CN112760262B

CN112760262B - Erythromycin degradation composite microbial inoculum and preparation method and application thereof

Info

Publication number: CN112760262B
Application number: CN202110126039.8A
Authority: CN
Inventors: 郑华宝; 许双燕; 张涛; 张�成; 宋成芳
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-05-17
Anticipated expiration: 2041-01-29
Also published as: CN112760262A

Abstract

The invention relates to an erythromycin degradation composite bacterial agent, which comprises the following microorganisms: acidovorax texatilis Ery-6A (Delftia acidovans sp. Ery-6A) is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2020960, Flavobacterium indolyticum Ery-6B (Chryseobacterium indicum sp. Ery-6B) and China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2020959. The invention also provides a preparation method and application of the erythromycin degradation composite bacterial agent. The erythromycin degradation composite bacterial agent has high erythromycin degradation efficiency, and can degrade chloramphenicol simultaneously.

Description

Erythromycin degradation composite microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to an erythromycin degradation complex microbial inoculum, and a preparation method and application thereof.

Background

Antibiotics are low to medium molecular weight compounds with a variety of chemical and biological properties. Antibiotics are drugs used to treat bacterial infections. Antibiotics are widely applied to animals in the process of large-scale livestock and poultry breeding and comprise tetracyclines, sulfonamides, fluoroquinolones, macrolides, amino and the like. About 21 million tons of antibiotics are produced annually in china, accounting for about 60% of the world's total amount, and 36 different antibiotic compounds are contained, of which the majority of antibiotics are used in livestock and poultry farming, and residual antibiotics in livestock and poultry manure enter farmlands along with fertilizers. Almost 90% of the antibiotics used by livestock and poultry are reported to be used at sub-therapeutic concentrations, with 70% being for disease prevention and 30% for growth promotion. Antibiotics applied to livestock and poultry mainly enter the environment through direct and indirect discharge of excrement, sewage irrigation, sludge composting and the like, and have adverse effects on a natural ecological system. Heretofore, the negative impact of antibiotics on ecosystem and human health has attracted considerable attention in the widespread use of humans and animals. Environmental contamination with antibiotics can occur from several sources, such as drug manufacturing processes, discarding unused drugs and containers, etc., or applying manure and waste residues. Antibiotic residues in the environment not only alter the structure and abundance of the environmental microbial community, but also affect the capacity of the microbial community. The presence and persistence of antibiotics poses serious environmental pollution problems. In recent years, a series of environmental problems caused by the potential influence of residual antibiotics in the environment and the like have been the focus of global attention due to the emergence of drug-resistant bacteria in the environment. Long-term exposure to low-toxicity and sub-toxic antibiotics can alter microbial ecology and promote the development and spread of antibiotic-resistant bacteria. In addition, the toxic effects on aquatic species may even directly threaten human health through the food chain. Currently, due to the diversity of antibiotic properties, microbial degradation by nature may be necessary for antibiotic depletion.

Erythromycin belongs to macrolide antibiotics, has strong antibacterial effect and wide application. Research reports from bangladesh, india, indonesia and talan reported that erythromycin was detected in mariculture water at concentrations up to 180ng/L, and that the United States Environmental Protection Agency (USEPA) first listed erythromycin as one of the candidate contaminants for drinking water that needs to be preferentially detected and controlled in 2009. The existing biological treatment process such as a biological aerated filter can only reduce trace erythromycin in wastewater, and the erythromycin residue in the environment is effectively degraded by depending on specific microorganisms. So far, few studies on the microbial degradation of erythromycin are made at home and abroad.

Disclosure of Invention

The invention provides a composite microbial inoculum capable of efficiently degrading erythromycin, a preparation method and application thereof for solving the technical problems.

The technical scheme for solving the technical problems is as follows:

in a first aspect, the invention provides an erythromycin degradation complex microbial inoculum, which comprises the following microorganisms:

the acid-eating bacterium Delftia acifluorfen Ery-6A (Delftia acidovirans sp. Ery-6A) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2020960;

chryseobacterium indogenes Ery-6B (Chryseobacterium indicans sp. Ery-6B) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2020959.

Furthermore, the effective viable bacteria ratio of the acidovorax delbrueckii Ery-6A to the chrysobacillus indolenini Ery-6B is 1: 0.1-10.

In a second aspect, the invention provides an application of the erythromycin degradation composite bacterial agent in degrading erythromycin.

In a third aspect, the invention provides a preparation method of the erythromycin degradation complex microbial inoculum, which is obtained by respectively culturing and mixing the Chryseobacterium indolerum Ery-6B and the acidovorax delbrueckii Ery-6A in culture media.

Further, the culture medium is an LB culture medium.

In a fourth aspect, the invention also provides a method for degrading erythromycin, which is to add the erythromycin degradation composite bacterial agent in

claim

1 or 2 into a water sample containing erythromycin to degrade the erythromycin in the water sample.

Further, the degradation conditions were: the temperature is 25-40 ℃, the pH is 5.5-7.5, and the shaking culture is carried out at 100-200 r/min.

