KR20160018140A - Bacillus subtilis BK418 with nitrogen fixation for soil and environmental improvement and cultivation methods thereof - Google Patents

Abstract

The present invention relates to a composition for soil improvement and, more specifically, to a composition for soil improvement including a Bacillus subtilis BK418 (KACC91903P) strain having nitrogen fixation power, cellulose-decomposing capabilities and antifungal activities or a culture medium thereof as an active ingredient. The composition for soil improvement including the Bacillus subtilis BK418 (KACC91903P) strain or the culture medium thereof as the active ingredient can convert nitrogen present in the environment or atmosphere into ammonia nitrogen so as to improve the soil or supply nitrogen to crops while non-biodegradable materials such as cellulose present in the soil or water are converted into organic matters, and can suppress pathogenic microorganisms by using antifungal activities to improve the soil, thereby being cultivated by using only molasses as a growth energy source.

Description

Technical Field [0001] The present invention relates to a Bacillus subtilis strain BK418 having nitrogen fixation ability for soil improvement and environmental purification, and a method for culturing the Bacillus subtilis strain BK418,

The present invention relates to a Bacillus subtilis strain BK418 having a nitrogen fixing ability and a method for culturing the same. More particularly, the present invention relates to a Bacillus subtilis BK418 strain having nitrogen fixation ability, and more particularly, to a method for improving the soil by converting nitrogen existing in the environment or the atmosphere into ammonia nitrogen, Bacillus subtilis strain BK418, which is capable of culturing the bacillus subtilis strain BK418 using only molasses as a growth energy source.

Soil has an important function to sustain the ecosystem as well as the food supply along with the material cycle. This function is highly dependent on the physical properties of the soil as well as on the biological properties, especially the action of microorganisms. Among the microorganisms present in the environment, various microorganisms present in the soil are used in many parts such as promotion of crop growth, material circulation and environmental purification. In particular, rhizosphere microorganisms present in the roots of crops have many nitrogen-fixing bacteria with ability to fix nitrogen in the air while forming crops and symbiotic crops. Therefore, there is a need for separation and identification of various microorganisms having nitrogen fixation ability Research is being done in a variety of ways.

What is essential for a living organism to live is DNA, a genetic material, and protein. Nitrogen is a necessary element for making two essential substances. Nitrogen is a very abundant resource because it takes up more than 80% of the air in the form of nitrogen molecules, but it can not be used to make DNA or proteins in the form of nitrogen molecules. Nitrogen molecules are naturally unbonded because their bonds are strong because the nitrogen element is linked by a triple bond. Therefore, even though the organism is absolutely necessary, it can not be used in the form of this nitrogen molecule. However, stable forms of nitrogen molecules are converted to ammonia nitrogen by roots bacteria or nitrogen-fixing bacteria, and converted ammonia nitrogen is used as a nutrient for growing crops. Nitrogen-fixing bacteria refer to these root-knot bacteria, oxygenated bacteria of Rhizobium.

When living organisms enter the soil, sugar, cellulose and lignin are gradually degraded by various microorganisms. In organic materials, substances that are easily decomposed, such as sugars, are used, and then, hemicellulose, cellulose and lignin are decomposed gradually. In addition, soil microorganisms have an important function to convert pollutants such as various chemical substances present in the soil into harmless substances and to self-environment. The role of these microorganisms is an important factor in keeping the soil ecosystem healthy.

In addition, microorganisms with excellent antimicrobial activity promote the growth of useful microorganisms and inhibit pathogenic microorganisms, thereby improving soil microorganisms and improving the soil ecosystem.

On the other hand, Bacillus subtilis is a bacterium belonging to the genus Bacillus that lives in soil and is gram-positive apo-forming bacteria. It is aerobic and has heat resistance. It is an organic substance decomposing microorganism which is metabolized by various saccharides such as glucose and starch as an energy source and is also used for fermented foods such as meju and chungkukjang.

