[0097] Example 6 Species identification: Phenotypic characteristics of strain 5-11-6-3 The characteristics of strain 5-11-6-3, which was suggested to belong to the genus Covetia by 16S rRNA sequence analysis, were analyzed. Motility tests were performed by inoculating the bacteria into test tubes containing agar medium with a 2% NaCl concentration, a modified version of the medium composition of Rudolph Hugh et al. (The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria, J Bacteriol. 1953 Jul;66(1):24-26.), and observing motility under a microscope. Motility tests were performed using sodium alginate and mannitol as carbon sources, and in both cases, the bacteria did not spread throughout the medium and growth was not observed, but rather growth was observed along the puncture line. It is believed that strain 5-11-6-3 is not motile on agar.

[0098] To investigate the temperature range in which bacteria can grow, a culture experiment was conducted using SW-5 medium (the composition of which is shown in Table 19). 30% Marine salts (the composition of which is shown in Table 20) was filter-sterilized (Minisart®-RC25, Sartorius, RC, pore size 0.45 μm) and added to the medium to a final concentration of 5%. A fixed amount of cultured 5-11-6-3 bacteria was added to SW-5 medium and cultured at temperatures of 4, 15, 25, 37, 42, or 45°C. Bacterial growth was confirmed in the temperature range of 4 to 42°C. Culture was continued for one week at 45°C, but no growth of 5-11-6-3 bacteria was observed.

[0099] [Table 19] [Table 20]

[0100] To determine the range of NaCl concentrations at which growth is possible, we conducted a culture experiment in SW medium supplemented with 0-25% marine salts. A fixed amount of bacterial strain culture was added to SW-5 medium, and the growth was monitored after one week of incubation at 37°C with NaCl concentrations of 0, 0.5, 3, 5, 10, 15, 20, or 25%. The 5-11-6-3 strain was confirmed to grow at 0.5-20% marine salts, but no growth was observed at 0%.

[0101] Cytochrome oxidase test filter paper (Nissui Pharmaceutical) was used for the oxidase test. Strain 5-11-6-3 bacteria were cultured in fresh PHA synthetic medium for four days. The test paper was placed in a petri dish, and a few drops of MQ water were added to wet the entire filter paper. The cultured bacteria on solid medium was immediately applied to it with a platinum loop. If the applied area turned deep blue within one minute, it was judged to be a positive bacterium. The color of the test paper did not change even after one minute had passed. Therefore, this strain was considered to be oxidase negative.

[0102] Nutritional tests were performed using media with the compositions shown in Table 21. The carbon sources used were L-arabinose, D-glucose, glycerol, myo-inositol, D-mannitol, D-mannose, D-sorbitol, sucrose, and lactose. The carbon source concentration was adjusted to 10% and filter-sterilized before use. Growth of the 5-11-6-3 strain of bacteria was confirmed on carbon sources other than L-arabinose, D-sorbitol, and lactose (D-glucose, glycerol, myo-inositol, D-mannitol, D-mannose, and sucrose).

[0103] [Table 21]

[0104] The results of analyzing the properties of the 5-11-6-3 strain of bacteria are shown in Table 22. Table 22 also shows the phenotypic properties of a previously reported Cobetia bacterium (Cobetia marina DSM4741 strain (type strain)) (Arahal DR et al., 2002, supra).

[0105] [Table 22]

[0106] Cobetia bacteria are Gram-negative bacteria, and strain 5-11-6-3 is also a Gram-negative bacterium, so the characteristics matched those of the genus Cobetia. Furthermore, the growth temperature and oxidase negativity of strain 5-11-6-3 were also consistent with those of the genus Cobetia. However, regarding the NaCl concentration at which they can grow, Cobetia marina can grow in a medium with a 0% NaCl concentration, while strain 5-11-6-3 could not. Furthermore, in nutritional tests, the types of sugars used for growth differed between strain 5-11-6-3 and Cobetia marina. Therefore, it was suggested that this strain is a Cobetia bacterium but may not be Cobetia marina.

