CN115125178A - Paenibacillus with tetracycline antibiotic degradation function, method and application - Google Patents
Paenibacillus with tetracycline antibiotic degradation function, method and application Download PDFInfo
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- CN115125178A CN115125178A CN202210930946.2A CN202210930946A CN115125178A CN 115125178 A CN115125178 A CN 115125178A CN 202210930946 A CN202210930946 A CN 202210930946A CN 115125178 A CN115125178 A CN 115125178A
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Abstract
The invention discloses a Paenibacillus (Paenibacillus) with a tetracycline antibiotic degradation function, wherein the strain is named as: aepi 0-1, classification name: the paenibacillus, the preservation number is: CGMCC No.23627, preservation date: 10/19/2021, depository: china general microbiological culture Collection center, West Lu No.1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China. The invention enriches, domesticates, separates and screens a strain with tetracycline antibiotic degradation efficacy from a facility vegetable field which is applied with organic fertilizer for a long time, provides a theoretical basis for the industrial application of tetracycline efficient degradation bacteria, is beneficial to enriching a strain resource library of the tetracycline antibiotic degradation bacteria, and provides an effective biodegradation method for the treatment of tetracycline antibiotic pollution.
Description
Technical Field
The invention belongs to the technical field of biological technology, relates to biodegradation of antibiotics and biological treatment of environmental pollutants, and particularly relates to a paenibacillus strain with a tetracycline antibiotic degradation function, a method and application thereof.
Background
Tetracycline antibiotics are a broad spectrum antibiotic produced by actinomycetes and have since been discovered widely used in the human pharmaceutical industry to reduce the incidence of disease. With the development of society, antibiotics are also gradually applied to animal veterinary drugs, aquaculture and feed additives. In recent years, due to the massive and unreasonable abuse of antibiotics, large amounts of tetracycline-containing antibiotics are being produced in antibiotic production facilities and around farms. It is reported that antibiotics are not completely absorbed after entering animals, and about three to nine percent of antibiotics are discharged out of the body in the form of raw drugs, resulting in a large amount of antibiotics remaining in animal feces. Biological manure in farms is generally used as an organic fertilizer applied to agricultural land, and along with the washing circulation of rainwater, antibiotics enter environments such as soil, surface water and underground water. Therefore, antibiotics are detected in different concentrations in soil, surface water and sediment environments, which pose a great threat to the environment and human health.
Antibiotics entering the environment accumulate continuously and finally exceed the self-cleaning capacity of the environment, so that the antibiotics have different degrees of harm to plants, microorganisms and even human beings. Antibiotics in the soil accumulate at the roots of the plants, and the high-concentration antibiotics have a certain inhibiting effect on the growth of the roots of the plants; in addition, the antibiotics absorbed by the plants from the soil are accumulated in the plant parts, which may affect the normal growth of the plants, and the accumulated parts in the plant plants may be transmitted through food chains and finally enter the bodies of animals and human bodies, thereby causing harm to the human bodies. When a certain amount of tetracycline antibiotics are accumulated in a human body for a long time, acute and chronic poisoning of human beings is caused, and certain organs of the human body can be irreversibly damaged, such as tetracycline teeth. Meanwhile, accumulation of tetracycline antibiotics in human bodies can affect the ability of leucocyte phagocytes and inhibit the transformation of lymphocytes, thereby affecting the immune system of human bodies. The antibiotics in the soil can inhibit the functional diversity of microbial communities, and meanwhile, the microbes in the soil are easy to generate drug resistance under the action of the antibiotics for a long time, so that antibiotic-resistant bacteria are formed, the effectiveness of the antibiotics is reduced, and the resistance genes of the antibiotics can be followed. Resistance genes are spread among microorganisms, so that the drug resistance of bacteria is increased, the ineffectiveness of antibiotics is increased, the antibiotics cannot play the original functions, diseases are difficult to treat, and the health of human beings is seriously threatened. Therefore, the residue of antibiotics in the environment is a global problem to be solved.