As a conventional technique, Patent Document 1 proposes a Bacillus subtilis BK418 (KACC91903P) strain having a complex digestive enzyme-producing ability and an excellent antimicrobial activity against pathogenic bacteria, but it has excellent decomposability and antimicrobial activity Microorganisms can not fix Bacillus subtilis which has soil fixation ability because of nitrogen fixation ability for nitrogen present in the atmosphere.

1. Korean Patent Application No. 10-2014-0023497

The present invention has been devised in view of the above-mentioned needs, and it is an object of the present invention to provide a method for converting a poorly decomposable substance such as fibrin in soil or water quality into an organic substance, inhibiting pathogenic microorganisms by antibacterial action, Bacillus subtilis BK418 strain and its culture which have the ability to improve soil and purify the environment.

In addition, the present invention provides a novel culture method using molasses as a single nutrient source for the Bacillus subtilis strain BK418.

The present invention also provides a Bacillus subtilis BK418 strain, a culture thereof, an environmental purification composition containing the same as an active ingredient, and a composition for soil improvement.

In order to solve the above-mentioned problem, the soil improvement composition according to the present invention comprises Bacillus subtilis strain BK418 (KACC91903P) having a nitrogen fixing power, a fibrin degrading ability and an antibacterial activity or a culture thereof as an active ingredient .

In one embodiment of the present invention, the Bacillus subtilis strain BK418 (KACC91903P) is characterized in that nitrogen present in air or the environment is converted into ammonia nitrogen.

In one embodiment of the present invention, the Bacillus subtilis strain BK418 (KACC91903P) is characterized in that it has a digestive enzyme-producing ability against fibrin and transforms a refractory substance such as fibrin present in the soil or water quality into an organic material.

In one embodiment of the present invention, the Bacillus subtilis strain BK418 (KACC91903P) is characterized by having an antibacterial activity against a pathogenic microorganism.

The method for culturing the culture of the strain Bacillus subtilis BK418 (KACC91903P) according to the present invention for the above-mentioned problem is carried out by inoculating the strain Bacillus subtilis BK418 (KACC91903P) into the TSB medium sterilized at 121 ° C for 15 minutes Preparing a culture medium containing molasses in distilled water, culturing the culture medium at 30 DEG C and 180 rpm for 18 hours, preparing a culture medium containing distilled water, and culturing the culture medium at 30 DEG C and 180 rpm inoculated with 1% And culturing the cells for one week.

The Bacillus subtilis strain of the present invention has nitrogen fixation ability to convert free nitrogen present in the atmosphere or soil into ammonia nitrogen by nitrogen fixation enzyme.

In addition, the Bacillus subtilis strain BK418 can convert a poorly decomposable substance such as fibrin present in soil or water quality into an organic substance, and inhibit pathogenic microorganisms by an antibacterial action.

In addition, the Bacillus subtilis strain BK418 can be cultured in a simple and convenient new culture method using molasses as a single nutrient source.

In addition, it is possible to provide the Bacillus subtilis BK418 strain and the culture thereof, the composition for environmental purification containing the same as an active ingredient, and the composition for soil improvement.

1 is a photograph showing a Bacillus subtilis strain BK418 grown in a NFB solid medium according to the present invention.
FIG. 2 is a photograph showing the reaction of the supernatant of Bacillus subtilis strain BK418 according to the present invention with a Nessler reagent. FIG.
FIG. 3 is a photograph showing activity results of protease, amylase and cellulase through supernatant of Bacillus subtilis strain BK418 according to the present invention. FIG.
FIG. 4 is a photograph showing the result of measuring the antimicrobial activity of the supernatant of Bacillus subtilis strain BK418 according to the present invention against Escherichia coli . FIG.
5 is a nucleotide sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention.
6 is a systematic diagram showing phylogenetic relationships with other bacteria based on the base sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings illustrate only the essential features of the invention in order to facilitate clarity of the invention, and the accompanying drawings are not intended to be construed in a limiting sense.

