CN116731923A

CN116731923A - Pseudomonas ZKJ for nitrate reduction and application thereof in degrading organic pollutants

Info

Publication number: CN116731923A
Application number: CN202310716718.XA
Authority: CN
Inventors: 寿登; 周凯杰; 成卓韦; 陈建孟; 陈东之; 於建明
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-06-16
Filing date: 2023-06-16
Publication date: 2023-09-12

Abstract

The invention discloses a nitrate reduction pseudomonas ZKJ and application thereof in degrading organic pollutants, and the nitrate reduction pseudomonas ZKJ has high-efficiency degradation effect on linalool ester pollutants and can convert the pollutants into CO ₂ 、H ₂ And the harmless substances such as O and the like have wide application prospect in biological purification of industrial waste gas and wastewater. The nitrate-reducing pseudomonas ZKJ can completely degrade linalyl acetateMineralization is realized for inorganic matters and cellular biomass, and the removal rate of linalyl acetate within 216mg/L is higher than 95%.

Description

Pseudomonas ZKJ for nitrate reduction and application thereof in degrading organic pollutants

Field of the art

The invention relates to a nitrate-reducing pseudomonas (Pseudomonas nitritireducens) ZKJ and application thereof in degrading organic pollutants such as linalyl acetate.

(II) background art

Linalyl acetate is an organic compound found in natural bergamot, lavender, savoury red sage and betadox essential oils. In addition, cocoa, celery, grape, peach, kelp are also present. The linalool is added into a mixture of acetic anhydride and phosphoric acid (phosphoric acid and acetic anhydride form a complex catalyst), and esterification reaction is carried out at a lower temperature. After the esterification reaction, washing with water, washing with brine to neutrality, drying with anhydrous sodium carbonate, and vacuum fractionating to obtain the product with highest ester content (more than or equal to 95%) and pure fragrance. Or adding linalool into acetic anhydride diluted by solvent and anhydrous sodium acetate for esterification reaction, and fractionating. It is a colorless transparent liquid with pleasant floral and fruity notes. It is volatile, slightly soluble in water, insoluble in glycerol, slightly soluble in water, soluble in ethanol, propylene glycol, diethyl ether, diethyl phthalate, benzyl benzoate, non-volatile oils and mineral oil fragrances. The chemical property is stable, the color is not changed, and the product is commonly used in middle-high grade fragrant products and essence for soap.

Linalyl acetate is a low toxicity, but is still hazardous to the human body due to its volatility. Acute exposure to high concentrations of linalyl acetate can cause symptoms such as skin irritation, ocular inflammation, and airway inflammation.

Therefore, the efficient degradation of linalyl acetate in the research environment is necessary for human health, and no report about the efficient degradation of pseudomonas by taking linalyl acetate as the only carbon source is found through literature search.

(III) summary of the invention

The invention aims to provide nitrate-reduced pseudomonas ZKJ and application thereof in degrading organic pollutants, wherein nitrate-reduced pseudomonas ZKJ can degrade linalyl ester pollutants such as linalyl acetate and the like serving as the only carbon source, has high-efficiency and strong linalyl acetate degradation capability, and is mild in growth environment and easy to expand and culture. The discovery of the degradation bacteria has important significance for the efficient purification of linalyl pollutants in industrial wastewater and waste gas.

The technical scheme adopted by the invention is as follows:

the invention provides a novel linalyl acetate degrading bacterium, namely nitrate reducing pseudomonas (Pseudomonas nitritireducens) ZKJ, which is preserved in China center for type culture collection, with the preservation number: cctccc NO: m2023672, date of preservation: 2023, 05 month 04, address: 430072, university of martial arts, wuhan, china.

The basic characteristics of the Pseudomonas nitrate-reducing bacteria ZKJ of the invention are as follows: the colony is light yellow, the surface is smooth, and no flagellum exists. The edge is more regular, light-tight and easy to pick, the lawn grows basically along the scribing line, and a layer of halation is uniformly distributed around the colony; aerobic, gram-negative.