The removal of tetracycline antibiotics is usually photo-degradation under natural conditions, and besides the photo-degradation under natural conditions, the removal of tetracycline antibiotics generally involves human intervention, such as physical, chemical, and biological degradation. The physical degradation is mainly physical adsorption, and usually, activated carbon and bentonite are used for adsorbing and removing antibiotics, and researches show that the antibiotic removal efficiency of the activated carbon and the bentonite can reach 95% and 88%, respectively. However, the physical adsorption method can only transfer antibiotics from one medium to another medium, and although harmful substances are not generated, the antibiotics cannot be completely removed, and the adsorption material cannot be reused. The chemical method mainly comprises electrochemical treatment and oxidation, but the chemical method has the defects of high cost, incapability of large-area popularization and the like. Based on the numerous disadvantages of physical and chemical methods, more and more scholars are looking to methods of biodegradation, especially microbial degradation. Microorganisms isolated in situ from soil have strong adaptability, microbial degradation is rapid and efficient, secondary pollution and high cost can be avoided, and therefore the microorganisms are favored by many scholars. Microorganisms having the potential to degrade tetracycline antibiotics have been isolated by the present scholars and include: advenella sp.4002, Achromobacter xylosoxidans TJ-2, Raoultella XY-1, etc., the degradation efficiency of tetracycline can reach 57.8%, 63.9% and 70.68% respectively. There are few reports on the relevant studies of paenibacillus degradation of antibiotics. Paenibacillus is aerobic or facultative anaerobic gram-positive bacillus, and has great research significance in various fields such as biomedicine, environmental pollution treatment and the like aiming at the research of the paenibacillus.
The paenibacillus polymyxa mainly participates in nitrogen fixation, phosphorus dissolution, formation of plant hormones, antibacterial substances and the like, has broad-spectrum antagonistic activity, can effectively prevent various plant fungi, bacteria, nematodes and other diseases, and simultaneously promotes plant growth and improves crop yield. Research indicates that some growth-promoting bacteria of the paenibacillus polymyxa have the effects of preventing diseases and promoting growth. The paenibacillus polymyxa can kill or dissolve pathogens by secreting a large amount of active substances to inhibit the activity of bacteria and fungi or producing various metabolites, thereby improving the disease resistance of plants. In the aspect of soil remediation, Paenibacillus can change the composition and stability of soil aggregates by producing exopolysaccharides. In the biomedical field, researches show that paenibacillus can produce various protein antibacterial substances (antibacterial proteins, peptide substances, exopolysaccharides and enzyme active substances) to act on cell walls and cell membranes of pathogenic bacteria and interact with receptor proteins related to the membranes, so that various plant fungal pathogens are antagonized. These studies all indicate the potential of Paenibacillus for soil remediation.
The microbial treatment method is rapid and efficient, has the advantages of no pathogenicity, no toxicity, good environmental compatibility and no residue, and can effectively avoid secondary pollution caused by chemical treatment and high cost of a physical method. However, the precondition for the degradation treatment of antibiotics by using the microbiological method is that the strain resources are high-quality, and although strains with the potential for degrading tetracycline are obtained at present, the number of the degraded strains separated at present is small, especially the pure-cultured high-quality microbial germplasm resources are lacked, and the obtained strain resources still have certain defects, such as low degradation efficiency, harsh strain growth conditions, incapability of ensuring the activity of the strain and the like.
Therefore, there is a need to further find degrading strains with good properties. The invention takes the strain as an intervention point, aims to screen out high-efficiency degradation strain resources from soil in situ, and provides theoretical and implementation basis for future biological methods for treating tetracycline antibiotic pollution in the environment.
Through searching, the following patent publications related to the patent application of the invention are found:
1. a tetracycline antibiotic degradation strain, a microbial inoculum containing the strain and an application (CN107058156A) thereof are disclosed, wherein the strain is Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB42C005 which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) in 2016, 7 and 22, and the preservation number is CGMCC No. 12812. The strain JZB42C005 has a very good degradation effect on tetracycline antibiotics in soil or water, especially terramycin. For the oxytetracycline with the concentration of 10mg/L in the inorganic salt culture solution, the degradation rate reaches 67.8 percent after 5 days of treatment.
2. The degrading bacterium YWF1 is identified as Methylobacterium (Methylobacillus), is preserved in China general microbiological culture Collection center in 2017, 04 and 17 months, and has a preservation number of CGMCC No. 14040. YWF1 can degrade three tetracycline antibiotics of tetracycline, oxytetracycline and aureomycin simultaneously, can prepare the strain into microbial agent suspension, is applied to degradation and removal of the tetracycline antibiotic pollutants in different environment media such as livestock wastewater, tetracycline pharmaceutical enterprise wastewater, medical wastewater or soil and the like, and has good industrial application prospect and environmental benefit.