Compared with the prior art, the invention has the following beneficial effects:

the erythromycin degradation composite bacterial agent can grow by taking erythromycin as a unique carbon source, and the concentration of the erythromycin substrate is 100 mg.L^-1On the basis of (1), the optimal degradation conditions are as follows: the temperature is 35 ℃, and the rotating speed is 120 r.min^-1The degradation rate of 48h at pH 7.0 was 79.91%. Further, the concentration was 100 mg.L for the substrate^-1The degradation rate of the chloramphenicol in 48 hours reaches 31.64 percent. At present, most researches on erythromycin degradation bacteria are not high in tolerance concentration, but the composite microbial inoculum can grow in an environment with high concentration of erythromycin as a unique carbon source, and the concentration is 1000 mg.L^-1The degradation rate of the erythromycin reaches up to 31.95 percent. The complex microbial inoculum can grow in the environment of 45 ℃ and can grow to 100 mg.L^-1The temperature of the degradation rate of the erythromycin is 30-35 ℃, the pH value is 7-7.0, the 120r/min shaking culture reaches 45.11%, and the composite bacteria can tolerate various metal ions, so that the composite bacteria can provide a treatment scheme and a solution way for factories with high-concentration erythromycin pollution, such as erythromycin pharmaceutical factories and the like.

Drawings

FIG. 1 is a photograph showing the morphology of the colonies of Chryseobacterium indolens Ery-6B and Acidovorax delbrueckii Ery-6A of the present invention;

FIG. 2 shows the single bacterium forms of Chryseobacterium indolerum Ery-6B and Acidovorax delbrueckii Ery-6A under transmission electron microscope;

FIG. 3 is a phylogenetic tree of acidovorax delbrueckii Ery-6A and Chryseobacterium indolens Ery-6B of the present invention;

FIG. 4 is a growth curve of the erythromycin degradation complex bacterial agent of the present invention;

FIG. 5 is a biological degradation kinetic curve of the erythromycin degradation complex inoculant of the present invention to Erythromycin (ERY) and Chloramphenicol (CAP);

FIG. 6 shows the effect of the initial concentration of erythromycin on the ability of the erythromycin degradation complex bacterial agent to degrade erythromycin;

FIG. 7 is a graph showing the effect of initial concentration of chloramphenicol on the ability of erythromycin degradation complex bacteria to degrade chloramphenicol;

FIG. 8 is the effect of temperature on the erythromycin degradation ability of the erythromycin degradation complex inoculant;

FIG. 9 is the effect of temperature on the ability of an erythromycin degradation complex bacterial agent to degrade chloramphenicol;

FIG. 10 is a graph showing the effect of rotational speed on the erythromycin degradation ability of the erythromycin degradation complex microbial inoculum;

FIG. 11 shows the effect of rotational speed on the ability of erythromycin degradation complex bacteria to degrade chloramphenicol

FIG. 12 is a graph showing the effect of pH on the ability of an erythromycin-degrading complex inoculant to degrade erythromycin;

FIG. 13 is a graph showing the effect of pH on the ability of an erythromycin degradation complex inoculant to degrade chloramphenicol;

FIG. 14 is a graph showing the effect of an external carbon nitrogen source on the erythromycin degradation ability of the erythromycin degradation complex bacteria agent;

FIG. 15 is the effect of an external carbon nitrogen source on the ability of an erythromycin degradation complex bacterial agent to degrade chloramphenicol;

FIG. 16 is a graph showing the effect of added metal ions on the erythromycin degradation ability of the erythromycin degradation complex microbial inoculum;

FIG. 17 shows the effect of added metal ions on the ability of the erythromycin degradation complex bacteria agent to degrade chloramphenicol.

Detailed Description

The principles and features of this invention are described in connection with the drawings and the detailed description of the invention, which are set forth below as examples to illustrate the invention and not to limit the scope of the invention.

Experimental Material

The soil separated by the degrading bacteria is taken from organic fertilizer production company of Deqing city in Zhejiang province, and the soil of the chicken manure field polluted by erythromycin is stacked for a long time.

Culture medium

Microelement solution (g/L): FeCl₃1.6g，CoCl₂·6H₂O 0.1g，MnCl₂·4H₂O 0.425g，ZnCl₂0.05g，NiCl₂·6H₂O 0.01g，CuSO₄·5H₂O 0.015g，H₃BO₃0.05g，Na₂MoO₄·2H₂O 0.01g，CaCl₂0.05g, distilled water to 1000 mL.

Inorganic salt liquid culture medium: NH (NH)₄Cl 1g/L，KH₂PO₄0.5 g/L，K₂HPO₄1.5 g/L，MgSO₄0.2 g/L, NaCl 1g/L, 2mL/L of trace element solution, and adding ultrapure water to 1000 mL. Sterilizing at 121 deg.C for 30min and pH 7.0.

LB liquid medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, and adding ultrapure water to 1000 mL. Sterilizing at 121 deg.C for 30min and pH 7.0.

Solid medium: to the liquid medium was added 1.5% agar.

