KR102051873B1 - Bacillus siamensis 1HC-NA strain having nitrogen removal activation, and microbial agent comprising the same for removing nitrogen - Google Patents

Abstract

The present invention Bacillus cyamensis having a nitrogen reducing activity ( Bacillus siamensis ) 1HC-NA strain and a microorganism preparation for nitrogen reduction using the same, Bacillus siamensis ( Bacillus siamensis ) 1HC-NA strain has a nitrite oxidation ability, denitrification capacity, and nitrogen reduction ability, for nitrogen treatment It can be usefully used as an environmentally friendly nitrogen reducing microbial agent in water treatment process.

Description

Bacillus siamensis 1HC-NA strain having nitrogen removal activation, and microbial agent comprising the same for removing nitrogen}

The present invention relates to a novel Bacillus siamensis 1HC-NA strain (KCTC 13372BP) having a nitrogen reducing activity, a culture thereof, and a microbial agent for reducing nitrogen containing the same as an active ingredient.

Water pollution is known to have natural and artificial causes, and in particular, water pollution by nitrogen may be an artificial cause. Most of the nitrogen comes from domestic wastewater, agricultural wastewater and livestock wastewater.

Nitrogen in water contains proteins, amino acids, organic nitrogen such as urea, ammonia nitrogen (NH ₄ ⁺ -N), and nitrite nitrogen (NO ₂ ^-- N). ) And nitrate nitrogen (NO ₃ ^-- N), and organic nitrogen in water is decomposed into ammonia nitrogen by the action of microorganisms and then oxidized to nitrate nitrogen through nitrite nitrogen. Algae that live in water multiply by using inorganic nitrogen such as ammonia nitrogen, nitrite nitrogen and nitrate nitrogen as nutrients. Therefore, when the nitrogen content in the water is high, it is considered as nitrogen wastewater when the algae proliferation is caused by the algae proliferation and environmental pollution such as odor, and when the total nitrogen content in the wastewater exceeds 60 mg / L. At present, wastewater treatment processes are known to reduce nitrogen content in wastewater through chemical treatment such as oxidation. In the case of the oxidation method, not only the pH decreases due to the acid treatment, but also the reagent used also flows into the stream.

In recent years, attempts have been made in various places to reduce the nitrogen content of water biologically, such as utilizing anaerobic microorganisms in an anaerobic tank during the water treatment process (environmental microbial sludge for high efficiency nitrogen and phosphorus removal in contaminated seawater and Polluted seawater treatment method using the same, Korea Patent No. 1733130).

Biological nitrogen abatement is achieved by nitrifying and denitrifying microorganisms with ammonia nitrogen (NH ₄ ⁺ -N) as nitrate nitrogen (NO ₃ ^-- N) (nitrification) and nitrate nitrogen (NO ₃ ^-- N) as nitrogen gas ( N2) (denitrification) to remove nitrogen in stages.

Microbial preparations are direct use of living microorganisms, and their vitality must be maintained until the moment they are consumed, and the living function must be exerted. Microbial agents are currently used in a wide variety of fields such as deodorants, air purifiers, herbicides, soil pesticides, soil improvers, growth promoters, water purification agents. Jungyimyeo various strains, such as Bacillus (Bacillus), Pseudomonas (Pseudomonas), photosynthetic bacteria, for use as agents to improve the environment have been developed, in recent years, and also as the development of bacteria immobilization technology sold by immobilizing the microbial agent to a carrier.

Currently, the development technology of microbial preparations has been improved in Korea, but the level of development is still insignificant compared to advanced foreign countries, and microbial preparations consumed in Korea tend to be imported and used mainly in foreign countries.

On the other hand, Bacillus sp . Microorganisms are aerobic or anaerobic gram-positive bacillus, generally recognized as safe (GRAS), and have various activities such as denitrification, iron reduction, and manganese oxidation. It is known to inhabit extensively.

