KR20170099199A - Novel Bacillus species CKY1202 and uses thereof - Google Patents

Abstract

The present invention relates to a new type of Bacillus sp. CKY1202 flora and a purpose thereof. More specifically, the present invention relates to Bacillus sp. CKY1202 flora for removing phosphorus, a method to cultivate the flora, and a method to remove phosphorus by using the flora. As the Bacillus sp. CKY1202 flora of the present invention is able to remove phosphorus, the present invention is able to be applied to the purification of underground water, surface water, industrial water, waste water, and sewage, which contain phosphorous.

Description

Novel Bacillus sp. CKY1202 strain and its use {Novel Bacillus species CKY1202 and uses thereof}

The present invention relates to a novel strain of Bacillus sp . CKY1202 and its use, and more particularly to a strain of Bacillus sp . CKY1202 deposited with deposit number KCTC18448P which removes phosphorus , A composition for removing phosphorus containing the strain, a method for culturing the strain, and a method for removing phosphorus using the strain.

As the characteristics of sewage and wastewater discharged from industrialization and population increase, there are many difficulties in the efficient treatment of these. These untreated pollutants flow into rivers and other waters, and cause many problems in efficient water quality management. In particular, nutrients such as nitrogen and phosphorus are the main cause of eutrophication and the importance of management is increasing. The average concentration of total phosphorus (T-P) in the wastewater is 5 to 20 mg / L, and most of it comes from synthetic detergents. Most of the existing wastewater treatment processes in the 1990s focused on removing biochemical oxygen demand (BOD) and suspended solids (SS). Therefore, the reduction of dissolved oxygen was caused by the eutrophication caused by discharging into the water system without removing nitrogen and phosphorous (Grady et al., 1999). The influx of nitrogen into the water system causes problems such as the economic loss due to the increase of the water purification cost and the difficulty in securing the safe and clean water resources in the public health. Therefore, it is the most important to block the influx of nutrients into the water system As a fundamental solution, the treatment of nitrogen and phosphorus is emphasized in addition to the removal of organic matter from wastewater, wastewater and livestock wastewater. However, in the case of nitrogen, the nitrogen in the air may be introduced into the water system by the nitrogen-fixing bacteria. Therefore, it is known that it is more effective to treat phosphorus rather than nitrogen to prevent eutrophication. This trend has already strengthened the emission regulations of nitrogen and phosphorus in various countries of the world including Switzerland and the United States, and researches are actively carried out to efficiently remove nitrogen and phosphorus in wastewater. In Korea, the Ministry of Environment is also in the process of announcing a legislative proposal to amend a part of the Enforcement Regulations of the Waterworks Act that adopts a new water quality standard that significantly reduces the concentration of total phosphorus discharged from sewage treatment plants to 1/10 to 1/4 of the current 2 mg / L level (Ministry of Environment Press, 2009).

On the other hand, there are physical and chemical methods to remove environmental pollutants, but biological purification technology (bioremediation) is less costly, environmentally friendly and does not cause secondary pollution. In recent years, approaches to these methods have been regarded as important. This technique exploits the ability of microorganisms to actively respond to soil responses. Biological methods to remove phosphorus from wastewater utilize the principle that microorganisms in the water absorb phosphorus as a nutrient during metabolism or are removed by some microbes by the Luxury Uptake (Levin and Sharpiro, 1965 ). Phosphorus removal by biological methods is safe and efficient. Phosphorus removal processes using this method include A / O, PhoStrip, and SBR (Sequencing Batch Reactor) processes.

However, despite many studies involving biological elimination, the microbiological mechanisms involved in phosphorus removal have not yet been established. For example, no clear mechanism has been identified for the source of the reducing power needed to store organic carbon sources in the form of polyhydroxyalkanotate (PHA) in the anaerobic stage where phosphate is released during biological removal. In addition, it is a reality that the microorganisms performing the phosphorus removal role until now are not completely revealed.

Under these circumstances, the inventors of the present invention have studied for the search for microorganisms having excellent phosphorus removal ability, and as a result, it has been confirmed that a novel Bacillus sp . CKY1202 isolated from a wastewater treatment plant effectively removes phosphorus and completed the present invention .

One object of the present invention is to provide a Bacillus sp . CKY1202 strain deposited with deposit number KCTC18448P, which removes phosphorus.

It is another object of the present invention to provide a phosphorus removal composition comprising the strain or a culture product thereof.

