KR101661546B1 - Mixed marine bacteria having excellent abillity of removing nitrogrn and phosphorus and method for removing nitrogrn and phosphorus using the same - Google Patents

Abstract

The present invention relates to a mixed marine microorganism strain having an excellent function of removing nitrogen and phosphorus, and to a method for removing nitrogen and phosphorus in seawater by using the same. More specifically, the present invention relates to a mixed strain having an excellent function of removing nitrogen and phosphorus, wherein the mixed strain comprises Bacillus aryabhattai and Vibrio neocaledonicus strains; a microorganism formulation for removing nitrogen and phosphorus, wherein the microorganism formulation comprises the same; and a method for removing nitrogen and phosphorus, wherein the method comprises a step of applying the mixed strain or the microorganism formulation to water contaminated by nitrogen and phosphorus.

Description

TECHNICAL FIELD The present invention relates to a mixed marine microorganism having excellent nitrogen and phosphorus removal ability, and a method for removing nitrogen and phosphorus from polluted seawater using the same. BACKGROUND ART [0002]

The present invention relates to a mixed marine microorganism having excellent nitrogen and phosphorus removal ability and a method for treating contaminated seawater using the same, and more particularly, to provide a mixed marine microorganism which can be applied for purification of pollution in a farm, It is about how you can do it.

With the rapid development of the industry and the pollution of the marine environment becoming a big problem, the catch of the aquatic products has been decreasing, and the interest in the aquaculture has been rising to provide a stable supply of high quality aquatic products.

Although many studies have been carried out on environmentally friendly and economical forms globally, it is known that improving the aquaculture environment is not easy. In Korea, almost all the coastal area is used for aquaculture, but overuse of aquaculture feed and deterioration of water quality environment are becoming a big problem.

In the aquaculture industry, it is essential to increase the weight of the fish and to feed the fish to produce high quality fish. The aquaculture feed consists of three major components of carbohydrate, protein and fat, ash, crude fiber, calcium, phosphorus, and feeds with different composition ratios depending on cultivation. In particular, to increase the weight of fish, high protein and fat content feed should be used, protein is an essential amino acid source, fat is an important nutrient that plays an important role as a source of essential fatty acid.

However, the remaining unfed feed is known to increase biochemical oxygen demand in the farm and to produce high levels of nitrogen and phosphorus, leading to eutrophication, algae growth, and serious water pollution such as the production of toxic substances. In addition, high levels of ammonia and nitrite from the remaining feed in the water cause a direct disease in the fish. High concentrations of non-ionized ammonia are known to pass through the cell walls of fish and inhibit oxygen transport capacity and osmotic control in the blood. High nitrite concentration is transmitted through the gill by the normal chloride transport mechanism and forms methemoglobin, It is known to inhibit ability.

Recently, the microbiological removal of nitrogen and phosphorus by using microorganisms for the purpose of accelerating deterioration of water quality in aquaculture, removing nitrogen and phosphorus which are directly toxic to fish, and improving the water quality environment from pollutants including nitrogen and phosphorus The need for research is emerging.

In the advanced countries, the development and application of microbial agents for improving the water quality in the aquaculture have been greatly promoted. Compared with this, in Korea, the microbial agents have been developed to a great extent. However, Most of the products are imported from foreign countries and disturb the micro ecosystem existing in the aquaculture system.

Therefore, a microorganism or a microorganism preparation capable of effectively purifying marine water or an effective microorganism for purifying nitrogen and phosphorous pollution in a farm, treating the polluted seawater of nitrogen and phosphorus effectively, and effectively removing polluted seawater of nitrogen and phosphorus It is expected that it can be usefully applied in related fields.

Korean Patent Application No. 2006-0005916 Korean Patent Application No. 2001-0021625

Accordingly, one aspect of the present invention is to provide a mixed marine microorganism having excellent nitrogen and phosphorus removal ability for treating contaminated seawater, and a microorganism preparation containing the same.

Another aspect of the present invention is to provide a method for treating contaminated seawater containing nitrogen and phosphorus in contaminated seawater using the mixed marine microorganism.

According to one aspect of the present invention, the Bacillus Ari ahbatayi (Bacillus aryabhattai ) and Vibrio neo caledonikus ( Vibrio Neocaledonicus ), and a mixed marine microorganism excellent in nitrogen and phosphorus removal ability are provided.

The mixed marine microorganism may be selected from the group consisting of Bacillus subtilis aryabhattai SJ8) and Vibrio neo Caledonia kusu (Vibrio neocaledonicus M100SG10) strain (Accession No. KACC92021P).

According to another aspect of the present invention, there is provided a microorganism preparation for nitrogen and phosphorus removal for contaminated seawater treatment comprising the mixed marine microorganism of the present invention.

According to another aspect of the present invention, there is provided a nitrogen and phosphorus removal method comprising the step of applying the mixed marine microorganism or microbial agent to seawater contaminated with nitrogen and phosphorus.

It is preferable that the step of applying to the seawater is performed at a temperature of 20 ° C or more and less than 37 ° C.

Preferably, the step of applying to the seawater is carried out at a pH of 6 to 8.

It is preferable that the step of applying to the seawater is carried out within a range of 0.2 to 1.0 g / L of the initial bacterial inoculation amount.

Preferably, the concentration of nitrogen (TN) in the contaminated water is more than 0 to 150 mg / L, and the concentration of phosphorus (TP) in the contaminated water is preferably not less than 0 and not more than 100 mg / L, More preferably greater than 0 and less than 400 mg / L.