The invention also provides an application of the nitrate-reduced pseudomonas ZKJ in degrading organic pollutants, and particularly relates to an application of adding a bacterial liquid or resting cells obtained by expanding and culturing the nitrate-reduced pseudomonas ZKJ into an inorganic culture liquid containing the organic pollutants, wherein the pH value of the inorganic culture liquid is=4-9, and culturing the inorganic culture liquid at 15-30 ℃ and 100-200rpm to degrade the organic pollutants.

Further, the organic contaminants include linalyl acetate, toluene.

Further, the initial concentration of the organic contaminant in the inorganic salt culture solution is 54 to 270mg/L, preferably 108mg/L.

Further, the addition amount of the resting cells of the pseudomonas reduction nitrate ZKJ is 20-80mg/L, preferably 50mg/L, based on dry weight; by OD of bacterial liquid ₆₀₀ Calculated as 0.01-0.05, preferably 0.02.

Further, the culture conditions were 15℃and 160rpm.

Further, the inorganic salt culture solution comprises the following components: k (K) ₂ HPO ₄ ·3H ₂ O 0.942g/L、KH ₂ PO ₄ 0.234 g/L、NaNO ₃ 1.7g/L、NH ₄ Cl 0.98g/L、MgCl ₂ ·6H ₂ O 0.2033g/L、CaCl ₂ ·2H ₂ O 0.011g/L、FeCl ₃ 0.0162g/L, 5ml/L of microelement mother liquor, deionized water as solvent, and pH 7.0; wherein the trace element mother liquor comprises the following components: cuSO ₄ ·5H ₂ O 0.02g/L、FeSO ₄ ·7H ₂ O 1.0g/L、MnSO ₄ ·4H ₂ O 0.1g/L、NaMoO ₄ ·2H ₂ O 0.02g/L、CoCl ₂ ·6H ₂ O 0.02g/L、H ₃ BO ₃ 0.014g/L、ZnSO ₄ ·7H ₂ O0.10 g/L, and deionized water as solvent.

Further, the resting cells of Pseudomonas nitrate-reducing bacteria ZKJ were prepared as follows:

(1) Slant culture:

inoculating Pseudomonas nitroreduction ZKJ to a slant LB solid culture medium, and culturing for 24-36 h at 30 ℃ to obtain slant thalli, wherein the final concentration composition of the LB solid culture medium is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value.

(2) And (3) performing expansion culture:

inoculating the inclined plane thallus obtained in the step (1) into an LB liquid culture medium by using an inoculating loop, culturing for 24-36 h at 15 ℃ and 160rpm to obtain a bacterial liquid with OD < 600 > = 0.1-0.2, centrifuging, collecting wet thallus, washing by using an inorganic salt culture solution, and obtaining a pseudomonas ZKJ resting cell; the final concentration composition of the LB liquid medium is as follows: 10g/L NaCl, 10g/L peptone, 5g/L yeast powder, deionized water as solvent and natural pH value.

Compared with the prior art, the invention has the beneficial effects that:

the nitrate-reduced pseudomonas ZKJ provided by the invention is obtained from sewage plant sludge, has high-efficiency degradation effect on linalyl pollutants, and can be used for completely converting the pollutants into CO ₂ 、H ₂ Harmless substances such as O and the like have wide application prospects in biological purification of industrial waste gas and wastewater.

The invention relates to Pseudomonas Z for nitrate reductionKJ can completely degrade linalyl acetate into inorganic substances (CO) ₂ 、H ₂ O) and cellular biomass, achieving complete mineralization, and a removal rate of linalyl acetate of greater than 95% for up to 216 mg/L.

(IV) description of the drawings

FIG. 1 is a photograph showing colony morphology of strain ZKJ on LB medium.

Fig. 2 is a transmission electron micrograph of strain ZKJ.

FIG. 3 is a phylogenetic tree of strain ZKJ.

FIG. 4 is a degradation curve of strain ZKJ for linalyl acetate at various concentrations.

FIG. 5 shows the degradation rate of strain ZKJ for linalyl acetate at 108mg/L at various pH values.

