CN112779189A

CN112779189A - Bacillus proteus soil and application thereof

Info

Publication number: CN112779189A
Application number: CN202110111233.9A
Authority: CN
Inventors: 刘婕; 钟国华; 朱祺; 卢琪琪; 李锦鸿
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-05-11
Anticipated expiration: 2041-01-27
Also published as: CN112779189B

Abstract

The invention discloses a strain of agrobacterium tumefaciens and application thereof. The Proteus tumefaciens Proteus terrae ZQ02 strain is preserved in Guangdong province microorganism strain preservation center in 2019 at 11 and 01 months, and the preservation number is GDMCC NO: 18782 and the deposit address is: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5. The strain has the degradation rate of 90.54% for tetracycline and 96.84% for chlorothalonil, has the advantages of high efficiency, rapidness, no residue and no secondary pollution in the aspect of degrading antibiotics and pesticides such as tetracycline and chlorothalonil, has stable degradation performance, and has wide application prospect in the aspect of ecologically restoring pesticide-antibiotic composite pollution.

Description

Bacillus proteus soil and application thereof

Technical Field

The invention belongs to the technical field of environmental pollution remediation, and particularly relates to a strain of agrobacterium tumefaciens and application thereof.

Background

Scientific use of agricultural chemicals (Agrochemicals) such as pesticides, fertilizers and antibiotics is an important basis for guaranteeing sustainable development of modern agricultural production, and the agricultural chemicals have remarkable achievements in the aspects of preventing and treating crop diseases and insect pests, promoting crop growth, preventing and treating livestock diseases and the like. China is a large country for using pesticides and antibiotics, and the phenomenon of long-term excess and frequent use of agricultural chemicals in China is common, so that the agricultural ecological environment presents brand new characteristics of pesticide and antibiotic composite pollution, the agricultural ecological environment becomes one of main sources of agricultural non-point source pollution, the ecological stability of soil is damaged, the agricultural non-point source pollution is accumulated on edible parts of crops through the suction and conduction effect in plants, and the risk of human health is easily caused. In the face of increasingly stringent agricultural product safety detection standards and wide public concern about ecological environment, a new efficient and safe technology and a new method are urgently needed to be developed, so that the problem of compound pollution of pesticides, antibiotics and the like in agricultural ecological environment is synchronously solved, the pressure of soil environment is relieved, the development of bacterial drug resistance is inhibited, and the quality of agricultural products and the safety of ecological environment are guaranteed.

Under the development situation of advocating green agriculture, how to effectively treat agricultural composite pollution is a key problem to be solved urgently. At present, the approaches for treating agricultural non-point source pollution mainly comprise physical removal, chemical decomposition and biodegradation. Substances such as an adsorbent and a catalyst are added into a polluted environment, so that the adsorption rate of pollutants is enhanced, the decomposition efficiency of the pollutants is improved through catalytic actions such as oxidation, reduction and photolysis, and the effect of quickly reducing the pollution is achieved. However, the repair technology using physical removal and chemical decomposition as the core has the disadvantages of high development difficulty, high cost, poor flexibility and the like, and particularly has high difficulty in treating compound pollutants in non-moving media such as soil, agricultural products and the like, so that the repair technology is difficult to popularize and apply. Compared with the prior art, the natural microbial resources are extremely rich, and the microbes can utilize exogenous pollutants as carbon and nitrogen sources required by the metabolism of the microbes, so that the pollutants are mineralized into simple inorganic matters without environmental stress. Therefore, the biodegradation technology based on the microbial metabolism function is a research hotspot for pollution control at home and abroad, has the advantages of simple and convenient operation, economy, high efficiency, no secondary pollution, strong sustainability and the like, has good degradation effect on various low-concentration high-risk compound pollutions in agricultural ecological environment, has very wide application prospect, and is worthy of deep research and development.

The nature of biodegradation is an enzymatic reaction, and usually the active microorganism has a specific character for the reaction substrate. Because the pesticide and antibiotic composite pollution contains various pollutants, the physicochemical property, the biocompatibility and the like of the pollutants are different, and the substrate diversity can increase the development difficulty of the biodegradation technology. The Chinese patent with publication number CN106434430A provides a compound microbial agent capable of degrading antibiotics and pesticide residues, but at present, a single strain for pesticide and antibiotic compound pollution is only reported, and the screening of active strains and the maintenance of degradation activity are the core difficulties of bioremediation of farmland compound pollution and are also important bases for practice and popularization of bioremediation technology.

Disclosure of Invention

The invention aims to provide a Proteus tumefaciens Proteus terrae ZQ02 strain and application thereof. The invention separates out a strain of the agrobacterium tumefaciens ZQ02 from a soil sample collected from a farmland, the strain has high-efficiency degradation effect on single tetracycline and single chlorothalonil, and has very obvious degradation effect on pesticide-antibiotic composite pollution, and particularly, the mixed degradation effect of the tetracycline-chlorothalonil composite pollution is more than 75 percent.

The invention mainly aims to provide a Proteus agrobacterium Proteus terrae ZQ02 strain.

The second purpose of the invention is to provide a microbial inoculum.