The nitrogen and phosphorous removal method is preferably carried out by a method selected from the group consisting of the A2 / 0 method, the SBR method and the MBR method.

According to the mixed marine microorganism of the present invention, it is possible to accelerate deterioration of water quality in seawater and aquaculture, to remove nitrogen and phosphorus which are directly toxic to fish, to improve the water quality environment from contaminants including nitrogen and phosphorus, Can be effectively restored.

1 is Bacillus Ari ahbatayi as a function of temperature (Bacillus lt; RTI ID = 0.0 > SJ8 ) < / RTI >
FIG. 2 is a graph showing changes in the pH of Bacillus subtilis lt; RTI ID = 0.0 > SJ8 ) < / RTI >
FIG. 3 is a graph showing changes in the concentration of Bacillus subtilis lt; RTI ID = 0.0 > SJ8 ) < / RTI >
4 is a vibrio as a function of temperature neo Caledonia kusu (Vibrio neocaledonicus M100SG10) strain of the present invention.
5 is a vibrio according to the pH change neo Caledonia kusu (Vibrio neocaledonicus M100SG10) strain of the present invention.
6 is a graph showing the ammonia removal effect of Vibrio neocaledonicus M100SG10 strain according to the initial bacterial concentration change.
Figure 7 (a) depicts the organic substances, nitrogen and phosphorus removal results in mixed marine bacteria of the present invention, Fig. 7 (b) is a Bacillus Ari ahbatayi (Bacillus strains remove nitrogen will showing an organic matter, nitrogen and phosphorous removal results of aryabhattai SJ8), Figure 7 (c) is the removal of neo strain Vibrio Caledonia kusu (shows an organic material, nitrogen and phosphorous removal results of Vibrio neocaledonicus M100SG10).
8 is a graph showing the experimental results in the A2 / 0 reactor using the mixed strain of the present invention.
9 is a graph showing the results of an SBR reactor experiment using a mixed strain of the present invention.
10 is a graph showing the results of an MBR reactor experiment using a mixed strain of the present invention.
Fig. 11 (a) is a view of an A2 / 0 series reactor, Fig. 11 (b) is a view of a SBR series reactor, and Fig. 11 (c) is a view of an MBR series reactor.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, a mixed marine microorganism having excellent ability of removing nitrogen and phosphorus is provided. The mixed marine microorganism of the present invention can be effectively applied to the contamination of nitrogen and phosphorus in water, especially when seawater such as aquaculture is contaminated with nitrogen and phosphorus, and can be effectively applied to the removal of organic matter. The nitrogen is preferably ammonia nitrogen.

More specifically, the mixed culture of the present invention is Bacillus Ari ahbatayi (Bacillus aryabhattai) and Vibrio neo Caledonia kusu (Vibrio neocaledonicus , more preferably the mixed strain is selected from the group consisting of Bacillus aryabhattai SJ8 and Vibrio neocaledonicus M100SG10). ≪ / RTI >

The mixed strain MNP1 of Bacillus aryabhattai SJ8 and Vibrio neocaledonicus M100SG10 was deposited on January 13, 2015 at the National Institute of Agricultural Science and Technology (KACC) On the 23rd day, a deposit certificate was obtained with the deposit number KACC92021P.

On the other hand, each of the strains contained in the mixed strain of the present invention was individually deposited in Korea Environmental Microorganisms Bank (KEMB).

More specifically, the Bacillus Ari ahbatayi (Bacillus aryabhattai SJ8) is KEMB 3001-129, Vibrio neo Caledonia kusu (Vibrio neocaledonicus M100SG10) was deposited with KEMB 3401-006.

According to the present invention, there is provided a microorganism preparation for removing nitrogen and phosphorus, which comprises a mixed strain having excellent nitrogen and phosphorus removal ability in the contaminated seawater. The microorganism preparation of the present invention may be provided with a strain fixed on a carrier.

The carrier which can be applied to the mixed strain of the present invention may be selected from the group consisting of ceramic aggregate, tidal flats and sludge, and the sludge is preferably granular sludge. When the granular sludge is used as the carrier, it is more effective in removing nitrogen and phosphorus.

Meanwhile, the ceramic aggregate preferably includes red mud, sludge sludge, and recycled soil, and may further include an electric furnace (EAF) dust. An example of the ceramic aggregate that can be used in the present invention is one having a specific gravity of 1.5 to 1.6, including 55 to 65 wt% of red mud, 25 to 35 wt% of sludge sludge and 5 to 15 wt% 55 to 65% by weight, 20 to 30% by weight of sludge sludge and 5 to 15% by weight of waste lime powder, and 1 to 5% by weight of EAF dust, the specific gravity being 0.7 to 0.9.

Further, the ceramic aggregate preferably has a water absorption rate of 15 to 25% and an apparent porosity of 17 to 26%.

Further, according to the present invention, there is provided a nitrogen and phosphorus removal method comprising the step of applying the mixed marine microorganism or microorganism preparation of the present invention as described above to water contaminated with nitrogen and phosphorus. At this time, the water is not particularly limited, but the present invention can be effectively applied to the decontamination of seawater.

That is, the mixed marine microorganism of the present invention accelerates deterioration of water quality and improves the water quality environment from contaminants including nitrogen and phosphorus which directly show toxicity to fish. Step, and more particularly, the nitrogen and phosphorous removal method of the present invention can be applied to an aquaculture farm.

Since the mixed marine microorganism of the present invention is an aerobic strain, the nitrogen and phosphorus removal method is preferably carried out under aerobic conditions.