FIG. 6 shows the degradation rate of strain ZKJ for 108mg/L linalyl acetate at different pH values at 22 h.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The inorganic salt culture solution comprises the following components: k (K) ₂ HPO ₄ ·3H ₂ O 0.942g/L、KH ₂ PO ₄ 0.234g/L、NaNO ₃ 1.7g/L、NH ₄ Cl 0.98g/L、MgCl ₂ ·6H ₂ O 0.2033g/L、CaCl ₂ ·2H ₂ O 0.011g/L、FeCl ₃ 0.0162g/L, 5ml/L of microelement mother liquor, deionized water as solvent, and pH 7.0; wherein the trace element mother liquor comprises the following components: cuSO ₄ ·5H ₂ O 0.02g/L、FeSO ₄ ·7H ₂ O 1.0g/L、MnSO ₄ ·4H ₂ O 0.1g/L、NaMoO ₄ ·2H ₂ O 0.02g/L、CoCl ₂ ·6H ₂ O 0.02g/L、H ₃ BO ₃ 0.014g/L、ZnSO ₄ ·7H ₂ O0.10 g/L, and deionized water as solvent.

The final concentration composition of the LB solid medium is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value.

The final concentration composition of the LB liquid medium is as follows: 10g/L NaCl, 10g/L peptone, 5g/L yeast powder, deionized water as solvent and natural pH value.

Example 1: isolation, purification and identification of strain ZKJ.

1. Isolation and purification of strain ZKJ.

The strain ZKJ is a gram-negative bacterium which is domesticated and separated from activated sludge collected from a municipal sewage treatment plant, and comprises the following specific steps:

adding 50mL of inorganic salt culture solution into 300mL of shaking flask, adding 10mL of activated sludge and 54mg/L of linalyl acetate, sealing the shaking flask, performing enrichment culture at 15 ℃ and 160rpm, detecting linalyl acetate concentration by adopting the method of example 3, taking out 5mL of enrichment solution from the shaking flask when linalyl acetate concentration is 50% of the initial linalyl acetate concentration, adding the same amount of linalyl acetate (54 mg/L) into 50mL of fresh inorganic salt culture solution, repeating the enrichment process for 5 times, and finally carrying out sterile water gradient dilution on the enrichment solution until 10 ^-6 Coating an LB solid culture medium, culturing for 1-2 days at 15 ℃, selecting single colony, inoculating to the LB solid culture medium again, carrying out separation flat panel line drawing purification, inoculating the obtained alternative bacteria to an inorganic salt culture solution taking 54mg/L linalyl acetate as a unique carbon source and energy source, culturing for 2 days at 15 ℃, detecting the linalyl acetate concentration by adopting the method of the embodiment 3, and selecting the alternative bacteria corresponding to the culture solution with the highest linalyl acetate degradation rate, and recording as a strain ZKJ.

2. Identification of Strain ZKJ

(1) Strain ZKJ characteristics: inoculating strain ZKJ to LB solid medium, culturing at 15deg.C for 1 day, and collecting colony with light yellow color and disc shape as shown in figure 1; the edges are neat, light-tight and easy to pick. The form of the cells was observed under a transmission electron microscope (FIG. 2) to be elliptical, flagellum-free, 4600X 3500nm in size, and gram-negative.

(2) 16S rRNA sequence analysis

The DNA of the strain ZKJ is extracted and purified by using an Ezup column type bacterial genome DNA extraction kit and stored at 4 ℃. The purified DNA was PCR amplified with bacterial universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT), respectively, and the PCR reaction procedure was set to 94℃for 4min, then 94℃for 45s,55℃for 45s,72℃for 1min extension, 30 cycles of cycles, and finally 72℃for 10min of repair extension. The PCR product was purified and recovered and then sequenced (Zhejiang Tianke Gaoxin technology development Co., ltd. (the institute of microorganisms of Yuanjiang province)), and the 16S rRNA sequencing result (shown as SEQ ID NO. 1) was uploaded to NCBI to obtain accession number OQ829420, and at the same time Blast comparison was performed on the sequence with the gene sequence in the NCBI database. It was found to belong to the genus Pseudomonas, having 99% homology with Pseudomonas entomophila str.L48 chromosomeme, pseudomonas hamedanensis strain SWRI 65.65 chromosomeme and Pseudomonas crudulactis strain UCMA17988 16Sribosomal RNA gene. From the results, 10 strains of Pseudomonas with representativeness were selected, and based on the homology of the 16SrRNA gene sequence, phylogenetic tree was constructed using MEGA7.0 software, as shown in FIG. 3. Is identified as Pseudomonas nitritireducens by genetic distance and 16S rRNA sequence comparison.