Antibiotic standard substance

Erythromycin (purity 98%, original leafy organism), chloramphenicol (purity 97-103%, biological)

Erythromycin liquid chromatography detection conditions: ulti MDAD detector for high performance liquid chromatograph model ate 3000, XTERRA RP C18 column (250X 4.6mm, 5 μ M), and 0.02M ammonium dihydrogen phosphate (phase A)/acetonitrile (phase B) at pH 3.0 as mobile phase. Initial 90% a; the temperature is reduced to 80% A in 2 min; 77% A at 3.5 min; 76% A at 5 min; 76.5% A at 8 min; maintaining 70% A for 9 min; reducing to 50% A at 20 min; gradually increasing to 90% A at 24min and maintaining for 1 min. The flow rate is 1 mL/min^-1The column temperature was 35 ℃, the sample size was 20 μ L, and the detection wavelength was 210 nm. .

And (3) detecting conditions of chloramphenicol by liquid chromatography: agilent model 1260 high performance liquid chromatograph DAD detector, the chromatographic column is XTERRA RP C18 column (250X 4.6mm, 5 μm), and the mobile phase is 0.1% formic acid water solution (phase A)/acetonitrile (phase B). Initial 90% a; the solution is reduced to 73.5 percent A in 2 min; 72.5% A at 5 min; 72% A at 8 min; 65% A at 9 min; keeping 40% A for 11 min; reducing to 20% A at 16 min; 10% A at 18min and hold for 1 min. The flow rate is 1 mL/min^-1The column temperature was 35 ℃, the amount of sample was 10 μ L, and the detection wavelength was 278 nm.

Calculating the content of the erythromycin according to a linear regression equation, and calculating the degradation rate by adopting the following formula:

degradation rate (control sample residue-full sample residue)/control sample residue × 100%

Example 1 screening and identification of erythromycin-degrading bacteria

1. Screening and identification of erythromycin-degrading bacteria

Adding 10g of soil into an Erlenmeyer flask filled with 100mL of inorganic salt culture medium, and simultaneously adding 50 mg.L of erythromycin^-10.2 percent of yeast extract, 0.1 percent of glucose, 30 ℃, 120 r.min^-1Performing shaking culture for 3 days; supplementing 100 mg. L^-1Erythromycin at 30 deg.C and 120r min^-1Performing shaking culture for 3 days; supplementing 100mL of inorganic salt culture medium and 100 mg.L^-1Culturing erythromycin at 30 deg.C under shaking at 150r min-1 for 3 days; supplementing 100 mg.L^-1Erythromycin at 30 deg.C and 120r min^-1The culture was performed for 3 days with shaking. Inoculating the bacteria solution after 4 rounds of acclimatization into a test tube containing 5mL of inorganic salt culture medium at an inoculum size of 10%, and continuing acclimatization by a gradient pressure type acclimatization method, wherein the erythromycin concentration is 100, 200, 300, 400, 500, 600, 800, 900 and 1000 mg.L in sequence^-1Erythromycin as the only carbon source at 30 deg.c and 120r min^-1Culturing for 48h in dark condition. Obtaining domesticated bacteria liquid, scratching on inorganic salt solid culture medium with erythromycin concentration, placing in 30 deg.C constant temperature incubator, culturing in dark for 48h, observing growth condition, picking single colony, and streaking to obtain strain liquid containing 1000 mg.L^-1And (3) carrying out four times of separation and purification on an inorganic salt plate of erythromycin to obtain pure culture of the strain.

Through multiple domestication separation and enrichment screening, two degradation bacterial strains Ery-6A and Ery-6B which can degrade high-concentration erythromycin and chloramphenicol under the combined action are obtained.

2. Identification of erythromycin-degrading bacteria

a. Morphological characteristics of the strains Ery-6A and Ery-6B

As shown in the figures 1 and 2, Ery-6A bacterial colony is circular, the diameter is about 1-2mm, the Ery-6A bacterial colony is milky, opaque, smooth in surface, moist and neat in edge, the bacterial colony is rod-shaped, flagellum-free and spore-free when the bacterial colony is observed under a transmission electron microscope, black white spots exist in cells, the Ery-6B bacterial colony is circular, the diameter is about 2-3mm, the Ery-6B bacterial colony is golden yellow, opaque, smooth and convex in surface and neat in edge, and the bacterial colony is rod-shaped under the transmission electron microscope, and flagellum and spores are not found.

b. Molecular characterization of the strains Ery-6A and Ery-6B

The amplification products obtained by PCR amplification of Ery-6A and Ery-6B degrading bacteria are close to 1.5 kb.