The step of applying in water is preferably carried out at a temperature of 20 ° C to 37 ° C, preferably 22 ° C to 35 ° C, and more preferably 25 ° C to 28 ° C

If the temperature is less than less than 20 ℃ has a tendency to slow the nitrogen and phosphorus jeseo speed, the temperature is 37 ℃ is Ari ahbatayi Bacillus (Bacillus aryabhattai) to reduce the nitrogen removal effect by the strain, there is a tendency that the concentration of nitrogen increased, rather, that if the temperature rises Bacillus Ari ahbatayi (Bacillus aryabhattai ) metabolism seems to use different pathways.

The step of applying in water is preferably carried out at a pH of from 6 to 8, more preferably at a pH of from 6 to 7 when the concentration of nitrogen in the contaminated water is high, and at a pH of from 7 to 8 when the concentration of phosphorus is high . When the pH exceeds 8, Bacillus < RTI ID = 0.0 > aryabhattai ) strain tends to be degraded.

In the meantime, the step of applying to the water is preferably carried out within the range of 0.2 or more, preferably 0.2 to 1.0 g / L of the initial inoculum amount. When the initial inoculum amount is less than 0.2 g / L, The effect is insignificant, and a long time may be required to remove these contaminants. However, when the initial inoculation amount is more than 1.0 g / L, the removal of nitrogen and phosphorus tends to be accelerated. However, when the initial inoculation amount is increased, The inoculation amount is preferably in the range of 0.2 to 1.0 g / L.

The marine microorganism of the present invention is characterized in that the concentration of nitrogen (TN) in the contaminated water is in the range of more than 0 to 150 mg / L, the concentration of phosphorous (TP) in the contaminated water is in the range of 0 to 100 mg / L or less, The organic substance concentration can be effectively applied in a range of more than 0 to 400 mg / L or less.

That is, the pollution that can be applied and treated by the marine microorganism of the present invention is 5.0 mg / L of low concentration and 150 mg / L of high concentration in the case of total nitrogen (TN) L to 100 mg / L in the case of organic matter (COD), and 100 mg / L to 400 mg / L in the case of low concentration (100 mg / L or less) It is very good.

The nitrogen and phosphorus removal method may be carried out by a method selected from the group consisting of the A2 / 0 method, the SBR method and the MBR method.

As described above, the present invention accelerates deterioration of water quality in seawater and aquaculture, eliminates nitrogen and phosphorous which are directly toxic to fish, improves the water quality environment from contaminants including nitrogen and phosphorus, It can be effectively restored.

Hereinafter, the present invention will be described more specifically by way of specific examples. The following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Sample collection

Four areas (Masan Bay, Jinhae Bay, Jamsom, Caguri) were selected from the offshore Tongyeong coast of the South Sea, and seawater and sediments were collected from each region as shown in Table 1 below. In the case of seawater, the conditions of dissolved oxygen and temperature are different according to the depth of seawater. Therefore, various depths of seawater were collected and mixed in consideration of different microbial distribution. Samples collected in each area were stored in an ice box and transported.

Cage Masan Bay 돝 Jinhae Bay Latitude 34 ° 59'38.19 "N 35 ° 6'12.88 "N 35 ° 11'0.44 "N 35 ° 6'55.77 "N Hardness 128 ° 40'26.38 "E 128 ° 36'14.31 "E 128 ° 34'59.31 "E 128 ° 41'7.16 "E Depth of sea water 8, 10, 15m 8, 10, 15m 8, 10, 15m 8, 10, 15m Sediment depth Sediment surface Sediment surface Sediment surface Sediment surface

The seawater samples were sampled at 20 L from each spot using a sump. For anaerobic conditions, the seawater samples were injected into sealed bottles using a 50 ml syringe. The bottles used for sampling were previously anaerobically prepared and sealed with a septa and an aluminum lid. The surface of the sediment is sampled using a crab, and the sediment is placed in a sampling bottle prepared beforehand. The sediment is stored in an anaerobic jar with an anaerobic gas pack to maintain the anaerobic condition, Respectively. Sampling at each site was carried out three times in total (November 2013, March and April 2014), and screening was carried out by continuously separating the samples from the collected samples.

2. Strain isolation

In order to remove the nutrients required to grow marine algae, it was most efficient to use microorganisms isolated from the field, so microorganisms were isolated from the samples collected at the site.

The surface layer of sea water is in contact with the air layer and is in a state of aerobic condition due to continuous supply of oxygen, but as the depth increases, the light and oxygen become depleted and the anaerobic state appears. Therefore, microorganisms distributed in the surface layer and microorganisms distributed in the deep layer are different depending on the dissolved oxygen concentration. Since the concentration of dissolved oxygen in the sampling area of the two samples (seawater and sediment) was different, it was divided into aerobic condition and anaerobic condition. Different media were used to separate microorganisms of various species.

Particularly, in this experiment, microbial separation was performed under aerobic conditions. At this time, the aerobic conditions were 1/10 diluted marine agar (Difco 2216, Table 2), marine agar, 1/10 diluted R2A agar (MB cell, Table 3) and R2A agar, modified marine broth, Table 4) were used. The anaerobic conditions were marine agar diluted 1/10, marine agar diluted 1/15, R2A agar diluted 1/10, R2A agar diluted 1/15, 27S agar (Table 5), modified agar broth modified marine broth) was used. L-cysteine (0.035 g / ml) was used as a reducing agent in the anaerobic culture medium. The culture medium was prepared by adding 35 g / L of NaCl to all the culture media for culturing microorganisms isolated from seawater.

