CN110904002A - Method for biologically removing tetracycline antibiotics - Google Patents
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- CN110904002A CN110904002A CN201911168281.0A CN201911168281A CN110904002A CN 110904002 A CN110904002 A CN 110904002A CN 201911168281 A CN201911168281 A CN 201911168281A CN 110904002 A CN110904002 A CN 110904002A
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Abstract
The invention discloses a method for biologically removing tetracycline antibiotics, which comprises the following steps: (1) seed culture: culturing the secondary seeds of the bacillus clausii and the bacillus amyloliquefaciens; (2) removing: mixing water containing tetracycline antibiotics with fermentation medium to obtain degradation solution, inoculating Bacillus clausii secondary seed and HM-618 secondary seed into the degradation solution, and fermenting. The invention establishes a co-culture system of bacillus clausii and bacillus amyloliquefaciens, and adds the co-culture system into a water body containing tetracycline antibiotics, so that the bacillus amyloliquefaciens can produce surface active substances in situ to promote the biological removal of the tetracycline antibiotics, and a series of complicated steps such as separation and purification of a surfactant are omitted; the method not only improves the biological removal efficiency of the tetracycline antibiotics, but also obviously reduces the biological toxicity of the tetracycline antibiotic conversion products.
Description
Technical Field
The invention belongs to the field of environmental remediation, and relates to a method for biologically removing tetracycline antibiotics by using a surfactant as an auxiliary agent.
Background
The tetracycline antibiotics represented by Oxytetracycline (OTC) and chlorotetracycline (CTC) are broad-spectrum antibiotics, are widely applied to animal husbandry and aquaculture, account for 14% of the total antibiotic usage, and are detected in different water body environments in China. It not only causes serious water pollution threatening the survival of aquatic plants, animals and microorganisms, but also causes the emergence of tetracycline resistance in bacteria and the spread of resistance genes, and thus the search for effective methods to remove tetracycline antibiotics from water bodies is imminent.
The traditional physical methods such as adsorption, photocatalysis and the like have high cost and are easy to cause secondary pollution. While biodegradation is reported in many documents as the most important way to remove tetracycline antibiotics, the current removal of tetracycline antibiotics is mostly dependent on microbial communities in the environment, or pure bacterial cultures concentrated on fungal species, and there are also a few strains identified with tetracycline antibiotic-degrading ability isolated, but the biological removal efficiency is not high, and the biological removal mechanism is still not well understood.
Surfactants are substances which can obviously reduce surface (interface) tension even at very low concentration, are widely applied to industries such as agriculture, food production, chemistry and chemical industry, medical pharmacy and the like at present, particularly to special fields such as petroleum industry, environmental engineering and the like, and can improve the bioavailability of organic pollutants through the processes of emulsification, pseudophase solubility improvement, microbial membrane degradation permeability enhancement, transportation promotion and the like.
At present, no report exists for removing tetracycline antibiotics by mixed fermentation of bacillus clausii and a surfactant-producing bacterium.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for biologically removing tetracycline antibiotics.
The technical scheme of the invention is summarized as follows:
a method for the biological removal of a tetracycline antibiotic comprising the steps of:
(1) seed culture: culturing Bacillus clausii Bacillus clausii T to obtain a first-stage Bacillus clausii seed; culturing the first-stage seeds of the bacillus clausii to obtain second-stage seeds of the bacillus clausii;
culturing Bacillus amyloliquefaciens HM-618, CGMCC7097, abbreviated as HM-618 to obtain first-grade seeds of HM-618; culturing the first-level seeds of HM-618 to obtain second-level seeds of HM-618;
(2) removing: adding 70-80mL of water containing tetracycline antibiotics into 20-30mL of fermentation medium according to a proportion to prepare degradation liquid; inoculating the secondary seed of the bacillus clausii obtained in the step (1) and the secondary seed of HM-618 into 100mL of degradation liquid to ensure that the total initial OD6000.15-0.23, wherein the initial inoculation ratio of the second-level seed of the Bacillus clausii to the second-level seed of the HM-618 is 1: 1; fermenting for 96-120h at the rotating speed of 200-220 rpm and the temperature of 36-38 ℃.
The formula of the fermentation medium is preferably as follows: 55-65g/L of glucose; 3-8g/L of yeast extract powder; 3-8g/L of peptone; 3-8g/L NaCl; KCl 0.3-0.8 g/L; k2HPO41-5 g/L；KH2PO41-5 g/L；MgSO4·7H2O 0.3-0.8g/L；FeSO4·7H2O(0.5-1.5)×10-3g/L；CuSO4·5H2O(0.5-1.5)×10-5g/L；MnSO4(5-8)×10-5g/L；ZnSO4·7H2O(0.5-1.5)×10-3g/L; the balance of water.
