CN110819553A - Bacillus aryabhattai and application thereof in acrylic acid degradation - Google Patents
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Abstract
The invention discloses a bacillus asypoda and application thereof in acrylic acid degradation, belonging to the technical field of biological treatment of environmental pollutants, wherein the bacillus asypoda is named as CY and is preserved in China center for type culture collection with the preservation number: CCTCC NO: m2019526, preservation date of 2019, 7 months and 5 days. The Bacillus aryabhattai CY provided by the invention can react to the initial concentration of 100 mg.L in 2d‑1The acrylic acid degradation rate reaches 99.8 percent, and the discovery of the degrading bacteria has important significance for the high-efficiency purification of the acrylic acid-containing industrial wastewater.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a Bacillus aryabhattai CY and application thereof in acrylic acid degradation.
Background
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 ten thousand tons every year, and according to the calculation that 1.2 tons of waste water are generated when 1 ton of acrylic acid is produced, about 140 ten thousand tons of acrylic acid waste water are generated in China every year. Therefore, the efficient treatment of acrylic acid wastewater has become an important content in the field of industrial wastewater pollution control.
In recent years, a great deal of research and application has been made in the aspect of acrylic acid wastewater treatment at home and abroad. The catalytic wet oxidation method 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, potential pollution of heavy metal catalysts and the like; the supercritical water oxidation method has the advantages of short reaction time, thorough pollutant removal, small occupied area, cleanness, environmental protection and the like, but the main problems of the technology are equipment corrosion and inorganic salt deposition caused by high-concentration dissolved oxygen and high temperature and high pressure; the fiber adsorption method, the ion exchange fiber method, the photoelectron wave decomposition method and the like have the characteristics of mild reaction conditions, high reaction speed and high removal efficiency, but the deep research is less at present, the treatment cost is high, and the popularization of the practical engineering application is limited. The acrylic acid wastewater treated by the biological method has the characteristics of low energy consumption, mild reaction conditions, high degradation efficiency and the like, but the research on the strains with acrylic acid degradation capability is rarely reported at home and abroad. Therefore, the invention provides a new method for screening available strains for acrylic acid degradation and biologically degrading acrylic acid, and has important significance for treating acrylic acid wastewater.
Disclosure of Invention
The invention aims to provide a bacillus aryabhattai CY and application thereof in acrylic acid degradation.
The technical scheme adopted by the invention is as follows: a strain of Bacillus aryabhattai, named as Bacillus aryabhattai CY, is preserved in China center for type culture Collection with the address: china, wuhan university, accession number: CCTCC NO: M2019526, preservation date 2019, 7 months and 5 days.
Said Bacillus aryabhattai CY strain is characterized in that: the colony color is yellowish, and the colony is small-size single colony, and the colony is inside to be the mucus form, has the spore, and is opaque, no flagellum, and the form of observing this thallus under the transmission electron microscope is the bacillus. The 16S rDNA sequence of the strain is shown in SEQ ID No. 1.
The application of the Bacillus aryabhattai in acrylic acid degradation.
Further, a fermentation liquid obtained by fermentation culture of the bacillus aryabhattai CY is a bacterium-containing suspension, and the bacterium-containing suspension is inoculated into an acrylic acid liquid selection culture medium to obtain the acrylic acid degradation liquid, wherein the bacterium-containing suspension accounts for 5 vt% of the acrylic acid degradation liquid. Acrylic acid is used as a carbon source, and is cultured under the conditions of 25-40 ℃ and 160rpm to degrade the acrylic acid. The acrylic acid liquid selective medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl500mg·L-1Yeast extract powder 1000 mg.L-1Acrylic acid 100-200 mg.L-1， MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41 mg·L-1The solvent is water, and the pH value is 5.0-9.0.