 The medium composition of the Marine Broth 2216 (control pH 7.6 ± 0.2) peptone 5 g Yeast extract 1 g Ferric citrate 0.1 g Sodium chloride 35 g Magnesium chloride 5.9 g Magnesium sulfate 3.24 g Calcium chloride 1.8 g Potassium chloride 0.55 g Sodium bicarbonate 0.16 g Potassium bromide 0.08 g Strontium chloride 34 mg Boric acid 22 mg Sodium silicate 4 mg Sodium fluoride 2.4 mg Ammonium nitrate 1.6 mg Disodium phosphate 8 mg D.W. 1000 ml

Composition of R2A broth (adjusted pH 7.2 ± 0.2) Proteose peptone 0.5 g Yeast extract 0.5 g The casein oxygen digest (acid digest) 0.5 g Glucose 0.5 g Soluble starch 0.5 g Dipotassium phosphate 0.3 g Magnesium sulfate 0.024 g Sodium pyruvate 0.3 g D.W. 1000 ml

The modified marine broth medium composition (adjusted pH 7.6 ± 0.2) Glucose 0.068 g Disodium succinate 0.100 g Ferric citrate (0.5%) 1 ml Sodium chloride 35 g Magnesium chloride 5.9 g Magnesium sulfate 3.24 g Calcium chloride 1.80 g Potassium chloride 0.55 g Sodium bicarbonate 0.16 g Potassium bromide 0.08 g Strontium chloride 34 mg Borik Mountain 22 mg Sodium silicate 4 mg Sodium fluoride 2.4 mg Ammonium nitrate 1.6 mg Disodium phosphate 8 mg D.W. 1000 ml

27s medium composition (adjusted pH 6.8) Yeast extract 1.0 g Disodium succinate hexahydrate 1.0 g Anhydrous ethanol 0.5 ml The ferric citrate solution (0.5%) 1.0 mL KH ₂ PO ₄ 0.5 g MgSO ₄ .7H ₂ O 0.4 g NaCl 35 g NH ₄ Cl 0.4 g CaCl ₂ .2H ₂ O 0.05 g 1x trace element solution SL-6 ^† 1 ml L-cysteine 0.035 g D.W. 1000 ml

^† trace element solution SL-6: Per 1 ^st distilled water _{1 L 0.1 g ZnSO 4 · 7H} 2 O, 0.03 g MnCl 2 · 4H 2 O, 0.3 g H 3 BO 3, 0.2 g CoCl 2 · 6H 2 O, 0.01 g CuCl ₂ .2H ₂ O, 0.02 g NiCl ₂ .6H ₂ O, 0.03 g NaMoO ₄ .2H ₂ O.

In order to separate the microorganisms from the collected seawater, the collected seawater was firstly filtered using a filter having a pore size of 8 μm (Whatman 1), and a 0.45 μm pore size filter (Whatman cellulose nitrate membrane , Diameter 47 mm) was used for secondary filtration. The filters used were collected in 250 ml bottles containing 200 ml of filtered seawater. After removing the microorganisms from the filter using a vortex or homogenizer, the supernatant was used. The supernatant was serially diluted to 10 ^-4 with 3% NaCl solution. 100 [mu] l of diluted samples were plated on each solid medium using a tripod, and the medium was cultured in a 28 < 0 > C incubator. Three days after the culture, a single strain was isolated according to the shape of the colonies and subcultured continuously in the same solid medium.

For sediment samples, 1.0 g / L of sediment samples were added to 3.0% NaCl solution, followed by vortexing for 30 minutes to remove microorganisms, and the supernatant was diluted to 10 ^-2 . 100 μl of the diluted sample was plated on a 10% marine agar, 10% R2A agar, 100% marine agar, 100% R2A agar plate in advance. The plated plates were incubated in a 28 ° C incubator. Three days after the culture, the strains were separated according to the shape of the colonies and continuously subcultured in the same solid medium.

As a result, a total of 3,490 microorganisms were isolated over three sampling cycles under aerobic conditions, and the number of strains isolated in March and April 2014 are shown in parentheses (Table 6). Many species were separated from diluted marine broth, and many were separated from sediments rather than from sea water.

Seawater sample Sediment sample gun 10% MB 10% R2A 100% MB 100% R2A 10% MB 10% R2A 100% MB 100% R2A Masan Bay 63 (51) 86 (95) 88 (75) 3 (50) 113 (81) 17 (25) 15 (50) 20 (46) 405 (473) Jinhae Bay 48 (83) 28 (42) 16 (61) 16 (22) 104 (107) 24 (32) 56 (61) 16 (71) 268 (478) Cage 54 (91) 144 (125) - (18) - (41) 126 (81) 12 (18) 54 (72) - (52) 390 (598) 돝 32 (72) 24 (32) 60 (54) 60 (69) 126 (145) 12 (21) 39 (41) 39 (48) 392 (482) gun 197 (297) 282 (293) 164 (208) 79 (182) 469 (514) 65 (96) 124 (224) 75 (217) 3490

Then, 16s rRNA gene sequencing analysis was performed to identify the isolated microorganisms. The isolated single colonies were assigned to Macrogen (Korea) for the sequencing analysis, and the nucleotide sequences were analyzed using 518F and 800R, which are used for identification of microorganisms, and the related species were identified using the Ezbiotexon homepage .

