CN115125178B - 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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- CN115125178B CN115125178B CN202210930946.2A CN202210930946A CN115125178B CN 115125178 B CN115125178 B CN 115125178B CN 202210930946 A CN202210930946 A CN 202210930946A CN 115125178 B CN115125178 B CN 115125178B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a Paenibacillus (Paenibacillus) with a tetracycline antibiotic degradation function, which is named as: paenibacillus sp.AEPI 0-1, class name: paenibacillus, deposit No.: CGMCC No.23627, preservation date: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center. The strain with the tetracycline antibiotic degradation effect is obtained by enrichment, domestication, separation and screening from facility vegetable fields for long-term application of the organic fertilizer, a theoretical basis is provided for industrial application of the tetracycline antibiotic degradation bacteria, strain resource libraries of the tetracycline antibiotic degradation bacteria are enriched, and an effective biodegradation method is provided for treatment of tetracycline antibiotic pollution.
Description
Technical Field
The invention belongs to the technical field of biology, relates to biodegradation of antibiotics and biological treatment of environmental pollutants, and in particular relates to paenibacillus strain with tetracycline antibiotic degradation function, a method and application thereof.
Background
Tetracyclines are a broad-spectrum antibiotic produced by actinomycetes and have been found to be widely used in the human pharmaceutical industry to reduce the incidence of disease. With the development of society, antibiotics are also increasingly used in animal veterinary medicine, aquaculture and feed addition. In recent years, due to the large and unreasonable abuse of antibiotics, a large amount of waste containing tetracycline antibiotics appears in the vicinity of antibiotic manufacturers and farms. It has been reported that antibiotics are not completely absorbed after entering the animal body, and about three to nine amounts are expelled from the body as raw medicines, resulting in a large amount of antibiotics remaining in the animal feces. Biological manure in farms is commonly used as an organic fertilizer applied to agricultural lands, and antibiotics are introduced into the soil, surface water, groundwater and other environments along with the flushing cycle of rainwater. Thus, different concentrations of antibiotics are detected in soil, surface water and sediment environments, which pose a great threat to the environment and human health.
Antibiotics entering the environment are continuously accumulated and finally exceed the self-cleaning capability of the environment, so that the antibiotics have different degrees of harm to plants, microorganisms and even human beings. The antibiotics in the soil are accumulated in the roots of the plants, and the high-concentration antibiotics have a certain inhibition effect on the growth of the roots of the plants; in addition, the plant absorbs antibiotics from soil and accumulates in plant parts, which may affect the normal growth of the plant, and the accumulated parts in plant plants may be transferred through food chains and finally enter animals and human bodies, thereby causing harm to human bodies. When the tetracycline antibiotics accumulate for a certain amount in the human body for a long time, acute and chronic poisoning of the human body can be caused, and irreversible harm can be caused to certain organs of the human body, such as tetracycline teeth and the like. Meanwhile, the accumulation of the tetracycline antibiotics in the human body can influence the phagocytic capacity of leucocytes and inhibit the transformation of lymphocytes, thereby influencing the immune system of the human body. The antibiotics in the soil can inhibit the functional diversity of the microbial community, and meanwhile, the microorganisms in the soil are easy to generate drug resistance under the action of the antibiotics for a long time, so that antibiotic drug resistant bacteria are formed, the effectiveness of the antibiotics is reduced, and the resistance genes of the antibiotics can follow. The resistance genes are mutually transmitted among microorganisms, so that the drug resistance of bacteria is increased, the invalidity of antibiotics is increased, the antibiotics cannot exert the original functions of the antibiotics, diseases are difficult to treat, and serious threat is caused to the health of human beings. Thus, the residue of antibiotics in the environment is a global problem to be solved.