The 16S rDNA sequences of the strains Ery-6A and Ery-6B are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2,

SEQ ID NO:1

tagcgccctccttgcggttaggctacctacttctggcgagacccgctcccatggtgtgacgggcggtgtgtacaagacccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccgacttcacgcagtcgagttgcagactgcgatccggactacgactggttttatgggattagctccccctcgcgggttggcaaccctctgtaccagccattgtatgacgtgtgtagccccacctataagggccatgaggacttgacgtcatccccaccttcctccggtttgtcaccggcagtctcattagagtgctcaactgaatgtagcaactaatgacaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacagccatgcagcacctgtgtgcaggttctctttcgagcacgaatccatctctggaaacttcctgccatgtcaaaggtgggtaaggtttttcgcgttgcatcgaattaaaccacatcatccaccgcttgtgcgggtccccgtcaattcctttgagtttcaaccttgcggccgtactccccaggcggtcaacttcacgcgttagcttcgttactgagaaaactaattcccaacaaccagttgacatcgtttagggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgtgcatgagcgtcagtacaggtccaggggattgccttcgccatcggtgttcctccgcatatctacgcatttcactgctacacgcggaattccatccccctctaccgtactctagccatgcagtcacaaatgcagttcccaggttgagcccggggatttcacatctgtcttacataaccgcctgcgcacgctttacgcccagtaattccgattaacgctcgcaccctacgtattaccgcggctgctggcacgtagttagccggtgcttattcttacggtaccgtcatgggccccctgtattagaaggagctttttcgttccgtacaaaagcagtttacaacccgaaggccttcatcctgcacgcggcattgctggatcaggctttcgcccattgtccaaaattccccactgctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggctggtcgtcctctcagaccagctacagatcgtcggcttggtaagcttttatcccaccaactacctaatctgccatcggccgctccaatcgcgcgaggcccgaaggtcccccgctttcatcctcagatcgtatgcggtattagctactctttcgagtagttatcccccacgactgggcacgttccgatgtattactcacccgttcgccactcgtcagcgtccgaagacctgttaccgttcgactgca

SEQ ID NO:2

gcagctcctgttacggtcaccgacttcaggtaccccagacttccatggcttgacgggcggtgtgtacaaggcccgggaacgtattcaccgcgccatggctgatgcgcgattactagcgattccagcttcatagagtcgagttgcagactccaatccgaactgagaccggctttcgagatttgcatcacatcgctgtgtagctgccctctgtaccggccattgtattacgtgtgtggcccaaggcgtaagggccgtgatgatttgacgtcatccccaccttcctctctacttgcgtaggcagtctcactagagtccccaacttaatgatggcaactagtgacaggggttgcgctcgttgcaggacttaacctaacacctcacggcacgagctgacgacaaccatgcagcaccttgaaaaatgtccgaagaaaagtctatttctaaacctgtcatttcccatttaagccttggtaaggttcctcgcgtatcatcgaattaaaccacataatccaccgcttgtgcgggcccccgtcaattcctttgagtttcaaacttgcgttcgtactccccaggtggctaacttatcactttcgcttagtctctgaagcttacgccccaaaaacgagttagcatcgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgtccatcagcgtcagttgttgcttagtaacctgccttcgcaattggtgttctaagtaatatctatgcatttcaccgctacactacttattccagctacttcaacaacactcaagacctgcagtatcaatggcagtttcacagttaagctgtgagatttcaccactgacttacagatccgcctacggaccctttaaacccaataaatccggataacgcttgcaccctccgtattaccgcggctgctggcacggagttagccggtgcttattcgtatagtaccttcagctactctcacgagagtaggtttatccctatacaaaagaagtttacaacccatagggccgtcgtccttcacgcgggatggctggatcaggctctcacccattgtccaatattcctcactgctgcctcccgtaggagtctggtccgtgtctcagtaccagtgtgggggatcaccctctcaggccccctaaagatcgcagacttggtgagccgttacctcaccaactatctaatcttgcgcgtgcccatctctatccaccggagttttcaatatcgaatgatgccattcaatatattatggggtattaatcttcctttcgaaaggctatcccccagataaaggcaggttgcacacgtgttccgcacccgtgcgccgctctcaagtctccgaagagactctaccgctcggctgcatgtgtagc

the obtained 16S rDNA fragment sequences (1399bp and 1392bp) were imported into GenBank alignment window, and sequence homology comparison was performed with BLAST software, which revealed that the sequence of Ery-6A and Delftia acicloranes were on the same branch and the similarity was 99.5%, and the sequence of Ery-6B and Chryseobacterium indole genes strain were on the same branch and the similarity was 98.27%. Phylogenetic evolutionary trees were constructed using the MEGA 7.0 program, as shown in figure 3.

By integrating the morphological characteristics and molecular identification, the strain Ery-6A is judged to belong to the genus Delftia, is named as Delftia acidovorans sp.Ery-6A, is preserved in China Center for Type Culture Collection (CCTCC) at 23 months in 2020, 12 and 23 months, has the address of Wuchang mountain Lopa in Wuhan, Hubei and has the preservation number of M2020960;

the strain Ery-6B belongs to the genus Chryseobacterium indolerum, is named as Chryseobacterium indolenum Ery-6B (Chryseobacterium indolenes sp. Ery-6B), and has been preserved in China Center for Type Culture Collection (CCTCC) at 12-23 months in 2020, with the address of Wuchang Loojia mountain in Wuhan, Hubei province and the preservation number of M2020959.