3. Establishment of nitrogen degradation strain

(1) Method for measuring ammonia nitrogen - Phenate method

The standard method (4500-NH ₃ F. Phenate Method) was used to measure ammonia nitrogen. This method forms chloramine (NH ₂ Cl) when ammonia reacts with sodium hypochlorite (NaOCl) contained in an oxidation solution (including 100 mL of alkaline citrate and 25 mL of sodium hypochlorite). At this time, the alkaline citrate contains 200 g of trisodium citrate and 10 g of sodium hydroxide. Phenol and sodium nitroprusside in a phenol solution containing 11.1 mL of liquid phenol (≥89%) and 95% allyl alcohol and a final volume of 100 mL were combined to form sodium phenoxide (C ₆ H ₅ NaO) It meets with sodium hypochlorite to form a 4-chlorophenol sodium salt (C ₆ H ₄ ClNaO). The formed 4-sodium chloride phenolate reacts with sodium phenolate to form indophenol (C ₁₂ H ₉ NO ₂ ) and the amount of ammonia nitrogen can be calculated by measuring the indene phenol formed. Indian phenol can be measured at 640 nm and a spectrophotometer DR / 4000U was used.

(2) Method for measuring nitrate nitrogen and nitrite nitrogen-HACH kit

HACH kit (140321-99, 26083-45) was used to measure nitrate nitrogen (NO ₃ ^- ) and nitrite nitrogen (NO ₂ ^- ), respectively, and HACH program was measured using 2530 and 2630, respectively.

(3) Nitrogen control strain isolation by oxygen condition

In aerobic conditions microorganisms convert ammonia to nitrate nitrogen. This is called the nitrification process. However, this process can not smoothly control nitrogen in land and marine farm wastewater. For this reason, in order to control ammonia nitrogen in the aerobic condition, it is necessary to develop a microorganism used as a raw material for absorbing ammonia nitrogen into the body and producing protein. In order to do this, we selected ammonia nitrogen removal strains first and then confirmed the presence of nitrate nitrogen during the culture.

To develop ammonia nitrogen control strains under aerobic conditions, the isolated strains were tested by inoculating the modified mineral salt medium with 0.01% yeast extract. The culture conditions were as follows: 28 ° C and 150 rpm for 3 days. Ammonia nitrogen was measured after initial and 3 days incubation to confirm the removal rate of ammonia nitrogen.

Modified mineral salt medium composition (adjusted pH 6.8 ± 0.2) KH ₂ PO ₄ 0.1 g K ₂ HPO ₄ 0.1 g Ammonium sulfate 0.05 g NaCl 30 g Glucose 1 g Magnesium sulfate 0.2 g Calcium chloride 0.02 g Ferric chloride 0.01 g 1X trace element solution SL-6 ^† 2 ml 1X Vitamin Solution ^‡ 1 ml D.W. 1L

_{0.1 g ZnSO 4 · H 2 O} , 0.03 g MnCl 2 · H 2 O, 0.3 g H 3 BO 3, 0.2 g CoCl 2 · H 2 O, 0.01 per 1 ^st distilled water 1 L: ^† trace element solution SL-6 g CuCl ₂ .H ₂ O, 0.02 g NiCl ₂ .H ₂ O, 0.03 g NaMoO ₄ .H ₂ O.

^‡ Vitamin solution: 10 mg biotin, 35 mg nicotinamide, 30 mg thiamin dichloride, 20 mg p-aminobenzoic acid, 10 mg pyridoxal chloride, 10 mg Ca-sodium pantothenate, 5 mg vitamin B12 per 1 L of 1 ^st distilled water.

4. Establishment of a phosphorylation strain

(1) Total method of measurement - HACH kit

Total Phosphate 0-3.5 mg / L PO ₄ ^{3 -} HACH kit (product number 2742645) was used to determine phosphorus removal rate. Phosphorus is also included in organic matter, and it is composed of poly-phosphate and ortho-phosphate as minerals. In order to detect total phosphor, phosphate should be converted into ortho-phosphate. Acid solution and potassium persulfate reagent should be converted into ortho-phosphate by hydrolysis when the sample is heated. The giver plays a role. 1.54 NaOH is neutralized to adjust the pH. When a phosphate reagent is added, molybdenum and ortho-phosphate react to form a phosphor-molybdate ) Is dyed blue. Total phosphorus can be measured by detecting phosphor-molybdate using 890 nm of DR / 4000U.

(2) In control strain isolation under exhalation conditions

The phosphorous accumulating organism (PAO) in the aerobic condition is a microorganism with EPS (extra-cellular polymeric substances), which accumulates in the form of polyhydroxyalkanoate in bacterial cells, and stored in poly-phosphate form. When these microorganisms meet oxygen, they are transported to EPS in polymerized phosphate state and then hydrolyzed into ortho-phosphate. In the EPS layer, hydrolyzed phosphorus in the form of orthophosphate has a circuit to store the phosphorus phosphate form inside the cell again. In this case, phosphorus is removed by absorbing the phosphate from the outer layer of EPS.

For the screening of phosphorus control strains under aerobic conditions, complex media with different yeast concentration and presence of peptone were used and 3.5% NaCl was used. The microorganisms were inoculated to OD (600 nm) of 1.0 and then inoculated in the medium at 1.0%, respectively, and cultured at 28 ° C and 150 rpm for 3 days.

On the other hand, the phenomenon of biologically phosphorus removal in anaerobic conditions has been found, but the complete circuit has not yet been revealed and is known to be biologically phosphorylated through the circuits of anaerobic / aerobic conditions.

Under anaerobic conditions, the phosphate-accumulating microorganisms take up fatty acids such as acetic acid and accumulate them in the form of polyhydroxyalkanoate, which energy is obtained by hydrolyzing polyphosphoric acid in the cells, and the degraded phosphoric acid is released to the outside of the cell . It has been found that in the succeeding aerobic conditions, the phosphorus accumulating microorganisms proliferate using the accumulated polyhydroxyalkanoate, while the phosphorus is excessively accumulated as polyphosphoric acid to remove total phosphorus.