16S rRNA sequences

1tgggagcttgctcccggattcagcggcggacgggtgagtaatgcctaggaatctgcctgg

61tagtgggggacaacgtttcgaaaggaacgctaataccgcatacgtcctacgggagaaagc

121aggggaccttcgggccttgcgctatcagatgagcctaggtcggattagctagttggtgggg

181taaaggcctaccaaggcgacgatccgtaactggtctgagaggatgatcagtcacactgga

241actgagacacggtccagactcctacgggaggcagcagtggggaatattggacaatgggcg

301aaagcctgatccagccatgccgcgtgtgtgaagaaggtcttcggattgtaaagcacttta

361agttgggaggaagggcagtaagttaataccttgctgttttgacgttaccaacagaataag

421caccggctaacttcgtgccagcagccgcggtaatacgaagggtgcaagcgttaatcggaa

481ttactgggcgtaaagcgcgcgtaggtggtttggtaagatggatgtgaaatccccgggctc

541aacctgggaactgcatccataactgcctgactagagtacggtagagggtggtggaatttc

601ctgtgtagcggtgaaatgcgtagatataggaaggaacaccagtggcgaaggcgaccacct

661ggactgatactgacactgaggtgcgaaagcgtggggagcaaacaggattagataccctgg

721tagtccacgccgtaaacgatgtcgactagccgttgggatccttgagatcttagtggcgca

781gctaacgcgataagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaat

841tgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaacc

901ttacctggccttgacatgtccggaatcttgcagagatgcgagagtgccttcgggaatcgg

961aacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgt

1021aacgagcgcaacccttgtccttagttaccagcacgttatggtgggcactctaaggagact

1081gccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacggcca

1141gggctacacacgtgctacaatggtcggtacagagggttgccaagccgcgaggtggagcta

1201atcccataaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcgga

1261atcgctagtaatcgtgaatcagaatgtcacggtgaatacgttcccgggccttgtacacac

1321cgcc。

(3) The strain ZKJ has the utilization capacity of 47 carbon sources on Mei Liai GN cards.

The metabolic conditions of the strain ZKJ on 47 different carbon sources (delegated to Zhejiang Tianke Gao Xin technology development Co., ltd. (original Zhejiang province of microorganisms) were examined by using a Mei Liai full-automatic identifier). The results of the identification are shown in Table 1. Through the biochemical reaction of the VITEK by a Mei Liai full-automatic identifier, the strain ZKJ can strongly utilize 11 carbon sources and cannot utilize other 36 carbon sources.

Table 1 Strain ZKJ Mei Liai full-automatic identifier VITEK Biochemical reaction results (GN card)

And (3) table notes: positive reaction; -: negative reaction

The strain ZKJ is Pseudomonas nitritireducens and named as Pseudomonas nitrate-reducing bacteria (Pseudomonas nitritireducens) ZKJ and is preserved in China center for type culture collection (China center), and addresses are determined by colony morphology, 16S rRNA sequence analysis and physiological and biochemical experiment identification: china, university of Wuhan, 430072, accession number: cctccc NO: m2023672, date of preservation: 2023, 05, 04.

EXAMPLE 2 acquisition of resting cells of Pseudomonas nitrate-reducing bacteria ZKJ

1. Slant culture:

inoculating Pseudomonas nitroreduction ZKJ into LB liquid medium, culturing at 15 ℃ and 160rpm for 24-36 h, drawing activated bacteria lines in a solid LB flat plate incubator at 30 ℃, drawing single bacterial colonies continuously to detect the purity of bacteria, and preserving the inclined plane of the LB test tube conventionally (4 ℃).