It is a third object of the present invention to provide a biological cleanser.

The fourth purpose of the invention is to provide a biodegradation agent.

The fifth purpose of the invention is to provide the application of the Proteus agrobacterium Proteus terrae ZQ02 strain in degrading tetracycline and/or pesticides.

The sixth purpose of the invention is to provide the application of the strain of the agrobacterium tumefaciens ZQ02 in the aspect of restoring the environment polluted by tetracycline and/or pesticides.

The seventh purpose of the invention is to provide the application of the microbial inoculum in degrading tetracycline and/or pesticides.

An eighth object of the present invention is to provide the use of the biological cleanser for degrading tetracycline and/or pesticides.

A ninth object of the present invention is to provide the use of the biodegradation agent for degrading tetracycline and/or pesticides.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a Proteus tumefaciens strain Proteus terrae ZQ02, wherein the Proteus tumefaciens strain ZQ02 is deposited in Guangdong province microorganism culture collection center in 2019, 11 and 01, and the deposit number is GDMCC No: 18782 and the deposit address is: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

ZQ02 was gram stained and determined to be a gram positive bacterium. On LB agar medium, its colony appears white, round, moist, smooth and glossy, and its edge grows a lawn with special foul smell. The size of the 16S rRNA fragment obtained by PCR amplification with the extracted total DNA of the microorganism as a template and a universal primer for bacteria is 1394 bp. Performing Blast comparison analysis on the sequence of the NCBI database, downloading related sequences with higher homology, performing comparison analysis by using Clustal X1.8.1, and constructing a phylogenetic tree by using Neighbor-Joining of MEGA7.0 as an algorithm. The 16S rRNA sequence of the degrading bacteria has high similarity with the bacteria of multiple Proteus genera (Proteus sp) in NCBI, the homology reaches more than 99 percent, and the degrading bacteria ZQ02 belongs to the Proteus genera (Proteus sp). By combining the results of the morphological observation and the physiological and biochemical experiments, the strain ZQ02 is the strain of the agrobacterium tumefaciens (Proteus terrae) and is identified as the Proteus terrae, and the strain is named as ZQ 02.

The strain is separated from a soil sample of a farmland in Boruo city, Guangdong province, can grow and propagate by using tetracycline or chlorothalonil as a unique carbon source and energy source, and can rapidly degrade the tetracycline and chlorothalonil, when the concentration of the tetracycline is 50mg/L, the inoculation amount is 5.36%, the temperature is 29.89 ℃, the pH value is 9, and the degradation rate of the tetracycline can reach 90.54%; the strain can also grow and propagate by using chlorothalonil as a unique carbon source and energy, the degradation rate can reach 96.84% in 7 days when the concentration of the chlorothalonil is 50mg/L, and the strain has the advantages of high efficiency, rapidness, no residue and no secondary pollution in the aspect of degrading tetracycline and the chlorothalonil, and has stable degradation performance. The invention enriches the resource library of pesticide degrading bacteria, provides a new development approach for breaking the bottleneck of the existing pesticide residue pollution treatment, provides a theoretical basis and a scientific basis for developing a new technology for the pollution treatment of tetracycline and chlorothalonil, and has wide application prospects in microbial agents for degrading chlorothalonil and tetracycline or preparing degrading microbial agents for ecologically restoring pesticide-antibiotic composite pollution.

The invention also claims a microbial inoculum containing the strain ZQ02 of the agrobacterium tumefaciens and/or a bacterial solution thereof.

The invention also claims a biological cleaning agent containing the agrobacterium tumefaciens ZQ02 strain and/or a bacterial solution thereof.

The invention also claims a biodegradation agent containing the agrobacterium tumefaciens ZQ02 strain and/or a bacterial liquid thereof.

Preferably, the preparation method of the bacterial liquid comprises the following steps: taking 0.2mL of frozen degrading bacteria ZQ02 bacterial liquid, transferring and inoculating the bacterial liquid to a conical shake flask containing 50mL of fresh LB culture medium, and carrying out shake culture at the culture temperature of 30 ℃ and 180r/min for 24h until the logarithmic phase of growth; taking 25mL of bacterial liquid to a sterilized centrifugal tube, centrifuging at 5000r/min for 5min, collecting thalli, and washing twice with 25mL of sterilized normal saline; finally adding sterilized normal saline, shaking evenly to ensure that the inoculation liquid OD₆₀₀The value is 1, and the bacterial liquid is obtained.

In addition, the application of the strain of the agrobacterium tumefaciens ZQ02 in degrading tetracycline and/or pesticides, the application of the strain of the agrobacterium tumefaciens ZQ02 in repairing tetracycline and/or pesticide polluted environments, the application of the microbial inoculum in degrading tetracycline and/or pesticides, the application of the biological cleaning agent in degrading tetracycline and/or pesticides, and the application of the biological degrading agent in degrading tetracycline and/or pesticides are all within the protection scope of the invention.

Preferably, the pesticide is chlorothalonil, carbofuran, diuron and thiamethoxam.

Most preferably, the pesticide is chlorothalonil.

