KR20130097347A - Mixed microorganisms having degradation activity of alginate and laminarin - Google Patents

Abstract

PURPOSE: A complex microorganism is provided to effectively decompose alginate and laminarin which are byproducts generated through processing and extracting brown algae, thereby producing reducing sugar such as glucose and producing a physiologically active substance or bioethanol. CONSTITUTION: A complex microorganism (KACC 91706P) includes Bacillus megaterium, Pantoea agglomerans, Stenotrophomonas terrae, Microbacterium oxydans, Bacillus bataviensis, and Bacillus amyloliquefaciens which decompose alginate and laminarin. The alginate and laminarin are a byproduct generated during processing and extracting brown algae.

Description

Mixed Microorganisms Having Degradation Activity of Alginate and Laminarin}

The present invention relates to a complex microorganism having an alginic acid and laminarin resolution, more specifically Bacillus megaterium ( Bacillus) that decomposes alginic acid and laminarin by-products generated during the brown algae processing and extraction process megaterium ), Pantoea agglomerans ), Stenotrophomonas terrae ), Microbacterium oxydans oxydans ), Bacillus bataviensis ) and Bacillus amyloliquefaciens ( Baccillus amyloliquefaciens ) and six kinds of complex microorganisms and a method for decomposing by-products of brown algae processing and extraction process using the complex microorganisms.

Currently, researches on renewable energy such as bio, hydrogen and solar are being actively conducted to develop stable and environmentally friendly energy that can replace oil and coal by strengthening the climate change convention and environmental regulations. The raw materials for bioenergy production are mainly grains such as sugar cane and corn, and wood-based resources such as forestry and agricultural by-products.However, there are food shortage problems, limited control area and difficulty in supplying nutrients, and cellulose other than cellulose. Problems such as economics due to additional pretreatment processes such as hemicellulose and lignin are revealing the limitations of biofuel production using grains and wood-based resources.

     In order to solve these problems, marine algae, which is composed of many fibers and various polysaccharides, has recently attracted attention as a new bioenergy raw material. Algae grow faster than wood and plant-based cellulose, and absorb carbon dioxide in the air through photosynthetic reactions, thus reducing greenhouse gases, and the low lignin content in seaweeds can simplify the pretreatment process. It has potential. Large seaweeds have various types and binding forms of polysaccharides according to species. Especially, brown algae such as seaweed and seaweed have many similarities to green plants, and they are 5.7% to 14.3% of cellulose and viscous polysaccharides between cells. Phosphorus alginic acid, fucoidan, and laminarin, a hypotonic polysaccharide.

     Laminarine, a by-product from brown algae processing and extraction, is composed of β-1,3-glucose bonds and is partially composed of β-1,6-glucose bonds. Alginic acid is D-manneuronic acid and L-. Gluronic acid is composed of β-1,4 bonds, and the polymanneuronic acid (MM), polygluronic acid (GG) form, or heteropolymer (MG) mixture of the two components in the homopolymer form is not uniform. It's mixed together. Until now, alginic acid and fucoidan, which are algae polysaccharides, have been extracted from brown algae through processing methods such as alkali, acid, or enzyme treatment, but they have been used industrially as food additives and health food resources. Due to this, it is difficult to use as a substrate for biofuel production, and treatment of by-products and wastes generated in addition to the materials currently used remains a problem.

Recently, new microorganisms producing brown algae enzymes and methods using enzymes have been reported at home and abroad (Franklin et. al . , J. Bacteriol . , 186: 4759, 2004; Cao et al . , J. Agric . Food Chem . , 55: 5113, 2007; CGC Chesters et al., Biochem . J. , 86:28, 1963; Korean Patent Application No. 2004-0021662; Korean Patent No. 10-0985841), there is little research on decomposing brown algae by-products using complex microorganisms having excellent alginic acid and laminarin degradability, and most of the studies do not target all the brown algae components but only specific ingredients. Because it was a target, it does not have great effectiveness.

Thus, the present inventors have made efforts to develop a method for decomposing alginic acid and laminarin, which are by-products that are difficult to process due to the brown algae processing and extraction process, resulted in six purely separated species capable of effectively decomposing alginic acid and laminarine. To confirm the complex microorganisms mixed with microorganisms of the present invention was completed.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method of decomposing by-products of brown algae processing and extraction process using a complex microorganism mixed with six kinds of microorganisms capable of effectively decomposing alginic acid and laminarin and the complex microorganisms.

