KR101894284B1 - Manufacturing powder and bead form of microbe mixed culture for phenol dissolution - Google Patents

Abstract

The present invention relates to a microorganism culture manufactured in a powder or bead form having a phenol decomposing ability containing single culture and mixed culture of Rhodococcus sp. 24, Microbacterium sp. 28, or Pseudomonas sp. GM1 as an active ingredient. The single culture and mixed culture of the present invention have the phenol decomposing ability, and can effectively decompose phenol according to temperature and time.

Description

TECHNICAL FIELD [0001] The present invention relates to a microorganism cultured in powder or bead formulations having phenol degradation ability,

The present invention relates to the use of Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) or Pseudomonas sp. GM1 ( Pseudomonas sp. GM1), or a mixed culture thereof, as an active ingredient, in a powder or bead formulation having a phenol-resolving power.

The present invention relates to the use of Rhodococcus sp. 24, microbacterium sp. 28 or Pseudomonas sp. GM1, or a mixed culture of GM1, or a mixed culture of GM1, or a mixed culture thereof.

The present applicant has found that the three types of microorganisms having excellent phenol decomposing ability are in the form of a mixed culture in which the phenol decomposition efficiency is effective (KCTC 13261BP, FBCC 501805) in the form of a mixed culture, and the microorganism- Application No. 10-2017-0076432 ".

In reference to the prior art literature, Korean Patent Registration No. 10-0563207 describes a method of biological degradation of alkyl phenols causing environmental pollution. Claim 1 of the present invention relates to a method for decomposing alkylphenols which are used as a surfactant and pollute the environment and which comprises decomposing an alkylphenol or a mixture of a large mushroom or a pore mushroom alone or a mixture thereof with an alkylphenol Quot; biological degradation method " Korean Patent Laid-Open Publication No. 10-2014-0119856 describes a new strain of Rhodococcus sp. Derived from a tidal flat for the purification of water in a bioflav system and a microbial agent. Claim 1 discloses a microbial agent for water purification of a bioflavon system containing as an active ingredient a culture of Rhodococcus pyridinivorans EDB2, which is a deposit number KACC91762P identified in coastal tidal flats with salinity tolerance and organic decomposition ability "Is described.

Korean Registered Patent No. 10-0563207 (published on Mar. 27, 2006) Korean Patent Publication No. 10-2014-0119856 (published on October 13, 2014)

DISCLOSURE OF INVENTION Technical Problem The present invention provides a method for producing phenol-decomposing microorganisms ( Rhodococcus sp. 24, Microbacterium sp. 28 and Pseudomonas sp. GM1 strain) by preparing a single culture or a mixed culture form in powder or bead formulations, The present invention is to provide a microorganism cultured product which is produced as an effective powder or bead formulation as a microorganism mixed culture for environmental pollution abatement using the three strains in an environment polluted with water.

The present invention relates to a method for producing Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) or Pseudomonas sp. GM1 ( Pseudomonas sp. GM1) and a mixed culture of the single cultured product in a powder or bead formulation to provide a microorganism culture having phenol degradation ability, thereby solving the technical problem.

The present invention solves the technical problems by providing a microorganism culture product in which each of the single cultures is mixed in a volume ratio of 1: 2: 1 or 2: 1: 1 to produce a powder or a bead formulation.

The present invention relates to a microorganism culture prepared by inoculating each of the above single cultures into a liquid culture broth and culturing the cells at 20 to 27 ° C for 30 to 40 hours at a ratio of 1: 2: 1, followed by lyophilization and pulverization To solve technical problems.

The present invention solves the technical problem by culturing the single cultured organism in an artificial fresh water medium for 30 to 40 hours to cure it in a bead form to provide a microorganism cultured in a bead formulation.

The present invention relates to a process for the preparation of Rhodococcus sp. 24, microbacterium sp. 28 or Pseudomonas sp. GM1 single cultures and mixed culture of the single cultures, each of the single cultures was inoculated in a liquid culture broth and cultured at 20 to 27 ° C for 30 to 40 hours to obtain a 1: 2: 1 ratio , Followed by lyophilization and pulverization to provide a microorganism cultured product as a powder, thereby solving the technical problem.

