CN111269848B - Rhodococcus ruber JJ-3 and application thereof in acrylic acid degradation - Google Patents
Rhodococcus ruber JJ-3 and application thereof in acrylic acid degradation Download PDFInfo
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Abstract
The invention disclosesA new strain-Rhodococcus ruber JJ-3 and its application for degrading acrylic acid are disclosed, the Rhodococcus ruber JJ-3 is preserved in China center for type culture Collection with the address: china, wuhan university, zip code: 430072, deposit number: CCTCC NO: m2019546, date of deposit 2019, month 07, day 12; the invention provides Rhodococcus erythropolis JJ-3 with acrylic acid degradation performance and application thereof in degrading acrylic acid wastewater, wherein the strain can react with initial concentration of 1000 mg.L within 16h‑1The acrylic acid degradation rate reaches 100%, and the discovery of the degrading bacteria has important significance for the efficient purification of acrylic acid in industrial wastewater.
Description
(I) technical field
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a new strain with acrylic acid degradation capacity, namely Rhodococcus ruber JJ-3(Rhodococcus ruber JJ-3), and application thereof.
(II) background of the invention
Acrylic acid is the simplest unsaturated carboxylic acid, consisting of one vinyl group and one carboxyl group, and is a vinyl monomer that polymerizes very quickly. Acrylic acid is a colorless liquid with a characteristic pungent odor, is miscible with water, alcohols, ethers, and chloroform, and is prepared from propylene. Acrylic acid is an important organic synthetic raw material and synthetic resin monomer, and is mostly used for producing acrylic esters such as methyl acrylate, ethyl acrylate, butyl acrylate, hydroxyethyl acrylate and the like. The acrylic acid and acrylic ester can be homopolymerized or copolymerized, and the polymer can be used in the production processes of synthetic resin, synthetic fiber, super absorbent resin, building material, paint and the like. Acrylic acid is one of the 3-class carcinogen lists published by international cancer research institution of world health organization in 2017.
Acrylic acid is an important chemical raw material and is widely applied to industries such as paint, coating, leather, textile, printing and the like. At present, the total yield of acrylic acid in China is about 120 million t, and the total amount of acrylic acid wastewater in China is about 140 million t every year according to 1.2t wastewater generated by producing 1t acrylic acid.
The catalytic wet oxidation method for treating the acrylic acid wastewater has the advantages of high removal efficiency, low energy consumption and small secondary pollution, is a cleaner treatment technology, but has the problems of high operation cost, high control difficulty, secondary pollution of heavy metal catalysts and the like; the supercritical water oxidation method for treating the acrylic acid wastewater has the advantages of short reaction time, thorough pollutant removal, small occupied area, cleanness, environmental protection and the like, but high-concentration dissolved oxygen, equipment corrosion and inorganic salt deposition caused by high temperature and high pressure are the main problems faced by the technology; the fiber adsorption method, the ion exchange fiber method, the Fen-ton oxidation method and the photoelectron decomposition method for degrading the acrylic acid wastewater have the characteristics of mild reaction conditions, high reaction speed and high removal efficiency, but cannot be applied to the field of actual engineering treatment because of less deep research and high treatment cost at present. The method for treating the acrylic acid wastewater by using the biological method has the advantages of low energy consumption, mild reaction conditions and higher efficiency, and is more in line with the national strategy of energy conservation, emission reduction and sustainable development, but the research on the acrylic acid degradation functional strains at home and abroad is only rarely reported, so the research has important significance for screening the available strains for acrylic acid degradation and performing acrylic acid biodegradation.
Disclosure of the invention
The invention aims to provide a Rhodococcus ruber with acrylic acid degradation performance and application thereof, and provides a new method for treating acrylic acid wastewater.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-Rhodococcus ruber (Rhodococcus ruber) JJ-3, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2019546, date of deposit 2019, month 07, day 12, address: china, wuhan university, zip code: 430072.
the Rhodococcus ruber JJ-3 strain is characterized in that: the colony is orange yellow, the round opaque surface is rough and dry, the cell shape is spherical, and the cell has no flagellum and regular edges. The optimal growth pH value is 7.0, the optimal growth temperature is 35 ℃, and the 16S rDNA sequence of the strain is shown in SEQ ID No. 1.
The invention also provides application of the Rhodococcus ruber JJ-3 in biodegradation of acrylic acid. Acrylic acid is the simplest unsaturated carboxylic acid, consisting of one vinyl group and one carboxyl group, and is pure, colorless and clear with a characteristic pungent odor.