5. Identification of microorganisms

After isolating the microorganisms isolated from a single strain, the strains showing efficiency through screening were first selected and 16s rRNA gene sequencing analysis was performed to identify the strains. Among them, Bacillus aryabhattai SJ8 ) and Vibrio neo caledonikus ( Vibrio neocaledonicus M100SG10) strains showed a high efficiency in the removal of ammonia and total phosphorus removed as shown below in Table 8, respectively.

Bacteria Homology (%) Efficiency (%) To remove Oxygen condition Ari ahbatayi Bacillus (Bacillus aryabhattai SJ8) 100 86 ammonia Expiration Vibrio Neo Caledonics Vibrio neocaledonicus M100SG10) 99.64 95 T-P Expiration

The process of establishing optimal conditions for the above-mentioned strains will be described in more detail below.

6. Establishment of Optimum Conditions for Nitrogen Degradation Strain

Based on the screen results of isolated microorganisms, Bacillus ariabata, the strain with the highest ammonia nitrogen removal efficiency ( Bacillus aryabhattai SJ8) were selected and the temperature, pH and bacterial concentration were varied to establish optimal conditions. Ammonia nitrogen removal efficiency was measured at 3 hour intervals.

In each of the drawings related to the present experiment, the control group (Control, con) shows the results when the strain was not applied under the same conditions.

(1) Temperature

Experiments were carried out on the strain Bacillus aryabhattai SJ8, which showed 86% ammonia removal efficiency, at 20, 25, 28 and 37 ℃, respectively. The pH was 6.8 and the initial inoculation concentration was 0.5 g / L.

As shown in FIG. 1, ammonia was completely removed at 28 ° C. after 6 hours. As a result, it was confirmed that 28 ° C. was the optimal condition. The lower the temperature, the slower the growth of the strain and the poorer the efficiency. At 37 ℃, the ammonia was increased after 3 hours. The higher the temperature, the higher the Bacillus aryabhattai SJ8 ) can be judged to use different pathways.

(2) pH

Bacillus aryabhattai SJ8 was tested under pH conditions with 1N HCl and 1N NaOH. The incubation temperature was 20 ℃ and the initial bacterial inoculum was 0.5 g / L.

As a result, as shown in FIG. 2, it was confirmed that 7 mg / L of ammonia was removed at pH 6 for 9 hours, thereby confirming the highest removal efficiency.

(3) Bacterial concentration

In order to measure the ammonia removal efficiency according to the initial inoculum amount, experiments were conducted by varying the initial inoculum amount.

As a result, as shown in FIG. 3, the sample inoculated at 0.5 g / L shows that the ammonia is not detected after two days, and the higher the concentration of the bacteria, the faster the ammonia nitrogen is removed. It is also possible to assume that ammonia is absorbed into the cells because nitrate nitrogen is not produced. Bacillus these advantages Ari ahbatayi (Bacillus aryabhattai SJ8) can be judged as a very reasonable strain when applied in the field.

7. Establishment of Optimum Conditions for Phosphorylation Strain

Based on the results of marine microorganism screening, Vibrio ( Vibrio) neocaledonicus M100SG10) were tested for optimum temperature, optimum pH and optimal bacterial concentration.

(1) Temperature

Among the strains that showed more than 5% phosphorus removal efficiency, the most efficient Vibrio neo caledonics ( Vibrio to screen the neocaledonicus M100SG10) it was carried out by temperature experiment to establish the optimum conditions. The established temperature conditions were 10, 20, 28 and 37 ℃, and the other pH was 8 and the initial inoculum concentration was 0.5 g / L.

As can be seen in FIG. 4, it was confirmed that the phosphorus removal rate was hardly observed for 9 hours at 10 ° C, but then phosphorus was removed. However, the optimum condition was established at 28 ℃ considering that it fell below 3 mg / L after 4 hours at 28 ℃.

(2) pH

The pH of the medium was adjusted to 5, 6, 7, and 8, and the incubation temperature was 28 ℃ and the initial inoculum was 0.5 g / L. Each pH was controlled with HCl (1N) and NaOH (1N), and phosphorus removal efficiency was confirmed at 4 hour intervals.

As a result, it was confirmed that the phosphorus was removed at a pH of 8 to 3 mg / L or less within 4 hours. On the other hand, the difference of phosphorus removal efficiency was small at pH 5, 6 and 7. Based on the above results, it was confirmed that the removal efficiency was the best at pH 8

(3) Bacterial concentration

In order to measure the phosphorus removal rate according to the initial inoculum amount, the inoculation amount was inoculated at 0.05, 0.1, 0.25, and 0.5 g / L. Phosphorus removal efficiency was measured at 2 hour intervals.

As can be seen from FIG. 6, when the bacteria were inoculated at 0.05 and 0.1 g / L after 4 hours, the residual total concentration was less than 3 mg / L. When the bacteria were inoculated at 0.25 and 0.5 g / L The concentration of residual total phosphorus was measured to be less than 1 mg / L.

After 7 hours, the ND value was recorded for all samples. In view of this, Vibrio neocaledonicus M100SG10) you can see that the initial bacterial concentration is higher to make sure that the initial removal was seen quickly, but after two hours the OD value increases in the medium to remove all the people. Thus, Vibrio neocaledonicus M100SG10) was found to be a highly economical strain showing very good efficiency even in small amounts.