The present invention relates to a process for the preparation of Rhodococcus sp. 24, microbacterium sp. 28 or Pseudomonas sp. GM1 monoculture and mixed cultures of the single cultures, the single cultured organism is cultured in Artificial Fresh water medium for 30 to 40 hours and cured in a bead form to provide a microorganism cultured in a bead formulation To solve technical problems.

As evidenced by the experimental examples of the present invention, the single microorganism cultures and mixed cultures of three species ( Rhodococcus sp. 24, Microbacterium sp. 28 and Pseudomonas sp. GM1) have the function of decomposing phenol, When used as a powder or bead formulation of a mixed culture, an effective phenol decomposing effect is exhibited depending on temperature and time.

1 is a photomicrograph of three kinds of phenol degrading strains ( Rhodococcus sp. 24, Microbacterium sp. 28, Pseudomonas sp. GM1) used in the present invention.
FIG. 2 is a graph showing phenol decomposition effects of a single cultured organism through an experimental example of the present invention. FIG.
FIG. 3 is a graph showing phenol decomposition effects of the mixed cultures as shown in Experimental Example of the present invention.
4 is a phylogenetic relationship diagram of three strains of the phenol-degrading strains ( Rhodococcus sp. 24, Microbacterium sp. 28, Pseudomonas sp. GM1) used in the present invention.
FIG. 5 is a view showing a culture of a bead formulation through an experimental example of the present invention. FIG.
FIG. 6 is a graph showing decomposition effect of phenol using a bead-form cultured organism as a result of an experiment of the present invention.
FIG. 7 is a graph showing degradation rates of phenol according to temperature of a bead-form cultured organism through experimental examples of the present invention.
FIG. 8 is a graph showing degradation rates of phenol degradation with time in the bead-form cultures observed through the experimental examples of the present invention.
Figure 9 is a block diagram of a method of manufacturing a mixed culture as an embodiment of the present invention.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and the inventor can properly define the concept of the term to describe its invention in the best possible way And should be construed in accordance with the principles and meanings and concepts consistent with the technical idea of the present invention.

Therefore, the embodiments described in the present specification, the reference examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be present.

Experimental Example 1. Phenol-decomposing microorganism culture and efficacy test

1) Culture and recovery of microorganisms

1 is a photomicrograph of three kinds of phenol degrading strains ( Rhodococcus sp. 24, Microbacterium sp. 28, Pseudomonas sp. GM1) used in the present invention.

Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. Three microorganisms of GM1 ( Pseudomonas sp. GM1) were cultured in 1 L of R2A medium (200 ml X 5 flask) containing phenol for 36 hours at 220 rpm, centrifuged (10,000 rpm, 10 minutes) Recall.

Each of the recovered cells is centrifuged after artificial freshwater or filtered water to remove the R2A medium components.

2) Microbial cultures and mixture composition

In order to determine the number of microorganisms and the mixing ratio between individuals, 200 ml of Artificial Freshwater or a filtered river is suspended in each recovered organism, and the O.D value is measured using a UV-vis spectrophotometer. The O.D values of each suspended microorganism are adjusted to 1.0 by diluting with artificial freshwater or filtered river, taking into account the measured O.D. value.

The phenol resolving ability can be measured by using each cell fixed with the same OD value by dilution. In the case of mixed cultures, the volume ratio can be adjusted according to the mixing ratio of the three cells.

3) Method for verifying phenol resolution

10 ml of artificial freshwater or filtered water with 200 ppm of phenol dissolved in a sealable glass test tube is dispensed, and 100 μl of a single microorganism culture or mixed culture with a constant O.D value is inoculated. In this experiment the control (control) is 200 ppm artificial freshwater or filtered river without inoculation. After inoculation, each test tube is fixed with a rubber cap and an aluminum cap which are insoluble in organic solvents, and then cultured at 0, 12, 18, 24, and 36 hours at 25 ° C and 220 rpm.

Use a GC chromatograph system to determine the amount of residual phenol in the incubated culture tube. 5 ml of dibutyl chloride is added to recover the remaining phenol from the test tube in which the culture or the mixed culture has been cultured. Transfer a portion of the extracted organic solvent layer to a vial for 1.5ml GC chromatography, and remove the physical components to prepare a sample. Prepared samples were analyzed using an HP-5 column in a GC chromatograph system and phenol chromatograms of control samples without standard solutions containing phenol and cells were used to determine the amount of phenol remaining in each test group .