Further, the application is as follows: inoculating a fermentation liquid, namely a bacterium-containing suspension, obtained by carrying out fermentation culture on Rhodococcus ruber JJ-3 into an acrylic acid liquid selection culture medium, and culturing at the temperature of 20-45 ℃ and the speed of 140-160rpm by taking acrylic acid as a carbon source to realize the degradation of the acrylic acid; the final concentration composition of the acrylic acid liquid selective medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Acrylic acid 100-2000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl24mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, and the pH value is 5.0-10.0.
Further, the OD of the suspension containing the bacterial cells is preferred600The value is 0.7 to 1.0, preferably 0.8.
Further, the acrylic acid is added at a concentration of 0.1 to 2g/L, preferably 0.1 to 1g/L, and most preferably 1 g/L.
Further, it is preferable that the culture pH is 7.0, 35 ℃ and 160 rpm.
Further, the volume inoculation amount of the bacterial suspension is preferably 3-20%, and preferably 10-15%.
The bacteria-containing suspension is prepared by the following steps:
(1) slant culture: inoculating Rhodococcus ruber JJ-3 to a slant culture medium, and culturing at 30 deg.C for 3 days to obtain a thallus slant; the final concentration of the slant culture medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO4 1000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Peptone 400 mg. L-1 Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl24mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, the pH value is 7.0, and the agar is 18-20 g.L-1(preferably 18 g. L)-1)；
(2) Seed culture: selecting colonies from the thallus slant, inoculating the colonies to an LB culture medium, and culturing at 30 ℃ for 18h to obtain a seed solution; the LB culture medium has the following final concentration composition: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0;
(3) fermentation culture: inoculating the seed solution to a fermentation culture medium by an inoculation amount with the volume concentration of 3-20%, and culturing at 30 ℃ to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1 Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl24mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, and the pH value is 7.0.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a Rhodococcus erythropolis JJ-3 with acrylic acid degradation performance and application thereof in acrylic acid degradation, wherein the strain can react to the initial concentration of 1000 mg.L in 5d-1The acrylic acid degradation rate reaches 100%, and the degrading bacteria have important significance for treating acrylic acid in industrial wastewater.
(IV) description of the drawings
FIG. 1 is a transmission electron micrograph of strain JJ-3.
FIG. 2 is a phylogenetic tree diagram of strain JJ-3.
FIG. 3 is a comparison of the acrylic acid degradation performance of strain JJ-3 at different pH.
FIG. 4 is a comparison of the acrylic acid degradation performance of strain JJ-3 at different temperatures.
FIG. 5 is a comparison of the acrylic acid-degrading performance of strain JJ-3 at different inoculum sizes.
FIG. 6 is a comparison of the degradation performance of strain JJ-3 at different initial acrylic acid concentrations.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: isolation, purification and characterization of Rhodococcus ruber JJ-3
Isolation and purification of Rhodococcus ruber JJ-3
Rhodococcus ruber JJ-3 is screened from sludge in a wastewater treatment tank of Zhejiang satellite energy Co., Ltd, and the specific steps are as follows:
the acrylic acid liquid selective culture medium is prepared by the following method: NH (NH)4Cl 540mg·L-1，K2HPO4 1000mg·L-1，KH2PO4 1000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl2 4mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, pH value is 7.0, and sterilization is carried out for 20min at 121 ℃.
Acrylic acid solid selection medium: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Peptone 400 mg. L-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl24mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, the pH value is 7.0, and 18 g.L are added simultaneously-1Agar, sterilized at 121 ℃ for 20 min.
Taking sludge in an acrylic acid wastewater treatment pool of a satellite energy company, standing for 24h, taking 10mL, inoculating into a sterile water culture bottle (250mL) containing 100mL, carrying out shaking culture at 30 ℃ and 160rpm for 30min, standing for 2min after shaking is stopped, taking 5mL of suspension, inoculating into a selective culture medium containing 100mL of acrylic acid liquid, carrying out shaking culture at 30 ℃ and 160rpm for 3d, taking 5mL of suspension, inoculating into a selective culture medium containing 100mL of fresh acrylic acid liquid, carrying out shaking culture at 30 ℃ and 160rpm for 3d, repeating the culture for 3 times, and preparing a bacterial liquid with a certain concentration by using sterile water. And (3) separating and purifying the obtained bacterial liquid by using an acrylic acid solid selective medium through multiple flat plate streaking to obtain a single bacterial colony, namely the acrylic acid degrading strain, which is marked as a strain JJ-3.