The acid-producing bacterium Ery-6A and the Chryseobacterium indolerum Ery-6B degrade the erythromycin together, so that the mixed bacterium agent for degrading the erythromycin is obtained by culturing and mixing the two strains of the bacterium seeds. The inoculation ratio of the two strains is 1: 0.1-10.

Example 2 degradation characteristics of erythromycin degradation Mixed bacteria

a. Growth curve and degradation kinetics curve

Respectively mixing and inoculating the acid-feeding bacterium Delftia Ery-6A and the Chryseobacterium indolerum Ery-6B according to the inoculation amount of 1 percent to 100 mg.L^-1Erythromycin and 100 mg. L^-1In inorganic salt culture medium of chloramphenicol, at 30 deg.C and 120 r.min^-1Culturing under the condition, sampling at different time, measuring OD value of bacterial liquid under 600nm wavelength by spectrophotometry, and determining growth amount of two bacteria.

The growth curve is shown in figure 4, wherein 0-14h is the growth delay period of the strain, the strain grows to the logarithmic phase within 14-60h, the number of the strain increases logarithmically, after 60h, the thallus grows to the stationary phase, and the number of the strain tends to be saturated.

Under the condition, the degradation kinetics curve of the complex microbial inoculum to the erythromycin and the chloramphenicol is shown in figure 5. The degradation rate of the erythromycin is increased along with the culture time, the erythromycin is rapidly increased within 0-48h, the maximum degradation rate reaches 74.10%, and the erythromycin tends to be stable after 48 h; the degradation rate of chloramphenicol also increases with the culture time, but the increase amplitude is small, the increase is rapid between 0 and 60 hours, and the degradation rate is kept unchanged after 60 hours. The erythromycin and chloramphenicol degradation process under the conditions of inoculation and non-inoculation is fitted by adopting a quasi-first order kinetic equation, except for a chloramphenicol biodegradation curve, R2 is over 90 percent, and the equation fitting degree is good, so that the degradation follows the quasi-first order reaction kinetics, and the rate constant k (h) is high^-1) Half life t_1/2(h) See table 1. The results show that the natural degradation of the two antibiotics is weak, the erythromycin degradation rate is remarkably improved by inoculating the composite microbial inoculum, the half-life period is reduced by more than half, and the influence is small compared with that of chloramphenicol.

TABLE 1 kinetic equations and kinetic parameters for ERY and CAP biodegradation

b. Influence factors of erythromycin and chloramphenicol degradation effect

Adopting single factor test to degrade the compound bacteria agent in different substrate concentrations (the initial concentration of erythromycin is set to 10-110 mg.L respectively)^-1And 10-1000 mg.L^-1Two intervals, the initial concentration of the chloramphenicol is set to be 10-500 mg.L^-1Interval of (1), temperature, rotation speed, pH, and carbon-nitrogen source (50 mg. L. is added according to the mass-to-volume ratio)^-1Glucose, sucrose, yeast extract and peptone) and metal ions (10 mg. L. in terms of mass/volume ratio)^-1Fe (b) of³⁺、Ga²⁺、Mg²⁺、Zn⁺、Cu²⁺And Mn²⁺) Culturing under the same conditions, and measuring the residual quantity of erythromycin or chloramphenicol in the culture medium. 1 no inoculation control and 3 replicates were set for each treatment.

Effect of b-1 substrate concentration on degradation Effect

Inoculating degrading bacteria into culture medium with different substrate concentrations according to inoculation amount of 5%, and setting erythromycin concentration as two gradients of 10, 50, 100, 200, 400, 800, 1000 mg.L^-1And 10, 30, 50, 70, 90, 110 mg.L^-1The chloramphenicol concentration was set at 10, 50, 100, 200, 300, 400, 500 mg.L^-1. At 30 ℃ and pH 7.0, 120 r.min^-1Was cultured under shaking for 48 hours.

The degradation rate of erythromycin at different concentrations is shown in FIG. 6, when the substrate concentration is 90-200 mg.L^-1The degradation effect is most remarkable. OD of bacteria in this concentration range₆₀₀Both are greater than 1, while too low or too high a substrate concentration inhibits bacterial growth, if in units of OD₆₀₀The removal amount of the erythromycin is compared with that of the bacteria, the difference of the removal amounts of the erythromycin with different substrate concentrations is small, and the fact that the different substrate concentrations influence the bacteria growth amount of the mixed bacteria agent to a certain extent is shown, so that different erythromycin degradation rates are obtained. When the concentration of erythromycin reaches 1000 mg.L^-1And the degradation rate is 31.95%, which shows that the erythromycin degradation complex microbial inoculum can survive under the condition of high concentration erythromycin and can generate considerable degradation effect.

