JP7614481B2 - Acinetobacter venezianus PFZR-1 and its use in degrading organic pollutants - Google Patents

Description

CCTCC CCTCC M 2022719

The present invention relates to Acinetobacter venezianus PFZR-1 and its use in degrading organic pollutants.

Hydrocarbons are organic compounds composed of two elements, carbon and hydrogen. They are insoluble in water, but easily soluble in organic solvents. They have a lower density than water, so they exist as oily insolubles in water. Cyclohexane (C ₆ H ₆ ) is a colorless liquid with a pungent odor and is a cycloalkane that is easily volatile. Cyclohexane has a wide range of uses and can be used as a raw material to produce products such as cyclohexanol, cyclohexanone, and adipic acid. It can be used as a solvent in chromatographic analysis and organic synthesis processes, and can be used as an adhesive in the production of resins, paints, and other materials. Therefore, cyclohexane is widely present in environments related to industrial production, analytical applications, transportation leaks, and other processes. Although cyclohexane is a low-toxicity substance, it has a mild irritating effect on the eyes and respiratory tract, can cause itching when in contact with the skin, and can cause anesthetic symptoms such as dizziness, nausea, and irritability when inhaled.

Cyclohexane has a certain viscosity, so it easily adheres to adsorbates in the environment, such as soil, surface water, and ocean, and is often difficult to remove. Microorganisms are also widely present in the environment, and the treatment of pollutants using microorganisms has attracted widespread attention due to its advantages of low cost, simple operation, clean technology, and harmlessness. At present, a large number of bacterial genera have been reported that degrade pollutants with high efficiency, including Pseudomonas, Flavobacterium, Corynebacterium, Acmobacter, Arthrobacter, Pediococcus, and Vibrio. There are almost no microorganisms in nature that grow using cyclohexane as the sole carbon source, but many microorganisms that use cyclohexane as the sole carbon source have been reported through laboratory acclimation, selection, and isolation culture. In 1919, Tausz and Pete isolated the first hydrocarbon-liquefying bacterium (Bacterium aliphaticumliquifaciens) that decomposes cyclohexane. In 1948, Imelik isolated Pseudomonas aeruginosa that can decompose cyclohexane and studied its metabolic products. In 1999, Li Daenian et al. in China isolated a decomposition strain that can use cyclohexane and cyclopentanone as the sole carbon source. Since then, more and more cyclohexane-decomposing bacteria have been reported. Acinetobacter has already been widely reported as one of the important microorganisms that decompose diesel oil.

In the present invention, a new type of Acinetobacter venetianus that uses cyclohexane as the sole carbon source was isolated from the environment, providing strong support for the application of microbial purification processes to soil, exhaust gas, and wastewater containing the pollutants.

The object of the present invention is to provide Acinetobacter venetianus PFZR-1, which is a new type of bacterium that is highly efficient at decomposing cyclohexane and has excellent environmental adaptability while also being able to effectively decompose other organic pollutants.

The technical techniques used in this invention are as follows:

The present invention provides a new cyclohexane-degrading bacterial strain-Acinetobacter venetianus PFZR-1, which has been deposited in China Typical Culture Depository Center with deposit number CCTCC NO:M 2022719, date of deposit on May 25, 2022, and address: Wuhan University, Wuhan, China 430072.

The basic characteristics of Acinetobacter venezianus PFZR-1 of the present invention are that the colonies are white, aerobic, smooth, moist, and oval in shape.

The present invention also relates to the use of the above-mentioned Acinetobacter venezianus PFZR-1 in the decomposition of organic contaminants by microorganisms. The above-mentioned application involves inoculating Acinetobacter venezianus PFZR-1 into an inorganic salt medium containing organic contaminants, and carrying out a decomposition reaction under conditions of pH = 5-10 (preferably pH = 6-8), 20-40°C, and 100-200 rpm, thereby realizing the decomposition of the organic contaminants, where the organic contaminants include cyclohexane, toluene, methanol, limonene, ethyl acetate, cyclohexanol, or cyclohexanone, and preferably cyclohexane.

Preferably, the decomposition reaction conditions are 30°C and 160 rpm.