2. Identification of Strain JJ-3
a. Physiological and biochemical characteristics of Strain JJ-3
The colony color is orange yellow, the round opaque surface is rough and dry, and has no flagellum and no spore. The shape of the cells was observed as spherical or short rod under a transmission electron microscope (FIG. 1). The optimum pH value for growth is 7.0, and the optimum temperature is 35 ℃.
b. 16S rRNA sequence analysis of Strain JJ-3
The strain JJ-3 is determined to be Rhodococcus ruber by 16S rRNA sequence analysis and physiological and biochemical experimental identification.
The method comprises the following specific steps:
the DNA of the strain JJ-3 was extracted and purified using a 3S column centrifugal type environmental sample DNA recovery kit (V2.2, Biotech, Inc., Zhejiang Tian Ke., Ltd.), and stored at 4 ℃. The purified DNA was PCR amplified using bacterial universal primers F27 and 1492R, the primer sequences were:
F27：5’-AGA GTT TGA TCC TGG CTC AG-3’
1492R：5’-GGT TAC CTT GTT ACG ACT T-3’
the PCR reaction system was (50. mu.L): 1.75. mu.L of template DNA, 1. mu.L each of primer F27 and primer R1492, MgCl2(25mmol·L-1)3μL，Taq enzyme (5U. mu.L)-1) 0.25. mu.L, 10 XPCR buffer 5. mu.L, dNTP (2.5 mmol. multidot.L)-1) mu.L, 34. mu.L of redistilled water.
The PCR reaction program was set as: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 59 ℃ for 1min, extension at 72 ℃ for 1.5min, and circulating for 35 cycles; then extending for 10min at 72 ℃; finally, the temperature is kept at 4 ℃ for 10 min. Sequencing the PCR product (Zhejiang family), and the sequencing result is shown in sequence SEQ ID NO: 1 is shown.
The 16S rDNA sequence of the strain JJ-3 was uploaded to the Genbank for homology comparison, and found to belong to the genus Rhodococcus, having the highest homology to Rhodococcus ruber of 100%, and FIG. 2 is a phylogenetic tree diagram of the strain. In order to further determine the reliability of the identification result, the strain JJ-3 is finally determined to belong to Rhodococcus ruber through physiological and biochemical experiments, therefore, the strain JJ-3 is named as Rhodococcus ruber JJ-3 and is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2019546, date of deposit 2019, month 07, day 12, address: china, wuhan university, zip code: 430072.
example 2 Rhodococcus ruber JJ-3 fermentation broth
(1) Slant culture
Inoculating Rhodococcus ruber JJ-3 to a slant culture medium, and culturing at 30 deg.C for 3 days to obtain a thallus slant; the final concentration of the slant culture medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Peptone 400 mg. L-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl24mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, the pH value is 7.0, and 18 g.L are added simultaneously-1Agar.
(2) Seed culture: selecting colonies from the thallus slant, inoculating the colonies to an LB culture medium, and culturing at 30 ℃ for 12h to obtain a seed solution; the LB mediumThe concentration composition is as follows: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0;
(3) fermentation culture: inoculating the seed solution to a fermentation medium at an inoculum size of 5 vol%, culturing at 30 deg.C and 160rpm to OD600When the concentration is 0.8, obtaining a fermentation culture solution, namely the bacterium-containing suspension; the final concentration of the fermentation medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO4 1000mg·L-1，KH2PO4 1000mg·L-1，NaCl 500mg·L -1200 mg/L yeast extract powder-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl2 4mg·L-1，CuSO4 4mg·L-1，FeSO44mg·L-1，MnSO4 4mg·L-1The solvent is water, the pH value is 7.0, and the solvent is water.
Example 3: rhodococcus ruber JJ-3 acrylic acid degradation performance detection
1. Investigating the acrylic acid degrading performance of Rhodococcus ruber JJ-3 under different initial pH conditions
The experiments of the Rhodococcus ruber JJ-3 on degrading acrylic acid are carried out at different initial pH values, and the pH value of 7.0 is found to be the optimal pH value, and the degradation rate is the highest at the time, and the concrete implementation steps are as follows:
acrylic acid was used as a carbon source (added at a concentration of 1000 mg. multidot.L)-1) OD was inoculated at a volume concentration of 5%600The culture broth was obtained by inoculating 0.8 suspension containing bacteria (prepared by the method of example 2) to an acrylic acid liquid selection medium (having the same composition as in example 2) having different pH values (pH values of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively), culturing at 35 ℃ and 160rpm under shaking for 16 hours, and the concentration of acrylic acid was measured by high performance liquid chromatography, i.e., 1.5mL of the culture broth was used as a sample, the sample was centrifuged, the supernatant was extracted by a 5mL disposable syringe, the remaining microorganisms were removed by filtration through a disposable organic syringe filter having a pore size of 0.22 μm, and the concentration of acrylic acid was measured by high performance liquid chromatography.