The present invention provides a soil improving composition comprising Bacillus subtilis BK418 (hereinafter referred to as 'BK418') having a nitrogen fixing power, a fibrin degrading ability and an antibacterial activity or a culture thereof as an active ingredient.

The Bacillus subtilis BK418 was deposited at the National Institute of Genetic Resources (KACC) under accession number KACC91903P. The Bacillus subtilis BK418 morphologically corresponds to Gram-positive bacilli and has a homology of 99% with Bacillus subtilis.

FIG. 5 is a nucleotide sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention, and FIG. 6 is a diagram showing the sequence of 16s rRNA based on the base sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention It is a systematic diagram showing the genetic relationship.

As shown in FIG. 5 and the sequence listing, the 16s rRNA base sequence was analyzed and found to be 1,433 bases. As shown in FIG. 6, a phylogenetic analysis using the 16S rRNA nucleotide sequence revealed that Bacillus Bacillus subtilis BK418 was named as the isolate because it showed a similarity rate of 99% (1430/1431) with 16s rRNA gene of subtilis strain D13 (accession No: KC441757).

The composition for improving soil containing the Bacillus subtilis BK418 or a culture solution thereof as an active ingredient can convert nitrogen into ammonia nitrogen, which is present in the air or the environment, with nitrogen fixing ability.

In addition, the composition for soil improvement can convert a refractory material such as fibrin in the soil or water quality into an organic material, and can inhibit pathogenic microorganisms by antibacterial action to improve the soil and purify the environment.

The present invention provides a culture of the Bacillus subtilis strain BK418. The culture includes the culture medium or culture medium in which the BK418 strain is cultured, and the culture medium cultured in the culture medium or culture, and the culture may or may not include the BK418 strain. The culture is not particularly limited in its formulation, and may be a formulation conventionally used in the art. For example, the culture may be liquid or solid, and may be in the form of a round, or a concentrated form, or a dried form of the culture.

More specifically, the method for culturing the culture of the Bacillus subtilis strain BK418 may include culturing the seed culture, preparing the culture medium, and culturing the seed culture inoculating the culture medium.

The Bacillus subtilis strain BK418 was inoculated on a TSB medium sterilized for 15 minutes at 121 DEG C and cultured at 30 DEG C and 180 rpm for 18 hours to prepare seeds. 10 to 50 g / L of molasses or sugar was added to distilled water To prepare a culture medium, and 1% of the seeds can be inoculated in the culture medium and cultured at 30 DEG C and 180 rpm for 1 week.

The soil improvement composition means a microorganism or a component thereof that shows a beneficial effect on the environment such as soil. It converts nitrogen contained in the soil or air into ammonia nitrogen to facilitate environmental purification and easy absorption and utilization of the crop as a nitrogen source. At the same time, it can convert soil-decomposing substances such as fibrin in the soil or water quality into organic matter and inhibit pathogenic microorganisms by antibacterial action, so that the soil can be improved.

In addition, since the Bacillus subtilis BK418 strain can be cultured in a simple and easy new culture method using molasses as a single nutrient source, it can be easily applied to various industries such as a composition for soil improvement with an inexpensive medium raw material have.

Hereinafter, the present invention will be described in more detail by describing experimental examples.

Experimental Example 1

Bacillus subtilis ( Bacillus subtilis ) Confirmation of nitrogen fixation ability of strain BK418

(1) Culture of strain and viable cell count

Bacillus subtilis strain BK418 was inoculated into a liquid medium of TSB (tryptic soy broth, BD, USA) sterilized at 121 ° C for 15 minutes and cultured at 30 ° C and 180 rpm for 18 hours.

Growth of Bacillus subtilis strain BK418 was confirmed in distilled water by using Nitrogen Fixing Bacteria (NFB) liquid medium and 2% agar as a solid medium containing nitrogen source as shown in Table 1. The medium was sterilized at 121 占 폚 for 15 minutes.