Further, the carbon source further comprises an auxiliary carbon source, specifically glucose, glycerol, yeast or sucrose, and the carbon concentration of the auxiliary carbon source in the acrylic acid degradation solution is 100 mg.L-1。
Further, a fermentation liquid, i.e. a bacterial-containing suspension, obtained by fermentation culture of the bacillus aryabhattai CY is prepared by the following method:
(1) slant culture: inoculating Bacillus aryabhattai CY to a slant culture medium, and culturing at 30 ℃ for 3 days to obtain slant thalli; the slant culture medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1， KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1C, COlefine acid 100 mg.L-1， MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO 41 mg·L-1The solvent is water, the pH value is 7.0, and the agar is 18 g.L-1。
(2) Seed culture: selecting a bacterial colony from the inclined-plane thallus, inoculating the bacterial colony to a seed culture medium, and culturing at 30 ℃ for 18h to obtain a seed solution; the seed culture medium comprises the following components: 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 5%, and culturing at 30 ℃ to obtain a fermentation culture solution, namely a bacterium-containing suspension; the fermentation medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO 41 mg·L-1，MnSO41mg·L-1The solvent is water, and the pH value is 7.0.
The invention has the following beneficial effects: the invention provides a bacillus ajoensis strain with acrylic acid degradation performance and application thereof in acrylic acid degradation, wherein the strain can react to the initial concentration of 100 mg.L in 2d-1The acrylic acid degradation rate of the bacteria reaches more than 99 percent, and the bacteria have important significance for treating acrylic acid in industrial wastewater.
Drawings
FIG. 1 is a transmission electron micrograph of strain CY;
FIG. 2 is a phylogenetic tree diagram of strain CY;
FIG. 3 is a comparison of the acrylic acid degradation performance of strain CY under different auxiliary carbon sources;
FIG. 4 is a comparison of the acrylic acid degradation performance of the strain CY at different initial acrylic acid concentrations;
FIG. 5 is a comparison of the acrylic acid degradation performance of the strain CY at different temperatures;
FIG. 6 is a comparison of the acrylic acid degradation performance of the strain CY at different pH.
Detailed Description
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 Bacillus aryabhattai CY
Isolation and purification of Bacillus aryabhattai CY
The Bacillus aryabhattai CY is screened from sludge in a wastewater treatment tank of Zhejiang satellite energy Co., Ltd, and comprises the following specific steps:
the acrylic acid liquid basic culture medium is prepared by the following method: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1， MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, pH is 7.0, and sterilization is carried out at 121 ℃ for 20 min.
Acrylic acid solid selection medium: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4 mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, pH is 7.0, and 18 g.L are added-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 250mL culture bottle containing 100mL of sterile water, carrying out shaking culture at 30 ℃ and 160rpm for 30min, standing for 2 min after stopping shaking, 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 after 3d, inoculating into a selective culture medium containing 100mL of fresh acrylic acid liquid, carrying out shaking culture at 30 ℃ and 160rpm for 3d, and preparing a bacteria liquid with a certain concentration by using sterile water after 3 times of culture. 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 bacterial strain CY.
2. Identification of the Strain CY
a. Morphological characteristics of the Strain CY
The colony color is yellowish, and the colony is small-size single colony, and the colony is inside to be the mucus form, has the spore, and is opaque, no flagellum. The form of the cells was observed as bacilli under a transmission electron microscope (FIG. 1). The optimum pH value for growth is 7.0, and the optimum temperature is 30 ℃.
b. 16S rRNA sequence analysis of Strain CY
The strain CY is determined to be Bacillusaryabhattai by 16S rRNA sequence analysis and physiological and biochemical experimental identification. The method comprises the following specific steps:
the DNA of the strain CY is extracted and purified by a 3S column centrifugal environment sample DNA recovery kit (V2.2, Zhejiang department of Biotechnology, Ltd.), and is 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)3 μ L of Taq enzyme (5U. μ 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 CY is uploaded to the gene sequence in Genbank for homology comparison, and the gene sequence is found to belong to the genus Bacillus, has the highest homology with Bacillus aryabhattai B8W22 and reaches 100 percent, and FIG. 2 is a phylogenetic tree diagram of the strain. In order to further determine the reliability of the identification result, the strain CY was finally determined to belong to Bacillus aryabhattai through physiological and biochemical experiments, and therefore, the strain was named as Bacillus aryabhattai (Bacillus aryabhattai) CY.