The removal of tetracycline antibiotics is usually photodegradation under natural conditions, and generally includes physical, chemical, biological degradation, and the like, in addition to photodegradation under natural conditions. 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 removing efficiency of the activated carbon and bentonite on the antibiotics can reach 95% and 88% respectively. However, the physical adsorption method can only transfer antibiotics from one medium to another medium, and although no harmful substances are generated, the antibiotics cannot be thoroughly removed, and the adsorption material cannot be reused. The chemical method mainly comprises an electrochemical treatment method and an oxidation method, but the chemical method has the defects of high cost, incapability of large-area popularization and the like. Based on a number of drawbacks of physical and chemical methods, more and more scholars have focused their attention on methods of biodegradation, in particular microbial degradation. Microorganisms isolated from soil in situ have strong adaptability, and microbial degradation is rapid and efficient, secondary pollution and high cost can be avoided, so that the method is favored by many students. Microorganisms which have been isolated by the present scholars and which have the potential to degrade tetracycline antibiotics include: advenella sp.4002, achromobacter xylosoxidans TJ-2, raoul XY-1 and the like, and the degradation efficiency of the Achromobacter xylosoxidans, raoul XY-1 and the like on tetracycline can reach 57.8%, 63.9% and 70.68% respectively. There are also few reports on the related studies of the degradation of antibiotics by paenibacillus. Paenibacillus is an aerobic or facultative anaerobic gram-positive bacillus, and has great research significance in the fields of biological medicine, environmental pollution control and the like.
Paenibacillus polymyxa is mainly involved in nitrogen fixation, phosphorus dissolution, formation of plant hormone, antibacterial substances and the like, has broad-spectrum antagonistic activity, can effectively prevent diseases such as various plant fungi, bacteria, nematodes and the like, and simultaneously promotes plant growth and improves crop yield. Researches indicate that some growth-promoting bacteria of Paenibacillus polymyxa have the effects of preventing diseases and promoting growth. Paenibacillus polymyxa kills or dissolves pathogens by secreting large amounts of active substances to inhibit bacterial and fungal activity or to produce various metabolites, improving plant disease resistance. In terms of soil remediation, paenibacillus can alter the composition and stability of soil aggregates by producing exopolysaccharides. In the biomedical field, studies have shown that paenibacillus produces a variety of proteinaceous antibacterial substances (antibacterial proteins, peptides, extracellular polysaccharides, enzyme actives), which act on the cell walls and cell membranes of pathogenic bacteria and interact with membrane-associated receptor proteins, thereby antagonizing a variety of plant fungal pathogens. These studies all demonstrate the potential of paenibacillus for soil remediation.
The microorganism treatment method is quick 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 of the degradation treatment of antibiotics by using a microbiological method is that the strain has high quality strain resources, although strains with tetracycline degrading potential are obtained at present, the number of the separated degradation strains is small, especially the germplasm resources of the purely cultured high quality microorganisms are lacking, and meanwhile, the obtained strain resources still have certain defects, such as low degradation efficiency, severe strain growth conditions, incapacity of guaranteeing strain activity 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, and aims to screen out high-efficiency degradation strain resources in situ from soil, thereby providing theory and implementation basis for a biological method for treating tetracycline antibiotic pollution in the environment in the future.
By searching, the following patent publications related to the present patent application are found:
1. the strain is Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB C005, which is preserved in China general microbiological culture collection center (CGMCC) for short and has the preservation number of CGMCC No.12812 in the year 2016, 7 and 22. The strain JZB C005 has very good degradation effect on tetracycline antibiotics, especially terramycin, in soil or water. For terramycin with the concentration of 10mg/L in the inorganic salt culture solution, the degradation rate reaches 67.8% after 5 days of treatment.
2. The strain of tetracycline antibiotic degrading bacteria and application thereof (CN 107523516A) is characterized in that the degrading bacteria YWF are identified as methyl bacillus (methyl) and are preserved in China general microbiological culture collection center (CGMCC No. 14040) at the date of 04 and 17 in 2017. The methyl bacteria sp.YWF1 can degrade three kinds of tetracycline antibiotics, namely tetracycline, oxytetracycline and aureomycin simultaneously, can be prepared into microbial inoculum suspension, is applied to degradation and removal of tetracycline antibiotic pollutants in different environmental media such as livestock wastewater, tetracycline pharmaceutical enterprise wastewater, medical wastewater or soil, and has good industrial application prospect and environmental benefit.
3. The tetracycline antibiotic degrading strain is Kluyveromyces Kluyvera intermedia centered in China, and has been preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 20117 in 7/15/2020. The strain can be applied to the preparation of tetracycline antibiotic degrading bactericides, is favorable for enriching strain resource libraries of tetracycline antibiotic degrading bacteria, and provides an effective biodegradation method for the treatment of tetracycline antibiotic pollution. The strain used in the invention has high efficiency for degrading minocycline, and the degradation rate of the strain reaches more than 90% in 72 hours under the condition that the initial content of minocycline is 50mg/L under laboratory conditions. The invention provides an effective biological way for removing minocycline in environmental water.