3. A tetracycline antibiotic degradation strain, a preparation method and an application thereof (CN112143668A), wherein the tetracycline antibiotic degradation strain is Kluyvera intermedia of Kluyvera intermedia, which is preserved in the China general microbiological culture Collection center in 7-15.2020, and the preservation number is CGMCC No. 20117. The strain can be applied to preparation of a tetracycline antibiotic degrading agent, is beneficial to enriching a strain resource library of the tetracycline antibiotic degrading bacteria, and provides an effective biodegradation method for treatment of tetracycline antibiotic pollution. The strain used in the invention has high minocycline degradation efficiency, and the 72h degradation rate of the strain reaches over 90 percent under the condition of laboratory conditions and the initial minocycline content of 50 mg/L. The invention provides an effective biological approach for removing minocycline in the environmental water body.
By contrast, the strains of the present patent application are substantially different from the strains disclosed in the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a paenibacillus strain with a tetracycline antibiotic degradation function, a method and application thereof.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a Paenibacillus (Paenibacillus) with a tetracycline antibiotic degradation function is named as: paenibacillus sp.aepi 0-1, classification name: the paenibacillus, the preservation number is: CGMCC No.23627, preservation date: 10/19/2021, depository: china general microbiological culture Collection center, Hospital No. 3 of West Lu No.1, on the North of the Chao Yang district, Beijing, China.
Further, the degrading bacterial strain is obtained by the following steps:
(1) enrichment and domestication of tetracycline antibiotic-resistant bacteria: screening strains by using a tetracycline screening culture medium with the initial concentration of 20mg/L in a suspension of a greenhouse soil sample (the soil sample in the invention is collected from a certain greenhouse soil for long-term application of an organic fertilizer in Tianjin), which is applied with the organic fertilizer for a long time, sequentially transferring by using tetracycline screening culture media with the concentrations of 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 400mg/L, enriching and domesticating to obtain a tolerant strain;
(2) and (3) separation and purification of paenibacillus: preparing tolerant strain into tolerant strain culture solution, and diluting with sterile water to 10% -6 、10 -7 And 10 -8 Then carrying out separation and purification of the strains to obtain a single strain, wherein the separation and purification of the strains use a traditional flat plate coating method and a traditional flat plate marking method;
(3) screening potential degrading bacteria: inoculating the single strain obtained by separation and purification into a culture medium containing tetracycline antibiotics for degradation experiment screening to obtain a strain with the capability of degrading the tetracycline antibiotics in the culture medium, and identifying the strain as Paenibacillus sp.AEPI 0-1 by 16S rDNA.
The application of the paenibacillus in degrading tetracycline antibiotics is disclosed.
The application of the Paenibacillus in the aspect of serving as and/or preparing a tetracycline antibiotic degrading agent.
Further, the tetracycline antibiotic is tetracycline.
Further, the tetracycline antibiotic is tetracycline hydrochloride.
The method for removing the antibiotics by using the paenibacillus comprises the following steps:
activating the paenibacillus, inoculating the preserved paenibacillus in a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 24 hours in a constant-temperature shaking table at 25-35 ℃ and 250r to obtain a seed solution, inoculating the seed solution into a culture medium containing tetracycline hydrochloride antibiotic with the pH value of 6.0-10.0, and culturing at the temperature of 25-37 ℃ so as to realize the rapid degradation and removal of the antibiotic.
Further, the paenibacillus can degrade tetracycline antibiotics with the drug concentration of 400mg/L or less;
alternatively, the culture medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH of 7, sterilization at 121 ℃ for 15min, and cooling to room temperature.
The method for preparing the tetracycline antibiotic bacterial agent by using the paenibacillus comprises the following steps:
inoculating the paenibacillus into a liquid culture medium, wherein the liquid culture medium is a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 12-24h in a 180r shaking table at the temperature of 25-30 ℃, and collecting a culture solution to obtain the tetracycline antibiotic bactericide.
Further, the liquid medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH of 7, sterilization at 121 ℃ for 15min, and cooling to room temperature.