Preferably, the initial concentration of the organic contaminant in the inorganic salt medium is 63.3 to 316.4 mg/L (preferably 63.3 to 158.2 mg/L). The amount of Acinetobacter venezianus PFZR-1 added to the inorganic salt culture solution is 0.01 to 0.1, preferably 0.02, calculated based on the bacterial cell concentration OD ₆₀₀ value.

The composition of the inorganic salt culture solution is 0.942 g/ _{L K2HPO4} , 0.234 g/ _{L KH2PO4} , 1.7 g/L _NaNO3 , 0.98 g/L _NH4Cl , 0.2033 g/ _{L MgCl2.6H2O} , 0.0111 g/ _{L CaCl2.2H2O} , 0.0162 g/L _FeCl3 , and 5 ml/L trace elements, with deionized water as the solvent and pH=5-10. The composition of the trace elements was _ZnCl2 0.088 g/L, _{MnCl2.4H2O 0.060} g/L, KI _0.01 g/L, _{Na2MoO4.2H2O 0.1} g/L, and _{H3BO3 0.05} g/L, and the solvent was deionized water.

Before inoculating the Acinetobacter venezianus PFZR-1 of the present invention, activation and expansion are first performed, and the expanded quiescent cells are inoculated into an inorganic salt medium. The quiescent cells are prepared by the following steps.

(1) Slant culture:
Acinetobacter venezianus PFZR-1 is inoculated into LB solid medium and cultured in a culture box at 30° C. to obtain slant cultures. The composition of the LB solid medium is 5 g/L yeast extract, 10 g/L NaCl, 10 g/L peptone, and 15 to 20 g/L agar, with no adjustment to pH, and deionized water as the solvent.

(2) Expansion culture:
The slant cells from step (1) are inoculated into an LB liquid medium and cultured at 30°C and 160 rpm for 24 hours to obtain an expansion culture solution, which is centrifuged to collect wet cells and washed with an inorganic salt culture solution to obtain stationary cells of Acinetobacter venezianus PFZR-1. The LB liquid medium has a composition of 5 g/L yeast extract, 10 g/L NaCl, and 10 g/L peptone, with no adjustment to pH, and deionized water as the solvent.

Compared with conventional techniques, the main beneficial effects of the present invention are as follows:

The present invention provides Acinetobacter venezianus PFZR-1, a highly efficient bacterium for decomposing organic pollutants, especially cyclohexane, which is obtained from sludge in wastewater treatment plants and can grow using cyclohexane as the sole carbon and energy source, with a decomposition concentration of 316.4 mg/L. The pH range suitable for the growth of the bacterium is quite wide, and cyclohexane can be decomposed relatively well at a pH of 6.0 to 8.0, with the decomposition rates reaching 90% or more in both cases. Moreover, Acinetobacter venezianus PFZR-1 has a high decomposition efficiency for cyclohexane, low cost, simple operation, and no secondary pollution, making it more advantageous than conventional decomposition methods using adsorption and photocatalysis. Compared to existing cyclohexane-degrading bacteria, the PFZR-1 strain has a wide range of decomposition concentrations (63.3-316.4 mg/L), strong adaptability (pH: 6.0-8.0), and a certain degree of decomposition activity against various other contaminants (toluene, methanol, limonene, ethyl acetate, cyclohexanol, and cyclohexanone). In other words, it provides valuable technical support for the application of microbial purification processes to soil, exhaust gas, and wastewater containing the contaminants.

1 is a photograph of the colony morphology of strain PFZR-1 in LB medium. 1 is a transmission electron micrograph of strain PFZR-1. Phylogenetic tree of strain PFZR-1. 1 shows the degradation curves of strain PFZR-1 for different concentrations of cyclohexane within 48 hours. 1 is a degradation curve of strain PFZR-1 towards cyclohexane at different pH values in 48 hours.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be further described in conjunction with specific examples, but the scope of protection of the present invention is not limited thereto.