By way of comparison, the strains of the present patent application differ substantially from the strains disclosed in the above patents.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides paenibacillus strain with tetracycline antibiotic degradation function, a method and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
paenibacillus (Paenibacillus) with tetracycline antibiotic degradation function, and the name of the strain is: paenibacillus sp.AEPI 0-1, class name: paenibacillus, deposit No.: CGMCC No.23627, preservation date: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Further, the degrading bacterial strain is obtained through the following steps:
(1) Enrichment and domestication of tetracycline antibiotic resistant bacteria: screening strains in a suspension of a soil sample of a facility vegetable field (the soil sample is taken from a facility vegetable field of Tianjin city where the organic fertilizer is applied for a long time) with a tetracycline screening culture medium with an initial concentration of 20mg/L, and sequentially transferring with the tetracycline screening culture medium with the concentration of 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 400mg/L, enriching and domesticating to obtain a tolerant strain;
(2) Isolation and purification of paenibacillus: preparing a tolerant strain obtained by acclimatization into a tolerant strain culture solution, and diluting with sterile water to 10 -6 、10 -7 And 10 -8 Then separating and purifying the strain to obtain a single strain, wherein the separation and purification of the strain adopts a traditional flat plate coating method and a flat plate streaking method;
(3) Screening of potential degrading bacteria: and inoculating the single strain obtained by separation and purification into a culture medium containing tetracycline antibiotics for degradation experiment screening to obtain the strain with the capacity of degrading the tetracycline antibiotics in the culture medium, and identifying the strain as Paenibacillus sp.AEPI 0-1 through 16S rDNA.
Use of paenibacillus as described above in the degradation of a tetracycline antibiotic.
Use of a paenibacillus as described above as and/or in the preparation of a tetracycline degrading antibiotic agent.
Further, the tetracycline antibiotic is tetracycline.
Further, the tetracycline antibiotic is tetracycline hydrochloride.
The method for removing antibiotics by using paenibacillus as described above comprises the following steps:
activating paenibacillus, inoculating seed-retaining paenibacillus in a culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/LNaCl, culturing in a constant temperature shaking table at 25-35 ℃ and 150-250r for 24 hours to obtain seed liquid, inoculating the seed liquid into a culture medium containing tetracycline hydrochloride antibiotics with the pH value of 6.0-10.0, and culturing at 25-37 ℃ to realize rapid degradation and removal of the antibiotics.
Further, the paenibacillus can degrade tetracycline antibiotics with the drug concentration of below 400 mg/L;
alternatively, the medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder and 5g/L sodium chloride, sterilizing at 121 ℃ for 15min, and cooling to room temperature.
The method for preparing the tetracycline antibiotic microbial inoculum by using the paenibacillus comprises the following steps:
inoculating paenibacillus into a liquid culture medium, wherein the liquid culture medium is a culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/LNaCl, culturing in a 180r shaking table at 25-30 ℃ for 12-24 hours, and collecting the culture solution to obtain the tetracycline antibiotic microbial inoculum.
Further, 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 and 5g/L sodium chloride, sterilizing 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 effect from a facility vegetable field for applying organic fertilizer for a long time, the strain is identified as paenibacillus by 16S rDNA sequencing, the strain is named AEPI 0-1, the strain has the function of degrading the tetracycline antibiotic, the invention provides a theoretical basis for the industrialized application of the tetracycline antibiotic high-efficiency degradation strain, is favorable for 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 culture medium components through single factor experiments and orthogonal experiment designs at the laboratory level of the efficiency of degrading tetracycline, and the degradation efficiency can reach about 90%. The strain can be applied to the preparation of the tetracycline antibiotic microbial inoculum, is favorable for enriching strain resource libraries of tetracycline antibiotic degrading bacteria, provides an effective biodegradation method for the treatment of tetracycline antibiotic pollution, and simultaneously provides a safe and environment-friendly solution for the problem of tetracycline drug residue.
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 such as mixed waste water polluted by the tetracycline antibiotics and soil to degrade the tetracycline antibiotics pollutants in the environment polluted by the mixed tetracycline antibiotics.