Another object of the present invention is to provide a method for culturing the strain.

It is yet another object of the present invention to provide a method for removing phosphorus comprising reacting the strain with a sample comprising phosphorus.

In order to achieve the above object, the present invention provides, in one aspect, a strain of Bacillus sp . CKY1202 deposited with Deposit No. KCTC18448P, which removes phosphorus.

The Bacillus sp . CKY1202 strain that removes the phosphorus of the present invention is not limited thereto . As a specific example, the strain CKY1202 may be a strain derived from at least one selected from groundwater, surface water, industrial water, sewage, Lt; / RTI >

The term " phosphorus "of the present invention is the main cause of eutrophication with nitrogen, and the average concentration of TP (total phosphorus) in the wastewater is 5 to 20 mg / L, It comes out. Most of the existing wastewater treatment processes in the 1990s focused on removing biochemical oxygen demand (BOD) and suspended solids (SS). Therefore, the reduction of dissolved oxygen was caused by the eutrophication caused by discharging into the water system without removing nitrogen and phosphorous (Grady et al., 1999). The influx of nitrogen into the water system causes problems such as the economic loss due to the increase of the water purification cost and the difficulty in securing the safe and clean water resources in the public health. Therefore, it is the most important to block the influx of nutrients into the water system Since it is a fundamental solution, it is necessary to remove nitrogen and phosphorus along with the removal of organic matter from wastewater, wastewater and livestock wastewater. According to the untreated nitrogen and phosphorus, contamination by groundwater and soil has been reported.

In addition, the strain may remove phosphorus contained in at least one selected from groundwater, surface water, industrial water, sewage, wastewater and sewage, but is not limited thereto.

The term "16S rRNA" of the present invention, also referred to as 16S ribosomal RNA, means an rRNA that constitutes a 30S subunit of a prokaryotic ribosome. The sequence of the 16S rRNA is used for identification of the organism because there is almost no diversity among the same species, but diversity among other species appears. Therefore, identification of organisms which are impossible or difficult to cultivate, It is particularly useful for classification.

According to one embodiment of the present invention, the 16S rRNA gene was amplified to determine the nucleotide sequence and then analyzed by molecular systematics. As a result, 100% similarity to other strains belonging to the genus Bacillus sp . Was a novel strain belonging to the genus Bacillus (Fig. 1).

Thus, the strain was designated as " Bacillus sp . CKY1202" and deposited on Feb. 18, 2016, with the deposit number KCTC18448P deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology.

The strain of the present invention can remove phosphorous.

According to one embodiment of the present invention, the Bacillus sp . (20, 30 and 40 mg / l), oxygen (aerobic, anaerobic / aerobic) and carbon (glucose) concentrations were measured to determine the phosphorus removal efficiency of CKY1202 , acetate, and glucose and acetate). As a result, Bacillus sp. CKY1202 showed the highest phosphorus removal efficiency at 30 ° C and about 99% phosphorus at 20 mg / L (Figs. 2a and 2b)

In another aspect, the present invention provides a phosphorus removal composition comprising a strain of Bacillus sp . CKY1202 or a culture product thereof.

The description of the term " Bacillus sp . CKY1202 strain "of the present invention is as described above.

Since the Bacillus sp . CKY1202 strain provided by the present invention can remove phosphorus, the culture product of the Bacillus sp . CKY1202 strain contained in the composition is not particularly limited as long as it exhibits phosphorous removal activity As a specific example, the culture may be a culture of the strain, a culture supernatant, a lysate, fractions thereof and the like. The culture supernatant may be obtained by centrifuging a culture of a strain of Bacillus sp . CKY1202, and the lysate may be obtained by physically or ultrasonically treating Bacillus sp . CKY1202, The fractions can be obtained by applying the culture, the culture supernatant, the crushed material, etc. by centrifugation, chromatography or the like.

In another aspect, the present invention provides a method for culturing the Bacillus sp . Strain CKY1202 in a medium containing glucose and acetate.

In one embodiment of the present invention, the growth efficiency was the best when the strain was subjected to anaerobic conditions and mixed with acetate and glucose as a carbon source (FIG. 4).

In addition to the glucose and the acetate, the strain of the present invention can be cultured in a variety of conditions such as temperature, pH, inoculation amount, and the like. As a specific example, the temperature can be cultured at 20 to 40 ° C, To 0.5% by weight, preferably 0.5% to 20% by weight.