The invention has the following beneficial effects:

the Proteus terrae ZQ02 strain provided by the invention can utilize tetracycline or chlorothalonil as a unique carbon source and energy source for growth and propagation, and can rapidly degrade the tetracycline and the chlorothalonil, wherein the tetracycline degradation rate can reach 90.54%; the degradation rate of chlorothalonil can reach 96.84%, and the degradation agent has the advantages of high efficiency, rapidness, no residue and no secondary pollution in the aspect of degrading tetracycline and chlorothalonil, and has stable degradation performance.

The invention enriches the resource library of pesticide degrading bacteria, provides a new development approach for breaking the bottleneck of the existing pesticide residue pollution treatment, provides a theoretical basis and a scientific basis for developing new technologies for the tetracycline and chlorothalonil pollution treatment, and has wide application prospects in microbial agents for degrading chlorothalonil and tetracycline or preparing degrading microbial agents for ecologically restoring pesticide-antibiotic composite pollution.

Drawings

FIG. 1 is a diagram of the morphology of Agrobacterium tumefaciens (Proteus terrae sp) on LB solid plates and under a microscope;

FIG. 2 shows a phylogenetic tree constructed based on 16S rRNA by strain ZQ02, and phylogenetic analysis was performed based on the 16S rRNA sequences of strain ZQ02 and other strains with high homology. The phylogenetic tree is constructed by using Neighbor-joining (NJ) as an algorithm. The numbers in parentheses indicate the sequence accession numbers in GenBank.

FIG. 3 shows the tetracycline biodegradation by strain ZQ02 under medium conditions.

FIG. 4 shows the effect of different conditions on the removal of tetracycline by the degrading bacterium ZQ 02. The single factors are respectively: (a) incubation temperature (b) inoculum size (c) pH (d) starting concentration.

FIG. 5 is a graph of a 3D response surface and a contour plot, and a 3D response surface (a) and a contour plot (b) of tetracycline degradation rate change under the effect of the inoculation amount and the culture temperature; and (c) a 3D response curved surface (c) and a contour map (D) of the change of the degradation rate of the tetracycline under the action of the environmental pH value and the culture temperature.

FIG. 6 shows the degradation of tetracycline in the contaminated soil simulated by the strain, the initial residual concentration of tetracycline is 50mg/kg, the culture time is 20d, and the treatment groups are respectively: natural soil; natural soil + degrading bacteria ZQ 02; sterilizing the soil; sterilized soil + degrading bacteria ZQ 02.

FIG. 7 shows the biodegradation of chlorothalonil by strain ZQ02 under medium conditions.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

1. Culture medium

The basal salt medium comprises: (NH)₄)₂SO₄ 2g，MgSO₄·7H₂O 0.2g，CaCl₂·2H₂O 0.01g，FeSO₄·7H₂O 0.001g，Na₂HPO₄·12H₂O 1.5g，KH₂PO₄ 1.5g，H₂O 1L。

Luria-Bertani medium: 10g of trypsin, 5g of yeast extract, 10g of NaCl, and H₂O 1L。

Solid media were prepared by adding 2% agar to the above media.

The final pH of all media was adjusted to 7 and then autoclaved at 121 ℃ for 20 min.

2. Drug and reagent formulations

Tetracycline was purchased from alatin (shanghai, china); 9 tested pesticides: high-efficiency cypermethrin, chlorpyrifos, clothianidin, thiamethoxam, carbofuran, pyraclostrobin, chlorothalonil, diuron and atrazine, the purity of which is more than 95 percent, and the high-efficiency cypermethrin, the chlorpyrifos, the clothianidin, the thiamethoxam, the carbofuran, the atrazine are purchased from Shanghai spectral laboratory science and technology company Limited; chromatographic grade acetonitrile and methanol were purchased from semer technologies; other reagents were analytically pure.

Preparation of tetracycline and pesticide mother liquor: the weighed raw medicine mass is calculated according to the actual content of the raw medicine, an organic solvent is added to fully dissolve the raw medicine (tetracycline raw medicine is dissolved by methanol, 9 kinds of pesticides are dissolved by acetonitrile), and finally the raw medicine is dissolved in a 100mL brown bottle, wherein the concentration of working mother liquor is 1000 mg/L.

0.9% physiological saline: 9g NaCl was weighed accurately, 1000mL distilled water was added, the solution was stirred with a glass rod to obtain a sufficient solution, and the solution was poured into a volumetric flask and sealed and sterilized at high temperature.

0.1% aqueous formic acid: formic acid (1 mL) was added and dissolved in 1000mL of triple distilled water.

Mcllvaine buffer solution: 625mL of 0.2mol/L disodium hydrogen phosphate solution was mixed with 1000mL of 0.1mol/L citric acid solution and the pH was adjusted to 4.

0.1mol/L Na2 EDTA-Mclvaine buffer solution: 60.5g of disodium edetate are weighed into 1625mL of Mclvaine buffer solution, dissolved and shaken uniformly.