In order to achieve the above object, the present invention is Bacillus megaterium ( Bacillus) having alginic acid and laminarin resolution megaterium ), Pantoea agglomerans , Stenotrophomonas terrae ), Microbacterium oxydans oxydans), Bacillus Bar Tavira N-Sys (Bacillus bataviensis) and Bacillus amyl Lori query Pacifico Enschede (Bacillus amyloliquefaciens ) to provide a complex microorganism consisting of.

The present invention also provides a method for decomposing the by-product of the brown algae processing and extraction process using the complex microorganisms.

It is possible to provide a complex microorganism having excellent degradability of alginic acid and laminarin of the present invention, the complex microorganism can effectively reduce alginic acid and laminarin by-products of brown algae processing and extraction process to produce reducing sugars such as glucose and bioactive substances It may be applied to a recycling method such as bioethanol production.

Figure 1 relates to a graph showing the laminarin resolution of the six strains separated in the laminarin liquid medium (pH (▲); Cell density (●); Total reducing sugars (□).
Figure 2 is a cross-plating of each of the six isolated strains and other bacteria relates to the antagonism between the bacteria ((a) EJ1, (b) EJ2, (c) EJ3, (d) EJ4, (e) EJ5 , (f) EJ6).
Figure 3 relates to a graph showing the laminarin decomposition reaction of (a) a complex microorganism mixed six strains and (b) EJ4 single strain (pH (▲); Cell density (●); Total reducing sugars (■) ; Glucose (◆)).
Figure 4 relates to the formation of a clear zone by degradation when the complex microorganism of the six strains isolated on the alginic acid plate medium (a) 0 days and (b) 6 days incubation at 37 ℃.
FIG. 5 relates to the formation of a clear zone by degradation when the complex microorganisms of six strains isolated on laminarin plate medium were cultured at 37 ° C. (a) for 0 days and (b) for 6 days. .

Unless defined otherwise, all technical and scientific terms used herein are commonly referred to by those skilled in the art to which this invention belongs.

It has the same meaning as understood. In general, the nomenclature used herein

It is well known in the art and commonly used.

In one aspect of the invention, the invention is Bacillus MEGATHERIUM (Bacillus with alginic acid and laminarin resolution megaterium ), Pantoea agglomerans ), Stenotrophomonas terrae ), Microbacterium oxydans oxydans ), Bacillus bataviensis ) and Bacillus amyloliquefaciens ( Bacillus) amyloliquefaciens ).

In the present invention, the alginic acid and laminarin may be characterized as a by-product of the brown algae processing and extraction process.

In one embodiment of the present invention, six strains forming pure colonies in alginic acid and laminarin agar medium in samples taken from the intestines of soil, mud flats and marine animals were identified, and the six isolates were isolated from EJ1 ~. Named EJ6.

As a result of observing the ability to decompose the alginic acid and laminarin of the six isolated strains, it was confirmed that the strain showing the highest resolution in the laminarin liquid medium is EJ4 (FIG. 1). In addition, the ring formation around the colonies of each strain cultured in alginic acid and laminarin plate medium As a result, the alginic acid plate medium, EJ1, EJ4 and EJ6 strains, laminarin plate medium EJ4 strains formed a higher ring than the other strains (Table 2), the alginic acid and laminarin degradation capacity for each species isolated It was confirmed that there is a difference.

In the present invention, the complex microorganism may be characterized in that the KACC 91706P.

As a result of the identification of the complex microorganism, EJ1 is Bacillus megaterium, EJ2 is Pantoa eglomerans, EJ3 is Stenotropomonas tere, EJ4 is Microbacterium oxydans, EJ5 is Bacillus bataviensis and EJ6 has been identified as Bacillus amyloliquefaciens.

The complex microorganisms identified above were deposited with the Korea Agricultural Microbial Resources Center on February 7, 2012 (Accession Number: KACC 91706P).

In another aspect of the present invention, the present invention relates to a method for decomposing byproducts of brown algae processing and extraction processes using complex microorganisms.

When decomposing alginic acid and laminarin using the above-mentioned complex microorganisms, it was confirmed that a useful substance was produced. As a result, the total reducing sugar and glucose concentrations generated by the decomposition of the laminarin by the complex microorganism were 6.14 g / l, respectively. And 3.39g / ℓ, it was found that when the laminarin is decomposed, an intermediate metabolite such as oligosaccharide can be produced in addition to glucose as a reducing sugar (FIG. 3b). In comparison, total reducing sugars and glucose concentrations were 4.23 g / l, respectively, in the digestion reaction of a single strain of EJ4 having excellent laminarine degrading ability. And 2.23 g / l (FIG. 3A). Therefore, when using a microorganism than the EJ4 single strain, it was confirmed that the total reducing sugar produced 1.45 times more, glucose was 1.71 times more, compared with the results of a single strain in alginic acid and laminarin plate medium, 6 strains mutually Synergism of the liver confirmed that the mixed microorganisms mixed therewith had superior alginic acid and laminarin degrading ability (FIGS. 4 and 5).