Bacillus subtilis strain BK418 was plated on NFB solid medium and cultured at 30 ° C. for 1 week. As a result, it was confirmed that the growth was observed in the solid medium excluding the nitrogen source as shown in FIG. In addition, it was confirmed that by inoculating the microorganisms of the 1% in the number of NFB broth out of about 30 ℃, for one week under the conditions of 180rpm it carries out a number of viable cells measured after culture results 10 ³ cfu / ㎖ cells. These results indicate that Bacillus subtilis strain BK418 can grow through nitrogen fixation even in the environment where nitrogen source is excluded.

(2) Nitrogen fixing performance test

Nitrogen fixing ability of Bacillus subtilis strain BK418 was confirmed by Nestlé reagent. The Nessler reagent is a reagent for identification of nitrogen compounds, and its limit detection concentration is about 0.2 ppm for detection or quantification of high sensitivity to ammonia gas or ammonium ion. Dissolve iodide (HgI ₂ ) in an aqueous solution of potassium iodide and add an aqueous solution of potassium hydroxide to make it strongly alkaline. When it reacts with ammonia salt, it precipitates brown. When the amount of ammonia salt is small, it dissolves in excess reagent, . Particularly when the amount is small, a colorimetric assay is carried out. NFB liquid medium was inoculated with 1% of seed culture and cultured at 30 ° C and 180 rpm for 1 week. After centrifugation at 8,000 xg, the supernatant was filtered with a 0.2 ㎛ membrane filter, and then a small amount of Nessler reagent was added to brown And the formation of ammonia nitrogen in the culture liquid can be confirmed by confirming the discoloration of yellowish brown. As shown in FIG. 2, when a small amount of the Nessler reagent was reacted with the culture solution, it was confirmed that discoloration to yellowish brown and precipitation of brown were formed. Thus, the strain of Bacillus subtilis BK418 transforms nitrogen contained in air into ammonia nitrogen Nitrogen fixation ability.

Experimental Example 2

Bacillus subtilis ( Bacillus subtilis ) Culture method of BK418 strain

Bacillus subtilis strain BK418 was inoculated on a TSB liquid medium sterilized for 15 minutes at 121 ° C and cultured at 30 ° C and 180 rpm for 18 hours.

The amount of molasses in distilled water was varied to 10, 20, 30, and 50 g / L, and the growth of Bacillus subtilis BK418 strain was confirmed by the amount of molasses. The medium was sterilized at 121 占 폚 for 15 minutes.

1% of the seeds were inoculated into the medium containing molasses and cultured for 1 week at 30 ° C and 180 rpm. The number of viable cells during the culture was diluted by a 10-fold dilution method and 100 μl was plated on a TSB solid medium to confirm the number of colonies.

As a result, the viable cell count of the Bacillus subtilis strain BK418 cultured in the molasses medium is shown in Table 2.

As shown in Table 2, when 10 g / L of molasses was added at the time of preparing the medium, viable cell counts of 10 ⁷ cfu / ml or more were observed. When 20 g / L was added, viable cell counts of 10 ⁸ cfu / As a result, it was found that the production of a composition containing the Bacillus subtilis BK418 strain or a culture thereof as an active ingredient satisfies the optimum growth conditions when the molasses is added to the Bacillus subtilis strain BK418 at a concentration of 20 g / Can be used.

Experimental Example 3

Bacillus subtilis ( Bacillus subtilis ) Confirmation of resolution of BK418 strain

SM medium containing 1% sodium chloride, 0.5% yeast extract, 1% peptone, and 2% skim milk and 2% soluble starch instead of skim milk in the SM medium were used to identify the enzyme produced by Bacillus subtilis strain BK418 And CMC medium containing 1% of carboxy methyl cellulose were prepared. Bacillus subtilis strain BK418 was inoculated into each solid medium and incubated at 30 ° C for 48 hours. The resulting clear zone was treated with protease, amylase and fibrinolytic enzyme cellulase) were investigated.