3. Supply test consumptive material and main instrument

Example 1 isolation, identification and preservation of strains

1. Separating and purifying strain

Collecting a soil sample from a farmland system in Boruo city, Guangdong province, taking 5g of the soil sample, placing the 5g of the soil sample into a 250mL triangular flask containing 50mL of a basic salt culture medium, adding a proper amount of tetracycline mother liquor as a single carbon source to ensure that the final concentration of tetracycline in the culture medium is 5mg/L, keeping the temperature at 30 ℃, and performing shake-table acclimation culture for 5d at 180 r/min. Setting tetracycline concentration of 4 gradients as acclimatization culture concentration (25, 50, 75, 100mg/L), and inoculating into culture medium of next concentration at 5d according to inoculum size of 5mL until the concentration is 100 mg/L.

Transferring 1mL of a bacterial solution domesticated under the concentration of 100mg/L tetracycline into 9mL of physiological saline, diluting the bacterial solution into 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 in a gradient manner, and taking 100uL of the liquid in 10-5-10-8 as an inoculum size to coat the liquid on an LB solid culture medium. After 5d, single colony on the plate is picked, streaked and separated to obtain pure strain. Inoculating the purified strain in a fresh liquid culture medium, adding tetracycline mother liquor to enable the initial concentration of tetracycline to be 50mg/L, and detecting the concentration of antibiotics by HPLC after 5 days to determine the degradation capability of the strain on tetracycline. Finally, the strain with tetracycline degradation ability is preserved in glycerol tubes at-80 ℃.

2. Morphological observation and physiological and biochemical experiment of strain

(1) Experimental methods

0.2mL of liquid was withdrawn from the glycerol tube and transferred to fresh LB liquid medium for activation. When the logarithmic phase of growth is reached, selecting a bacterial liquid by using an inoculating rod, streaking the bacterial liquid on an LB solid culture medium, culturing at a constant temperature of 30 ℃, and observing morphological characteristics such as color, shape, size, transparency and the like of a bacterial colony after a single bacterial colony appears on a flat plate.

The physiological and biochemical tests of the microorganisms are carried out according to general bacteria identification methods and Bergey bacteria identification manuals. Specific physiological and biochemical indicators are: gram staining, anaerobic growth, catalase, oxidase, nitrate reduction, glucose fermentation, maltose fermentation, turanose fermentation, sucrose fermentation, salicin fermentation, rhamnose fermentation, trehalose fermentation, esculin fermentation, urease, ONPG assay, citrate utilization, indole, hydrogen sulfide, ornithine decarboxylase. The various microorganisms have different enzyme systems and different decomposition capacities for the nutrient substrates, so that the metabolites are more or less different from each other and can be used for identifying bacteria. The metabolism of bacteria on various substrates and metabolites thereof are detected by a biochemical test method, so that the species of the bacteria can be identified.

(2) Results

The strain was gram-stained and determined to be a gram-positive bacterium. On LB agar medium, its colony appears white, round, moist, smooth and glossy, and its edge grows a lawn with special foul smell. As shown in fig. 1.

The main physiological and biochemical characteristics of the strain separated by the invention are shown in the following table 1:

TABLE 1

The main physiological and biochemical characteristics of degrading bacteria ZQ02

"+" is a positive reaction and "-" is a negative reaction.

3. Molecular biological identification of 16SrDNA of strain

The size of the 16S rRNA fragment obtained by PCR amplification with the extracted total DNA of the microorganism as a template and a universal primer for bacteria is 1394 bp. Performing Blast comparison analysis on the sequences with NCBI database, downloading related sequences with higher homology, performing comparison analysis by Clustal X1.8.1, and constructing a phylogenetic tree by using Neighbor-Joining of MEGA7.0 as an algorithm, as shown in FIG. 2. As is clear from FIG. 2, the 16S rRNA sequence of the degrading bacteria has high similarity to bacteria of the genus Proteus (Proteus sp) among NCBI strains, and the homology is 99% or more, and the degrading bacteria belong to the genus Proteus (Proteus sp).

And the results of morphological observation and physiological and biochemical experiments are combined to obtain the strain as the agrobacterium tumefaciens (Proteus terrae). Therefore, the strain was named Proteus terrae ZQ 02.

4. Strain preservation

The strain of the agrobacterium tumefaciens ZQ02 is preserved in Guangdong province microorganism culture collection center in 2019, 11 and 01, the preservation number is GDMCC No.18782, and the preservation address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

Example 2 Effect of the strains on the degradation of Tetracycline

1. Experimental methods

(1) Preparation of inoculum

0.2mL of the frozen degrading bacteria ZQ02 bacterial liquid is taken and transferred to be inoculated toA conical shaking flask containing 50mL of fresh LB culture medium is cultured at the temperature of 30 ℃ and is shake-cultured at 180r/min for 24h until the logarithmic phase of growth. And (3) taking 25mL of bacterial liquid into a sterilized centrifugal tube, centrifuging at 5000r/min for 5min, collecting thalli, and washing twice with 25mL of sterilized normal saline. Finally adding sterilized normal saline, shaking evenly to ensure that the inoculation liquid OD₆₀₀The value is 1, and a inoculum is prepared.