In addition, as a result of cross-plating and comparing the alginic acid and laminarin plate medium in order to confirm whether the complex microorganisms produce antagonists against each other, each of the six strains isolated each other and the antagonists It could be confirmed that it does not produce (FIG. 2).

In the present invention, Pseudomonas sp. Known to produce the complex microorganism and alginic acid decomposing elements. Lee et al . , Kor. J. Microbiol . Biotechnol ., 37: 350, 2009), Streptomyces sp. (Cao et. al ., J. Agric . Food Chem . , 55: 5113, 2007), Bacillus lilicheniformis AL-577 (Uo et al . , J. Korean . Soc . Food . Sci . Nutr. , 35: 231, 2006) and Erwinia tasmaniensis Lee et al . , Bioresour . Technol ., 102: 5962, 2011), etc. When comparing the decomposing ability of alginic acid of microorganisms, such as microorganisms, the optimal reaction conditions for producing alginic acid degrading enzymes differ according to their characteristics, and as alginic acid is decomposed, Degradation of alginic acid ability of each microorganism is reduced due to different reaction conditions, but the present invention is to decompose alginic acid and laminarin using six kinds of complex microorganisms without mutual antagonism, which is changed by degrading alginic acid and laminarin. Less impact on the reaction conditions can produce more reducing sugars.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example  1. Alginic acid and Laminarin  Pure separation of degraded microorganisms

In this example, microorganisms were separated from samples taken from the intestines of soil, tidal flats, and marine animals to isolate microorganisms that effectively decompose alginic acid and laminarin.

Samples were collected and inoculated in a 250 ml flask of kelp addition medium (NH ₄ Cl 0.1 g / l, kelp pieces 1 cm × 1 cm or less, pH 6.8), followed by shaking culture at 37 ° C. and 180 rpm for 8 days, respectively. It was plated on a solid medium to which 1.5% agar was added to the nutrient medium (Nutreient broth, pH 6.8). Each microorganism incubated was subsequently plated in fresh solid medium until isolated into pure colonies, resulting in final pure separation of each strain. The isolated pure strains were subcultured every 2 weeks with storage at 4 ℃.

To screen useful microorganisms in the isolated pure strains, the pure strains were plated in the following two agar media.

Agar medium for the selection of laminarin-degrading microorganisms (Laminarin 1g / l, MgSO ₄ 0.1g / l, NaCl 0.1g / l, CaCl ₂ 0.1g / l, (NH ₄ ) ₂ SO ₄ 2 g / l, KH ₂ PO ₄ 0.5 g / l, Mineral 0.5 ml, Vitamin 0.5 ml, pH 6.8)

Ii) Agar medium for the selection of alginic acid-decomposing microorganisms (Alginate 1g / L, Yeast extract 0.5g / L, Peptone 0.5g / L, (Mg ₄ ) ₂ SO ₄ 0.5 g / l, K ₂ HPO ₄ 0.5 g / l, pH 6.8)

As a result, six strains forming pure colonies in the alginic acid and laminarin agar medium were identified, and the six isolates were named as EJ1 to EJ6.

Example  2. Alginic acid and Laminarin  Determination of resolution

In order to confirm that the six strains isolated in Example 1 were capable of degrading alginic acid and laminarin, the polysaccharides of brown algae, each of the six strains isolated was 8 ml laminarin liquid medium in a 15 ml tube. After inoculation on, shaking culture for 8 days at 37 ℃, 180rpm, the growth curve, total reducing sugar and pH of the strain was measured to test the laminarin resolution.