2. Expansion culture

Inoculating the slant thallus in the step 1 into LB liquid culture medium, culturing for 24-36 h at 15 ℃ and 160rpm to obtain bacterial liquid with OD < 600 > = 0.1-0.2, centrifuging, collecting wet thallus, washing with inorganic salt culture liquid, and obtaining the resting cells of the nitrate-reduced pseudomonas ZKJ.

Example 3: the degradation performance of the nitrate-reducing pseudomonas ZKJ on linalyl acetate with different concentrations is detected.

The inorganic salt culture solution is subpackaged in shake flasks with the volume of 300mL, 50mL of each flask is sterilized at 110 ℃ for 40min. And (5) standing for 2d at room temperature after sterilization is finished, and determining the growth of the sterile impurities. Resting cells obtained in example 2 were added to a final concentration of 50mg/L (based on dry cell weight), linalyl acetate was then added as the sole carbon source to a final concentration of 54, 108, 162, 216, 270mg/L, respectively, and shake flasks were shake-sealed and shake-cultured at 15℃at 160rpm, and a blank without bacteria was made. Analysis strip for determining concentration of linalyl acetate in gas phase above shake flask by Agilent gas chromatographyThe parts are as follows: the column was HP-INNOWax Polyethylene Glycol (30 m. Times.320. Mu.m.times.0.50 μm), column temperature 240 ℃, column flow rate: 0.5mL min ^-1 The temperature of the sample inlet is 250 ℃, the split ratio is 40:1, the detector is a hydrogen Flame Ionization Detector (FID), the temperature is 250 ℃, and the hydrogen flow is 40 mL-min ^-1 Air flow rate is 450 mL.min ^-1 The sample injection amount is 0.8mL. The concentrations of linalyl acetate remaining in the shake flask were measured at regular time, and the removal rate curves of linalyl acetate of different initial concentrations of the strain with time were plotted, and the results are shown in fig. 4. The results show that strain ZKJ can rapidly degrade all added substrate when linalyl acetate concentration is below 216 mg/L; at a linalyl acetate concentration of 270mg/L, strain ZKJ had a degradation rate of about 80% of the substrate over the time tested.

Example 4: detection of degradation Properties of 108mg/L linalyl acetate by Pseudomonas nitrate-reducing bacteria ZKJ under different initial pH conditions.

With 1mol/L NaOH aqueous solution or 1mol/L H ₂ SO ₄ The inorganic salt culture solution is adjusted to different pH values (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0) by the aqueous solution, and the bacterial solution prepared in the method of the example 2 is connected under the condition that the initial linalyl acetate concentration is 108mg/L, so that the initial bacterial solution concentration in each parallel sample is OD ₆₀₀ Calculated as 0.02. Shake flask was sealed and samples were shake cultured in a constant temperature shaker at 15 ℃,160rpm, and a blank without bacteria was made. The concentrations of linalyl acetate remaining in the shake flask were measured at regular time by the method of example 3, and the results of plotting the removal rate curves of linalyl acetate with time under different pH environments of the strain and the degradation rates of linalyl acetate at different pH at 22h for 108mg/L are shown in FIG. 5 and FIG. 6. The results show that at each pH, pseudomonas ZKJ can degrade linalyl acetate and has the best effect on linalyl acetate degradation at pH 7 and 8.

Example 5: the degradation performance of the nitrate-reducing pseudomonas ZKJ on different organic matters is detected.

The inorganic salt culture solution is subpackaged in shake flasks with the volume of 300mL, 50mL of each flask is sterilized at 110 ℃ for 40min. And (5) standing for 2d at room temperature after sterilization is finished, and determining the growth of the sterile impurities. Resting cells obtained in example 2 were added to a final concentration of 50mg/L (based on dry cell weight), linalyl acetate, toluene, chlorobenzene, dichloromethane were then added as the sole carbon source to a final concentration of 80mg/L, shake flask was sealed, shake cultured at 15℃at 160rpm, and a blank without bacteria was made. The concentration of the four contaminants in the shake flask gas phase was determined using Agilent gas chromatography as described in example 3.