In the present invention, the strain may be specifically 20 to 40 占 폚, more specifically 25 to 35 占 폚, and still more specifically 30 占 폚.

According to one embodiment of the present invention, it was confirmed that the best growth was achieved at 30 ° C by the temperature-dependent (15, 25 and 30 ° C) growth in order to confirm the phosphorus removal efficiency of Bacillus sp . CKY1202 ).

In another aspect, the present invention provides a method for removing phosphorus, comprising reacting the strain of Bacillus sp . CKY1202 with a sample containing phosphorus.

The phosphorus removal method of the present invention includes a step of reacting a sample containing phosphorus to be removed with a strain of Bacillus sp . CKY1202 to remove phosphorus contained in the sample.

The term "sample" of the present invention means a contaminated water containing phosphorus, and may be a contaminated water further containing nitrogen. Specific examples thereof include ground water, surface water, industrial water, sewage, wastewater and sewage, But is not limited thereto.

At this time, the reaction temperature may be specifically 20 to 40 ° C, more specifically 25 to 35 ° C, and more specifically 30 ° C; The reaction time may be specifically from 5 minutes to 24 hours, more specifically from 10 minutes to 2 hours, and still more specifically from 70 minutes.

In one embodiment of the present invention, it was confirmed that most of phosphorus was removed by Bacillus sp . CKY1202 at 30 DEG C in 70 minutes (Fig. 2B).

The Bacillus sp . CKY1202 strain of the present invention can remove phosphorus, and thus can be widely used for the purification of phosphorus-containing groundwater, surface water, industrial water, sewage, wastewater and sewage.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a molecular-scale genealogical classification scheme using the 16S rRNA gene sequence of Bacillus sp . CKY1202 strain.
FIG. 2 is a graph showing changes in temperature of Bacillus sp . (Fig. 2A) and phosphorus removal ability (Fig. 2B) of CKY1202.
FIG. 3 is a graph showing the effect of Bacillus sp . (Fig. 3A) and phosphorus removal ability (Fig. 3B) of CKY1202.
FIG. 4 is a graph showing the effect of oxygen concentration on Bacillus sp . (Air G: 1% glucose treatment, air A: aerobic condition and 1% acetate treatment, air AG: aerobic condition, 0.5% acetate, 0.5% glucose treatment, non-air G : Anaerobic / aerobic conditions and 1% glucose treatment, non-Air A: anaerobic / aerobic conditions and 1% acetate treatment, non-Air AG: anaerobic / aerobic conditions and 0.5% acetate, 0.5% glucose treatment).
FIG. 5 is a graph showing the distribution of Bacillus sp . (Air G: aerobic condition and 1% glucose treatment, air A: aerobic condition and 1% acetate treatment, air AG: aerobic condition, 0.5% acetate, 0.5% glucose treatment, air B: CKY1202) Bacillus sp . CKY1202 strain, non-air G: anaerobic / aerobic and 1% glucose treatment, non-Air A: anaerobic / aerobic and 1% acetate treatment, non-Air AG: anaerobic / 0.5% acetate, 0.5% glucose treatment, non-Air B: Bacillus sp . CKY1202 strain)

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example  One. New strain Bacillus  genus( Bacillus sp . ) Isolation and Identification of Strain

Example  1-1. Isolation and Culture of Strain

The microorganisms were separated from the wastewater collected at the wastewater treatment plant. 5 mL of wastewater collected at the wastewater treatment plant is filtered with a 0.22 μm pore size membrane filter under reduced pressure and washed with sterilized distilled water. The filtrate is discarded and 5 mL of sterile distilled water is added to the remaining cells of the filter. Cells are collected, 2 mL was inoculated into a 250 mL Erlenmeyer flask containing the culture medium, and cultured with shaking (30 ° C, 150 rpm) for 48 hours. The phosphorus concentration of the medium was set at 20 mg / L. The culture medium used for the microbial culture was a modified Zafiri medium (Zafiri, 1995). Table 1 shows the composition of the solutions in the test media.

Trace metal solution was filtered using membrane filter (Pore size: 0.45 μm, Diameter: 25 mm). 1 M Tris was adjusted to pH 7.0 ± 0.2 with dilute sodium hydroxide solution. Experimental media were prepared by mixing Basic media solution, Trace metal solution, Phosphate solution and 1 M Tris buffer solution at appropriate mixing ratios.