In addition, Laminarin medium (Laminarin 0.1g / L, MgSO ₄ 0.1 g / l, NaCl 0.1 g / l, CaCl ₂ 0.1 g / l, (NH ₄ ) ₂ SO ₄ 2 g / l, KH ₂ PO ₄ 0.5 g / l, Mineral 0.5 ml, Vitamin 0.5 ml, pH 6.8) and Alginate medium (Peptone 0.5 g / l, Sodium alginate 0.1 g / l, pH 6.8) added 1.5% agar to the center of solid flat media 10 μl of each strain culture solution of EJ1 to EJ6 cultured in daily alginic acid and laminarin liquid medium was dropped and dried, and then cultured in a 37 ° C. incubator. After colonies were formed on the medium, the laminarin plate medium was stained with 10 ml of 0.5% (w / v) Congo red solution for 15 minutes, and then bleached twice with 1 M NaCl, and then stained red with Congo red solution. The formation of a yellow ring around the colonies on the medium and the change in the ring size over time were observed (Lee, DS, et. al ., Biotechnology letters , 17: 355, 1995). 10 ml of cetylpyridinium chloride monohydrate (CPC) was added to the alginate flat medium for 10 minutes, and the solution was removed. The surface of the medium was washed with distilled water and opaque color by 10% CPC. Changes in size and size of transparent rings around colonies were observed on the medium changed to (Peter, G., et al ., Amerian Society of Microbiology , 56: 2265, 1990).

As a result, it was confirmed that the strain showing the highest resolution in the laminarin liquid medium was EJ4 (FIG. 1), and whether the ring was formed around the colonies of each strain cultured in the laminarin and alginic acid flat medium. As a result, Alginic acid plate medium EJ1, EJ4 and EJ6 strains, laminarin plate medium EJ4 strains formed a relatively higher ring than other strains. Therefore, it was confirmed that there is a difference in the resolution of alginic acid and laminarin for each of the isolated species (Table 1).

Alginic Acid and Laminarin Resolution Comparison Polysaccharide substrate Strain Ring size (cm)

Alginic acid EJ1 2.5 EJ2 0.0 EJ3 0.0 EJ4 1.9 EJ5 1.3 EJ6 2.8

Laminarin EJ1 0.1 EJ2 0.4 EJ3 0.4 EJ4 1.8 EJ5 0.2 EJ6 0.4

Example  3. Alginic acid and Laminarin  Identification and Deposit of Degraded Microorganisms

Each strain of EJ1 to EJ6 isolated in Example 1 of the present invention was primarily identified through Gram staining, catalase test, colony morphology, and microscopic examination. Each of the six strains had vigorous motility in nutrition and all were positive for the catalase test (Table 2).

Identification of Alginic Acid and Laminarin Degrading Microorganisms microbe Characteristic Cell shape Colony color Gram stain Whether resistant spores are produced Catalase test EJ1 L: 1-2 µm, W: 1-1.5 µm White + + + EJ2 L: 1-2 µm, W: 0.5-1.5 µm Pale yellow - - + EJ3 L: 2-4 탆, W: 0.4-0.7 탆 Pale yellow - - + EJ4 L: 0.1-1.5 µm, W: 0.5-1 µm yellow + - + EJ5 L: 3-5 μm, W: 1-1.5 μm Beige + + + EJ6 L: 1-2 µm, W: 0.5-1 µm White + + +

Secondary identification was performed by molecular phylogenetic method based on 16S rRNA (16S rRNA coding gene) sequence. Chromosome DNA of each strain was isolated using AccuPrep chromosome DNA extraction kit (Bionia, Korea), and the 5'-CCAGCAGCCGCGGTAATACG-3 '(SEQ ID NO: 1) and 5'-TACCAGGGTATCTAATCC-3' ( PCR was performed using the primer of SEQ ID NO: 2).

Initial modification of the PCR process was carried out for 5 minutes at 95 ℃, 30 cycle amplification was performed for 10 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃, the final stretching step was carried out for 7 minutes at 72 ℃. As a result of PCR, about 1500 bp fragment of 16S-rRNA gene was amplified in each strain.

The amplified PCR product was commissioned by Macrogen Co., Ltd. for 16S-rRNA sequencing analysis, and then confirmed 16S-rRNA subsequences (SEQ ID NOs. 3 to 8) of each strain, and BioEdit Sequence Alignment Editor (ver. 5.0.9, Hall, T., Nucleic Acids Symposium Homology searches were performed in GenBank's Advanced BLAST (http://www.ncbi.nlm.nih.gov) using the series (41:95, 1999) (Altschul et al. al ., Nucleic Acids Res . , 25: 3389, 1997).

As a result, EJ1 is Bacillus megaterium, EJ2 is Pantoa Eglomerans, EJ3 is Stenotropomomonas tere, EJ4 is Microbacterium oxidans, EJ5 is Bacillus bataviensis, and EJ6 is Bacillus amylolyticus. It was identified as (Table 3).