Therefore, the present inventors isolate the Bacillus genus xiamensis species microorganisms having a nitrogen reducing activity, and confirm that the separated microorganisms have nitrite oxidizing ability, denitrification ability, and nitrogen reducing ability, thereby treating the microorganisms in wastewater treatment. It can be used as a microbial agent for environmentally friendly nitrogen reduction in biological nitrogen treatment.

The present inventors endeavored to develop an environment-friendly nitrogen reduction microbial agent, and as a result, by confirming that the novel Bacillus siamensis 1HC-NA strain of the present invention has nitrite oxidation, denitrification, and nitrogen reduction ability The present invention has been completed.

Accordingly, an object of the present invention is to provide a Bacillus siamensis 1HC-NA strain (KCTC 13372BP) having a nitrogen reducing activity and a microbial agent for reducing nitrogen using the same.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides Bacillus siamensis 1HC-NA strain (KCTC 13372BP) having a nitrogen reducing activity.

In one embodiment of the invention, the strain is characterized in that it comprises a 16S rRNA gene represented by SEQ ID NO: 1.

In another embodiment of the present invention, the strain is characterized by having a nitrite oxidizing ability, denitrification ability or nitrogen reducing ability.

In addition, the present invention is effective at least one selected from the group consisting of the Bacillus siamensis 1HC-NA strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof. Provided as a component, it provides a microbial agent for reducing nitrogen.

In one embodiment of the present invention, the dried material is characterized in that the lyophilized powder of the Bacillus siamensis 1HC-NA strain.

In addition, the present invention comprises the steps of (a) culturing the Bacillus siamensis 1HC-NA strain, (b) suspending the cells of the cultured strain in a medium and then lyophilized to prepare a lyophilized powder Step, and (c) provides a method for producing a microbial preparation for nitrogen reduction, comprising the step of preparing a microbial preparation comprising the lyophilized powder.

In one embodiment of the present invention, the freeze-drying in the step (b) is frozen for 1 to 10 days after suspending the cells in skim milk medium containing 0.05 to 1% w / v yeast extract It is characterized by drying.

In another embodiment of the present invention, the microbial preparation in step (c) is characterized in that it comprises 0.1 to 10% by weight of lyophilized powder, 90 to 99.9% by weight of glucose.

Bacillus siamensis 1HC-NA strain according to the present invention has been found to have nitrite oxidation, denitrification, and nitrogen reducing ability, it is expected that it can be usefully used for biological nitrogen treatment in wastewater treatment process do.

1 is a view showing the nitrite oxidation activity of Bacillus siamensis 1HC-NA strain according to an embodiment of the present invention.
Figure 2 is a view showing the denitrification activity of Bacillus siamensis 1HC-NA strain according to an embodiment of the present invention.
3 is a diagram showing the total nitrogen reduction activity of Bacillus siamensis 1HC-NA strain according to an embodiment of the present invention.
4 is a diagram showing the nucleotide sequence of the gene (rDNA) encoding 16S rRNA of Bacillus siamensis 1HC-NA strain according to an embodiment of the present invention.
Figure 5 is a phylogenetic schematic diagram comparing the base sequence (bp) of the gene (rDNA) encoding 16S rRNA of Bacillus siamensis 1HC-NA strain according to an embodiment of the present invention with a known strain It is a figure shown.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, the present invention will be described in detail.

An object of the present invention is to provide a Bacillus siamensis 1HC-NA strain (KCTC 13372BP) having a nitrogen reducing activity.

The sequence of the gene (rDNA) encoding the 16S rRNA of the strain is represented by SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1 has 99% homology with Bacillus siamensis KCTC 13613 ^T , which is a published strain, and 1% difference between the two strains was recognized, and it was found to be a novel strain.

Accordingly, the present inventor named the novel strain obtained as Bacillus siamensis 1HC-NA strain, deposited on October 20, 2017 to the Korea Research Institute of Bioscience and Biotechnology (KCTC) accession number KCTC Was given 13372BP.

The strain of the present invention can be obtained by separating and identifying from soil samples, and has nitrite oxidizing ability, denitrification ability, nitrogen reducing ability.