Example 2 fermentation broth of Bacillus aryabhattai CY
(1) Slant culture
Inoculating Bacillus aryabhattai CY to a slant culture medium, and culturing at 30 ℃ for 3 days to obtain slant thalli; the slant culture medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000 mg·L-1，NaCl500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, the pH value is 7.0, and the agar is 18 g.L-1。
(2) Seed culture: selecting bacterial colonies from the bacterial slant, inoculating the bacterial colonies to a seed culture medium, and culturing at 30 ℃ for 18h to obtain a seed solution; the final concentration of the seed culture medium 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 culture medium by an inoculation amount with the volume concentration of 5%, and culturing at 30 ℃ to obtain a fermentation culture solution, namely a bacterium-containing suspension; the fermentation medium comprises the following components: NH (NH)4Cl 540mg·L-1， K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, and the pH value is 7.0.
The fermentation broth of Bacillus aryabhattai CY obtained in example 2 was cultured for 18 hours, and OD was determined600Reaching 1.5, which shows higher activity.
Example 3: bacillus aryabhattai CY acrylic acid degradation performance detection
1. Investigating the acrylic acid degrading performance of the Bacillus aryabhattai CY under different auxiliary carbon sources
The experiment that the Bacillus aryabhattai CY degrades acrylic acid is carried out under different auxiliary carbon sources, and the result shows that the optimal auxiliary carbon source is yeast, and the specific embodiment is as follows:
respectively taking glucose, glycerol, yeast, sucrose and sodium acetate as the only auxiliary carbon sources (carbon concentration is 100 mg. L)-1) The OD is added according to the inoculation amount of 5 percent of volume concentration of acrylic acid degradation liquid600The bacterial-containing suspensions (prepared by the method of example 2) of 1.5 were inoculated into 5 media, namely, an acrylic acid liquid selective medium a (the auxiliary carbon source is glucose), an acrylic acid liquid selective medium B (the auxiliary carbon source is glycerol), an acrylic acid liquid selective medium C (the auxiliary carbon source is yeast), an acrylic acid liquid selective medium D (the auxiliary carbon source is sucrose), and an acrylic acid liquid selective medium E (the auxiliary carbon source is sodium acetate), and the compositions of the liquid selective media were: NH (NH)4Cl 540mg·L-1，K2HPO41000 mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1Acrylic acid 100 mg. L-1The carbon concentration of the auxiliary carbon source is 100 mg.L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO 41 mg·L-1，MnSO41mg·L-1The solvent is water and the pH value is7.0, shaking and culturing at 30 ℃ and 160rpm for 2d to respectively obtain culture solutions, measuring the concentration of acrylic acid by adopting high performance liquid chromatography, namely taking 1.5mL of the culture solution as a sample, firstly centrifuging the sample, extracting supernatant by using a 5mL disposable syringe injector, filtering by using a disposable organic syringe filter with the pore diameter of 0.22 mu m to remove residual microorganisms, and then taking filtrate and measuring the concentration of acrylic acid by adopting high performance liquid chromatography.
The carbon source provides an important energy source for the growth of the microorganisms and constitutes the cellular material of the microorganisms. Different carbon sources have different degrees of influence on microbial reduction due to different structures and molecular weights.