The results are shown in Table 2. It can be seen that pseudomonas ZKJ has a certain degradation capacity for toluene in addition to linalyl acetate, but has little degradation effect for chlorine-containing substrates such as chlorobenzene, dichloromethane.

TABLE 2 test effects of various substrates

Substrate(s)	36h residual concentration (mg/L)	Degradation rate (%)
			Linalyl acetate	4.8	94
Toluene (toluene)	37.6	53
			Chlorobenzene (Chlorobenzene)	72	10
Dichloromethane (dichloromethane)	73.6	8

Although the present invention has been described with reference to the above embodiments, it should be understood that the invention is not limited to the embodiments described above, but is capable of modification and variation without departing from the spirit and scope of the present invention.

Claims

1. Pseudomonas (Pseudomonas nitritireducens) ZKJ, deposited with China center for type culture Collection, accession number: cctccc NO: m2023672, date of preservation: 2023, 05 month 04, address: 430072, university of martial arts, wuhan, china.

2. Use of pseudomonas nitrate reduction ZKJ according to claim 1 for degrading organic pollutants.

3. The use according to claim 2, wherein the use is to add bacterial liquid or resting cells obtained by expanding pseudomonas nitrate reduction ZKJ into an inorganic culture liquid containing organic pollutants at ph=4-9, and culture the bacterial liquid or resting cells at 15-30 ℃ and 100-200rpm to degrade the organic pollutants.

4. The use according to claim 3, wherein the organic contaminant comprises linalyl acetate, toluene.

5. The use according to claim 3, wherein the initial concentration of organic contaminants in the mineral salts broth is 54-270mg/L.

6. The use according to claim 3, wherein the culture conditions are 15℃and 160rpm.

7. The use according to claim 3, wherein the mineral salts broth consists of: k (K) ₂ HPO ₄ ·3H ₂ O0.942g/L、KH ₂ PO ₄ 0.234g/L、NaNO ₃ 1.7g/L、NH ₄ Cl 0.98g/L、MgCl ₂ ·6H ₂ O 0.2033g/L、CaCl ₂ ·2H ₂ O 0.011g/L、FeCl ₃ 0.0162g/L, 5ml/L of microelement mother liquor, deionized water as solvent, and pH 7.0; wherein the trace element mother liquor comprises the following components: cuSO ₄ ·5H ₂ O 0.02g/L、FeSO ₄ ·7H ₂ O 1.0g/L、MnSO ₄ ·4H ₂ O 0.1g/L、NaMoO ₄ ·2H ₂ O 0.02g/L、CoCl ₂ ·6H ₂ O 0.02g/L、H ₃ BO ₃ 0.014g/L、ZnSO ₄ ·7H ₂ O0.10 g/L, and deionized water as solvent.

8. The use according to claim 3, wherein the resting cells of pseudomonas nitrate-reducing bacteria ZKJ are added in an amount of 20 to 80mg/L on a dry weight basis; by OD of bacterial liquid ₆₀₀ Calculated as 0.01-0.05.

9. The use according to claim 3, wherein the resting cells of pseudomonas nitrate-reducing bacteria ZKJ are prepared by the steps of:

(1) Slant culture:

inoculating Pseudomonas nitroreduction ZKJ to a slant LB solid culture medium, and culturing for 24-36 h at 30 ℃ to obtain slant thalli, wherein the final concentration composition of the LB solid culture medium is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;

(2) And (3) performing expansion culture:

inoculating the slant bacterial cells obtained in the step (1) into an LB liquid culture medium by using an inoculating loop, and culturing at 15 ℃ and 160rpm for 24-36 h to obtain OD ₆₀₀ Bacterial liquid with the concentration of 0.1-0.2, centrifuging, collecting wet bacterial cells, washing with an inorganic salt culture medium, and obtaining pseudomonas ZKJ resting cells; the final concentration composition of the LB liquid medium is as follows: 10g/L NaCl, 10g/L peptone, 5g/L yeast powder, deionized water as solvent and natural pH value.