Chromatographic conditions are as follows:
chromatographyColumn:
C18columns (4.6 mm. times.250 mm, 5 μm); mobile phase: 0.02% phosphoric acid (pH 2.5): 70 parts of methanol: 30, of a nitrogen-containing gas; flow rate 0.6 mL/min-1(ii) a Ultraviolet detection wavelength: 210 nm; column temperature: 26 ℃; sample introduction amount: 10 μ L.
As shown in FIG. 3, the environmental suitability of the strain JJ-3 to weak acids is relatively poor, and when the pH is 7.0, the acrylic acid degradation effect of the strain JJ-3 is the best, and reaches 98.0%.
2. Investigating the acrylic acid degrading performance of Rhodococcus ruber JJ-3 under different temperature conditions
The experiment of degrading acrylic acid by Rhodococcus ruber JJ-3 is carried out at different temperatures, and the temperature is found to be the optimal temperature at 35 ℃, and the degradation rate is the highest at the time, and the specific implementation steps are as follows:
acrylic acid as carbon source (concentration 1000 mg. L)-1) OD was inoculated at a volume concentration of 5%6000.8 of the suspension containing the bacteria (prepared by the method of example 2) was inoculated into an acrylic acid liquid selection medium (pH 7.0, composition same as in example 2) at 25.0, 30.0, 35.0, 40.0, 45.0 ℃ respectively, and cultured with shaking at 160rpm for 16 hours to obtain a culture solution, the concentration of acrylic acid was measured by high performance liquid chromatography (same as in step 1),
as shown in FIG. 4, the environmental suitability of the strain JJ-3 to low temperature is relatively poor, and the acrylic acid degradation effect of the strain JJ-3 is the best, reaching 98.7%, when the temperature is 35.0 ℃.
3. Investigating the acrylic acid degrading performance of Rhodococcus ruber JJ-3 under different inoculation amounts
The acrylic acid degradation experiment of Rhodococcus ruber JJ-3 is carried out under different inoculation amounts, and the result shows that the optimal inoculation amount is 15% (v/v), and the specific embodiment is as follows:
acrylic acid was used as a carbon source (added at a concentration of 1000 mg. multidot.L)-1) OD was graded by 6 inoculations at different volume concentrations (3%, 5%, 8%, 10%, 15%, 20%) respectively600The bacterial suspension (prepared in example 2) containing 0.8 was inoculated into an acrylic acid liquid selection medium (composition: same as in example 2) at 35 ℃ under 160rpmCulturing for 16h by shaking to obtain a culture solution, measuring the concentration of acrylic acid by adopting a high performance liquid chromatography (same as the step 1), wherein the result is shown in figure 5, and the data show that when the inoculation amount is more than 3%, because nutrient substances in the culture medium are sufficient at the beginning, the inoculation amount directly influences the degradation of the substrate, the degradation rate slowly rises along with the increase of the inoculation amount, when the inoculation amount is 15%, the degradation rate reaches 100%, and along with the continuous increase of the inoculation amount, a competition relation to the nutrient substances is formed among the degrading bacteria, so that the reduction rate is reduced. As can be seen from the figure, the optimal inoculation amount of the strain JJ-3 is about 10-15%.
4. Investigating the acrylic acid degrading performance of Rhodococcus ruber JJ-3 under different initial acrylic acid concentrations
The degradation experiment of Rhodococcus ruber JJ-3 is carried out under different initial acrylic acid concentrations, and the result shows that the degradation experiment of Rhodococcus ruber JJ-3 is carried out under the condition that the acrylic acid concentration is 100--1The acrylic acid can be effectively degraded, and the specific embodiment is as follows:
using acrylic acid as carbon source, inoculating OD according to 15% volume concentration600The bacterial-containing suspensions (prepared in example 2) of 0.8 were inoculated respectively at an initial acrylic acid concentration of 100 mg. multidot.L-1、200mg·L-1、400mg·L-1、600mg·L-1、800mg·L-1、1000mg·L-1、2000mg·L-1The culture broth was obtained by shaking-culturing in 7 acrylic acid liquid selective media (composition same as example 2) at 35 ℃ and 160rpm for 16 hours, and the concentration of acrylic acid was measured by high performance liquid chromatography (same as step 1), with the results shown in FIG. 6: at 100--1Under the initial acrylic acid concentration, the Rhodococcus ruber JJ-3 can effectively degrade acrylic acid, and the degradation rate is 100%.