The following examples and drawings are for illustrative purposes only and should not be construed as limitations on the present invention. Unless otherwise specified, the reagent materials used in the following examples are biochemical reagent materials generally available from the market or obtained through commercial routes, and the laboratory equipment used is equipment commonly used in laboratories unless otherwise specified, and the methods and equipment used in the following examples are methods and equipment commonly used in the same field.

The composition of the medium used in the examples of the present invention is as follows:

The composition of the inorganic salt culture solution was 0.942 g/L K ₂ HPO ₄ , 0.234 g/L KH ₂ PO ₄ , 1.7 g/L NaNO ₃ , 0.98 g/L NH ₄ Cl, 0.2033 g/L MgCl ₂ ·6H ₂ O, 0.0111 g/L CaCl ₂ ·2H ₂ O, 0.0162 g/L FeCl ₃ , and 5 ml/L trace elements, with deionized water as the solvent and pH = 5-10. The composition of the trace elements is _ZnCl2 0.088 g/L, _MnCl2.4H2O 0.060 g _/ L, KI 0.01 g/L, _Na2MoO4.2H2O 0.1 g/L, and _H3BO3 0.05 g/ _L , and the _solvent is deionized water _.

Composition of LB solid medium: yeast extract 5 g/L, NaCl 10 g/L, peptone 10 g/L, agar 15-20 g/L, pH unchanged, solvent is deionized water.

LB liquid medium composition: yeast extract 5 g/L, NaCl 10 g/L, peptone 10 g/L, pH unchanged, solvent is deionized water.

Example 1: Isolation, purification and identification of Acinetobacter venetianus PFZR-1

1. Sample collection and acclimation (1) Primary screening: Activated sludge from some domestic wastewater treatment pool is collected on-site, left to settle for 2 hours, and then the supernatant and suspended impurities are removed, leaving fine-grained sludge. The bottom layer sludge after settling is taken and mixed with the inorganic salt medium at 1:1 (v/v) to form a mixed medium, which is then poured into a sludge acclimation tank with a total volume of 4L. Cyclohexane is used as the substrate, which is the only carbon source and energy, and the sludge is acclimated at a constant temperature of (30±1°C). During the acclimation experiment, the aeration tank is stably aerated, the pH value of the acclimation tank is maintained at 7.0, and when the substrate concentration falls to 30 mg/L or less during the day, the substrate is added once to make the substrate concentration after addition reach 100 mg/L (a sample is taken before the substrate is added and the residual substrate concentration is measured), and the mixed medium is replaced at 5-day intervals. After about 2 months, the acclimated sludge becomes able to stably decompose 150 mg/L of cyclohexane per day, and this sample is placed in a shake flask and further concentrated to obtain an acclimated sample.

The above conditioned sample is inoculated into 50 mL of inorganic salt medium at a volume concentration of 10%. Using cyclohexane as the substrate, the initial concentration is 63.3 mg/L. The culture is cultivated on a shaker at 30°C and 160 r/min. The remaining substrate concentration is measured within 48 hours using gas chromatography as described in Example 3. After the substrate decomposition rate reaches approximately 80%, the culture liquid is transferred to a fresh inorganic salt medium containing cyclohexane at the same concentration (63.3 mg/L) at a volume concentration of 10% and subjected to subculture concentration. After that, the initial concentration of cyclohexane is gradually increased (63.3 to 316.4 mg/L) and subculture concentration is repeated several times. The culture liquid with a cyclohexane input concentration of 316.4 mg/L is collected and used as the primary selection sample.

(2) Rescreening: The sample from the first screening is diluted and applied to an inorganic salts solid medium containing 63.3 mg/L cyclohexane, grown for 24 hours at 30°C using the plate inversion method, and a single colony is picked up. The single colony is streaked several times in LB solid medium, and then inoculated again into an inorganic salts medium containing cyclohexane (initial concentration: 63.3 mg/L) as the only carbon and energy source, and cultured at 30°C for 48 hours. The residual substrate concentration is measured using gas chromatography as described in Example 3, and the decomposition activity is calculated.

A single colony with cyclohexane decomposition ability is selected, and the reselection step is repeated to further separate and purify the bacteria. When the decomposition rate reaches 80% or more, a strain with cyclohexane decomposition activity is obtained, and this strain is designated as strain PFZR-1.