8. Using mixed strains Batch  Experiment

The CNP removal experiment was performed under the optimum condition for the mixed strain MNP1 of the present invention. The ratio of CNP (COD, TN, TP) in the culture medium was 200: 5: 1, 28 ℃, pH6, and the initial mixed strain inoculated amount was 1.0 g / L.

As shown in Fig. 7 (a), phosphorus was completely decomposed in one hour, and ammonia was removed after 2.5 hours by 2.5 mg / L. In case of COD, 70 mg / L was removed until 4 hours, and only 66 mg / L remained.

As it is shown in Fig. 7 (b), the nitrogen removal gyunin Bacillus Ari ahbatayi (Bacillus aryabhattai SJ8), the concentration of COD _Cr , NH ₃ -N, and PO ₄ ^{3 - -} P was measured for each reaction time.

In the first experiment, COD _Cr was 232, 155, 125, 86 and 55 mg / L, NH ₃ -N was 10.2, 8.4, 5.2, 3.6 and 0.5 mg / L, PO ₄ ^3- , 1.2 and 0.4 mg / L, respectively. In the second experiment, COD _Cr 171, 174, 136, 98 and 44 mg / L, NH ₃ -N 11.8, 9.8, 6.6, 3.0 and 0.7 mg / L, PO ₄ ^{3 -} 0.7 mg / L, 3 car experiment, _{COD Cr 200, 144, 146,} 70, 62 mg / L, NH 3 -N 8.8, 7.2, 4.2, 2.2, 1.5 mg / L, PO 4 3 - -P 4.8, 4.2 , 2.4, 0.5 and 0.3 mg / L, respectively.

As shown in FIG. 7 (c), in order to evaluate the contaminant removal characteristics using Vibrio neocaledonicus M100SG10, which is a phosphorus removal germ, COD _Cr , NH ₃ -N, PO The concentration of ₄ ^{3 - -} P was measured.

The results of the first experiment showed that COD _Cr was 238, 165, 135, 100 and 86 mg / L, NH ₃ -N was 5.8, 4.8, 2.8, 1.4 and 0.2 mg / L and PO ₄ ^{3 -} , 1.1 and 0.5 mg / L, respectively. In the second experiment, COD _Cr 180, 184, 152, 116, 70 mg / L, NH ₃ -N 4.3, 4.0, 2.0, 0.6, 0.8 mg / L, PO ₄ ^{3 -} 0.9 mg / L in the first experiment, and COD _Cr 206, 164, 140, 88 and 60 mg / L in the third experiment, NH ₃ -N 5.0, 4.5, 3.6, 1.0 and 0.3 mg / L, PO ₄ ^{3 -} , 2.0, 2.0 and 1.0 mg / L, respectively.

9. Reactor experiment using mixed marine microorganism

The mixed strain MNP1 of the present invention was placed in the reactor and the removal rate of nitrogen phosphorus in the artificial medium was measured. The reactor was fabricated by the three methods of A2 / 0 series, SBR series and MBR (membrane bio reactor) series.

In case of microbial adhesion, pollutant removal characteristics were evaluated by applying two ceramic carriers (Ceramic Carrier - 1, Ceramic Carrier - 2), tidal flats, and granule sludge. In the case of granule sludge, it was applied to two processes of SBR and MBR. In the case of ceramic carriers and tidal flats, column reactor experiments were conducted.

(1) The A2 / 0 series reactor

Figure 8 shows the results when the A2 / 0 series reactor was operated. After 12 hours, the COD _Cr was removed from the initial 198.5 mg / L to 70.4 mg / L, TN was decreased from 9.6 mg / L to 1.1 mg / L, and TP from 5.2 mg / L to 0.4 mg / .

(2) SBR series reactor

Figure 9 shows the results when operating at a high concentration in an SBR-based reactor. As a result of the evaluation of pollutant removal characteristics, the removal efficiency of COD _Cr was about 79.8% at 400.6 mg / L of influent and 80.8 mg / L of effluent (FIG. 9 (a)). NH ₃ -N Influent is 10.1 mg / L, the effluent 0.9 mg / L showed a removal efficiency of about 91.1% (Fig. _{9 (b)), PO 4} 3 - in the case incoming water 5.1 mg / L, the effluent 1.9 -P mg / L showed a removal efficiency of about 62.7% (Fig. 9 (c)). In the case of COD _Cr of SBR reactor at low concentration, the removal efficiency was 203.5 mg / L for the influent and 57.5 mg / L for the effluent and the removal efficiency was 71.7%. NH ₃ -N was removed with 5.1 mg / L of influent and 1.0 mg / L of effluent and showed a removal efficiency of about 80.4%. In the case of PO ₄ ^{3 -} -P, removal of 2.6 mg / L of influent and 1.0 mg / And showed a removal efficiency of about 61.5%.

(3) MBR (membrane bio reactor) reactor

10 shows the results when the MBR series reactor was operated at a high concentration. As a result of evaluating the pollutant removal characteristics, COD _Cr showed a removal efficiency of about 77.9% with 404.6 mg / L of influent and 89.3 mg / L of effluent (FIG. 10 (a)). NH ₃ -N Influent is 10.1 mg / L, the effluent 1.0 mg / L showed a removal efficiency of about 90.1% (Fig. _{10 (b)), PO 4} 3 - in the case incoming water 5.0 mg / L, the effluent 2.8 -P mg / L showed a removal efficiency of about 44.0% (Fig. 10 (c)). In the case of low concentration, the removal efficiency of COD _Cr was about 70.3% with 203.5 mg / L of influent and 60.4 mg / L of effluent. NH ₃ -N showed a removal efficiency of about 74.5% at 5.1 mg / L of influent and 1.3 mg / L of effluent. In the case of PO ₄ ^{3 -} -P, the effluent was 2.5 mg / L and the effluent 1.4 mg / Respectively.