(2) Determination of degradation Properties

Adding tetracycline mother liquor into a conical flask to enable the initial concentration to be 50mg/L and the pH value to be 7, inoculating degrading bacteria inoculation liquid according to the inoculation amount of 5% of the volume ratio, establishing sterile blank control, repeating each treatment group for 3 times, setting the culture temperature to be 30 ℃, and placing the treatment groups in a constant-temperature shaking table at the rotating speed of 180r/min for the culture time to be 7 d. Samples were taken daily on time and antibiotic residues were detected.

(3) Chromatographic conditions

The liquid chromatograph Agilent 1260 was equipped with a UV detector and used to measure the concentration of tetracycline and pesticide. The chromatographic conditions are as follows: c18 reverse phase chromatography column (4.6X 250mm, 5 μm, Athena), with controlled column temperature at 30 deg.C, sample size of 20 μ L, flow rate of 0.8mL/min, mobile phase 0.1% aqueous formic acid: acetonitrile 70:30, absorption wavelength 355.

The calculation formula of the degradation rate is as follows:

wherein X is the degradation rate and is expressed in percentage, C₀As the initial concentration, C_tIs the residual concentration at the reaction time t.

Quality control: and correcting the standard substance by adopting an external standard method to prepare a standard curve.

2. Results of the experiment

As shown in FIG. 3, the inoculation treatment group and the blank control group were set up to evaluate the degradation kinetics in the medium with an initial tetracycline concentration of 50mg/L for 7 days. When LB liquid medium is used as a medium for degradation experiments, hydrolysis also plays a crucial role in degradation. The non-inoculated blank group had a tetracycline degradation rate of 22.52% after 7d (see FIG. 3 a). In the treatment group inoculated with the degrading bacteria ZQ02, the tetracycline concentration is obviously reduced, and an HPLC result shows that the tetracycline content is obviously reduced at 3d, and the degradation rate is 69.79%; after 7d of culture, the degradation rate of tetracycline by the degrading bacteria ZQ02 is 82.37% (as shown in FIG. 3 b). Inoculation with strain ZQ02 significantly accelerated tetracycline degradation compared to the control. Therefore, the strain ZQ02 can degrade 50mg/L tetracycline, and is expected to become a candidate material and a scheme for repairing tetracycline pollution.

EXAMPLE 3 optimal degradation conditions for tetracycline by Strain ZQ02

1. Experimental methods

(1) Influence factors of degradation bacteria for degrading tetracycline

The single-factor experiment mainly researches the influence of temperature, initial pH value, inoculation amount and initial concentration on the biodegradation of tetracycline. The treatment groups of the influence factors are respectively as follows: the culture temperature (20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃); pH (5, 6, 7, 8, 9); initial concentrations (5, 10, 25, 50, 75, 100 mg/L); the inoculum size (1%, 3%, 5%, 7%, 9%).

(2) Response surface optimization design

In order to clarify the bioremediation potential of the degrading bacteria ZQ02(Proteus terrae) on tetracycline, a response surface method is applied to optimize the influence of various factors and the interaction of the factors. Based on the Box-Behnken design, main influence factors (culture temperature, pH value and inoculum size) are used as independent variables, and the degradation rate is used as a response value. The initial concentration was 50mg/L and the Box-Behnken experiment was designed using the software Minitab 17 for a total of 15 treatments, each treatment being 3 replicates, the specific experimental arrangement being shown in Table 2. After 5d of culture, sampling and analyzing the residual tetracycline, and calculating the degradation rate.

According to the actual degradation rate and the encoded independent variable level obtained by the Box-Behnken experiment, a binary polynomial fit is carried out on the data by Origin 8.0. The binary polynomial formula is: yi 0+ ∑ b i X i + ∑ b ij X iX + ∑ b ii Xi2Yi is the predicted response value, Xi and Xj are variables, b0 is a constant, bi is a linear coefficient, bij is an interaction coefficient, and bii is a quadratic coefficient.

2. Results of the experiment

As shown in FIG. 4(a), tetracycline was inoculated with degrading bacteria ZQ027d at 20, 25, 30, 35, and 40 ℃ respectivelyThe residual concentration of tetracycline in the treated group is respectively reduced from 50mg/L to 11.85mg/L, 5.93mg/L, 7.37mg/L, 21.56mg/L and 25.92mg/L, the degradation rates are respectively 76.28%, 88.14%, 85.26%, 48.16% and 56.87%, and the OD is OD₆₀₀The values are 0.775, 1.233, 1.377, 0.506 and 0.349 respectively. In the range of 20-40 ℃, the biodegradation efficiency begins to increase along with the increase of the temperature and then decreases after exceeding 30 ℃. In view of degradation of tetracycline and growth of degrading bacteria after 7 days of inoculation, the bioremediation conditions should not be set to a higher temperature. The growth of microorganisms is inhibited under the condition of high temperature, and the bioremediation process is influenced. Therefore, the strain ZQ02 prefers to degrade tetracycline under low temperature conditions, and the optimal temperature range for degradation of tetracycline is 25-30 ℃.