The invention has the advantages and positive effects that:
1. the invention enriches, domesticates, separates and screens a strain with tetracycline antibiotic degradation efficacy from a facility vegetable field which is applied with organic fertilizer for a long time, the strain is identified as paenibacillus by 16S rDNA sequencing, the strain is named as AEPI 0-1, the strain has the function of degrading tetracycline antibiotic, the invention provides theoretical basis for the industrial application of the tetracycline efficient degradation strain, is beneficial to enriching a strain resource library of the tetracycline antibiotic degradation strain, and provides an effective biodegradation method for the treatment of tetracycline antibiotic pollution.
2. The strain used in the invention optimizes the components of the culture medium through single-factor experiments and orthogonal experimental design at the laboratory level of the efficiency of degrading tetracycline, and the degradation efficiency can reach about 90%. The strain can be applied to preparation of tetracycline antibiotic degrading agents, is beneficial to enriching a strain resource library of the tetracycline antibiotic degrading bacteria, provides an effective biodegradation method for treatment of tetracycline antibiotic pollution, and provides a safe and environment-friendly solution for the residue problem of tetracycline drugs.
3. The paenibacillus can be used as a microbial agent to be applied to the environment polluted by single tetracycline antibiotics, and can also be added to the environment polluted by mixed tetracycline antibiotics, such as sewage, waste water, soil and the like to degrade the tetracycline antibiotic pollutants.
Drawings
FIG. 1 is a morphological diagram of Paenibacillus sp AEPI 0-1 in solid culture medium under microscope; wherein, the left figure is the morphological figure of paenibacillus AEPI 0-1 under the solid culture medium, and the right figure is the morphological figure of paenibacillus AEPI 0-1 under the microscope;
FIG. 2 is a 16S rDNA clade diagram of Paenibacillus sp AEPI 0-1 of the present invention;
FIG. 3 is a graph showing the effect of carbon and nitrogen source species on the tetracycline hydrochloride degradation efficiency of Paenibacillus sp AEPI 0-1 in accordance with the present invention;
FIG. 4 is a graph showing the effect of carbon and nitrogen source concentrations on the tetracycline hydrochloride degradation efficiency of Paenibacillus sp AEPI 0-1 in accordance with the present invention; wherein a is a graph of the influence of carbon source concentration on TC-HCl degradation efficiency, and b is a graph of the influence of nitrogen source concentration on TC-HCl degradation efficiency;
FIG. 5 is a graph showing the effect of NaCl concentration on the tetracycline hydrochloride degradation efficiency of Paenibacillus AEPI 0-1 in accordance with the present invention;
FIG. 6 is a graph showing the effect of initial pH of the medium on the tetracycline hydrochloride degradation efficiency of Paenibacillus sp AEPI 0-1 in accordance with the present invention;
FIG. 7 is a graph showing the degradation efficiency of tetracycline hydrochloride of Paenibacillus under the optimum conditions of the present invention, AEPI 0-1, as a function of time.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products unless otherwise specified, the methods used in the invention are all conventional methods in the field if not specified, and the quality of each substance used in the invention is conventional quality.
A Paenibacillus (Paenibacillus) with a tetracycline antibiotic degradation function is named as: aepi 0-1, classification name: the Paenibacillus has a preservation number of: CGMCC No.23627, preservation date: 10/19/2021, depository: china general microbiological culture Collection center, West Lu No.1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China.
Preferably, the paenibacillus is obtained by the following steps:
(1) enrichment and domestication of tetracycline antibiotic-resistant bacteria: screening strains by using a tetracycline screening culture medium with the initial concentration of 20mg/L in a suspension of a greenhouse soil sample (the soil sample in the invention is collected from a certain greenhouse soil for long-term application of an organic fertilizer in Tianjin), which is applied with the organic fertilizer for a long time, sequentially transferring by using tetracycline screening culture media with the concentrations of 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 400mg/L, enriching and domesticating to obtain a tolerant strain;
(2) and (3) separation and purification of paenibacillus: preparing tolerant strain into tolerant strain culture solution, and diluting with sterile water to 10% -6 、10 -7 And 10 -8 Then carrying out separation and purification of the strains to obtain a single strain, wherein the separation and purification of the strains use a traditional flat plate coating method and a traditional flat plate marking method;
(3) screening potential degrading bacteria: inoculating the single strain obtained by separation and purification into a culture medium containing tetracycline antibiotics for degradation experiment screening to obtain a strain with the ability of degrading the tetracycline antibiotics in the culture medium, and identifying the strain as bacillus like strain, namely Paenibacillus sp.AEPI 0-1 through 16S rDNA.