[0107] Example 7: Examination of culture conditions (sodium alginate concentration) A colony of the Covetia genus strain 5-11-6-3 was seed cultured on a PHA synthetic plate medium (medium described in Table 12, pH 5.0) for 2 days. After 2 days of culture, 3 mL of the seed culture was inoculated and main culture was performed in triplicate. To examine the effect of sodium alginate concentration on PHA synthesis, the main culture medium was set to a NaCl concentration of 2% and a sodium alginate concentration of 1%, 2%, or 3%, and the medium was cultured for 12, 24, or 36 hours. After main culture, the pH and OD 660After measuring the amount of microbial cells, the cells were transferred to a centrifuge tube and centrifuged at 7,670 × g for 15 minutes, after which the supernatant was discarded by decantation. The cells were suspended in RO water and centrifuged again under the same conditions, followed by decantation. The cells were suspended in RO water and centrifuged again, and finally the supernatant was discarded and stored at -30°C. The pre-frozen cells were freeze-dried for at least 24 hours using a freeze dryer (EYELA FDS-1000, Tokyo Rikakikai Co., Ltd.). They were then crushed in a mortar and pestle, and their weight was measured. Approximately 5 mL of chloroform was added per 200–300 mg of dried cells, and extraction was performed for 48 hours at 70°C using a heat block. After extraction, the cells were filtered using a filter (DISMIC®-25HP, Advantec, PTFE, pore size 45 μm) and heated to 55°C using a heat block to dry the extract. If the extract was dirty, it was dissolved in a small amount of chloroform, and then an excess amount (approximately 10 times or more) of hexane was added to precipitate P(3HB). The hexane was then removed and the extract was dried. The weight of the precipitated P(3HB) was calculated from the weight of the test tube before and after the experiment, and the P(3HB) accumulation rate was calculated from the dry cell weight. The results are shown in Table 23. [Table 23]

[0108] When sodium alginate concentrations were varied between 1 and 3%, the addition of 3% sodium alginate to the medium resulted in the highest growth and P(3HB) accumulation. The P(3HB) accumulation rate reached 46.6% after 36 hours, and the maximum amount of P(3HB) synthesis was 297.5 mg after 24 hours of culture. [Industrial Applicability]

[0109] The present invention is useful in the plastics industry. [Sequence List Free Text]

[0110] SEQ ID NO: 1: 16S rRNA gene sequence of the microbial strain 5-28-6-1 SEQ ID NO: 2: 16S rRNA gene sequence of the microbial strain 5-11-6-3 SEQ ID NO: 3: Primer for amplifying the 16S rRNA gene SEQ ID NO: 4: Primer for amplifying the 16S rRNA gene SEQ ID NO: 5: Primer for amplifying the 18S rRNA gene SEQ ID NO: 6: Primer for amplifying the 18S rRNA gene

Claims

1. A method for producing polyhydroxyalkanoic acid (PHA) using alginic acid as a carbon source, comprising culturing a Covetia bacterium having the ability to utilize alginic acid and synthesize PHA in a culture medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture.

2. The method according to claim 1, further comprising the steps of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or an acid as a pre-step.

3. The production method described in claim 1 or 2, wherein the Covetia bacterium is any one selected from Covetia bacteria that have a 16S rRNA gene containing the following base sequence (a) or (b) and biosynthesize polyhydroxyalkanoic acid (PHA) using alginic acid as a carbon source: (a) The base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the microorganism is an isolated microorganism, a deposited microorganism, or a genetically modified microorganism.

5. The method according to any one of claims 1 to 4, wherein the concentration of alginic acid or alginic acid decomposition product in the medium is in the range of 0.5% by mass to 20% by mass.

6. The method according to any one of claims 1 to 5, wherein the concentration of sodium chloride in the medium is in the range of 0.5% by mass to 20% by mass.

7. The method according to any one of claims 1 to 6, wherein the PHA comprises a 3-hydroxybutanoic acid monomer unit.

8. The method according to claim 7, wherein the PHA is poly(3-hydroxybutanoic acid).

9. Implementing the method according to any one of claims 1 to 8 to obtain polyhydroxyalkanoic acid (PHA); A method for producing a bioplastic product, comprising the step of obtaining a plastic product containing 25% by mass or more of the PHA obtained in the step above.

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