Identification of Alginic Acid and Laminarin Degrading Microorganisms microbe 16S-rDNA
size GenBank
Accession No. Strain name Homology EJ1 1670 bp GU252120.1 Bacillus megaterium 98% EJ2 1668 bp AM184214.1 Pantoea agglomerans 98% EJ3 1669 bp NR_042569.1 Stenotrophomonas terrae 99% EJ4 1429 bp HQ113206.1 Microbacterium oxydans 98% EJ5 1671 bp NR_036766.1 Bacillus bataviensis 99% EJ6 1461 bp GQ340479.1 Bacillus amyloliquefaciens 99%

Compound microorganisms identified as shown in Table 2 above were deposited with the Korea Agricultural Microbial Resources Center on February 7, 2012 (Accession Number: KACC 91706P).

Example   4: 6 strains isolated Mutual  Antagonism measurement

In order to confirm whether or not to produce an antagonist against each other among the six pure strains isolated in Example 1 of the present invention was plated to cross each other in alginic acid and laminarin solid medium and incubated for 2 days at 37 ℃.

As a result, it was confirmed that each of the six isolated strains do not produce antagonists with each other (Fig. 2).

Example  5. Alginic acid by complex microorganisms and Laminarin  Decomposition reaction measurement

Using the microorganisms of the above six strains isolated in the embodiment of the present invention was confirmed the reaction characteristics to produce useful substances by decomposing alginic acid and laminarin.

The complex microorganisms of the six strains used in the following examples were mixed in the same amount of the six strains identified in Example 3, and each of the six strains were cultured in 0.1% laminarin liquid medium and centrifuged at the end of the logarithmic growth phase. Each strain was harvested by separation, and then mixed and used.

Using a 100 ml flask containing 40 ml laminarin liquid medium, a total of 80 µg (wet cells) were inoculated into the flask at a ratio of 1: 1 in each of the six strains in the same amount, and then digested for 15 days at 37 ° C. and 180 rpm. Was performed. During the biodegradation reaction as described above, samples in the flask were taken at intervals of 3 days to analyze the characteristics of the decomposition reaction. During the degradation reaction, the pH change in the culture medium was measured using a pH meter (ISTECH, Korea). Measurement was made at the wavelength (375 nm). The total reducing sugars produced in the decomposition reaction were uniformly mixed with 100 µl of the supernatant centrifuged (70,00 rpm, 10 minutes) and 1 ml DNS (3.5-dinitrosalicylic acid) solution using vortex. Next, the mixture was reacted in boiling water for 10 minutes, cooled in cold water for 5 minutes, and then measured at a 570 nm wavelength using a spectrophotometer, and measured by a standard curve using glucose as a control.

The concentrations of glucose and other oligosaccharides produced as a result of the laminarin decomposition reaction were analyzed by HPLC / RID (Agilent Technologies 1200 series, USA) using a Sugar-pak ^TM 1 (Waters, 6.5 × 300 mm) column. As analytical solvent, water of 0.8 ml / min flow rate was used, and the detector and oven temperature were set to 35 ° C. and 80 ° C., respectively.

In addition, as shown in Example 2, after culturing for 6 days the mixed microorganisms mixed with six strains of isolation was confirmed whether the ring production in laminarin and alginic acid plate medium.

As a result, as a result of the decomposition of laminarin using a microorganism, the pH of the initial culture was 6.8 gradually decreased, the final pH value of the culture 15 days was 4.17. When laminarin was decomposed, it could be seen that in addition to glucose, intermediate metabolites such as oligosaccharides could be formed as reducing sugars, and the total reducing sugars and glucose concentrations were 6.14 g / l, respectively. And 3.39 g / L (FIG. 3B). In comparison, total reducing sugar and glucose concentrations were 4.23 g / l, respectively, in the digestion reaction of a single strain of EJ4, which has the highest laminarine degrading ability. And 2.23 g / l (FIG. 3A). Therefore, when using a microorganism than the EJ4 single strain, it was confirmed that the total reducing sugar produced 1.45 times more, glucose was 1.71 times more, compared with the results of a single strain in alginic acid and laminarin plate medium, 6 strains mutually Synergism of the liver confirmed that the mixed microorganisms mixed therewith had superior alginic acid and laminarin degrading ability (FIGS. 4 and 5).