4) Phenol resolving ability Result: Degradation effect of single culture

FIG. 2 is a graph showing phenol decomposition effects of a single cultured organism obtained through the experiment of the present invention. (Experiment 2)

2, it can be confirmed that all of the three single cultures have phenol resolution ability as compared with the control group.

5) Phenol resolving ability Result: Decomposition effect of mixed culture

FIG. 3 is a graph showing phenol decomposition effects of the mixed cultures as shown in Experimental Example of the present invention. (Experiment 2)

3, Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. 1: 1: 1, 1: 2: 1, and 1: 1: 2 in volume ratio of GM1 ( Pseudomonas sp. GM1) cultures.

6) Analysis of experimental results

Analysis of the results showed that single microorganism cultures and mixed cultures of three species ( Rhodococcus sp. 24, Microbacterium sp. 28 and Pseudomonas sp. GM1) had the function of decomposing phenol, and single cultures and microbial cultures were cultured in the laboratory It has been shown that it has the effect of effectively and rapidly decomposing phenol in a fresh water environment such as river or ground water, not only in an artificial condition.

In particular, the decrease in phenol content was measured using a river, which is similar to the actual environment. As a result, it was confirmed that phenol reduction effect was faster and more stable than a single strain in mixed cultures (1: 2: 1 and 2: 1: 1 mixing conditions) I could.

Experimental Example 2. Powder production of a phenol-decomposing microorganism mixed culture

1) Powder production of phenol-decomposing microorganism mixed culture

Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. Three microorganisms of GM1 ( Pseudomonas sp. GM1) are cultured in the R2A liquid culture at 25 ° C for 36 hours, and the microorganisms cultured in PBS buffer 1X are centrifuged and washed. The OD value of the washed microbial suspension is measured using a UV-vis spectrophotometer. The OD value of each suspended cell is adjusted to 0.5 by taking into account the measured OD value. Each single culture was mixed at a ratio of (Rhodococcus sp. 24: Microbacterium sp. 28: Pseudomonas sp. GM1 = 1: 2: 1) according to the ratio of the mixed cultures to make a total volume of 500 ml. skim milk solution is mixed with skim milk for 24 hours at -80 ℃ and freeze-dried for 2 days using a large-capacity freeze dryer. Powdered using a pulverizer and vacuum packed.

Experimental Example 3. Preparation of bead formulation of phenol-degrading microorganism mixed culture and efficacy test

1) Culture and recovery of microorganisms

4 is a phylogenetic relationship diagram of three phenol degrading strains ( Rhodococcus sp. 24, Microbacterium sp. 28, Pseudomonas sp. GM1) used in the present invention.

The above three microorganisms are cultured in 200 ml of Artificial Fresh water medium containing Yeast Extract and Pyruvic acid for 36 hours, and only the cells are recovered by centrifugation.

      * Artificial fresh water composition

          CaCl2 - 0.45 mM

          Mg2SO4 - 0.2 mM

          KCl - 0.65 mM

          NaSO4 - 0.26 mM

          (NH4) 2SO4 - - - - 0.75 mM

          KH2PO4 ------------- 1.25 mM

2) Manufacture of bead formulation by microorganism immobilization

For microbial immobilization, 3 g of sodium alginate is mixed with 90 ml of phosphate buffer (pH 7.0) and 10 ml of the suspension. The beads are prepared by dropping the suspension in a 1% CaCl ₂ solution using a 3 ml syringe dropwise. After the beads are made, they are cured in 1% CaCl2 solution for 1 hour and then washed three times with phosphate buffer. FIG. 6 is a photograph of an immobilized microorganism prepared in a bead formulation prepared by the above method.

FIG. 5 is a view of a bead formulation through microorganism immobilization.

3) Phenol decomposition test of bead formulation

Sealable glass test tubes with a single strain and mixed strain prepared by the method in (Rhodococcus sp 24:.. Microbacterium sp 28:. Pseudomonas sp GM1) 0.4g beads and 200 ppm of phenol 5ml solution at 25 ℃ for about 30 minutes the reaction was The residual phenol content was measured.