Figure 4(b) shows the effect of inoculum size on tetracycline biodegradation. When the inoculum size is 1%, 3%, 5%, 7% and 9%, the tetracycline residual concentration in each treatment group is respectively reduced from 50mg/L to 8.47mg/L, 8.32mg/L, 7.37mg/L, 8.46mg/L and 9.05mg/L after the constant temperature shaking culture for 7d, the degradation rate is respectively 83.06%, 83.35%, 85.26%, 83.07% and 81.90%, and the OD is OD₆₀₀The values are 1.465, 1.534, 1.377, 1.361, and 1.287, respectively. When the initial inoculum size was 1%, a growth lag phase was observed in the initial phase and the degradation rate was significantly lower than in the other groups. This phenomenon may be due to the toxicity of tetracycline which inhibits cell viability and number. As for the final degradation result, the degradation efficiency of five treatment groups has no significant difference, and when the inoculation dose is 1-9%, more than 80% of tetracycline is removed. In practical applications of bioremediation, microbial inoculum size should be considered as an important issue. In order to ensure good biodegradation of environmental pollutants, good growth of microorganisms in the environment, and reduction of bioremediation cost, in practical applications, the optimal initial bacterial inoculum size is 5% of biodegradable tetracycline.

As shown in FIG. 4(c), after 7d of cultivation, the residual concentration of tetracycline in each treatment group was decreased from 50mg/L to 34.36mg/L, 9.39mg/L, 7.37mg/L, 5.52mg/L and 4.92mg/L at

pH

5, 6, 7, 8 and 9, respectively, and the degradation rates were 35.28%, 81.22%, 85.26%, 88.98% and 90.16%, respectively, and the OD was adjusted to 35.28%, 81.22%, 85.26%, 88.98% and 90.16%, respectively₆₀₀The values are 0.421, 1.139, 1.377, 1.387, and 1.498, respectively. The pH value greatly influences the biodegradation rate. Under acidic conditions, at a pH of 5, the bacterial growth was lower than that of the other treatment groups except for low biodegradation rate. This phenomenon may be associated with pH affecting the activity of its degrading enzymes and cell growth. Compared with the acidic environment, the alkaline environment and the neutral environment are beneficial to the biodegradation of tetracycline and the growth of degradation bacteria. According to the results, the optimal pH range of the degrading bacteria ZQ02 for the tetracycline biodegradation is 7-9.

The effect of different initial tetracycline concentrations on the biodegradation of strain ZQ02 is shown in FIG. 4 (d). After 7d of culture, the concentration of tetracycline residues in each treatment group was reduced from 5mg/L, 10mg/L, 25mg/L, 50mg/L, 75mg/L and 100mg/L to 0.67mg/L, 1.61mg/L, 4.04mg/L, 7.37mg/L, 26.16mg/L and 76.05mg/L, respectively, with degradation rates of 86.55%, 83.92%, 83.84%, 85.26%, 65.12% and 23.95%, and OD600 values of 1.519, 1.441, 1.466, 1.377, 0.784 and 0.369, respectively. When the concentration of tetracycline is lower than 50mg/L, the degrading strain ZQ02 is effective in removing more than 80% of tetracycline in the medium. However, at an initial concentration of 75mg/L or more, the growth of the microorganism shows a long growth retardation due to the toxic effect of the excessive tetracycline, resulting in a great limitation in the degradation ability. Therefore, considering the practical use condition and effective degradation, the optimal initial concentration of tetracycline should be less than 75 mg/L.

In order to explore the bioremediation potential of the agrobacterium tumefaciens ZQ02 on tetracycline, a response surface method is applied to optimize the influence of various factors and the interaction thereof. Based on Box-Behnken optimization design, the initial concentration of tetracycline is 50mg/L, the influence factors are temperature, inoculum size and pH value, three levels are set for each influence factor, the encoding is (-1, 0, 1), and the total number of 15 running treatments is. As shown in Table 2, after 5 days of culture, biodegradation of tetracycline was obtained and expressed as a percentage for each group.

TABLE 2 Box-Behnken design and the resulting response values

And performing polynomial regression analysis on the experimental data, and establishing a secondary response model through Minitab 17 software. Thus, the fitted and derived quadratic polynomial equation is as follows:

Yi＝84.69－0.62X₁+3.94X₂+24.37X₃－17.49X₁ ²－19.81X₂ ²－11.13X₃ ²+2.18X₁X₂－0.14X₁X₃－0.3X₂X₃

in the above equation, Yi is the predicted response value, X₁，X₂And X₃The encoded values are respectively corresponding to temperature, inoculum size and pH.

As shown in Table 3 below, statistical analysis showed that the model had high significance (p)<0.001) and its correlation coefficient R²Is 0.9912. Thus, there is a high correlation between the experimental measurements and the predicted values, indicating that this model is suitable for theoretical prediction of tetracycline degradation. Analysis of variance of the regression equation showed that the initial pH and the linear coefficient of the inoculum dose both reached significant levels (P)<0.05), which indicates that the above single condition has a significant effect on the degradation result. Meanwhile, secondary coefficients of culture temperature, pH value and inoculation dose show that the secondary coefficients have significant influence on the biodegradation of tetracycline. Interaction coefficients (P) of three influencing factors>0.05) temporarily did not see a significant effect.