(4) Column reactor test of ceramic carrier and tidal flat

The following ceramic aggregate 1 is composed of 55 to 65% by weight of red mud, 25 to 35% by weight of a sludge sludge and 5 to 15% by weight of waste sludge and the ceramic aggregate 2 comprises 55 to 65% by weight of red mud, 20 to 30% And 5 to 15% by weight of waste paper and 1 to 5% by weight of electric furnace (EAF) dust.

On the other hand, the granular sludge was made of aerobic microbial polymer, and the tidal flats were obtained from Jebudo.

Result of pollutant removal characteristics by carrier type Carrier type COD _Cr (mg / L) NH ₃ -N (mg / L) PO ₄ ^{3 -} -P (mg / L) Influent Effluent Influent Effluent Influent Effluent Granule sludge-SBR 400.6 80.8 10.1 0.9 5.1 1.9 Granule sludge-MBR 404.6 89.3 10.1 1.0 5.0 2.8 Ceramic aggregate 1 401.8 304.4 10.1 7.2 5.1 4.1 Ceramic aggregate 2 400.6 337.7 10.2 8.4 5.1 4.5 foreshore 403.1 385.6 10.4 9.5 5.0 4.8

As can be seen from Table 9, it was confirmed that the granule sludge among the carriers was the most efficient, and it was confirmed that the process was more efficient than the SBR application reactor. As can be seen from FIGS. 9 and 10, it was confirmed that the reactor efficiency was high even under the conditions of high concentration of organic matter, total nitrogen, and total phosphorus.

As a result, the removal efficiency of COD _Cr by using ceramic aggregate 1 was found to be about 24.2% with 401.8 mg / L of influent and 304.4 mg / L of effluent. NH ₃ -N showed about 28.8% removal efficiency with 10.1 mg / L of influent and 7.2 mg / L of effluent. In the case of PO ₄ ^{3 -} -P, the effluent was 5.1 mg / L and the effluent 4.1 mg / Respectively.

As a result of applying the ceramic aggregate 2, the removal efficiency of COD _Cr was about 15.7% at 400.6 mg / L of influent and 337.7 mg / L of effluent. NH ₃ -N showed a removal efficiency of 18.0% with 10.2 mg / L of influent and 8.4 mg / L of effluent. In the case of PO ₄ ^{3 -} -P, the effluent of 5.1 mg / L and the effluent of 4.5 mg / Respectively.

As a result of applying tideland carrier, COD _Cr showed a removal efficiency of about 4.4% with 403.1 mg / L of influent and 385.6 mg / L of effluent. NH ₃ -N showed a removal efficiency of 8.2% with 10.4 mg / L of influent and 9.5 mg / L of effluent. In the case of PO ₄ ^{3 -} -P, the effluent of 5.0 mg / L and the effluent of 4.8 mg / Respectively. However, the tidal flat carrier has a relatively large specific gravity and contains a large number of impurities, so that the possibility of application is somewhat low because the stirring and fluid movement are not smooth.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be obvious to those of ordinary skill in the art.

National R & D project supporting this invention

Assignment number: NRF-2013M3A2A1067498

Department: Future Creation Science Division

Research Management Institution: Korea Research Foundation

Research Project Name: Source Technology Development Project> Public Welfare Safety Technology Development Project

Research title: Development of system for pollution control and red tide control of polluted aquaculture farms using bio / nano ceramic membrane

Contribution rate: 80

Host organization: Kyonggi University

Research period: 2013.10.01-2018.07.31

Assignment number: NRF-2015M3A9B8029697

Department: Future Creation Science Division

Research Management Institution: Korea Research Foundation

Research Project Name: Source Technology Development Project> Bio / Medical Technology Development Project> Research Material Support Project

Research Project: Microbiology and gene conservation center for environmental industry

Contribution rate: 20

Host organization: Kyonggi University

Research period: 2015.05.01-2020.04.30

Claims (12)

A mixed marine microorganism strain having a nitrogen and phosphorus removal ability, consisting of a mixed strain MNP1 (accession number KACC 92021P) consisting of Bacillus aryabhattai SJ8 and V100 microorganism strain M100SG10 ( Vibrio neocaledonicus M100SG10).
delete A microorganism preparation for nitrogen and phosphorus removal comprising the mixed marine microorganism strain of claim 1.
A method for removing nitrogen and phosphorus, comprising applying the mixed strain or microbial agent of claim 1 or 3 in water contaminated with nitrogen and phosphorus.
5. The method of claim 4, wherein applying in water is applied in seawater.
5. The method of claim 4, wherein the step of applying in water is performed at a temperature of 20 占 폚 to 37 占 폚.
5. The method of claim 4, wherein the step of applying in water is carried out at a pH of from 6 to 8.
5. The method of claim 4, wherein the step of applying in water is carried out within a range of 0.2 to 1.0 g / L of initial bacterial inoculum.
5. The method of claim 4, wherein the contaminated water has a nitrogen (TN) concentration of greater than 0 to 150 mg / L.
5. The method of claim 4, wherein the concentration of phosphorus (TP) in the contaminated water is greater than 0 and less than or equal to 100 mg / L.
5. The method of claim 4, wherein the concentration of organic material in the contaminated water is greater than 0 and less than or equal to 400 mg / L.
5. The method of claim 4, wherein the nitrogen and phosphorous removal process is performed with a process selected from the group consisting of an A2 / 0 process, a SBR process, and an MBR process.

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