2. Identification of strain PFZR-1 (1) Characteristics of strain PFZR-1:
The strain PFZR-1 was inoculated into LB medium and incubated at 30° C. for 24 hours. The colonies were white, aerobic, smooth, moist, and elliptical. The colony morphology is shown in FIG. 1, and the transmission electron micrograph is shown in FIG. 2.

(2) 16S rRNA sequence analysis DNA of strain PFZR-1 was extracted using Prep Man Ultra Kits nucleic acid extraction agent and stored at 4 ° C. Amplification was performed using a bacterial universal primer (forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer: 5'-GGTTACCTTGTTACGACTT-3') in an ABI PCR amplifier. Nucleic acid sequencing was performed on the PCR purified product using an Applied Biosystems 3500 genetic analyzer. The sequencing work was completed by Zhejiang Tianke High-Tech Development Co., Ltd. The 16S rRNA sequence of the strain is as follows (Genebank accession number is ON692913).

SEQ ID NO:1:
CCTACTTCTGGTGCACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCC GCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGATCGGCTTTTTGAGATTAGCATCCTA TCGCTAGGTAGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCGTCCC CGCCTTCCTCCAGTTTGTCACTGGCAGTATCCTTAAAGTTCCCCATCCGAAATGCTGGCAAGTAAGGAAAAGGGTTGCGCTCGTTG CGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTATCTAGATTCCCGAAGGCACCAATCCCATC TCTGGAAAGTTTCTAGTATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGG CCCCCGTCAATTCATTTGAGTTTTAGTCTTGCGACCGTACTCCCCAGGCGGTCTACTTATCGCGTTAGCTGCGCCACTAAAGCCT CAAAGGCCCCCAACGGCTAGTAGACATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCCATGCTTTCGTACC TCAGCGTCAGTATTAGGCCAGATGGCTGCCTTCGCCATCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATT CTACCATCCTCTCCCATAACTCTAGCCATCCAGTATCGAATGCAATTCCCAAGTTAAGCTCGGGGATTTCACATTTGACTTAAATG GCCGCCTACGCACGCTTTACGCCCAGTAAAATCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCC GGTGCTTATTCTGCGAGTAACGTCCACTATCCAGTAGTATTAATACTAGTAGCCTCCTCCTCGCTTAAAGTGCTTTAACAACCATA AGGCCTTCTTTCACACACGCGGCATGGCTGGATCAGGGTTCCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCT GGGCCGTGTCTCAGTCCCAGTGTGGCGGATCATCCTCTCAGACCCGCTACAGATCGTCGCCTTGGTAGGCCTTTACCCCACCAAC TAGCTAATCCGACTTAGGCTCATCTATTAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCAT TCCTTTCGGAATGTTGTCCCCCACTAATAGGCAGATTCCTAAGCATTACTCACCCGTCCGCCGCTAGGTCCAGTAGCAAGCTACC

(3) Physiological and biochemical experiments Ability of strain ZRPF-1 to utilize 46 types of carbon sources on the Merieux CBC card: Using the Merieux fully automated identification device, the metabolic status of the strain to the 46 different carbon sources was investigated (commissioned by Zhejiang Tianke Hi-Tech Development Co., Ltd. (formerly Zhejiang Provincial Institute of Microbiology)). The identification results are shown in Table 1. Through biochemical reactions using the Merieux fully automated identification device VITEK, it was found that strain ZRPF-1 could utilize 15 types of carbon sources relatively well, and could not utilize the other 31 types of carbon sources.

The morphological and physiological/biochemical characteristics of strain PFZR-1 were studied and analyzed, and the sequence of the strain was blast-compared with the gene sequences in the NCBI database, and a phylogenetic tree (Figure 3) was constructed in conjunction with the homology analysis of 16s RNA, thereby confirming that strain PFZR-1 is Acinetobacter Venetianus, and named Acinetobacter Venetianus PFZR-1, which was deposited in the China Center for Typical Cultures Depositary. The deposit number is CCTCC NO:M 2022719, the date of deposit is May 25, 2022, and the address is Wuhan University, Wuhan, China, 430072.