TABLE 3 analysis of variance (ANOVA) fitting a quadratic polynomial model

DF is the degree of freedom, SS is the sum of the sequences, MS is the mean square, R²＝0.9912，Adj R²0.975. In Prob>In the model term of F, the P value<0.05 and P value>0.1 indicates that the model term is significant.

As shown in fig. 5, the 3D response surface map and the contour map can effectively and intuitively reflect various factors and their interactions in the test results. In this studyIn the method, a quadratic polynomial model is successfully fitted, the number of processing groups and the resource investment are reduced, and the purpose of optimizing degradation conditions is achieved. The theoretically optimal conditions for the degradation process can be obtained from the first partial derivative of the quadratic polynomial equation. At the theoretical optimum, the maximum degradation efficiency is 98.10%, X₁，X₂And X₃The code value of (a) is: 0.0101, 0.0909 and 1, decoded as: temperature (29.89 ℃), inoculum size (5.36%) and pH (9). To test the accuracy of the model predictions, validation experiments were performed using the above described optimization conditions. After 5 days of incubation, only 4.73mg/L tetracycline remained in the medium, corresponding to 90.54% of the initial dose of tetracycline was degraded, approaching the expected prediction of the model. Thus, the measured values are largely consistent with the predicted values, which indicates that the model is feasible and reliable for optimizing degradation conditions.

Example 4 microbial remediation of Tetracycline contamination in soil by Strain ZQ02

1. Experimental methods

(1) Preparing an inoculation solution: as in example 2.

(2) And (3) determining the degradation performance:

and observing the degradation dynamics of the tetracycline in the soil and the repair capability of the degrading bacteria to the tetracycline pollution. Weighing 100g of dry soil, placing the dry soil in a conical flask, adding water to keep the water content of the dry soil at 60%, adding tetracycline mother liquor to make the final concentration in the soil be 50mg/kg, inoculating degrading strains with the inoculation amount of 5%, taking non-inoculated natural soil as a blank control group, treating each group for 3 times, and placing the groups in a constant-temperature incubator at 30 ℃. The sterilized soil treatment requires weighing the soil into a volumetric flask and carrying out high-temperature dry heat sterilization. And setting sterile soil and degrading bacteria treatment and blank control thereof, wherein the specific operation is the same as that described above. Sampling at regular intervals for 0, 3, 5, 7, 10, 15 and 20 days, collecting samples, placing the samples in a sealed bag, freezing and storing the samples for waiting for pretreatment. In order to avoid the soil environment from being too dry, water is properly added to keep the water content of the soil to be about 60 percent.

(3) The chromatographic condition and degradation rate calculation method comprises the following steps: as in example 2.

2. Results of the experiment

Four ringsThe kinetics of the degradation of elements in soil are shown in figure 6. After the treatment process of 20 days, the degradation rate of tetracycline in the sterilized soil is 38.61%, and the degradation rate constant k is 0.0204d^-1The theoretical half-life is 33.91 d. And the degradation rate of the tetracycline in the natural soil treatment group is 53.32%, and the degradation rate constant and the half-life period are 0.0375d^-1And 18.84 d. Naturally occurring tetracycline in soil degrades faster than other contaminants, and the indigenous flora in soil may accelerate the degradation of the contaminants to some extent.

In the treatment group of the sterilized soil added with degrading bacteria, the degradation rate is 67.74 percent, and the degradation rate constant k is 0.0599d^-1The theoretical half-life is 11.57 d. The degradation rate of the natural soil added with the degradation bacteria treatment group is 81.65 percent, and the degradation rate constant k is 0.0901d^-1The theoretical half-life is 8.27 d. The degradation efficiency of tetracycline in soil can be promoted by adding the degradation bacterium ZQ 02. Among the four treatments, the degradation efficiency of the natural soil added with the degrading bacteria ZQ02 is optimal. The degrading bacteria can repair the tetracycline together with other microorganisms in the soil, so that the purpose of removing pollutants is achieved.

Example 5 degradation Activity of Strain ZQ02 on pesticides

1. Experimental methods

As farmland pollution, the residual antibiotics and pesticides have long duration in time, the coverage in space is wide, and the two kinds of pollution are mutually staggered. Aiming at the characteristics of complex and various pollutants, the degradation activity of the strain to 9 common pesticides is tested, the cognition of the degradation spectrum is widened, and feasible basis is provided for bioremediation under multiple organic pollution. The tested pesticides include 9 types, namely, beta-cypermethrin, chlorpyrifos, clothianidin, thiamethoxam, carbofuran, pyraclostrobin, chlorothalonil, diuron and atrazine. ZQ02 was inoculated into 50mL of fresh sterilized LB medium at 5% inoculum size by volume, and the stock solution of the pesticide to be tested was added so that the final concentration was 50 mg/L. Sampling 5mL in time, and detecting the pesticide residue condition in a liquid phase after pretreatment.