Drawings
FIG. 1 is a diagram showing the morphology of Paenibacillus AEPI 0-1 in solid medium and microscope according to the present invention; wherein, the left graph is a morphological graph of the paenibacillus AEPI 0-1 under a solid culture medium, and the right graph is a morphological graph of the paenibacillus AEPI 0-1 under a microscope;
FIG. 2 is a 16S rDNA evolutionary tree of Paenibacillus AEPI 0-1 in accordance with the present invention;
FIG. 3 is a graph showing the effect of carbon and nitrogen source species on the efficiency of the Paenibacillus AEPI 0-1 in terms of tetracycline hydrochloride degradation in accordance with the present invention;
FIG. 4 is a graph showing the effect of carbon and nitrogen source concentration on the tetracycline hydrochloride degradation efficiency of Paenibacillus AEPI 0-1 in accordance with the present invention; wherein a is an influence diagram of carbon source concentration on TC-HCl degradation efficiency, and b is an influence diagram 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 according to the present invention;
FIG. 6 is a graph showing the effect of the initial pH of the culture medium on the tetracycline hydrochloride degradation efficiency of Paenibacillus AEPI 0-1 according to the present invention;
FIG. 7 is a graph showing the degradation efficiency of Paenibacillus AEPI 0-1 in terms of tetracycline hydrochloride over time under optimal conditions in the present invention.
Detailed Description
The invention will now be further illustrated by reference to 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 conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.
Paenibacillus (Paenibacillus) with tetracycline antibiotic degradation function, and the name of the strain is: paenibacillus sp.AEPI 0-1, class name: paenibacillus, deposit No.: CGMCC No.23627, preservation date: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
Preferably, the paenibacillus is obtained by the following steps:
(1) Enrichment and domestication of tetracycline antibiotic resistant bacteria: screening strains in a suspension of a soil sample of a facility vegetable field (the soil sample is taken from a facility vegetable field of Tianjin city where the organic fertilizer is applied for a long time) with a tetracycline screening culture medium with an initial concentration of 20mg/L, and sequentially transferring with the tetracycline screening culture medium with the concentration of 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 400mg/L, enriching and domesticating to obtain a tolerant strain;
(2) Isolation and purification of paenibacillus: preparing a tolerant strain obtained by acclimatization into a tolerant strain culture solution, and diluting with sterile water to 10 -6 、10 -7 And 10 -8 Then separating and purifying the strain to obtain a single strain, wherein the separation and purification of the strain adopts a traditional flat plate coating method and a flat plate streaking method;
(3) Screening of potential degrading bacteria: and inoculating the single strain obtained by separation and purification into a culture medium containing tetracycline antibiotics for degradation experiment screening to obtain the strain with the capacity of degrading the tetracycline antibiotics in the culture medium, and identifying the strain as Paenibacillus sp.AEPI 0-1 through 16S rDNA.
Use of paenibacillus as described above in the degradation of a tetracycline antibiotic.
Use of a paenibacillus as described above as and/or in the preparation of a tetracycline degrading antibiotic agent.
Preferably, the tetracycline antibiotic is tetracycline.
Preferably, the tetracycline antibiotic is tetracycline hydrochloride.
The method for removing antibiotics by using paenibacillus as described above comprises the following steps:
activating paenibacillus, inoculating seed-retaining paenibacillus in a culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/LNaCl, culturing in a constant temperature shaking table at 25-35 ℃ and 150-250r for 24 hours to obtain seed liquid, inoculating the seed liquid into a culture medium containing tetracycline hydrochloride antibiotics with the pH value of 6.0-10.0, and culturing at 25-37 ℃ to realize rapid degradation and removal of the antibiotics.
Preferably, the paenibacillus can degrade tetracycline antibiotics with the drug concentration of below 400 mg/L;
alternatively, the medium is: 2g/L glucose, 1g/L yeast powder, 1.5g/L peptone, 10g/L sodium chloride, 2g/L sucrose, 25g/L yeast powder and 5g/L sodium chloride, sterilizing at 121 ℃ for 15min, and cooling to room temperature.
The method for preparing the tetracycline antibiotic microbial inoculum by using the paenibacillus comprises the following steps:
inoculating paenibacillus into a liquid culture medium, wherein the liquid culture medium is a culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/LNaCl, culturing in a 180r shaking table at 25-30 ℃ for 12-24 hours, and collecting the culture solution to obtain the tetracycline antibiotic microbial inoculum.
Preferably, 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 and 5g/L sodium chloride, sterilizing at 121 ℃ for 15min, and cooling to room temperature.