The degradation rate of chloramphenicol under different chloramphenicol concentrations is shown in FIG. 7, the degradation effect of the complex microbial inoculum on chloramphenicol is not significant, and the substrate concentration is 50-100 mg.L^-1The degradation rate is highest, and the bacterial concentration is gradually reduced along with the increase of the substrate concentration, and the degradation rate is correspondingly reduced.

b-2, influence of temperature on degradation Effect

The culture temperature was set to 20 ℃ under otherwise controlled conditions,25. 30, 35, 40 and 45 ℃. FIGS. 8 and 9 show the degradation effects of erythromycin and chloramphenicol at 48h, respectively, and it can be seen that OD of the degrading bacteria₆₀₀And the change trend of the degradation rate is similar to that of the degradation rate. For erythromycin, OD at 30-35 deg.C₆₀₀The growth temperature is more than 1, the optimum growth temperature is 35 ℃, and the degradation rate reaches 79.59%. In units of OD₆₀₀The comparison of the bacteria amount to the erythromycin removal amount shows that the erythromycin degradation rate is greatly influenced by the bacteria growth amount when the temperature reaches more than 30 ℃, and the OD of the bacteria is 20-25 DEG C₆₀₀The lower erythromycin degradation rate can reach about 65%, which shows that the growth condition of the bacteria has little influence on the degradation rate. For chloramphenicol, OD₆₀₀The degradation rate is closer to the overall trend of the degradation rate, the degradation rate reaches 29.64% at 30 ℃, and is obviously higher than the degradation rates at other temperatures, which shows that the temperature has larger influence on the degradation of the chloramphenicol by the degrading bacteria.

b-3 influence of rotational speed on degradation effect

Inoculating the composite microbial inoculum according to the inoculation amount of 5 percent to erythromycin with the initial concentration of 100 mg.L^-1In the culture medium of (1), and set at 100, 120, 140, 160, 180, 200 r.min^-1Six rotating speeds, at 35 ℃, pH 7.0 conditions under shaking culture for 48 hours. As shown in FIG. 10, we found that the change of the erythromycin degradation rate is identical to the growth curve of the complex microbial inoculum, i.e., the removal rate of erythromycin at each rotation speed is compared with the unit OD of the bacteria₆₀₀The results are similar, and the growth condition of the bacteria influences the change of the degradation rate of the erythromycin. At a rotation speed of 120r min^-1In the process, the OD concentration of the bacteria is the maximum, the erythromycin degradation effect is the most obvious, and the degradation rate reaches 79.17%.

Simultaneously, the compound microbial inoculum is inoculated to the solution containing 100 mg.L^-1The rotating speed of the medium for chloramphenicol is set at 100, 120, 140, 160, 180 r.min^-1And carrying out shake culture for 48h under the conditions of 30 ℃ and pH 7.0, and sampling to determine the degradation condition of the chloramphenicol. As shown in FIG. 11, the bacteria growth concentration was higher at a higher rotation speed, but the degradation rate was 120 r.min^-1The time is far higher than that under other rotation speed conditions, the rotation speed is expressed by the removal amount of the chloramphenicol to the unit OD of the bacteria, and the degradation efficiency is shownIs the best.

b-4, influence of pH on the degradation Effect

The influence of eight gradients of controlling variables and adjusting pH to 5, 5.5, 6, 6.5, 7, 7.5, 8 and 8.5 on the erythromycin degradation of the complex microbial inoculum is tested. The results shown in FIG. 12 are obtained after 48h, and it is known that the compound microbial inoculum can effectively degrade erythromycin at pH 5.5-7.5, but the compound microbial inoculum grows optimally under neutral conditions, and the degradation rate reaches 79.91% when the pH is 7.0. When the pH value is acidic, the compound microbial inoculum OD₆₀₀When the content of the erythromycin derivative is less than 0.4, the growth of bacteria is inhibited, but the erythromycin in the substrate still maintains a certain degradation rate, and the degradation of the erythromycin is directly influenced by the pH, which is presumed to be because the erythromycin is alkaline and is easily decomposed in acid. The strain is sensitive to alkaline pH, when the pH is 8.5, the strain hardly grows, and the degradation rate is only 7.32%.

For chloramphenicol, the test was performed by setting seven gradients of

pH

3, 4, 5, 6, 7, 8, and 9 under the control of other conditions as optimal conditions. As a result, as shown in FIG. 13, when the pH was less than 5, the strain hardly grew, but chloramphenicol still showed a certain degradation rate, indicating that chloramphenicol may have a certain amount of structural change under acidic conditions.

b-5, influence of external carbon and nitrogen source on degradation effect

Under the condition of controlling variables, respectively adding 50 mg.L into inorganic salt culture medium^-1The glucose, the sucrose, the yeast extract and the peptone are used as an additional carbon-nitrogen source, and the content of the antibiotics is determined after 48 hours. As shown in FIGS. 14 and 15, the degradation rates of the degrading bacteria growing in different carbon-nitrogen source culture media to erythromycin were different, the degradation effect of the sucrose-containing degrading bacteria was significantly higher than that of the control, and the degradation rates were sequentially sucrose>Yeast extract>Glucose>Peptone>And (6) comparison. At a carbon source of 50 mg.L^-1Under the condition of sucrose, 100 mg.L in the culture medium^-1The degradation rate of the erythromycin reaches 88.68 percent within 48 hours, and simultaneously, the OD of the bacteria₆₀₀1.4, which is obviously higher than the growth amount of bacteria without carbon and nitrogen sources, and the removal amount of the erythromycin is compared with the unit OD₆₀₀It can be shown that the added carbon and nitrogen source is beneficial to the growth of the complex microbial inoculum to a certain extent, thereby achieving the aim of improving the pairDegradation of erythromycin.