The cultured medium was plated on an agar plate and incubated at 30 ° C in an incubator to identify colonies. Eight microorganisms were selected considering the color and shape of the colony formed. The selected bacteria were inoculated 1% (v / v) into a 200-mL experimental medium contained in a 250-mL Erlenmeyer flask, and the growth and microinflora concentration of the microorganisms were measured with a spectrophotometer (SHIMADZU, UV mini-1240) The microorganisms were selected as one of the high species.

Example  1- 2. Measure the growth of strain

Cultures were collected by syringe using a spectrophotometer at 550 nm

The absorbance was measured.

Example  1- 3. Quantification of phosphoric acid

The concentration of phosphorus in the culture medium was measured by modifying the molybdenum method by ascorbic aicd (Callaway, 1995: 175-176). The culture broth was collected with a syringe and filtered with a membrane filter (Pore size: 0.20 μm, Diameter: 25 mm). 7 mL of distilled water and 1 mL of filtrate were added to the test tube and mixed well. 2 mL of ascorbic aicd-molybdate reagent was added as a coloring reagent, and the mixture was vortexed for 30 seconds and developed at 30 ° C for 30 minutes. The absorbance at 880 nm was measured with a spectrophotometer (SHIMADZU, UV mini-1240) using a control solution containing only a coloring reagent in water and compared.

Example  1- 4. Identification of strain

The selected microorganisms were identified by 16S rRNA gene sequencing. The nucleotide sequences of the microorganisms obtained by identification were compared and analyzed by using the NCBI BLAST program. At this time, the homology criterion was set at 95% or more, and the most homologous microorganisms were selected as the analysis result.

Example  2. Phosphorus removal evaluation of strains

Example  2-1. Evaluation of Phosphorus Removal of Strain

In a 250 mL Erlenmeyer flask, 200 mL of the experimental medium with the phosphorus concentration of 20 mg / L was added, and the microorganisms isolated from the wastewater were inoculated into Erlenmeyer flasks at 1% (v / v) and shaken at 30 ° C and 150 rpm. Cultures were collected at time intervals and the growth and phosphorus concentrations of microorganisms were measured using a spectrophotometer (SHIMADZU, UV mini-1240). The concentration of phosphorus was collected by filtration through membrane filter (pore size: 0.20 μm, Diameter: 25 mm) and the remaining concentration in the filtrate was measured. The experiment was repeated three times.

Example  2-2. Temperature change Bacillus sp . Of CKY1202  Removal of phosphorus

In a 250-mL Erlenmeyer flask, add 200 mL of the experimental medium with a phosphorus concentration of 20 mg / L, add 1% (v / v) Bacillus sp . CKY1202 was inoculated and cultured at 15 ° C, 25 ° C and 30 ° C with shaking at 150 rpm. Samples were taken at regular intervals and cultured using a SHIMADZU (UV mini-1240) Phosphorus concentration was measured. The experiment was repeated three times.

Example  2-3. At the initial phosphorus concentration  Following Bacillus sp . Of CKY1202  Removal of phosphorus

In a 250 mL Erlenmeyer flask, add 200 mL of the experimental medium with the respective phosphorus concentrations set at 20, 30, and 40 mg / L, add 1% (v / v) Bacillus sp . CKY1202 was inoculated and shake cultured at 30 ° C and 150 rpm. Samples were taken at regular intervals and growth and phosphorus concentrations were measured using a spectrophotometer (SHIMADZU, UV mini-1240). The experiment was repeated three times.

Example  2-4. Oxygen conditions and Carbon source  According to the change Bacillus sp . Of CKY1202  Removal of phosphorus

In a 250-mL stoppered Erlenmeyer flask containing the test medium at a phosphorus concentration of 20 mg / L, 1% (v / v) Bacillus sp . CKY1202 was inoculated. One group was incubated for 12 hours with a stopper. After changing to a silicone plug, the cells were shaken for 60 hours. The other group was incubated with a silicone stopper and incubated for 72 hours with shaking at 150 rpm. Samples were taken at regular intervals and the growth and phosphorus concentrations of microorganisms were measured using a spectrophotometer (SHIMADZU, UV mini-1240). 1% (v / v) of glucose, acetate, glucose and acetate were added to the test media. The experiment was repeated twice.

Experimental Example  1. Isolation and Identification of Experimental Microorganisms

The nucleotide sequence of the 16S rRNA gene was amplified to identify the strain isolated in Example 1, and the result of the molecular systematic analysis was shown in FIG. The base sequence of the strain amplified using the 16S rRNA gene was about 1111 bp in length.