The nitrite oxidizing ability refers to the ability to oxidize nitrite nitrogen (NO ₂ ^-- N) produced by the oxidation of ammonia nitrogen (NH ₄ ⁺ -N) in the waste water to nitrate nitrogen (NO ₃ ^-- N), When the nitrite oxidation ability was based on the growth of the strain in the nitrite oxidation activity measuring medium (nitrite oxidation agar), the Bacillus siamensis 1HC-NA strain was confirmed to grow in the nitrite oxidation activity measuring medium (See FIG. 1 and Experimental Example 1).

In addition, the denitrification capacity refers to the ability to remove the nitrate nitrogen (NO ₃ ^-- N) by converting the nitrogen gas (N2), wherein the denitrification capacity is the denitrification activity measuring medium (deitrification agar) around the strain Based on the color change, the color change from green to blue was observed around the Bacillus siamensis 1HC-NA strain in the denitrification activity measurement medium (see FIG. 2 and Experimental Example 2).

In addition, the nitrogen reduction capacity is treated by the Bacillus siamensis 1HC-NA strain in the synthetic nitrogen wastewater, and the water pollution process test method to measure the total nitrogen content of the sum of organic nitrogen and inorganic nitrogen from the Ministry of Environment It was carried out by applying the total nitrogen content measurement method shown in, and confirmed the reduction of nitrogen content in the wastewater treated with the strain (see Fig. 3 and Experimental Example 3).

Therefore, it was confirmed that the strain of the present invention has an effect of reducing nitrogen in the nitrogen wastewater, and accordingly, a gene (rDNA) encoding 16S rRNA of the Bacillus siamensis 1HC-NA strain was identified. It was. The gene encoding 16S rRNA of the strain (rDNA) was confirmed in 16S rRNA gene sequencing (see FIG. 4 and Example 2).

In addition, the phylogenetic schematic diagram using the gene (rDNA) encoding the 16S rRNA of the Bacillus siamensis 1HC-NA strain (see Fig. 5 and Example 2). The phylogenetic schematic of the strain was identified in Molecular Evolutionary Genetics Analysis 6.0 version.

Accordingly, the present invention provides Bacillus siamensis 1HC-NA strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof. It includes, it provides a microbial agent for reducing nitrogen.

At this time, the dried material is preferably a lyophilized powder of the Bacillus siamensis 1HC-NA strain, but is not limited thereto.

In the present invention, the microbial preparation may be prepared by adding additives such as additives, extenders, and nutrients to the strain or its culture. At this time, the additives include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl From aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 One or more selected may be used, and extenders and nutrients include skim milk, yeast extract, soy flour, rice, wheat, loess, diatomaceous earth, bentonite, dextrin, glucose and starch. Use one or more selected from, and as a disintegrant, bento One or more selected from the group consisting of bentonite, talc, dialite, kaolin and calcium carbonate can be used.

In addition, the present invention comprises the steps of (a) culturing the Bacillus siamensis 1HC-NA strain, (b) suspending the cells of the cultured strain in a medium and then lyophilized to prepare a lyophilized powder Step, and (c) provides a method for producing a microbial preparation for nitrogen reduction, comprising the step of preparing a microbial preparation comprising the lyophilized powder.

At this time, step (a) is a step of streaking the Bacillus siamensis 1HC-NA strain isolated from the soil in the culture medium and culturing under the same culture conditions. At this time, the medium is usually preferably agar medium (or nutrient agar), but is not limited now.

In step (b), the cells of the cultured strain are suspended in skim milk medium containing 0.05 to 1% w / v yeast extract, and then lyophilized for 1 to 10 days to freeze-dried powder. Manufacturing step. At this time, the skimmed milk medium (skim milk) medium using a skim milk medium containing a yeast extract of 0.4 to 0.6% w / v (skim milk) it is preferable that the survival rate and growth of the strain after lyophilization is more smooth Can be.

In addition, the step (c) is a step of preparing a microbial preparation comprising 0.1 to 10% by weight of the lyophilized powder and 90 to 99.9% by weight of the Bacillus siamensis 1HC-NA strain.

Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings. However, these Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples and Experimental Examples.

Example  One.

1-1. From soil Bacillus Xiamensis ( Bacillus siamensis ) 1HC Isolation of -NA Strains

Soil samples were collected from soybean cultivated soils in Sangju, Gyeongsangbuk-do, to isolate microorganisms that could be used as nitrogen-reducing microorganisms. Microbial separation from soil was carried out by diluting 1 g of a soil sample in 9 mL of 0.85% NaCl solution, followed by agitation using 9 mL of 0.85% NaCl solution. 100 μL was removed from each diluted sample section and plated in nutrient agar. The microorganisms grown into single colonies were separated by morphological classification using colony appearance, and the final isolate was selected by evaluating the activity as a nitrogen-reducing microbial agent using the isolated microorganisms. The final isolated strain was identified by analyzing the nucleotide sequence by amplifying the 16S rRNA gene region.

1- 2. Preparation of lyophilized powders and microbial preparations

The isolated strain of Example 1-1 was streaked on nutrient agar medium and cultured, and a lyophilized formulation was used for the experiment. In the preparation of lyophilized preparation, the cells were suspended in skim milk medium containing 0.5% (w / v) yeast extract, and then lyophilized for 5 days to prepare lyophilized powder. In addition, a microbial preparation using the strain was prepared by mixing 1% lyophilized powder and 99% glucose of the strain to be used in the experiment to make the same composition as the nitrogen-reducing microbial preparation used as a control overseas product.

Experimental Example

1. Measurement of nitrite oxidation activity

To confirm the nitrite oxidation activity, the isolated strain of Example 1-1 was streaked on nutrient agar medium and cultured to obtain a single colony. Isolated single colonies were nitrite oxidation agar (NaNO ₂ 0.006 g, MgSO _{4 ·} 7H ₂ O 0.1 g, FeCl _{3 ·} 7H ₂ O 0.03 g, CaCl _{2 ·} 2H ₂ O 0.3 g, KH ₂ PO ₄ 1 g, Na ₃ 0.3 g of C ₆ H ₅ O ₇ , 1 g of NaHCO ₃ , 15 g of Agar were streaked and confirmed nitrite oxidation activity based on growth and formation of a clear ring around the strain by griss test. The strain was found to have better growth and nitrite oxidation activity in nitrite oxidation agar than control strain E. coli DH5a, the results are shown in FIG.

2. Denitrification  Active measurement

To confirm denitrification activity, the isolated strain of Example 1-1 was streaked on nutrient agar medium and cultured to obtain a single colony. A single colony was separated _{denitrification agar (NaNO 3 1 g,} Asparagine 1 g, 1% BTB solution 5 mL, Na 3 C 6 H 5 O 7 8.5 g, MgSO 4 · 7H 2 O 1.0 g, FeCl 3 · 7H 2 O 0.05 g, CaCl _{2 ·} 2H ₂ O 0.2 g, KH ₂ PO ₄ 1 g, Agar 15 g) was streaked and confirmed denitrification activity based on growth. E. coli DH5a was used as a control strain, and the strain confirmed the color change from green to blue around the strain in the denitrification agar, and the results are shown in FIG. 2.

3. Nitrogen Reduction  Active measurement

Synthetic nitrogen wastewater with a total nitrogen content of 200 mg / L ((NH ₄ ) ₂ SO ₄ 0.47 g, NaNO ₂ 0.25 g, NaNO ₃ 0.65 g, FeCl ₃ 0.2 mg, CaCl ₂ 0.2 mg, 0.9 mg) of MnSO ₄ ) was prepared and sterilized, and 10 g of the microbial preparation of Example 1-2 and 10 g of the overseas microbial formulation corresponding to 1% of the synthetic nitrogen wastewater volume were treated.

The foreign microbial agent is a BiOWiSH-Aqua product manufactured by BiOWiSH technology, and the main strain component of the foreign microbial agent is Bacillus sp. to be.