Specifically, the preparation and detection of the correlation are as follows:
example 1: isolation and purification of tetracycline antibiotic degrading bacteria
5g of vegetable soil from Tianjin, which is a facility for applying organic fertilizer for a long time, is weighed into 50ml of liquid culture medium, then placed into a shaking table at 30 ℃ and 180r for culture for 12-24 hours, and domesticated to obtain 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 shaking table at 30deg.C and 180r, culturing in dark place for 4-7d, and performing circulation transfer. Wherein the concentration of tetracycline during switching is as follows: each concentration is transferred twice, and finally the tolerant bacteria culture solution is obtained through enrichment and domestication, wherein the concentration is 20mg/L, 50mg/L, 100mg/L, 200mg/L and 400 mg/L.
Diluting the acclimatized bacteria-tolerant culture solution with sterile water for 10 times 6 、10 7 And 10 8 Dilutions were obtained and then coated on plates containing tetracycline hydrochloride. The preparation method of the flat plate containing the tetracycline hydrochloride comprises the following steps: 1g/L yeast powder, 2g/L peptone, 10g/L sodium chloride, 15-20g/L agar powder, sterilizing at 121deg.C for 15min, cooling to about 60deg.C, addingThe tetracycline hydrochloride mother liquor makes the antibiotic concentration in the solid plate reach 200mg/L, and then the plate is poured in an ultra clean bench. The coated plate was placed in an incubator at 30℃and incubated in the dark for 48 hours to obtain a single colony after purification, followed by plate streaking of the single colony for purification. The form of the purified paenibacillus under a solid culture medium and a microscope is shown as figure 1, and the paenibacillus colony is milky white, neat in edge, round in bump, smooth in surface, moist, sticky and not easy to pick on the solid culture medium; under a microscope, the thallus is rod-shaped, sporulates, elliptical and larger.
Example 2: screening and identification of tetracycline antibiotic degrading bacteria
The obtained pure bacteria were picked up from a solid plate and inoculated into a liquid medium (same as in example 1), seed liquid was obtained after culturing for 24 hours in a shaking table at 30℃and 180r, 10% seed liquid was inoculated into a liquid medium containing 100mg/L tetracycline hydrochloride, followed by culturing for 4 days at 30℃and 180r under a dark condition, and then sampling was performed. Centrifuging the obtained bacterial liquid at 4000r, taking supernatant, passing through a 0.22 mu m water-based filter membrane, and measuring the content of tetracycline hydrochloride in the culture medium by using an ultra-high performance liquid chromatograph, so as to calculate the degradation efficiency of each bacterial strain, and finally screening out bacterial strains with high degradation efficiency.
Extracting genome DNA of the strain by using a bacterial genome extraction kit according to the strict operation of the specification, and then carrying out PCR amplification on 16S rDNA genes, wherein the upper and lower primers 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 result of electrophoresis, the PCR product is sent to Jing Yang Biotechnology Co., ltd (Tianjin) for DNA sequencing to obtain a 16S rDNA sequence, then the NCBI database is utilized for comparing the sequences, the comparison result shows that the bacteria belong to paenibacillus, and the phylogenetic tree is shown in figure 2. And finally, preserving the paenibacillus, wherein the preservation information is as follows: the microbial strain is preserved in China general microbiological culture Collection center (CGMCC) in the 10 th and 19 th of 2021, and the preservation number is CGMCC No.23627.
Example 3: effect of Paenibacillus on tetracycline degradation efficiency under different conditions
A loop of paenibacillus is selected from a solid flat plate by an inoculating loop to be inoculated into a liquid culture medium, the components of the liquid culture medium are 1g/L yeast powder, 2g/L peptone and 10g/L sodium chloride, the liquid culture medium is cultured for 24 hours at 30 ℃ and 180r to obtain seed liquid, 10% seed liquid is inoculated into the liquid culture medium containing 100mg/L tetracycline hydrochloride, then the liquid culture medium is subjected to light-shielding culture for 4 days at 30 ℃ and 180r, sampling and centrifugation are carried out, the supernatant of the culture liquid is subjected to a 0.22 mu m water-based filter membrane, the content of the tetracycline hydrochloride in the culture medium is measured by an ultra-high performance liquid chromatograph, and the degradation rate of each strain is calculated. Wherein, the influence of different carbon and nitrogen source types and concentrations, salt concentration, different pH values and substrate concentration on the degradation efficiency of tetracycline hydrochloride is explored.