Example 2: Obtaining stationary cells of Acinetobacter venezianus PFZR-1 (1) Slant culture:
Acinetobacter venezianus PFZR-1 was inoculated into LB liquid medium and cultured at 30°C and 160 rpm for 48 hours, after which the activated bacteria was streaked onto an LB solid plate and cultured in a culture box at 30°C for 24 hours, and a single colony was taken and subsequently streaked onto a plate to measure the purity of the bacteria, which was then stored normally (4°C) on a slant in an LB test tube.

(2) Expansion culture:
The slant cells of step (1) are inoculated into an LB liquid medium and cultured at 30°C and 160 rpm for 24 hours to obtain an expansion culture solution, which is centrifuged to collect the wet cells, which are then washed with an inorganic salt culture solution to obtain Acinetobacter venezianus PFZR-1 resting cells.

Example 3: Measurement of the degradation performance of Acinetobacter venezianus PFZR-1 against different concentrations of cyclohexane.

Under relatively suitable culture conditions (pH 7.0, temperature 30°C), the degradation of different concentrations of cyclohexane by Acinetobacter venezianus PFZR-1 was studied. Specifically, the degradation was as follows:

Different concentrations of substrate cyclohexane were added to 50mL of fresh inorganic salt medium, and the initial substrate concentrations were 63.3, 94.9, 126.6, 158.2, 189.8, 253.12, and 316.4mg/L, respectively, and the stationary cells of Acinetobacter venezianus PFZR-1 prepared by the method of Example 2 were inoculated, and the initial cell concentration in each parallel sample was 0.02 (calculated by OD ₆₀₀ ). The samples were cultured in a shaker at 30°C and 160 r/min, and samples were taken at regular intervals to measure the cyclohexane concentration using gas chromatography, and the cyclohexane degradation rate of Acinetobacter venezianus PFZR-1 was calculated. During the experiment, one parallel sample and one blank control group not inoculated with the strain were set. The results are shown in FIG. 4. Although Acinetobacter venezianus PFZR-1 was able to completely degrade cyclohexane even at a fairly high concentration (316.4 mg/L), it was unable to completely degrade it within 60 hours after the cyclohexane concentration exceeded 316.4 mg/L and reached 395.5 mg/L.

The concentration of cyclohexane in the gas phase was measured using a Fukuda 9790II gas chromatograph. The chromatography column was a KB-5 (30 m x 0.32 mm x 0.5 μm). The temperatures of the sample inlet, detector (FID), and column were 120°C, 200°C, and 80°C, respectively, the auxiliary furnace temperature was 100°C, and the split flow ratio was 100:1. The hydrogen flow rate was 30 mL/min, the air flow rate was 300 mL/min, the carrier gas nitrogen flow rate was 30 mL/min, and the gas injection amount was 1 mL.

Example 4: Measurement of the degradation performance of Acinetobacter venezianus PFZR-1 against cyclohexane at different pH levels.
50 mL of inorganic salt culture solution with pH 5, 6, 7, 8, 9, and 10 was placed in shake flasks with a volume of 330 mL, and each flask was sterilized at 110°C for 40 minutes, with 50 mL. After sterilization, the flasks were left at room temperature for 2 days to confirm that there was no growth of contaminating bacteria. Cyclohexane was used as the only carbon source for Acinetobacter venezianus PFZR-1, and the inorganic salt medium was inoculated. The initial concentration of the substrate was 63.3 mg/L, and the stationary cells of Acinetobacter venezianus PFZR-1 prepared by the method of Example 2 were inoculated, and the initial cell concentration was 0.02 (calculated by OD ₆₀₀ ), the temperature was 30°C, and the rotation speed was 160 r/min. A blank control was prepared without adding bacteria. The residual cyclohexane concentration in the shake flask was measured at regular intervals in the same manner as in Example 3, and a removal rate curve for cyclohexane of the strain at different pH levels within 48 hours was created. The results are shown in FIG. 5. It was found that the optimum pH for the decomposition of cyclohexane by Acinetobacter venezianus PFZR-1 was 7.