Sequence listing
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<120> Rhodococcus ruber JJ-3 and application thereof in acrylic acid degradation
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<213> Rhodococcus ruber (Rhodococcus ruber)
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actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct gatgcagcga 300
cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct ttcagtaccg acgaagcgca 360
agtgacggta ggtacagaag aagcaccggc caactacgtg ccagcagccg cggtaatacg 420
tagggtgcga gcgttgtccg gaattactgg gcgtaaagag ctcgtaggcg gtttgtcgcg 480
tcgtctgtga aaacccgcag ctcaactgcg ggcttgcagg cgatacgggc agacttgagt 540
actgcagggg agactggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac 600
accggtggcg aaggcgggtc tctgggcagt aactgacgct gaggagcgaa agcgtgggta 660
gcgaacagga ttagataccc tggtagtcca cgccgtaaac ggtgggcgct aggtgtgggt 720
ttccttccac gggatccgtg ccgtagctaa cgcattaagc gccccgcctg gggagtacgg 780
ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca caagcggcgg agcatgtgga 840
ttaattcgat gcaacgcgaa gaaccttacc tgggtttgac atacaccgga ccgccccaga 900
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Claims (8)
1.Rhodococcus ruber (C)Rhodococcus ruber) JJ-3, preserved in China center for type culture Collection, with the preservation number: CCTCC NO: m2019546, date of deposit 2019, month 07, day 12, address: china, wuhan university, zip code: 430072.
2. use of Rhodococcus ruber JJ-3 according to claim 1 for the biodegradation of acrylic acid.
3. The use according to claim 2, characterized in that said use is: inoculating a fermentation liquid, namely a bacterium-containing suspension, obtained by carrying out fermentation culture on Rhodococcus ruber JJ-3 into an acrylic acid liquid selection culture medium, and culturing at the temperature of 20-45 ℃ and the speed of 140-160rpm by taking acrylic acid as a carbon source to realize acrylic acid degradation; the final concentration composition of the acrylic acid liquid selective medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO4 1000mg·L-1，KH2PO4 1000mg·L-1，NaCl 500mg·L-1200 mg/L yeast extract powder-1Acrylic acid 100-2000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl2 4mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, and the pH value is 5.0-10.0.
4. Use according to claim 3, characterized in that the pH is 7.0.
5. Use according to claim 3, characterized in that the temperature is 35 ℃.
6. The use according to claim 3, wherein the OD of the suspension comprising bacterial cells is600The value is 0.7-1.0, and the volume inoculation amount of the bacterial suspension is 3-20%.
7. The use according to claim 3, wherein the initial concentration of acrylic acid is 100--1。
8. Use according to claim 3, wherein the bacterial-containing suspension is prepared by the steps of:
(1) slant culture: inoculating Rhodococcus ruber JJ-3 to a slant culture medium, and culturing at 30 deg.C for 3 days to obtain a thallus slant; the final concentration of the slant culture medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO4 1000mg·L-1，KH2PO4 1000mg·L-1，NaCl 500mg·L-1200 mg/L yeast extract powder-1Peptone 400 mg. L-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl2 4mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, the pH value is 7.0, and the agar is 18-20 g.L-1；
(2) Seed culture: selecting colonies from the thallus slant, inoculating the colonies to an LB culture medium, and culturing at 30 ℃ for 18h to obtain a seed solution; the LB culture medium has the following final concentration composition: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0;
(3) fermentation culture: inoculating the seed solution to a fermentation culture medium by an inoculation amount with the volume concentration of 3-20%, and culturing at 30 ℃ to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO4 1000mg·L-1，NaCl 500mg·L-1200 mg/L yeast extract powder-1Acrylic acid 1000 mg. L-1，MgSO4·7H2O 16mg·L-1，CaCl2 4mg·L-1，CuSO4 4mg·L-1，FeSO4 4mg·L-1，MnSO4 4mg·L-1The solvent is water, and the pH value is 7.0.
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