As described above, specific portions of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Agricultural Biotechnology Research Institute KACC91706P 20120207

<110> Pukyong National University Industry-University Cooperation Foundation <120> Mixed Microorganisms Having Degradation Activity of Alginate and          Laminarin <130> P12-B031 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer <400> 1 ccagcagccg cggtaatacg 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 2 taccagggta tctaatcc 18 <210> 3 <211> 1670 <212> DNA <213> Bacillus megaterium (EJ1) <400> 3 gttatatttt tgcggctagc tttgatctga acttatggtc ctaaagcggg ccaggccgtt 60 ttctaatctt ggagttacag tcctcggtca tcctngacgg tcgatggatc aggggtcttg 120 ccgcagagaa gagaagagaa cttccaccgt aaccaaaaat tttaaagant tttatagaat 180 tttttttata gctcagatga acgctggcgg cgtgcntaat acatgcaagt cgagcgaact 240 gattagaagc ttgcttctat gacgttagcg gcggacgggt gagtaacacg tgggcaacct 300 gcctgtaaga ctgggataac ttcgggaaac cgaagctaat accggatagg atcttctcct 360 tcatgggaga tgattgaaag atggtttcgg ctatcactta cagatgggcc cgcggtgcat 420 tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg catagccgac ctgagagggt 480 gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 540 tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga aggctttcgg 600 gtcgtaaaac tctgttgtta gggaagaaca agtacaagag taactgcttg taccttgacg 660 gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg 720 caagcgttat ccggaattat tgggcgtaag cgcgcgcagg cggtttctta agtctgatgt 780 gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga gtgcagaaga 840 gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg 900 cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag 960 gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc 1020 gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac 1080 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 1140 agcaacgcga agaaccttac caggtcttga catcctctga caactctaga gatagagcgt 1200 tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1260 tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc atttagttgg 1320 gcactctaag gtgactgccg gtgacaaacc ggagggaagg gtgggggatg acgtcaaatc 1380 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggatggtaca aagggctgca 1440 agaccgcgag gtcaagccaa tcccataaaa ccattctcag ttcggattgt aggctgcaac 1500 tcgcctacat gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gttgaatacg 1560 ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg 1620 tggagtaacc gtaaggagct agccgcctaa gtgggacaga tgattggggt 1670 <210> 4 <211> 1668 <212> DNA Pantoea agglomerans (EJ2) <400> 4 gggttctatg ctgtcattcc aaatttaacg cctacacgcc cccccaggtg ggcggccacg 60 tggatnttaa ttccttttcc tccacggggg gaactcccat ggaagtggct tgatagtttt 120 ttgcgggggg gggtggactt ccgaggtgaa ccgctaaaat tcgtattttt tttggggaac 180 tntagtgggg tagatgtgtc tcagtgagaa cggtgggcac gcgcctcaca cgcacgtgta 240 gcggtagcgc acaggagcct gcttttcggg tgacgagtgg cggacgggtg agtaatgtct 300 gggaaactgc ctgatggagg gggataacta ctggaaacgg tagctaatac cgcataacgt 360 cgcaagacca aagaggggga ccttcgggcc tcttgccatc agatgtgccc agatgggatt 420 agctagtagg tggggtaacg gctcacctag gcgacgatcc ctagctggtc tgagaggatg 480 accagccaca ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat 540 attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtatgaagaa ggccttcggg 600 ttgtaaagta ctttcagcga ggaggaaggc gttgaggtta ataacctcag cgattgacgt 660 tactcgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 720 aagcgttaat cggaattact gggcgtaagc gcacgcaggc ggtctgtcaa gtcggatgtg 780 aaatccccgg gctcaacctg ggaactgcat tcgaaactgg caggctagag tcttgtagag 840 gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa taccggtggc 900 gaaggcggcc ccctggacaa agactgacgc tcaggtgcga aagcgtgggg agcaaacagg 960 attagatacc ctggtagtcc acgccgtaaa cgatgtcgac ttggaggttg tgcccttgag 1020 gcgtggcttc cggagctaac gcgttaagtc gaccgcctgg ggagtacggc cgcaaggtta 1080 aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgatg 1140 caacgcgaag aaccttacct actcttgaca tccagagaac ttagcagaga tgctttggtg 1200 ccttcgggaa ctctgagaca ggtgctgcat ggctgtcgtc agctcgtgtt gtgaaatgtt 1260 gggttaagtc ccgcaacgag cgcaaccctt atcctttgtt gccagcggtt aggccgggaa 