The results are shown in FIG. 3, and the results show that the chemical structure and molecular weight of the carbon source used have great influence on the reduction efficiency, the bacillus aryabhattai CY has the best utilization effect on the yeast, and the degradation rate is 95%; glucose, glycerol and sucrose can also well promote the acrylic acid degradation of the bacillus asythus CY, and the degradation effect can reach more than 90%. Sodium acetate is a small molecular organic acid, the selective utilization of the strain CY is poor, the degradation effect is only 62 percent,
2. investigating the acrylic acid degrading performance of the Bacillus aryabhattai CY under different initial acrylic acid concentrations
The result of a degradation experiment of Bacillus aryabhattai CY carried out under different initial acrylic acid concentrations shows that the acrylic acid concentration of the Bacillus aryabhattai CY is 100-1000 mg.L-1The acrylic acid can be degraded as follows in the specific embodiment:
taking acrylic acid as a carbon source, and inoculating the OD according to the volume concentration of 5 percent of acrylic acid degradation liquid600The bacterial suspensions (prepared in example 2) of 1.5 were inoculated respectively at initial acrylic acid concentrations of 100 mg. multidot.L-1、200mg·L-1、 400mg·L-1、600mg·L-1、800mg·L-1、1000mg·L-1The 6 liquid selective media, wherein the other components in the liquid selective media are: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, pH value is 7.0, shaking culture is carried out at 30 ℃ and 160rpm for 2d to obtain culture solution, the concentration of acrylic acid is measured by adopting high performance liquid chromatography, namely 1.5mL of culture solution is taken as a sample, the sample is firstly centrifuged, a 5mL disposable syringe injector is used for extracting supernatant, then a disposable organic syringe filter with the aperture of 0.22 mu m is used for filtering to remove residual microorganisms, and then filtrate is taken to measure the concentration of acrylic acid by adopting high performance liquid chromatography.
The concentration of the acrylic acid liquid selective culture medium comprises: the results are shown in FIG. 4: at 100mg L-1At the initial concentration of acrylic acid, the Bacillus aryabhattai CY can degrade acrylic acid with the degradation rate of 99%, and the degradation rate of the Bacillus aryabhattai CY in other concentrations of acrylic acid is reduced because the acrylic acid has certain toxicity and the growth and metabolism of cells are inhibited when the concentration is too high.
3. Investigating the acrylic acid degrading performance of the Bacillus aryabhattai CY at different temperatures
The test of acrylic acid degradation by the bacillus aryabhattai CY is carried out at different temperatures, and the result shows that the optimal temperature is 30 ℃, and the specific embodiment is as follows:
will OD600The bacterial-containing suspension (prepared in example 2) of 1.5 was inoculated into an acrylic acid liquid selection medium having a composition of 5% by volume of the acrylic acid degradation solution: NH (NH)4Cl 540mg·L-1， K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water and the pH value is7.0, respectively at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C, 45 degrees C, 160rpm shaking culture 2d, obtain culture solution, adopt high performance liquid chromatography to determine the concentration of acrylic acid, namely take 1.5mL culture solution as the sample, centrifuge the sample first, draw the supernatant fluid with the disposable syringe of 5mL, filter through the disposable organic syringe filter of pore size 0.22 μm and remove the surplus microorganism, then take the filtrate and measure the concentration of acrylic acid with high performance liquid chromatography.
The results are shown in fig. 5, and the data show that the activity of the microorganisms is significantly inhibited when the temperature is greater than 40 ℃. As can be seen from the figure, the optimal temperature of the Bacillus aryabhattai CY is 25-40 ℃, and when the temperature is 30 ℃, the acrylic acid degradation rate of the Bacillus aryabhattai CY reaches 99.5%.