In contrast, different carbon-nitrogen sources are antagonistic to the degradation of chloramphenicol by the degrading bacteria, and it is presumed that the degradation of chloramphenicol is significantly reduced because the added nutrient source is more easily utilized by the degrading bacteria for self-activity.

b-6, influence of additional metal ions on degradation effect

Respectively adding 10 mg.L into the culture medium under the condition of no additional carbon and nitrogen source^-1Fe (b) of³⁺、Ga²⁺、Mg²⁺、Zn⁺、Cu²⁺And Mn²⁺Six kinds of additional metal ions are added, and the pH value is 7.0 at 35 ℃ and 120 r.min^-1The culture was carried out under shaking for 48 hours. The results show that Cu is removed²⁺Besides, the influence of other added metal ions on the degradation of erythromycin by the degrading bacteria is not obviously different, which indicates that the strain has stronger tolerance (as shown in FIG. 16). From unit OD₆₀₀The removal amount of erythromycin is known, and Zn is removed⁺And Cu²⁺Besides directly influencing the degradation of the erythromycin, other metal ions all reduce the removal amount of the erythromycin by inhibiting the growth of bacteria. Cu²⁺The growth of degrading bacteria is obviously inhibited, but the influence on the biodegradation of the erythromycin is not reached to a significant level. Compared with erythromycin, as shown in fig. 17, chloramphenicol is more affected by the total effect of the added metal ions, but the effect degree of each metal ion is not significantly different.

The erythromycin degradation composite bacterial agent can grow by taking erythromycin as a unique carbon source, and the concentration of the erythromycin substrate is 100 mg.L^-1On the basis of (1), the optimal degradation conditions are as follows: the temperature is 35 ℃, and the rotating speed is 120 r.min^-1The degradation rate of 48h at pH 7.0 was 79.91%. Further, the concentration was 100 mg.L for the substrate^-1The degradation rate of the chloramphenicol in 48 hours reaches 31.64 percent.

The composite microbial inoculum can grow in an environment at 45 ℃ and has the degradation rate of 45.11 percent, the highest temperature reaches more than 50 ℃ in the cow dung composting process, the removal rate of fluoroquinolone antibiotics (FQs) in sludge composting materials in a medium temperature period (35 ℃) is 38.43 percent, the removal rate of fluoroquinolone antibiotics in sludge composting materials in a high temperature period (50 ℃) is 29.97 percent, and the temperature is 29 percentIs generally considered as a main factor influencing the degradation of antibiotics, and shows that the higher the temperature which can be tolerated by antibiotic-degrading bacteria is, the more favorable the strain is for putting into use, and the OD of the composite microbial inoculum at 45 ℃ is₆₀₀Still reach about 0.7, can keep considerable activity in order to degrade erythromycin, have stronger environmental suitability, can contribute more feasibility for the compound antibiotic pollution problems such as microorganism restoration erythromycin, chloramphenicol, etc.. And the tolerance of the complex microbial inoculum to various metal ions can well relieve the negative effect of complex pollution on enzyme activity.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Zhejiang agriculture and forestry university

<120> erythromycin degradation complex microbial inoculum and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1399

<212> DNA

<213> acid bacterium Delftia acidovorans

<400> 1

tagcgccctc cttgcggtta ggctacctac ttctggcgag acccgctccc atggtgtgac 60

gggcggtgtg tacaagaccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 120

gcgattccga cttcacgcag tcgagttgca gactgcgatc cggactacga ctggttttat 180

gggattagct ccccctcgcg ggttggcaac cctctgtacc agccattgta tgacgtgtgt 240

agccccacct ataagggcca tgaggacttg acgtcatccc caccttcctc cggtttgtca 300

ccggcagtct cattagagtg ctcaactgaa tgtagcaact aatgacaagg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acagccatgc agcacctgtg 420