The application of the paenibacillus in degrading tetracycline antibiotics is disclosed.
The application of the paenibacillus as described above in the aspect of being used as and/or preparing a tetracycline antibiotic degrading bacterium agent.
Preferably, the tetracycline antibiotic is tetracycline.
Preferably, the tetracycline antibiotic is tetracycline hydrochloride.
The method for removing the antibiotics by using the paenibacillus comprises the following steps:
activating the paenibacillus, inoculating the preserved paenibacillus in a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 24 hours in a constant-temperature shaking table at 25-35 ℃ and 250r to obtain a seed solution, inoculating the seed solution into a culture medium containing tetracycline hydrochloride antibiotic with the pH value of 6.0-10.0, and culturing at the temperature of 25-37 ℃ so as to realize the rapid degradation and removal of the antibiotic.
Preferably, the paenibacillus can degrade tetracycline antibiotics with the drug concentration of below 400 mg/L;
alternatively, the culture medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH 7, sterilizing at 121 ℃ for 15min, and cooling to room temperature.
The method for preparing the tetracycline antibiotic bactericide by using the paenibacillus comprises the following steps:
inoculating the paenibacillus into a liquid culture medium, wherein the liquid culture medium is a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 12-24h in a 180r shaking table at the temperature of 25-30 ℃, and collecting a culture solution to obtain the tetracycline antibiotic bactericide.
Preferably, the liquid culture medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH of 7, sterilization at 121 ℃ for 15min, and cooling to room temperature.
Specifically, the preparation and detection are as follows:
example 1: separation and purification of tetracycline antibiotic degrading bacteria
Weighing 5g of vegetable soil of a certain long-term organic fertilizer application facility from Tianjin city into 50ml of liquid culture medium, then putting the vegetable soil into a shaking table with the temperature of 30 ℃ and the temperature of 180r for culturing for 12-24h, and domesticating to obtain a soil suspension. The liquid culture medium comprises the following components: 1g/L yeast powder, 2g/L peptone, 10g/L sodium chloride, sterilizing at 121 ℃ for 15min, and cooling to room temperature.
Inoculating 10% soil suspension into liquid culture medium containing 20mg/L tetracycline hydrochloride, placing into shaker at 30 deg.C and 180r, and culturing in dark for 4-7d for cyclic transfer. Wherein the concentration of tetracycline in the transfer is as follows in sequence: 20mg/L, 50mg/L, 100mg/L, 200mg/L and 400mg/L, and transferring for two times in each concentration, and finally enriching and domesticating to obtain a tolerant bacteria culture solution.
Respectively diluting the domesticated tolerogenic bacteria culture solution with sterile water 10 6 、10 7 And 10 8 Dilutions were obtained and then coated on plates containing tetracycline hydrochloride. The preparation method of the plate containing tetracycline hydrochloride comprises the following steps: 1g/L yeast powder, 2g/L peptone, 10g/L sodium chloride and 15-20g/L agar powder, sterilizing at 121 ℃ for 15min, cooling to about 60 ℃, adding tetracycline hydrochloride mother liquor to enable the antibiotic concentration in the solid plate to reach 200mg/L, and pouring the plate in a super clean bench. And (3) placing the coated plate in an incubator at 30 ℃, and culturing for 48h in the dark to obtain a purified single colony, and then carrying out plate streaking on the single colony for purification. The form of the purified paenibacillus under a solid culture medium and a microscope is shown in figure 1, on the solid culture medium, the paenibacillus colony is milky white, the edge is neat, the bulge is circular, the surface is smooth, moist and sticky, and the paenibacillus colony is not easy to pick; under a microscope, the thallus is rod-shaped, produces spores, and the spores are oval and large.
Example 2: screening and identification of tetracycline antibiotic degrading bacteria
The obtained pure bacteria were selected from the solid plate and inoculated into a liquid medium (same as example 1), cultured in a shaker at 30 ℃ and 180r for 24 hours to obtain a seed solution, 10% of the seed solution was inoculated into a liquid medium containing 100mg/L tetracycline hydrochloride, and then cultured at 30 ℃ and 180r in the dark for 4 days, followed by sampling. Centrifuging the taken bacterial liquid at 4000r, taking the supernatant, filtering the supernatant through a 0.22-micron water system filter membrane, and measuring the content of tetracycline hydrochloride in the culture medium by using an ultra-high performance liquid chromatograph, thereby calculating the degradation efficiency of each strain and finally screening out the strain with high degradation efficiency.