In the aspect of nitrogen source types, peptone and yeast powder are selected as basic nitrogen sources, the optimal nitrogen source is selected by examining the influence of the proportion of the peptone and the yeast powder on the degradation of tetracycline hydrochloride, the total concentration of the added nitrogen source is kept at 3g/L, meanwhile, 100mg/L of tetracycline hydrochloride, 10g/L of sodium chloride and no carbon source are added. In terms of the type of carbon source, the study was conducted by selecting and adding glucose, glycerol, sucrose and soluble starch, wherein the addition concentrations were 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, the degradation rate of tetracycline hydrochloride gradually increases with the increase of the concentration of yeast powder and the decrease of the concentration of peptone; in terms of the kind of the carbon source, the addition of sucrose makes the degradation efficiency of tetracycline hydrochloride highest compared with other carbon sources. Thus, sucrose and yeast powder were selected as the optimal carbon-nitrogen source species for subsequent investigation.
The optimal carbon source is determined, and the invention researches the degradation efficiency of sucrose under the conditions of six concentrations of 0, 5, 10, 15, 20 and 25g/L on tetracycline hydrochloride, and simultaneously adds 2g/L peptone, 1g/L yeast powder, 10g/L sodium chloride and 100mg/L tetracycline hydrochloride into a culture medium. As shown in FIG. 4a, the degradation rate of tetracycline hydrochloride is highest under the condition of low concentration, and gradually decreases with increasing concentration, which is probably due to the fact that the addition of sucrose forms a competition relationship with the paenibacillus to utilize the tetracycline hydrochloride, the utilization of the paenibacillus to the tetracycline hydrochloride is reduced, and further, the degradation rate of the tetracycline hydrochloride is reduced. Therefore, the addition of low concentration sucrose is more suitable for the degradation of the paenibacillus to the tetracycline hydrochloride. After determining the optimal nitrogen source, the invention researches the degradation efficiency of tetracycline hydrochloride under the conditions of six yeast powder concentrations of 3, 5, 10, 15, 20 and 30g/L, and simultaneously adds 100mg/L tetracycline hydrochloride and 10g/L sodium chloride into a culture medium to examine the optimal concentration of the yeast powder, as shown in figure 4b, the degradation rate of the yeast powder is firstly increased and then decreased along with the increase of the concentration of the yeast powder, and the degradation rate of the yeast powder reaches the highest when the concentration of the yeast powder is about 20 g/L.
In the aspect of examining the concentration of sodium chloride in a 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 into the culture medium, and the result is shown in figure 5, and the degradation rate of the tetracycline hydrochloride is increased and then reduced along with the increase of the concentration of sodium chloride, and the tetracycline hydrochloride has higher degradation efficiency under the concentration of 5-10g/L sodium chloride.
In the initial pH investigation, an initial culture medium added with 1g/L yeast powder, 2g/L peptone and 10g/L sodium chloride is used, 7 initial pH values of 4, 5, 6, 6.5 (without pH adjustment), 7, 8, 9 and the like are set, meanwhile 100mg/L tetracycline hydrochloride is added into the culture medium, the influence of the pH value of the culture medium on the degradation of the tetracycline hydrochloride by the paenibacillus is investigated, and the result is shown in figure 6. According to the graph, as the initial pH of the culture medium is increased, the degradation rate of tetracycline hydrochloride is increased and then reduced, and compared with an acidic environment, the degradation rate of tetracycline hydrochloride is more favorable for degradation of tetracycline hydrochloride in an alkaline environment, and the degradation efficiency of tetracycline hydrochloride reaches the highest when the culture medium is initially neutral.
In conclusion, the paenibacillus can degrade tetracycline hydrochloride in liquid culture mediums under different conditions, and particularly, 5-10g/L sodium chloride, 15-20g/L yeast powder and 0-5g/L sucrose are added into the culture mediums under the condition that the initial pH value is neutral, so that the degradation efficiency of tetracycline hydrochloride is higher.
Example 4: orthogonal test for improving degradation efficiency of paenibacillus to tetracycline
Based on the single factor experimental results in example 3, three nutrients in the culture medium were selected in the present invention: the concentration of carbon source, nitrogen source and NaCl is used as three factors, three-factor three-level orthogonal experiment design is carried out to improve the tetracycline degrading efficiency of the paenibacillus, wherein the carbon source selects sucrose with three concentration levels of 0g/L, 2g/L and 5g/L, the nitrogen source selects yeast with three concentration levels of 15g/L, 20g/L and 25g/L, the NaCl concentration selects three levels of 2g/L, 5g/L and 8g/L, the pH value of the culture medium is 7.0, the specific orthogonal experiment table is shown in Table 1, and 9 different experiment groups are designed to carry out orthogonal experiments. As shown in table 1, it can be seen that the degree of influence on the degradation rate of tetracycline hydrochloride among the three substances added to the culture medium is ordered as follows: : yeast powder > sucrose > sodium chloride. In conclusion, according to the orthogonal test, 2g/L sucrose, 25g/L yeast powder and 5g/L sodium chloride are added into the culture medium, and when the pH is regulated to about 7, the degradation efficiency of tetracycline hydrochloride is highest.