Example 5: Broad spectrum of substrate degradation by Acinetobacter venezianus PFZR-1 In actual use, not only is cyclohexane an organic pollutant, but industrial exhaust gas generally contains various volatile organic exhaust gases. Therefore, there is a great need to investigate the decomposition effect of Acinetobacter venezianus PFZ-1 on other substrates. As in Example 3, the carbon source was changed to toluene, methanol, limonene, chloroform, ethyl acetate, cyclohexanol, and cyclohexanone with an initial concentration of 50 mg/L, and other operations were performed in the same manner as in Example 3. The decomposition effect is shown in Table 2.

As shown in Table 2, except that Acinetobacter venezianus PFZR-1 has no decomposition effect on chloroform, it has decomposition effects on all the other selected contaminants to different degrees. The reason is that chloroform is a halogenated hydrocarbon compound that is difficult to decompose, so the decomposition effect is poor.

The present invention has been disclosed above by way of examples, but these do not limit the scope of the present invention, and any changes or modifications made by those skilled in the art without departing from the concept and scope of the present invention shall fall within the scope of protection of the present invention.

Claims (7)

Acinetobacter venetianus PFZR-1, deposited at the China Typical Culture Depository Center, with deposit number CCTCC NO:M 2022719, date of deposit May 25, 2022, and address Wuhan University, Wuhan, China 430072. Use of Acinetobacter venezianus PFZR-1 according to claim 1 in the decomposition of organic pollutants by microorganisms. The use of claim 2, wherein the organic contaminant comprises cyclohexane, toluene, methanol, limonene, ethyl acetate, cyclohexanol, or cyclohexanone. The use according to claim 3, in which Acinetobacter venezianus PFZR-1 is inoculated into an inorganic salt medium containing organic pollutants, and a decomposition reaction is carried out under conditions of pH = 5-10, 20-40°C, and 100-200 rpm, thereby achieving decomposition of organic pollutants. The use according to claim 3, wherein the initial concentration of the organic contaminant in the inorganic salt medium is 63.3-316.4 mg/L, and the amount of stationary cells input in the inorganic salt culture solution is 0.01-0.1 in terms of bacterial cell concentration OD ₆₀₀ value. The composition of the inorganic salt culture solution was 0.942 g/ _{L K2HPO4} , 0.234 g/ _{L KH2PO4} , 1.7 g/L _NaNO3 , 0.98 g/L _NH4Cl , 0.2033 g/ _L MgCl2.6H2O, 0.0111 g/L _CaCl2.2H2O , 0.0162 g/ _{L FeCl3} , and 5 ml/L trace elements, the solvent was deionized water, pH = 5 to 10, and the composition of the trace elements was 0.088 g/ _{L ZnCl2} , 0.060 g/L _MnCl2.4H2O , 0.01 _g /L _KI , 0.01 g/ _{L Na2MoO4.2H2O} , 0.1 g/L _H3BO 4. The use according to claim 3, wherein ₃ is 0.05 g/L and the solvent is deionized water. Before inoculating the Acinetobacter venezianus PFZR-1, activation and expansion culture are first performed, and the expanded resting cells are inoculated into an inorganic salt medium, and the resting cells are
(1) Slant culture:
inoculating Acinetobacter venezianus PFZR-1 into an LB solid medium and culturing it in a culture box at 30°C to obtain slant cultures, the composition of the LB solid medium being 5 g/L yeast extract, 10 g/L NaCl, 10 g/L peptone, 15-20 g/L agar, with no adjustment to pH, and using deionized water as the solvent;
(2) Expansion culture:
inoculating the slant culture medium of step (1) with the LB liquid medium, culturing it at 30°C and 160 rpm for 24 hours to obtain an expansion culture liquid, centrifuging the liquid, collecting the wet culture medium, and washing it with an inorganic salt culture liquid to obtain Acinetobacter venezianus PFZR-1 resting cells, the composition of the LB liquid medium being 5 g/L yeast extract, 10 g/L NaCl, and 10 g/L peptone, the pH of which is not modified, and the solvent is deionized water;
The use according to claim 3, wherein the composition is prepared by