Extracting the genome DNA of the strain by using a bacterial genome extraction kit and strictly operating according to the instruction, then carrying out PCR amplification on the 16S rDNA gene, wherein the upstream primer and the downstream primer of the 16S rDNA are bacterial universal primers, and finally carrying out electrophoresis on the obtained PCR product by using 1% agarose gel. According to the electrophoresis result, the PCR product is sent to Jingyang biotechnology limited (Tianjin) for DNA sequencing to obtain the 16S rDNA sequence thereof, and then the sequences are compared by using NCBI database, the comparison result shows that the bacillus belongs to the Paenibacillus, and the phylogenetic tree thereof is shown in figure 2. Finally, the paenibacillus is preserved, and the preservation information is as follows: is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23627 at 10 months and 19 days of 2021.
Example 3: effect of Paenibacillus on tetracycline degradation efficiency under different conditions
Inoculating monocyclic bacillus from a solid plate by using an inoculating loop, inoculating the monocyclic bacillus into a liquid culture medium, wherein the components of the liquid culture medium are 1g/L of yeast powder, 2g/L of peptone and 10g/L of sodium chloride, culturing the liquid culture medium for 24h at 30 ℃ and 180r to obtain a seed solution, inoculating 10% of the seed solution into the liquid culture medium containing 100mg/L of tetracycline hydrochloride, culturing for 4d in a dark place at 30 ℃ and 180r, sampling and centrifuging, taking a culture solution supernatant, passing through a 0.22 mu m water system filter membrane, measuring the content of the tetracycline hydrochloride in the culture medium by using an ultra-high performance liquid chromatograph, and further calculating the degradation rate of each strain. Wherein the influence of different types and concentrations of carbon and nitrogen sources, salt concentration, different pH values and substrate concentration on the tetracycline hydrochloride degradation efficiency is respectively researched.
In the aspect of nitrogen source variety, peptone and yeast powder are selected as basic nitrogen sources, the optimal nitrogen source is selected by observing the influence of the proportion on the degradation of tetracycline hydrochloride, the total concentration of the added nitrogen source is kept at 3g/L, and meanwhile, 100mg/L tetracycline hydrochloride, 10g/L sodium chloride and no carbon source are added. In terms of the type of carbon source, glucose, glycerol, sucrose and soluble starch were added to the mixture and the concentration of the mixture was 2g/L, and 2g/L peptone, 10g/L sodium chloride, 1g/L yeast powder and 100mg/L tetracycline hydrochloride were added. The results are shown in FIG. 3: in the aspect of nitrogen source variety, the degradation rate of tetracycline hydrochloride is gradually increased along with the increase of the concentration of the yeast powder and the decrease of the concentration of the peptone; in terms of the kind of carbon source, sucrose addition maximizes the degradation efficiency of tetracycline hydrochloride as compared to other carbon sources. Therefore, sucrose and yeast powder were selected as the optimal carbon and nitrogen source species for subsequent studies.
The optimal carbon source is determined, the degradation efficiency of sucrose to tetracycline hydrochloride under six concentrations of 0, 5, 10, 15, 20 and 25g/L is researched, and 2g/L peptone, 1g/L yeast powder, 10g/L sodium chloride and 100mg/L tetracycline hydrochloride are added into a culture medium. As shown in FIG. 4a, the degradation rate of tetracycline hydrochloride is the highest under the condition of low concentration, and the degradation rate of tetracycline hydrochloride gradually decreases with the increase of concentration, which is probably because the addition of sucrose forms a competitive relationship with the utilization of tetracycline hydrochloride by Paenibacillus, so that the utilization of tetracycline hydrochloride by Paenibacillus is reduced, and the degradation rate of tetracycline hydrochloride is further reduced. Therefore, the addition of low-concentration sucrose is more suitable for the degradation of tetracycline hydrochloride by the paenibacillus. After the optimal nitrogen source is determined, the degradation efficiency of tetracycline hydrochloride is researched under six yeast powder concentrations of 3g/L, 5g/L, 10g/L, 15g/L, 20g/L and 30g/L, and meanwhile 100mg/L tetracycline hydrochloride and 10g/L sodium chloride are added into a culture medium to investigate the optimal concentration of the yeast powder, as shown in fig. 4b, the degradation rate is firstly increased and then decreased along with the increase of the concentration of the yeast powder, and the degradation rate reaches the highest when the concentration of the yeast powder is about 20 g/L.