TABLE 1 orthogonal experiment table
Example 5: degradation efficiency of paenibacillus on tetracycline antibiotics under optimal condition
A Paenibacillus environmental-friendly on a solid plate is selected in a liquid culture medium (2 g/L glucose, 1g/L yeast powder, 1.5g/L peptone and 10g/L sodium chloride), seed liquid is obtained after culturing for 24 hours at 30 ℃ and 180r, 10% of seed liquid is inoculated in a liquid culture medium containing 100mg/L tetracycline hydrochloride, 2g/L sucrose, 25g/L yeast powder and 5g/L sodium chloride are added in the culture medium, and the initial pH is adjusted to 7. And then sampling is carried out at 0h, 12h, 24h, 36h, 48h, 72h, 96h, 120h and 144h, the content of tetracycline hydrochloride in the culture medium is respectively measured, and the degradation efficiency is calculated. The result is shown in figure 7, and the result shows that the paenibacillus has good degradation effect on tetracycline hydrochloride, the degradation rate can reach about 85% in the fourth day, and the degradation rate is close to 90% in the 6 th to 7 th days; compared with the tetracycline hydrochloride degradation efficiency of about 11% without the paenibacillus in the natural state, the paenibacillus provided by the invention has the capacity of degrading the tetracycline antibiotics, and simultaneously has the degradation rate of about 90% under the optimal condition, and has higher degradation rate. The invention provides a new degradation strain for degrading residual antibiotics in water and soil, and simultaneously provides a new idea for solving the problem of environmental pollution of antibiotics.
Although 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 disclosure of the embodiments.
Claims (5)
1. Paenibacillus strain with tetracycline antibiotic degradation functionPaenibacillus sp.), characterized in that: the name of the strain is:Paenibacillussp, AEPI 0-1, class name: paenibacillus, deposit No.: CGMCC No.23627, preservation date: 2021, 10, 19, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No.1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
2. Use of paenibacillus according to claim 1 for the degradation of tetracycline hydrochloride below 400 mg/L.
3. The use of paenibacillus according to claim 1 in the preparation of tetracycline hydrochloride bacterial agents that degrade below 400 mg/L.
4. A method for removing tetracycline hydrochloride using paenibacillus as defined in claim 1, wherein: the method comprises the following steps:
activating paenibacillus, inoculating seed-retaining paenibacillus in a culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/LNaCl, culturing 24h in a constant temperature shaking table at 25-35 ℃ and 150-250r to obtain seed liquid, inoculating the seed liquid into a culture medium containing tetracycline hydrochloride antibiotics with the pH value of 6.0-10.0, and culturing at 25-37 ℃ to realize rapid degradation and removal of the antibiotics.
5. A method for preparing a bacterial agent for degrading tetracycline hydrochloride by using the paenibacillus as defined in claim 1, which is characterized in that: the method comprises the following steps:
inoculating Paenibacillus into liquid culture medium containing 0-5g/L sucrose, 10-30g/L yeast powder and 5-10g/L NaCl, culturing in 180-r shaking table at 25-30 deg.C for 12-24h, and collecting culture liquid to obtain bacterial agent for degrading tetracycline hydrochloride.
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| CN109762773A (en) * | 2019-02-28 | 2019-05-17 | 中国农业科学院农业环境与可持续发展研究所 | A kind of tree-shaped series bacillus, immobilized microbial inoculum and its application |
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| CN109762773A (en) * | 2019-02-28 | 2019-05-17 | 中国农业科学院农业环境与可持续发展研究所 | A kind of tree-shaped series bacillus, immobilized microbial inoculum and its application |
| WO2020226482A1 (en) * | 2019-05-09 | 2020-11-12 | Hong Heng Lai | Synergistic compositions for controlling plant pathogens with chitinolytic microorganisms / organic acid combinations and method of use thereof |
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