 In this experiment, the control (control) is 200 ppm phenol without inoculation. (1: 2: 1) beads were incubated at 25 ℃, 30 ℃, 25 ℃, 40 ℃ and 45 ℃ for 30 min in the same manner as above to determine the optimal conditions for phenol decomposition of the beads of the most effective mixed strains in a short time. Lt; / RTI > Finally, phenol degradation rate of immobilized mixed strain (1: 2: 1) was measured over time.

To determine the amount of residual phenol, 2 ml of methylene chloride solution was used. 0.3 g of sodium sulfate was added to the extracted solvent to remove moisture from the extracted solution, and the residual phenol content was measured.

4) Phenol degradation results: phenol decomposition effect of single and mixed strain bead formulations

FIG. 6 is a graph showing phenol decomposition effects of the single strain and the mixed strain beads which have been examined through the experimental examples of the present invention.

6, it can be seen that a considerable amount of phenol was removed during a reaction time of 30 minutes for both the single strain and the mixed strain bead as compared with the control. In addition, the beads of the mixture of three strains ( Rhodococcus sp. 24: Microbacterium sp. 28: Pseudomonas sp. GM1) mixed at a ratio of 1: 2: 1 showed the fastest phenol removal function.

5) Results of phenol resolution: The optimum temperature for phenol decomposition of immobilized mixed strain (1: 2: 1) bead formulation

FIG. 7 is a graph showing degradation of phenol according to the temperature of the immobilized mixed strain (1: 2: 1) beads, which has been examined through the experimental example of the present invention.

7, the optimum conditions for phenol decomposition of the immobilized beads of the mixed strains in the shortest period of time were examined at 25 ° C., 30 ° C., 25 ° C., 40 ° C. and 45 ° C. for 30 minutes, The decomposition rate was confirmed.

6) Results of phenol degradation: phenol decomposition effect of immobilized mixed strain (1: 2: 1) bead formulation over time

FIG. 8 is a graph showing phenol decomposition effect of immobilized mixed strains (1: 2: 1) beads observed over time according to Experimental Example of the present invention.

Referring to FIG. 8, the phenol decomposition function over time shows a rapid reaction rate which decomposes more than 40% of phenol in 30 minutes. After 30 minutes, the decomposition rate of phenol gradually decreased, but phenol decomposition was continuously observed.

7) Analysis of experimental results

Analysis of the results showed that single microorganism cultures and mixed cultures of three species ( Rhodococcus sp. 24, Microbacterium sp. 28 and Pseudomonas sp. GM1) have the function of degrading phenol.

Single cultures and microbial cultures have been shown to effectively and rapidly degrade phenols not only in laboratory artificial conditions but also in freshwater environments such as rivers and groundwater.

In particular, the decrease in phenol content was measured using a river, which is similar to the actual environment. As a result, it was confirmed that phenol reduction effect was faster and more stable than a single strain in mixed cultures (1: 2: 1 and 2: 1: 1 mixing conditions) I could.

In addition, a single cultured product and a mixed cultured product produced by a bead formulation have the same phenol resolution ability, and have an effect of rapidly decomposing effectively according to time and temperature.

The beads of the single strain and the mixed strain were confirmed to remove a considerable amount of phenol for 30 minutes, and three strains ( Rhodococcus sp. 24: Microbacterium sp. 28: Pseudomonas sp. GM1) mixed at a ratio of 1: 2: Showed the fastest phenol removal function in the mixture.

The optimal conditions for phenol decomposition of the most effective mixed strain beads at temperature were 25 ℃. It can be judged that this shows the highest decomposition rate at room temperature.

The phenol decomposition function with time shows a rapid reaction rate which decomposes more than 40% of phenol in 30 minutes. After 30 minutes, the decomposition rate of phenol gradually decreased, but phenol decomposition was continuously observed.

As a result, when the bead type was prepared, three strains ( Rhodococcus sp. 24: Microbacterium sp. 28: 1 strain) mixed with a single strain and a mixed strain bead at a ratio of 1: 2: Pseudomonas sp. GM1) was decomposed continuously at 25 ℃ and about 40% phenol was decomposed continuously for 30 minutes at 25 ℃.

Example 1. Method for producing a mixed culture having phenol degrading ability

9 is a block diagram of a method for producing a mixed culture having a phenol resolving ability.