2. Degradation results

TABLE 4 kinetic parameters of the strain ZQ02 for the degradation of 9 pesticides

The biodegradation activity of the agrobacterium tumefaciens ZQ02 on 9 common pesticides and the analysis of the degradation kinetics thereof are shown in table 4. After the strains are co-cultured for 7 days, the strains have obvious degradation effects on chlorothalonil, carbofuran, thiamethoxam and diuron with the initial concentration of 50mg/L, wherein the degradation rate of the chlorothalonil reaches 96.84%, which indicates that the strains have the potential of reducing and removing other agricultural environment pollutants except ciclovir.

EXAMPLE 6 degrading Activity of Strain ZQ02 on chlorothalonil in culture Medium

1. The experimental method comprises the following steps:

(1) preparing an inoculation solution: as in example 2

(2) Determination of degradation Properties

ZQ02 was inoculated into 50mL of fresh sterilized LB medium at 5% inoculum size by volume, and the stock solution of chlorothalonil to be tested was added so that the final concentration was 50 mg/L. Sampling 5mL in time, and detecting the pesticide residue condition in a liquid phase after pretreatment.

(3) The chromatographic condition and degradation rate calculation method comprises the following steps: as in example 2

2. The experimental results are as follows:

as a result, as shown in FIG. 7, the inoculation-treated group and the blank control group were set up to evaluate the degradation kinetics in the medium at an initial concentration of chlorothalonil of 50mg/L for 7 days in succession. When an LB liquid culture medium is used as a medium for degradation experiments, in a treatment group inoculated with degrading bacteria ZQ02, the chlorothalonil concentration is obviously reduced, and an HPLC result shows that the chlorothalonil content is obviously reduced at 1d, and the degradation rate is 86.50%; after 7d of culture, the degrading bacteria ZQ02 has a degrading rate of 96.84 percent (as shown in figure 6) to chlorothalonil. Inoculation with strain ZQ02 significantly accelerated the degradation of chlorothalonil compared to the control. Therefore, the strain ZQ02 can degrade 50mg/L chlorothalonil and is expected to become a candidate material and a scheme for repairing chlorothalonil pollution.

Example 7 degradation Activity of Strain ZQ02 on pesticide-antibiotic Co-contamination in soil

1. Experimental methods

Simulating soil compositely contaminated by pesticide-antibiotic, and evaluating the bioremediation potential of the strain ZQ02 on pesticide-antibiotic under soil conditions. According to the results of the early-stage activity screening, the tested pesticides are carbofuran, thiamethoxam, chlorothalonil and diuron. In the soil test, the concentration of the pesticide and antibiotic compounds is 25mg/L pesticide +25mg/L tetracycline. Weighing 100g of dry soil sample, placing the dry soil sample in a conical flask, adding water to keep the water content of the dry soil sample at 60%, adding pesticide and tetracycline mother liquor to enable the initial concentration in the soil to reach a set value, inoculating degrading bacteria in an inoculation amount of 5%, and placing the soil sample in a 30 ℃ constant temperature box. Sampling for 0, 3, 5, 7, 10, 15 and 20 days, placing in a sealed bag, freezing and storing for waiting for pretreatment. During the culture period, in order to avoid the soil environment from being too dry, water is properly added into the soil to keep the water content of the soil to be about 60 percent.

2. Degradation results

TABLE 5 remediation of pesticide-antibiotic complexes in soil by degrading bacteria

According to the early-stage activity screening result, a pesticide X + tetracycline composite pollution system is experimentally constructed, and the synergistic degradation capability of the strain ZQ02 on composite pollution is tested. The results are shown in Table 5, and the strains respectively have obvious degradation effects on the composite residues of carbofuran, thiamethoxam, chlorothalonil, diuron and tetracycline in the soil. In the four groups of mixed treatment, the strain ZQ02 has 68.49-98.00% of tetracycline degradation bacteria and 33.32-77.19% of degradation rate of 4-class pesticides, and fully embodies the advantageous performance of the strain ZQ02 in the synergistic degradation of tetracycline and the composite pollution of pesticide residues.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A Proteus tumefaciens strain ZQ02, wherein the Proteus tumefaciens strain ZQ02 is deposited in the Guangdong province collection of microorganisms in 2019, 11/01, with the deposit number being GDMCC No: 18782.

2. a microbial preparation comprising the strain ZQ02 of Agrobacterium according to claim 1 and/or a bacterial solution thereof.

3. A biological detergent characterized by comprising the strain ZQ02 of Agrobacterium according to claim 1 and/or a bacterial solution thereof.

4. A biodegradable agent comprising the strain ZQ02 of Agrobacterium according to claim 1 and/or a bacterial solution thereof.

5. Use of the strain of agrobacterium strain ZQ02 as claimed in claim 1 for degrading tetracycline and/or pesticides.

6. The use of the strain of Agrobacterium ZQ02 according to claim 1 for the remediation of tetracycline and/or pesticide-contaminated environments.

7. The use of the bacterial agent of claim 2 for degrading tetracycline and/or pesticides.

8. Use of the biological cleaning agent according to claim 3 for degrading tetracycline and/or pesticides.

9. Use of the biodegradable agent of claim 4 for degrading tetracycline and/or pesticides.

10. The use according to any one of claims 5 to 9, wherein the pesticide is chlorothalonil, carbofuran, diuron, thiamethoxam.