In the aspect of investigating the concentration of sodium chloride in the culture medium, the invention sets 6 sodium chloride concentration gradients of 0, 2.5, 5, 10, 15, 20g/L and the like, and simultaneously adds 1g/L yeast powder, 2g/L peptone and 100mg/L tetracycline hydrochloride in the culture medium, and the result is shown in figure 5, and it can be seen from the figure that the degradation rate of the tetracycline hydrochloride is firstly increased and then decreased along with the increase of the concentration of the sodium chloride, and the tetracycline hydrochloride has higher degradation efficiency under the concentration of 5-10g/L sodium chloride.
In the initial pH examination, 7 initial pH values of 4, 5, 6, 6.5 (pH was not adjusted), 7, 8, 9, and the like were set using an initial medium to which 1g/L yeast powder, 2g/L peptone, and 10g/L sodium chloride were added, and 100mg/L tetracycline hydrochloride was added to the medium to examine the effect of the basic acid/base of the culture on degradation of tetracycline hydrochloride by Bacteroides, and the results are shown in FIG. 6. It is obtained from the figure that with the increase of the initial pH of the culture medium, the degradation rate of the tetracycline hydrochloride is increased firstly and then reduced, compared with the acid environment, the degradation of the tetracycline hydrochloride is more favorable under the alkaline environment, and when the initial pH of the culture medium is neutral, the degradation efficiency of the tetracycline hydrochloride reaches the highest.
In conclusion, the liquid culture medium of the Paenibacillus can degrade the tetracycline hydrochloride under different conditions, and particularly, under the condition that the initial pH is neutral, 5-10g/L of sodium chloride, 15-20g/L of yeast powder and 0-5g/L of sucrose are added into the culture medium, so that the degradation efficiency of the tetracycline hydrochloride is high.
Example 4: orthogonal test for improving tetracycline degradation efficiency of paenibacillus
On the basis of the results of the one-factor experiment in example 3, the present invention selected three nutrients in the medium: the concentration of a carbon source, a nitrogen source and NaCl is used as three factors, and a three-factor three-level orthogonal experiment design is carried out to improve the tetracycline degradation efficiency of the Paenibacillus, wherein the carbon source selects sucrose with the concentration levels of 0g/L, 2g/L and 5g/L, the nitrogen source selects yeast powder with the concentration levels of 15g/L, 20g/L and 25g/L, the concentration of NaCl selects the levels of 2g/L, 5g/L and 8g/L, the pH of the culture medium is 7.0, a specific orthogonal experiment table is shown in table 1, and 9 different experiment groups are designed for carrying out orthogonal experiments. As shown in Table 1, it can be seen that the influence degree on the degradation rate of tetracycline hydrochloride in the three substances added to the medium is ranked as follows: : yeast powder > sucrose > sodium chloride. In conclusion, the orthogonal experiment shows that the degradation efficiency of tetracycline hydrochloride is highest when 2g/L of sucrose, 25g/L of yeast powder and 5g/L of sodium chloride are added into the culture medium and the pH is adjusted to be about 7.