The tetracycline antibiotics are oxytetracycline and chlorotetracycline, and other tetracycline antibiotics can be selected.
The invention has the advantages that:
the invention establishes a co-culture system of bacillus clausii and bacillus amyloliquefaciens, and adds the co-culture system into a water body containing tetracycline antibiotics, so that the bacillus amyloliquefaciens can produce surface active substances in situ in fermentation liquor to promote the degradation of the tetracycline drugs, and a series of complicated steps such as separation and purification of a surfactant are omitted; the method not only improves the biological removal efficiency of the tetracycline antibiotics, but also obviously reduces the biological toxicity of the tetracycline antibiotic purification product.
Drawings
Figure 1 shows the removal rates for pure and co-cultured oxytetracycline.
Figure 2 is the biotoxicity of oxytetracycline conversion product.
Figure 3 shows the removal rate of chlorotetracycline for pure and co-cultures.
Figure 4 is the biotoxicity of the chlortetracycline conversion product.
In the above figures:
OTC represents oxytetracycline only and no bacteria;
t + OTC stands for b.clausii T pure culture to remove oxytetracycline;
HT + OTC stands for B.clausii T and HM-618 mixed culture (initial OD of two bacteria)600In a ratio of 1: 1) removing oxytetracycline;
CTC represents chloride tetracycline only, no bacteria;
t + CTC represents b.clausii T pure culture with removal of chlortetracycline;
HT + CTC stands for B.clausii T and HM-618 mixed culture (initial OD of two bacteria)600In a ratio of 1: 1) the chlortetracycline is removed.
Detailed Description
Bacillus clausii (b.clausii T) was purchased from: 2016. year 6, organization: sunofil (sanofi), country: italy, website: http:// www.sanofi.it/l/it/it/index
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618CGMCC NO.7097 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2013, 1 month and 8 days, No. 3 of No. 1 Xilu-Chen of the Chaoyang district, Beijing City, Microbiol research institute of Chinese academy of sciences, postal code 100101, telephone 010 plus 64807355, and survives.
The invention will be further illustrated with reference to specific examples:
example 1
The first seed culture medium, tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, the balance water.
A second seed culture medium, 60g/L of glucose, 10g/L of beef extract, 10g/L of peptone, 10g/L of yeast extract, 5g/L of NaCl and the balance of water.
Example 2
The formula of the fermentation medium is as follows: glucose 55 g/L; 8g/L of yeast extract powder; peptone 8 g/L; NaCl 8 g/L; KCl0.8g/L; k2HPO45 g/L；KH2PO45 g/L；MgSO4·7H2O 0.8g/L；FeSO4·7H2O 1.5×10-3g/L；CuSO4·5H2O 1.5×10-5g/L；MnSO48×10-5g/L；ZnSO4·7H2O 1.5×10-3g/L; the balance of water.
Example 3
The formula of the fermentation medium is as follows: glucose 65 g/L; 3g/L of yeast extract powder; 3g/L of peptone; NaCl3 g/L; KCl0.3g/L; k2HPO41 g/L；KH2PO41 g/L；MgSO4·7H2O 0.3g/L；FeSO4·7H2O 0.5×10-3g/L；CuSO4·5H2O 0.5×10-5g/L；MnSO45×10-5g/L；ZnSO4·7H2O 0.5×10-3g/L; the balance of water.
Example 4
A method for the biological removal of a tetracycline antibiotic comprising the steps of:
(1) seed culture: culturing bacillus clausii T in a first seed culture medium to obtain a first-stage bacillus clausii seed; culturing the first-stage seed of the bacillus clausii in a first seed culture medium to obtain a second-stage seed of the bacillus clausii;
culturing bacillus amyloliquefaciens B.amyloliquefaciens HM-618 abbreviated as HM-618 in a second seed culture medium to obtain HM-618 first-grade seeds; culturing the first-level seeds of HM-618 in a second seed culture medium to obtain second-level seeds of HM-618;
(2) removing: adding 80mL of water containing oxytetracycline with the concentration of 12.5mg/L into 20mL of the fermentation culture medium of the embodiment 2 to prepare degradation liquid; inoculating the secondary seed of the bacillus clausii obtained in the step (1) and the secondary seed of the HM618 into 100mL of degradation liquid to ensure that the total initial OD6000.15, Bacillus clausii IIThe initial inoculation ratio of the grade seeds and the HM-618 grade seeds is 1: 1; fermenting at the rotation speed of 220rpm and the temperature of 36 ℃ for 120 h.
Centrifuging at 12000rpm, collecting supernatant, and detecting oxytetracycline concentration in the supernatant by using a high performance liquid chromatograph.
Comparative example 1:
the second-order Bacillus clausii seed obtained in step (1) was used in place of the second-order Bacillus clausii seed and the second-order HM-618 seed of example 4, and the procedure was otherwise the same as in example 4.