The 16S-rRNA partial sequence was analyzed and the most homologous microbial species was selected using NCBI BLAST program . As a result, Bacillus sp . And 100% homology in the amplified nucleotide sequence . It was confirmed that Bacillus sp . CKY1202.

Experimental Example  2. Bacillus sp . Of CKY1202  Phosphorus removal evaluation result

Experimental Example  2-1. Temperature change Bacillus sp . Of CKY1202  Removal of phosphorus

Bacillus sp . To confirm the phosphorus removal rate according to the temperature change of CKY1202, 1% (v / v) of Bacillus sp . CKY1202 was inoculated and cultured at 15, 25, and 30 ℃ for shaking culture. Microbial growth and phosphorus concentration were measured at constant time intervals. As a result, no growth of bacteria was observed at 15 ℃ (Fig. 2A). Bacterial growth was observed at 25 ℃ and 30 ℃, and the best growth was observed at 30 ℃. As a result, it was confirmed that almost all the phosphorus was removed at 30 ° C (FIG. 2B).

Experimental Example  2-2. At the initial phosphorus concentration  Following Bacillus sp . Of CKY1202  Removal of phosphorus

Bacillus sp . To determine the degree of phosphorylation of CKY1202, 1% (v / v) Bacillus sp . Was added to the experimental medium with phosphorus concentrations set at 20, 30 and 40 mg / L, respectively . The growth and phosphorus concentrations of CKY1202 were inoculated and shaken, and samples were taken at regular time intervals. The growth and phosphorus concentrations were not significantly different between 20 mg / L and 30 mg / L, The growth rate could be confirmed (Fig. 3A). As a result, it was confirmed that almost all the phosphorus was removed with a phosphorus removal rate of about 99.9% in the medium of 20 mg / L, and about 75.42% and 40 mg / L in the medium of 30 mg / And a phosphorus removal rate of about 59.57% was confirmed in the medium (Fig. 3B).

Experimental Example  2-3. Oxygen conditions and Carbon source  Depending on the type Bacillus sp . Of CKY1202  Removal of phosphorus

In order to confirm the change of phosphorus removal by initial oxygen condition and carbon source type, the culture medium containing glucose, acetate and glucose and acetate was divided into 12 hours anaerobic condition and anaerobic condition. As a result of the measurement of concentration, it was confirmed that the growth was slowed at the early stage in the treatment with the mixture of acetate and glucose as the carbon source under the anaerobic condition, but the highest growth was observed in the late stage (FIG. 4). The growth rate of acetate and glucose as a carbon source in aerobic condition was higher than that of control. Growth of the treatment group with acetate as a carbon source was the lowest. That is, when two carbon sources, which are not one carbon source, were mixed, the growth was the best, followed by glucose> acetate. It was also found that the growth of the treated plants after the anaerobic residence time was better than that of the treated plants.

The removal rate of phosphorus was highest in the anaerobic condition where the growth was the highest, and the carbon source was the highest in the semi-treatment with the mixture of glucose and acetate (FIG. 5). Next, it was shown that the carbon source was increased by glucose and acetic acid treatment in aerobic condition, followed by glucose and acetate treatment in aerobic condition. That is, the phosphorus removal rate was more sensitive to oxygen than the carbon source type. The treatments with the lowest removal rate were found to be treated with the addition of acetate as a carbon source under anaerobic conditions.

Based on the above results, the novel strain Bacillus sp. CKY1202 can be used as a useful microorganism for inhaling in various wastewater treatment facilities.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Korea Biotechnology Research Institute KCTC18448P 20160218

Claims (7)

Bacillus sp . CKY1202 strain deposited with accession number KCTC18448P, which removes phosphorus.
The method according to claim 1,
Wherein the strain is derived from at least one selected from the group consisting of groundwater, surface water, industrial water, sewage, wastewater and sewage.
The method according to claim 1,
Wherein the phosphorus is contained in at least one selected from the group consisting of groundwater, surface water, industrial water, sewage, wastewater and sewage.
A composition for removing phosphorus comprising the Bacillus sp . CKY1202 strain of claim 1 or a culture product thereof.
A method for culturing the strain of claim 1 in a medium containing glucose and acetate.
6. The method of claim 5,
Wherein the strain is cultured at 20 to < RTI ID = 0.0 > 40 C. < / RTI >
A method for removing phosphorus, comprising reacting the strain of claim 1 with a sample comprising phosphorus.

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