4. Investigating the acrylic acid degrading performance of the Bacillus aryabhattai CY under different initial pH conditions
When the experiments of the bacillus aryabhattai CY on degrading acrylic acid are carried out at different initial pH values, 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 specific implementation steps are as follows:
the OD is added according to the inoculation amount of 5 percent of volume concentration of acrylic acid degradation liquid6001.5 bacterial suspensions (prepared as described in example 2) were inoculated into acrylic acid liquid selection media (pH 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, respectively) at different pH values, consisting of: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1， KH2PO41000mg·L-1，NaCl 500mg·L-1 Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1， MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1Performing shake culture at 30 deg.C and 160rpm for 2d to obtain culture solution, measuring acrylic acid concentration by high performance liquid chromatography, namely taking 1.5mL culture solution as sample, centrifuging the sample, extracting supernatant with 5mL disposable syringe injector, and passing through disposable organic syringe with aperture of 0.22 μmFiltering with a filter to remove residual microorganisms, and measuring the concentration of acrylic acid by high performance liquid chromatography.
As shown in FIG. 6, when the pH is 7.0, the acrylic acid degradation effect of the strain CY is the best, and reaches 99.8%, and when the pH is 10, the degradation effect of the Bacillus aryabhattai CY is obviously reduced, because the pH is too high, the enzyme is inactivated, and the activity of the microorganism is inhibited, which indicates that the environmental adaptability of the strain CY to strong base is relatively poor.
Sequence listing
<110> Zhejiang tree college (Zhejiang tree university)
<120> a strain of Bacillus aryabhattai and application thereof in acrylic acid degradation
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<170>SIPOSequenceListing 1.0
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gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttagaggg tttccgccct 780
ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa 840
ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 900
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ttcgggggac agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1020
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cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg 1370
Claims (5)
1. A strain of Bacillus aryabhattai, which is named as Bacillus aryabhattaiCY and is preserved in China center for type culture Collection with the preservation number: CCTCC NO of M2019526, and the preservation date is 7 months and 5 days in 2019.
2. Use of the Bacillus aryabhattai of claim 1 for acrylic acid degradation.
3. Use according to claim 2, characterized in thatThe method comprises the steps of carrying out fermentation culture on Bacillus aryabhattai CY to obtain fermentation liquor, namely bacterial suspension, and inoculating the bacterial suspension to an acrylic acid liquid selective culture medium to obtain the acrylic acid degradation liquid, wherein the bacterial suspension accounts for 5 vt% of the acrylic acid degradation liquid. Acrylic acid is used as a carbon source, and is cultured under the conditions of 25-40 ℃ and 160rpm to degrade the acrylic acid. The acrylic acid liquid selective medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1Yeast extract powder 1000 mg.L-1Acrylic acid 100-200 mg.L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, and the pH value is 5.0-9.0.
4. The use according to claim 3, wherein the carbon source further comprises an auxiliary carbon source, in particular glucose, glycerol, yeast or sucrose, and the carbon concentration of the auxiliary carbon source in the acrylic acid degradation solution is 100 mg-L-1。
5. The use of claim 3, wherein the fermentation broth, i.e. the bacterial-containing suspension, obtained by the fermentative culture of Bacillus aryabhattai CY is prepared by:
(1) slant culture: inoculating Bacillus aryabhattai CY to a slant culture medium, and culturing at 30 ℃ for 3 days to obtain slant thalli; the slant culture medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent isWater, pH 7.0, agar 18 g.L-1。
(2) Seed culture: selecting a bacterial colony from the inclined-plane thallus, inoculating the bacterial colony to a seed culture medium, and culturing at 30 ℃ for 18h to obtain a seed solution; the seed culture medium comprises the following components: 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 5%, and culturing at 30 ℃ to obtain a fermentation culture solution, namely a bacterium-containing suspension; the fermentation medium comprises the following components: NH (NH)4Cl 540mg·L-1，K2HPO41000mg·L-1，KH2PO41000mg·L-1，NaCl 500mg·L-1Yeast extract powder 1000 mg.L-1Acrylic acid 100 mg. L-1，MgSO4·7H2O 4mg·L-1，CaCl21mg·L-1，CuSO41mg·L-1，FeSO41mg·L-1，MnSO41mg·L-1The solvent is water, and the pH value is 7.0.
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