FIG. 3 is a photograph showing the results of activity of protease, amylase and cellulase through the supernatant of Bacillus subtilis strain BK418 according to the present invention. As a result, And the cleaved clear zone was formed on the surface of the cells.

Experimental Example 4

Bacillus subtilis ( Bacillus subtilis ) Antibacterial activity of strain BK418

Escherichia coli used in this experiment is a Gram-negative pathogenic bacterium which is present in human and animal intestines and causes abdominal pain, diarrhea and enteritis. It is inoculated into LB (Luria Bertani) liquid medium and is cultured under conditions of 30 ° C. and 180 rpm After culturing for 21 hours, the cells were adjusted to 1 x 10 < ⁷ > cfu / ml and 100 [mu] l was plated on LB solid medium. A paper disk (ADVANTEC, Japan) was placed on top of the stained LB solid medium to adsorb 50 μl of the antimicrobial substance and cultured at 30 ° C. for 24 hours. The diameter of the clear zone (mm) Respectively. The antimicrobial substance was obtained by inoculating Bacillus subtilis strain BK418 in a TSB liquid culture medium and culturing at 30 ° C. and 180 rpm for 18 hours. The supernatant obtained after centrifugation at 8,000 × g was filtered with a 0.2 μm membrane filter to measure antimicrobial activity And used as a sample.

FIG. 4 is a photograph showing the antimicrobial activity of Escherichia coli of the supernatant of Bacillus subtilis strain BK418 according to the present invention. As shown in FIG. 4, And Bacillus subtilis strain BK418 or a culture thereof showed strong antibacterial activity against pathogenic bacteria.

The Bacillus subtilis BK418 strain or the culture thereof according to the present invention can convert a refractory substance such as fibrin or lignin into an organic substance, inhibit microorganisms by an antibacterial action, and have nitrogen fixing ability, And the like.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined in the appended claims. It will be possible.

<110> SEO, BUM-GU <120> Bacillus subtilis BK418 strain having complex digestive          productivity and antifungal activity and uses thereof <130> 0001 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1433 <212> DNA <213> Bacillus subtilis <400> 1 gctataatgc agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60 tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa 120 taccggatgg ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt 180 acagatggac ccgcggcgca ttagctagtt ggtgaggtaa yggctcacca aggcaacgat 240 gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300 acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360 gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt 420 cgaatagggc ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540 aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600 ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660 gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960 tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1260 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380 gtaacacccg aagtcggtga ggtaaccttt aggagccagc cgccgaagtg aca 1433

Claims (5)

Bacillus subtilis strain BK418 (KACC91903P) having nitrogen fixation ability, fibrinolytic activity and antibacterial activity or a culture solution thereof as an active ingredient. The method according to claim 1,
Wherein the Bacillus subtilis strain BK418 (KACC91903P) transforms nitrogen existing in air or environment into ammonia nitrogen. The method according to claim 1,
Wherein the Bacillus subtilis strain BK418 (KACC91903P) has a digestive enzyme-producing ability against fibrin, and converts a refractory substance such as fibrin present in the soil or water quality into an organic material. The method according to claim 1,
Wherein the Bacillus subtilis strain BK418 (KACC91903P) has an antibacterial activity against a pathogenic microorganism. A method for culturing a culture of the strain of Bacillus subtilis BK418 (KACC91903P) according to claim 1,
Culturing the strain of Bacillus subtilis BK418 (KACC91903P) in a TSB medium sterilized at 121 ° C for 15 minutes at 30 ° C and 180 rpm for 18 hours;
Preparing a culture medium containing molasses in distilled water, and
Culturing the above-mentioned culture medium with 1% of said seeds inoculated and culturing at 30 DEG C and 180 rpm for 1 week; and culturing the culture of the strain of Bacillus subtilis BK418 (KACC91903P).

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