1320 ctcaaaggag actgccagtg ataaactgga ggaaggtggg gatgacgtca agtcatcatg 1380 gcccttacga gtagggctac acacgtgcta caatggcgca tacaaagaga agcgacctcg 1440 cgagagcaag cggacctcat aaagtgcgtc gtagtccgga ttggagtctg caactcgact 1500 ccatgaagtc ggaatcgcta gtaatcgtag atcagaatgc tacggtgaat acgttcccgg 1560 gccttgtaca caccgcccgt cacaccatgg gagtgggttg caaaagaagt aggtagctta 1620 accttcggga gggcgcttac cactttgtga ttcatgactg gggtgagt 1668 <210> 5 <211> 1669 <212> DNA <213> tenotrophomonas terrae (EJ3) <400> 5 tcagcacnnn nagtacggan gggtctatcg tattcatcaa ctaancacnt ctccaccgag 60 ggtgtggcca cgtctcaagc atagcgtgca ctcaccgaca gtctcgtgaa aagngaagta 120 agtgttagca gagagttgcg atctcaatgt gccccagcgt ccatgtttca tttttgaggc 180 ccgtctaaaa gtttgnntgc tcagagtgaa cgctggcggt aggcctaaca catgcaagtc 240 gaacggcagc acaggagagc ttgctctctg ggtggcgagt ggcggacggg tgaggaatgc 300 atcggaatct actctttcgt gggggataac gtagggaaac ttacgctaat accgcatacg 360 acctacgggt gaaagccggg gaccttcggg cctggcgcga atgaatgagc cgatgcccga 420 ttagctagtt ggcggggtaa gagcccacca aggcgacgat cggtagctgg tctgagagga 480 tgatcagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 540 atattggaca atgggcgcaa gcctgatcca gccataccgc gtgggtgaag aaggccttcg 600 ggttgtaaag cccttttgtt gggaaagaaa agcaatcggt taatacccgg ttgttctgac 660 ggtacccaaa gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt 720 gcaagcgtta ctcggattac tgggcgtaag cgtgcgtagg tggttgttta agtctgtcgt 780 gaaagccctg ggctcaacct gggaattgcg atggaaactg ggcgactaga gtgtggcaga 840 gggtagtgga attcctggtg tagcagtgaa atgcgtagag atcaggagga acatccgtgg 900 cgaaggcgac tgcctgggcc aacactgaca ctgaggcacg aaagcgtggg gagcaaacag 960 gattagatac cctggtagtc cacgccctaa acgatgcgaa ctggatgttg ggtgcaattt 1020 ggcacgcagt atcgaagcta acgcgttaag ttcgccgcct ggggagtacg gtcgcaagac 1080 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagtatgtgg tttaattcga 1140 tgcaacgcga agaaccttac ctggccttga catgtcgcga actttccaga gatggattgg 1200 tgccttcggg aacgcgaaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg 1260 ttgggttaag tcccgcaacg agcgcaaccc ttgtccttag ttgccagcac gtaatggtgg 1320 gaactctaag gagaccgccg gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc 1380 atggccctta cggccagggc tacacacgta ctacaatggt agggacagag ggctgcaagc 1440 cggcgacggt aagccaatcc cagaaaccct atctcagtcc ggattggagt ctgcaactcg 1500 actccatgaa gtcggaatcg ctagtaatcg cagatcagca ttgctgcggt gaatacgttc 1560 ccgggccttg tacacaccgc ccgtcacacc atgggagttt gttgcaccag aagcagtagc 1620 ttaaccttcg ggagggcgct tgccacggtg tggccgatga ctggggtga 1669 <210> 6 <211> 1507 <212> DNA <213> Microbacterium oxydans (EJ4) <400> 6 nnnngctcat ctnnnnnnnn agaccgnctg nngnattnnn nncgtcnnng ntnngcnnnn 60 nnnnncgnnn nnnnncnngc nnancgcaga acatannggc ctagntnntn aacgttcacc 120 gccncnnnnt ggagggntgn nnncggnnnt ntnccanaga gtcgnnnctt angncgggca 180 ncataggtac ggagaggttg gctgtgnnng tnnnaacnct acagtctgan agcnggagtg 240 nngacganca tgccacaact gttcngagtg ttccaaggag tgcncggtcc aaanntagga 300 gggaagtcaa gtntgaataa gtttgttcga gatagcatcg aagtcagtcc gcatgcgcag 360 actagagtgc ggtaggggag attggaattc ctggtgtagc ggtggaatgc gcagatatca 420 ggaggaacac cgatggcgaa ggcagatctc tgggccgtaa ctgacgctga ggagcgaaag 480 ggtggggagc aaacaggctt agataccctg gtagtccacc ccgtaaacgt tgggaactag 540 ttgtggggtc ctttccacgg attccgtgac gcagctaacg cattaagttc cccgcctggg 600 gagtacggcc gcaaggctaa aactcaaagg aattgacggg gacccgcaca agcggcggag 660 catgcggatt aattcgatgc aacgcgaaga accttaccaa ggcttgacat acacgagaac 720 actctggaaa caggggactc tttggacact cgtgaacagg tggtgcatgg ttgtcgtcag 780 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caaccctcgt tctatgttgc 840 cagcacgtaa tggtgggaac tcatgggata ctgccggggt caactcggag gaaggtgggg 900 atgacgtcaa atcatcatgc cccttatgtc ttgggcttca cgcatgctac aatggccggt 960 acaaagggct gcaataccgt gaggtggagc gaatcccaaa aagccggtcc cagttcggat 1020 tgaggtctgc aactcgacct catgaagtcg gagtcgctag taatcgcaga tcagcaacgc 1080 tgcggtgaat acgttcccgg gtcttgtaca caccgcccgt caagtcatga aagtcggtaa 1140 cacctgaagc ccgtggccta acccttgtgg aaggagccgt cgaagtggga tcggtaatag 1200 actatnnagg gggggggggg gggntnnggt tcgagggcgc cnttttgttt gcggtcataa 1260 aaacacatac ctttttgtcg aaaaaggaag tggtagaaac acgagagcgg