TABLE 1 orthogonal Experimental Table
Example 5: degradation efficiency of the Paenibacillus to the tetracycline antibiotics under the optimal conditions
Selecting an environment-friendly paenibacillus strain stored on a solid plate from a liquid culture medium (2g/L glucose, 1g/L yeast powder, 1.5g/L peptone and 10g/L sodium chloride), culturing for 24 hours at 30 ℃ and 180r to obtain a seed solution, inoculating 10% of the seed solution into the liquid culture medium containing 100mg/L tetracycline hydrochloride, adding 2g/L sucrose, 25g/L yeast powder and 5g/L sodium chloride into the culture medium, and adjusting the initial pH to 7. Then sampling is carried out for 0h, 12h, 24h, 36h, 48h, 72h, 96h, 120h and 144h, the content of tetracycline hydrochloride in the culture medium is respectively determined, and the degradation efficiency is calculated. The result is shown in figure 7, which shows that the paenibacillus has good degradation effect on tetracycline hydrochloride, the degradation rate can reach about 85% on the fourth day, and the degradation rate is close to 90% on the 6 th to 7 th days; compared with the natural state, the degradation efficiency of tetracycline hydrochloride without being inoculated with the paenibacillus is only about 11%, so that the paenibacillus provided by the invention has the capability of degrading tetracycline antibiotics, and meanwhile, under the optimal condition, the degradation rate can reach about 90%, and the degradation rate is higher. The invention provides a new degrading strain for degrading residual antibiotics in water and soil, and provides a new idea for solving the problem of environmental pollution caused by antibiotics.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (10)
1. A Paenibacillus (Paenibacillus) with tetracycline antibiotic degradation function is characterized in that: the name of the strain is as follows: paenibacillus sp.aepi 0-1, classification name: the paenibacillus, the preservation number is: CGMCC No.23627, preservation date: 2021, 10/19, depository: china general microbiological culture Collection center, West Lu No.1 Hospital No. 3, Beijing, Chaoyang, the area of the republic of China.
2. The Paenibacillus of claim 1, wherein: the degrading bacterial strain is obtained by the following steps:
(1) enrichment and domestication of tetracycline antibiotic-resistant bacteria: screening strains by using a tetracycline screening culture medium with initial concentration of 20mg/L in a soil sample suspension of a facility vegetable field for applying an organic fertilizer for a long time, sequentially transferring by using tetracycline screening culture media with concentrations of 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 400mg/L, and enriching and domesticating to obtain a tolerant strain;
(2) separation and purification of Paenibacillus: preparing tolerant strain into tolerant strain culture solution, and diluting with sterile water to 10% -6 、10 -7 And 10 -8 Then carrying out separation and purification of the strains to obtain a single strain, wherein the separation and purification of the strains use a traditional flat plate coating method and a traditional flat plate marking method;
(3) screening potential degrading bacteria: inoculating the single strain obtained by separation and purification into a culture medium containing tetracycline antibiotics for degradation experiment screening to obtain a strain with the ability of degrading the tetracycline antibiotics in the culture medium, and identifying the strain as bacillus like strain, namely Paenibacillus sp.AEPI 0-1 through 16S rDNA.
3. Use of a paenibacillus strain according to claim 1 or 2 for the degradation of tetracycline antibiotics.
4. Use of a paenibacillus strain according to claim 1 or 2 as and/or in the preparation of a tetracycline antibiotic-degrading agent.
5. Use according to claim 4, characterized in that: the tetracycline antibiotic is tetracycline.
6. Use according to claim 4, characterized in that: the tetracycline antibiotic is tetracycline hydrochloride.
7. The method for removing antibiotics using Paenibacillus as claimed in claim 1 or 2, wherein: the method comprises the following steps:
activating the paenibacillus, inoculating the preserved paenibacillus in a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 24 hours in a constant-temperature shaking table at 25-35 ℃ and 250r to obtain a seed solution, inoculating the seed solution into a culture medium containing tetracycline hydrochloride antibiotic with the pH value of 6.0-10.0, and culturing at the temperature of 25-37 ℃ so as to realize the rapid degradation and removal of the antibiotic.
8. The method of claim 7, wherein: the paenibacillus can degrade tetracycline antibiotics with the drug concentration of below 400 mg/L;
alternatively, the culture medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH of 7, sterilization at 121 ℃ for 15min, and cooling to room temperature.
9. A method of preparing a tetracycline antibiotic bacterial agent using a paenibacillus as in claim 1 or 2, characterized in that: the method comprises the following steps:
inoculating the paenibacillus into a liquid culture medium, wherein the liquid culture medium is a culture medium containing 0-5g/L of cane sugar, 10-30g/L of yeast powder and 5-10g/L of NaCl, culturing for 12-24h in a 180r shaking table at the temperature of 25-30 ℃, and collecting a culture solution to obtain the tetracycline antibiotic bactericide.
10. The method of claim 9, wherein: the liquid culture medium is as follows: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder, 5g/L sodium chloride, initial pH of 7, sterilization at 121 ℃ for 15min, and cooling to room temperature.
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| CN117586931A (en) * | 2024-01-19 | 2024-02-23 | 中国农业科学院蔬菜花卉研究所 | Achromobacter xylosoxidans IVF-WK240 and application thereof |
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