tgcaggttct ctttcgagca cgaatccatc tctggaaact tcctgccatg tcaaaggtgg 480

gtaaggtttt tcgcgttgca tcgaattaaa ccacatcatc caccgcttgt gcgggtcccc 540

gtcaattcct ttgagtttca accttgcggc cgtactcccc aggcggtcaa cttcacgcgt 600

tagcttcgtt actgagaaaa ctaattccca acaaccagtt gacatcgttt agggcgtgga 660

ctaccagggt atctaatcct gtttgctccc cacgctttcg tgcatgagcg tcagtacagg 720

tccaggggat tgccttcgcc atcggtgttc ctccgcatat ctacgcattt cactgctaca 780

cgcggaattc catccccctc taccgtactc tagccatgca gtcacaaatg cagttcccag 840

gttgagcccg gggatttcac atctgtctta cataaccgcc tgcgcacgct ttacgcccag 900

taattccgat taacgctcgc accctacgta ttaccgcggc tgctggcacg tagttagccg 960

gtgcttattc ttacggtacc gtcatgggcc ccctgtatta gaaggagctt tttcgttccg 1020

tacaaaagca gtttacaacc cgaaggcctt catcctgcac gcggcattgc tggatcaggc 1080

tttcgcccat tgtccaaaat tccccactgc tgcctcccgt aggagtctgg gccgtgtctc 1140

agtcccagtg tggctggtcg tcctctcaga ccagctacag atcgtcggct tggtaagctt 1200

ttatcccacc aactacctaa tctgccatcg gccgctccaa tcgcgcgagg cccgaaggtc 1260

ccccgctttc atcctcagat cgtatgcggt attagctact ctttcgagta gttatccccc 1320

acgactgggc acgttccgat gtattactca cccgttcgcc actcgtcagc gtccgaagac 1380

ctgttaccgt tcgactgca 1399

<210> 2

<211> 1392

<212> DNA

<213> Chryseobacterium indogenes (Chryseobacterium indolium)

<400> 2

gcagctcctg ttacggtcac cgacttcagg taccccagac ttccatggct tgacgggcgg 60

tgtgtacaag gcccgggaac gtattcaccg cgccatggct gatgcgcgat tactagcgat 120

tccagcttca tagagtcgag ttgcagactc caatccgaac tgagaccggc tttcgagatt 180

tgcatcacat cgctgtgtag ctgccctctg taccggccat tgtattacgt gtgtggccca 240

aggcgtaagg gccgtgatga tttgacgtca tccccacctt cctctctact tgcgtaggca 300

gtctcactag agtccccaac ttaatgatgg caactagtga caggggttgc gctcgttgca 360

ggacttaacc taacacctca cggcacgagc tgacgacaac catgcagcac cttgaaaaat 420

gtccgaagaa aagtctattt ctaaacctgt catttcccat ttaagccttg gtaaggttcc 480

tcgcgtatca tcgaattaaa ccacataatc caccgcttgt gcgggccccc gtcaattcct 540

ttgagtttca aacttgcgtt cgtactcccc aggtggctaa cttatcactt tcgcttagtc 600

tctgaagctt acgccccaaa aacgagttag catcgtttac ggcgtggact accagggtat 660

ctaatcctgt tcgctcccca cgctttcgtc catcagcgtc agttgttgct tagtaacctg 720

ccttcgcaat tggtgttcta agtaatatct atgcatttca ccgctacact acttattcca 780

gctacttcaa caacactcaa gacctgcagt atcaatggca gtttcacagt taagctgtga 840

gatttcacca ctgacttaca gatccgccta cggacccttt aaacccaata aatccggata 900

acgcttgcac cctccgtatt accgcggctg ctggcacgga gttagccggt gcttattcgt 960

atagtacctt cagctactct cacgagagta ggtttatccc tatacaaaag aagtttacaa 1020

cccatagggc cgtcgtcctt cacgcgggat ggctggatca ggctctcacc cattgtccaa 1080

tattcctcac tgctgcctcc cgtaggagtc tggtccgtgt ctcagtacca gtgtggggga 1140

tcaccctctc aggcccccta aagatcgcag acttggtgag ccgttacctc accaactatc 1200

taatcttgcg cgtgcccatc tctatccacc ggagttttca atatcgaatg atgccattca 1260

atatattatg gggtattaat cttcctttcg aaaggctatc ccccagataa aggcaggttg 1320

cacacgtgtt ccgcacccgt gcgccgctct caagtctccg aagagactct accgctcggc 1380

tgcatgtgta gc 1392

Claims

1. An erythromycin degradation composite bacterial agent is characterized by comprising the following microorganisms:

acid bacterium delftia Ery-6A (Delftiaacidovoranssp.Ery-6A) preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020960;

chryseobacterium indolerum Ery-6B (Chryseobacteriumindologenessp.Ery-6B) preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020959.

2. The erythromycin degradation complex bacterium agent according to claim 1, wherein an effective viable bacteria ratio of the acidovorax defueli Ery-6A to the chrysobacillus indolenini Ery-6B is 1: 0.1-10.

3. The use of the erythromycin-degrading complex bacterium agent of claim 1 or 2 in degrading erythromycin.

4. The method for preparing an erythromycin degradation complex bacterial agent as claimed in claim 1 or 2, wherein the complex bacterial agent is obtained by culturing and mixing the acidovorax defueli Ery-6A and the chrysobacillus indolenini Ery-6B in a culture medium, respectively.

5. The production method according to claim 4, wherein the medium is an LB medium.

6. A method for degrading erythromycin, which is characterized in that the erythromycin degradation composite bacterial agent of claim 1 or 2 is added into a water sample containing erythromycin to degrade the erythromycin in the water sample.

7. The method of claim 6, wherein the degradation conditions are: the temperature is 25-40 ℃, the pH is 5.5-7.5, and the shaking culture is carried out at 100-200 r/min.