Step 1) The freeze-dried formulation of Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. The microorganism strain of GM1 ( Pseudomonas sp. GM1) is solid-cultured.

Step 2) The solid cultures of the three microbial strains are liquid-cultured.

Step 3) Measure the number of cultures.

Step 4) Mix the culture media of three strains having the same populations in a volume ratio of 1: 2: 1 or 2: 1: 1.

Step 5) The mixture is concentrated and lyophilized.

Example 2. Microorganism mixed with a phenol degrading ability

As demonstrated in Experimental Example 1, a microorganism mixed culture having phenol degradation ability, Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. A single culture of GM1 ( Pseudomonas sp. GM1) is used.

Alternatively, Rhodococcus sp. 24, microbacterium sp. 28 and Pseudomonas sp. GM1 cultures were mixed at a volume ratio of 1: 2: 1 or 2: 1: 1.

Example 3. Biodegradation method for decomposing phenol

As demonstrated in Experimental Example 1 above, Rhodococcus sp. 24, microbacterium sp. 28 and Pseudomonas sp. A single culture of GM1 or a mixed culture thereof is used to carry out a biodegradation method for decomposing phenol contained in wastewater.

Example 4. Powder-type preparation of a phenol-digesting microorganism mixed culture

Step 1) Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. Three microorganisms of GM1 ( Pseudomonas sp. GM1) are cultured for 30 ~ 40 hours at 20 ~ 30 ℃ in liquid medium of R2A.

Step 2) Centrifuge the cultured microbial cells with PBS buffer 1X and measure the number of cultures washed.

Step 3) Three kinds of culture media having the same population are mixed at a volume ratio of 1: 2: 1 or 2: 1: 1. This corresponds to the above experimental example and is not limited thereto. This corresponds to the above experimental example and is not limited thereto.

Step 4) Mixed culture and 15% skim milk solution are mixed and lyophilized.

Step 5) Powdered and vacuum packaged.

Example 5. Preparation of bead formulation of a phenol digesting microorganism mixed culture

Step 1) Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. GM1 ( Pseudomonas sp. GM1) microbial strains were cultured in artificial fresh water medium containing Yeast Extract and Pyruvic acid for 36 hours, respectively, and only microbial cells were recovered by centrifugation.

Step 2) Mix the sodium alginate solution with the strain suspension.

Step 3) The beads were made by dropping the suspension in CaCl ₂ solution.

Step 4) Curing for 1 hour after manufacturing the beads.

Step 5) Wash three times with phosphate buffer.

Example 6. Biodegradation method for decomposing phenol

As demonstrated in Experimental Example 3 above, Rhodococcus sp. 24, microbacterium sp. 28 or Pseudomonas sp. A single culture of GM1 or a mixed culture thereof is prepared and a biological degradation method of decomposing the phenol contained in the wastewater is carried out.

Korea Biotechnology Research Institute KCTC13261BP 20170517 National Nakdong River Biological Resource Center FBCC501805 20170706

Claims (6)

delete delete delete delete delete Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. GM1 ( Pseudomonas sp. GM1) in a liquid culture medium containing Yeast Extract and Pyruvic acid, respectively, and culturing them at 20 to 27 ° C for 36 hours;
Each of the cultured Rhodococcus sp. 24 ( Rhodococcus sp. 24), microbacterium sp. 28 ( Microbacterium sp. 28) and Pseudomonas sp. Culturing the microbial strain of GM1 ( Pseudomonas sp. GM1) by centrifugation to collect only the microbial cells, and mixing the recovered strains in a volume ratio of 1: 2: 1 to prepare a microorganism mixed culture (accession number: KCTC13261BP);
Mixing the microorganism mixed culture (Accession No: KCTC13261BP) with a sodium alginate solution to prepare a strain suspension;
Dropping the suspension of the strain in a 1% (w / v) CaCl ₂ solution to prepare a bead;
The prepared beads were cured in 1% (w / v) CaCl ₂ solution for 1 hour; And
And washing the cured beads three times with phosphate buffer to prepare a microorganism mixed culture in a bead formulation,
Characterized in that the phenol is decomposed at a temperature of 25 DEG C for 0.5 to 12 hours in a wastewater.

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