Sampling was performed every 12 hours from the time of treatment and the experiment was conducted for 2 days. In order to measure the total nitrogen reduction rate, the sample was centrifuged at 12,000 rpm for 3 minutes, the supernatant was taken, and the solution diluted 1,000-fold was used as the sample for the experiment. The experiment was conducted by applying the total nitrogen content measurement method described in the water pollution process test method announced by the Ministry of Environment. After the measurement, the regression equation was obtained. As a result of the experiment, the microbial preparation using the strain showed 20% nitrogen reduction rate for 2 days, and the results are shown in FIG. 3.

Example  2: Identification of Isolated Strains

Genomic DNA was extracted from the recovered cells for identification of nitrite oxidation, denitrification, and nitrogen reduction activities (Thompson, 1980), followed by polymerase chain reaction using the isolated genomic DNA as a template. 16S rRNA gene was amplified by chain reaction (PCR). In this case, 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used as universal primers (Lane 1991). The amplified PCR product was purified using a PCR purification system (Solgent, Daejeon, Korea). Sequencing for analyzing the entire sequencing of the purified PCR product was performed by Solgent (Solgent, Daejeon, Korea).

The analyzed sequencing results were compared using NCBI's BLASTN, and the homology and phylogenetic tree of sequencing were analyzed using neighbor-joining methods through Bioedit and Mega6 programs. . The results are shown in FIGS. 4 and 5.

The isolated strain was confirmed to have 99% homology with Bacillus siamensis KCTC 13613 ^T , a previously reported strain. Since the strain is 1% different from the existing Bacillus siamensis KCTC 13613 ^T , it is recognized as a novel strain belonging to the Siamensis species, which is named as Bacillus siamensis 1HC-NA. The strain was deposited in the Korea Institute of Biotechnology and Biotechnology Center (Accession No. KCTC 13372BP). In addition, as shown in Figure 4, the base sequence of the gene (rDNA) encoding the 16S rRNA of the Bacillus siamensis 1HC-NA strain of the present invention was confirmed.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13372BP

Deposit Date: 20171020

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Bacillus siamensis 1HC-NA strain having nitrogen removal          activation, and microbial agent configuring the same for removing          nitrogen <130> MP17-262 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1394 <212> DNA <213> Bacillus siamensis 1HC-NA 16S rRNA <400> 1 atgcagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420 gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtga 1394

Claims (9)

delete delete delete Bacillus siamensis 1HC-NA strain (KCTC 13372BP) comprising the 16S rRNA gene represented by SEQ ID NO: 1, the culture of the strain, the concentrate of the culture, the dried product of the culture, and combinations thereof At least one selected from the group consisting of as an active ingredient, characterized in that it has nitrite oxidation ability, denitrification ability, and total nitrogen reduction ability, microbial agent for nitrogen reduction.
The method of claim 4, wherein
The dried material is a microbial agent for reducing nitrogen, characterized in that the lyophilized powder of the Bacillus siamensis 1HC-NA strain.
(a) culturing the Bacillus siamensis 1HC-NA strain (KCTC 13372BP) comprising the 16S rRNA gene represented by SEQ ID NO: 1;
(b) suspending the cells of the cultured strain in a medium and then lyophilizing to prepare lyophilized powder; And
(c) preparing a microbial preparation comprising the lyophilized powder;
As a method for producing a microbial agent for reducing nitrogen, comprising:
The nitrogen-reducing microbial agent has a nitrite oxidation ability, denitrification ability, and total nitrogen reduction ability, the production method.
The method of claim 6,
In the step (b), the lyophilization is characterized in that the cells are lyophilized for 1 to 10 days after the cells are suspended in skim milk medium containing 0.05 to 1% w / v yeast extract. A method for producing a microbial agent for reducing nitrogen.
The method of claim 6,
The microbial preparation in the step (c), characterized in that it comprises 0.1 to 10% by weight of lyophilized powder, 90 to 99.9% by weight of glucose, the method for producing a microbial preparation for reducing nitrogen.


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