ggactctcct 1320 ccccgnnnga tttgtgtctg agtgnncccc ccaagtggaa tattttccan nntnccnncc 1380 tcgataaaga gagtggnngt gggncaacnc tctatatggn nggtgtnnct tactngcngg 1440 nnnncccgnn ncnnnttatg tnnncnnnnn ncnncnntaa tgatanaagc ntagagnaca 1500 nntnnca 1507 <210> 7 <211> 1671 <212> DNA <213> Bacillus bataviensis (EJ5) <400> 7 gtgatagttg cnngcgtgct ctcntancac aggctacacc ggtgggcagn ccctctatca 60 ttctttgtgt atccccactg cgcccgtact gccccatgcg gagtacttga ggcatttgat 120 gcagcactaa aggacgggaa ccctccaacc cgtgcgaatc ttagtttatt agggggtatt 180 agttttttcn gctcagacga acgctggcgg cgtgcctaat acatgcaagt cgagcgaatc 240 aataggagct tgctcctgtt gattagcggc ggacgggtga gtaacacgtg ggcaacctgc 300 ctgtaagact gggataactt cgggaaaccg gagctaatac cggataatcc ttttcctctc 360 atgaggaaaa gctgaaagtc ggtttcggct gacacttaca gatgggcccg cggcgcatta 420 gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga 480 tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc 540 ttccgcaatg gacgaaagtc tgacggagca acgcgccgcg tgagcgatga aggccttcgg 600 gtcgtaaagc tctgttgtta gggaagaaca agtatcggag taactgccgg taccttgacg 660 gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg 720 caagcgttgt ccggaattat tgggcgtaag cgcgcgcagg cggtccttta agtctgatgt 780 gaaagcccac ggctcaaccg tggagggtca ttggaaactg ggggacttga gtgcagaaga 840 ggaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg 900 cgaaggcggc tttctggtct gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag 960 gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc 1020 gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg gccgcaaggc 1080 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 1140 agcaacgcga agaaccttac caggtcttga catcctctga cactcctaga gataggactt 1200 tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1260 tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc attcagttgg 1320 gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1380 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag ggctgcgaaa 1440 ccgcgaggtt tagccaatcc cataaaacca ttctcagttc ggattgtagg ctgcaactcg 1500 cctacatgaa gccggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1560 cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtggg 1620 gtaaccgtaa ggagccagcc gcctaagtgg gacagatgat tggggtgagc n 1671 <210> 8 <211> 1461 <212> DNA <213> Bacillus amyloliquefaciens (EJ6) <400> 8 nnnnngncgn gtnctataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt 60 tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120 gaaaccgggg ctaataccgg atgcttgttt gaaccgcatg gttcaaacat aaaaggtggc 180 ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240 accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360 cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420 gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540 cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600 agggtcattg gaaactgggg aacttgagtg cagaaganga gagtggaatt ccacgtgtag 660 cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagatacctg gtagtncacg 780 ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840 attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900 gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960 tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140 aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200 cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260 tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320 atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgcacg 1440 aatgtgcnca ngangnngnn n 1461

Claims (4)

Bacillus megaterium , Pantoea that can decompose alginic acid and laminarin agglomerans ), Stenotrophomonas terrae ), Microbacterium oxydans oxydans ), Bacillus bataviensis ) and Bacillus amyloliquefaciens .
The complex microorganism according to claim 1, wherein the complex microorganism is KACC 91706P.
The complex microorganism of claim 1, wherein the alginic acid and laminarin are by-products of brown algae processing and extraction.
A method for decomposing the by-product of brown algae processing and extraction process using the complex microorganism of any one of claims 1 to 3.

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