The biological removal rate of the bacillus clausii in the control example 1 to oxytetracycline in the water body at 120h was 72%, and the biological removal rate of the bacillus clausii and HM-618 in the mixed culture at 120h was 77% (significance analysis p <0.01), as shown in fig. 1. The mixed culture of bacillus clausii and bacillus amyloliquefaciens of example 4 significantly reduced the biotoxicity of oxytetracycline conversion products in water, as shown in fig. 2.
Example 5
A method for the biological removal of a tetracycline antibiotic comprising the steps of:
(1) seed culture: culturing bacillus clausii T in a first seed culture medium to obtain a first-stage bacillus clausii seed; culturing the first-stage seed of the bacillus clausii in a first seed culture medium to obtain a second-stage seed of the bacillus clausii;
culturing the HM-618 in a second seed culture medium to obtain HM-618 first-grade seeds; culturing the first-level seeds of HM-618 in a second seed culture medium to obtain second-level seeds of HM-618;
(2) removing: adding 70mL of water containing chlortetracycline with the concentration of 12.5mg/L into 30mL of the fermentation medium of the embodiment 3 to prepare a degradation liquid; inoculating the secondary seed of the bacillus clausii obtained in the step (1) and the secondary seed of HM-618 into 100mL of degradation liquid to ensure that the total initial OD600Is 0.23, the initial inoculation ratio of the second-level seed of the Bacillus clausii to the second-level seed of the HM-618 is 1: 1; fermenting at the rotation speed of 200rpm and the temperature of 38 ℃ for 96 h.
Centrifuging at 12000rpm, collecting supernatant, and detecting the concentration of the chlortetracycline in the supernatant by using a high performance liquid chromatograph.
Comparative example 2:
the second-order Bacillus clausii seed obtained in step (1) was used in place of the second-order Bacillus clausii seed and the second-order HM-618 seed of example 5, and the procedure was otherwise the same as in example 5.
The mixed culture of the Bacillus clausii of comparative example 2 and the Bacillus clausii of example 5 with HM-618 completely removed the chlortetracycline in the water at 96h, as shown in FIG. 3. And the mixed culture of the bacillus clausii and the bacillus amyloliquefaciens in the example 5 obviously reduces the biotoxicity of the chlortetracycline conversion product in the water body, and the figure is 4.
The high performance liquid chromatography used a C18 column (250X 4.6mm,5 μm), a Waterse2695 Alliance HPLC and a 2489UV/Vis detector, and a detection wavelength of Oxytetracycline (OTC) and chlorotetracycline (CTC) of 355 nm. The detection conditions are as follows: the temperature of the chromatographic column is 35 ℃, the injection volume is 20 mu L, and the flow rate is 1 mL/min-1. Mobile phase composition: 80% of the solution contains 0.01 mol.L-1Oxalic acid in water, 20% acetonitrile.
Claims (3)
1. A method for the biological removal of tetracycline antibiotics comprising the steps of:
(1) seed culture: culturing Bacillus clausii Bacillus clausii T to obtain a first-stage Bacillus clausii seed; culturing the first-stage seeds of the bacillus clausii to obtain second-stage seeds of the bacillus clausii;
culturing Bacillus amyloliquefaciens HM-618, CGMCC7097, abbreviated as HM-618 to obtain first-grade seeds of HM-618; culturing the first-level seeds of HM-618 to obtain second-level seeds of HM-618;
(2) removing: adding 70-80mL of water containing tetracycline antibiotics into 20-30mL of fermentation medium according to a proportion to prepare degradation liquid; inoculating the secondary seed of the bacillus clausii obtained in the step (1) and the secondary seed of HM-618 into 100mL of degradation liquid to ensure that the total initial OD6000.15-0.23, the initial inoculation of the second-level seed of Bacillus clausii and the second-level seed of HM-618The seed ratio is 1: 1; fermenting for 96-120h at the rotating speed of 200-220 rpm and the temperature of 36-38 ℃.
2. The method of claim 1, wherein the fermentation medium is formulated as: 55-65g/L of glucose; 3-8g/L of yeast extract powder; 3-8g/L of peptone; 3-8g/L of NaCl; KCl 0.3-0.8 g/L; k2HPO41-5 g/L；KH2PO41-5g/L；MgSO4·7H2O 0.3-0.8g/L；FeSO4·7H2O(0.5-1.5)×10-3g/L；CuSO4·5H2O(0.5-1.5)×10-5g/L；MnSO4(5-8)×10-5g/L；ZnSO4·7H2O(0.5-1.5)×10-3g/L; the balance of water.
3. The method of claim 1, wherein said tetracycline antibiotics are oxytetracycline and chlortetracycline.
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