CN114317324B - Alcaligenes and product and application thereof - Google Patents
Alcaligenes and product and application thereof Download PDFInfo
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- CN114317324B CN114317324B CN202111446726.4A CN202111446726A CN114317324B CN 114317324 B CN114317324 B CN 114317324B CN 202111446726 A CN202111446726 A CN 202111446726A CN 114317324 B CN114317324 B CN 114317324B
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Abstract
The invention discloses an Alcaligenes as well as a product and an application thereof, wherein the Alcaligenes is named Alcaligenes sp.R3, and the preservation number of the Alcaligenes in China center for type culture collection is CCTCC NO: m2020882. The alcaligenes in the invention is obtained from pig manure through enrichment, separation and purification, has the highest degradation rate of tetracycline reaching 80.16 percent, embodies the characteristics of good degradation effect, strong specificity, short time consumption and high removal efficiency, can be applied to removing or reducing the residue of tetracycline in the environment, provides safe and environment-friendly tetracycline degradation bacteria for the problem of tetracycline residue, and simultaneously provides excellent strains for further researching the removal of tetracycline by embedding and fixing microorganisms.
Description
Technical Field
The invention belongs to the technical field of biodegradation of tetracycline, and particularly relates to an alcaligenes strain, and a product and application thereof.
Background
Tetracycline antibiotics are widely used because of their low cost, and include tetracycline, aureomycin, oxytetracycline, and the like. Among them, tetracycline (TC) is widely used in daily life of people, and since only a small part of it is metabolized or absorbed in animals, more than 70% of it is excreted in the form of metabolites through urine or feces of humans and animals in the environment, tetracycline has high persistence, toxic effects on the ecosystem and the spread of drug resistance in microorganisms, and has high pollution hazard to the environment, especially water resources and soil. Tetracycline (TC) is one of the most commonly used antibiotics, has high persistence and biological activity, produces toxic effects on ecosystem and persistence in the spread of drug resistance in microorganisms, can accumulate in water and soil, and causes pollution problems to the environment, particularly water resources and soil. Because the water-soluble tetracycline hydrochloride is unstable and easy to degrade in a water environment, a large amount of tetracycline is detected in shallow water, drinking water and sludge, and cannot be completely removed by using the traditional sewage treatment method. Since the automatic biodegradation of tetracycline in the natural environment is very difficult, there is an urgent need to develop alternative methods for reducing tetracycline in aqueous solutions.
In order to reduce environmental pollution, researchers have explored various methods for removing tetracycline antibiotics from water bodies; at present, the methods for removing organic pollutants such as tetracycline in water mainly comprise physical methods, chemical oxidation methods, microbial degradation methods and the like.
The microbial degradation method is to convert organic pollutants in the wastewater by using the metabolism of microorganisms, the period is long, the removal efficiency of antibiotic pollutants is low, and the screening of high-efficiency antibiotic degrading bacteria is the difficult point of removing the pollutants by the microorganisms.
The Chinese invention patent CN112501075A provides the application of Alcaligenes faecalis in preparing microbial agent for efficiently degrading ammonia nitrogen and nitrite nitrogen in aquaculture water. The microbial agent comprises alcaligenes faecalis powder with the bacterium content not lower than 5.0 multiplied by 108CFU/g and a carrier, and the mass ratio of the alcaligenes faecalis powder to the carrier is 1: 50-100. The alcaligenes faecalis powder is prepared from alcaligenes faecalis GBW-HB1905 with the preservation number of CGMCC No.20364, and the alcaligenes faecalis powder has wide salt tolerance and the growth salinity range of 15-35 per mill. The microbial inoculum has the capability of efficiently and rapidly degrading ammonia nitrogen and nitrite nitrogen in the aquaculture water body, can continuously and stably maintain the ammonia nitrogen and the nitrite nitrogen in a lower concentration range, can effectively solve and improve the problem of high concentration of the ammonia nitrogen and the nitrite nitrogen in the aquaculture water body, and is beneficial to aquaculture and income. The alcaligenes in the reference can only be used for treating ammonia nitrogen and nitrite nitrogen in the wastewater, but can not be used for degrading antibiotics, especially tetracycline, in the wastewater.
Disclosure of Invention
In order to overcome the problems in the prior art, the application provides an alcaligenes and a product and application thereof, and the specific method is to screen a tetracycline degrading bacterium with strong specificity, short time consumption and high removal efficiency from pig manure, so as to provide an excellent strain for further researching the removal of tetracycline by embedding and fixing microorganisms and provide abundant microorganism resources for treating antibiotic wastewater.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides an Alcaligenes for degrading tetracycline, wherein the Alcaligenes is Alcaligenes sp.R3, and the preservation number of the Alcaligenes is CCTCC NO: m2020882.
As an alternative embodiment, the nucleotide sequence of the Alcaligenes provided by the invention is shown in SEQ ID NO. 1.
As an alternative embodiment, the Alcaligenes faecalis provided by the invention is obtained by enriching, separating and purifying pig manure.
As an alternative embodiment, the present invention provides a basic culture medium for Alcaligenes latus used in the enrichment, isolation and purification process, comprising Na 2 HPO 4 ，KH 2 PO 4 NaCl and peptone.
As an alternative embodiment, the present invention provides a selection medium for Alcaligenes latus used in the enrichment, isolation and purification process comprising Na 2 HPO 4 ，KH 2 PO 4 NaCl, peptone and tetracycline hydrochloride solutions.
As an alternative embodiment, in the alcaligenes provided by the present invention, the specific process of enriching, isolating and purifying the alcaligenes comprises the following steps:
the method comprises the following steps: enrichment, separation and purification of bacterial strain
Taking a pig manure sample from a certain pig farm, and storing the pig manure sample in a refrigerator at the temperature of-20 ℃ by using a self-sealing bag device. 10g of the sample is put into a triangular flask containing glass beads, added into 90mL of sterile water, and shaken at 30 ℃ for 20min at 170 r/min. Taking a treated sample, sucking 2mL of supernatant, and inoculating the supernatant into a liquid culture medium for culture for 2d; then inoculating to screening medium containing 20mg/L tetracycline, culturing for 2d, sequentially inoculating to 40, 80, 120, 160, 200, 240, 280, 320mg/L tetracycline screening medium, culturing for 2d under constant conditions, and diluting by 10-fold dilution after enrichmentDiluting in line, taking 10 -4 、10 -5 、10 -6 、10 -7 And (3) coating the diluent, inverting the coated plate into a constant-temperature incubator at 30 ℃ for culturing for 24h, and carrying out plate streaking on the grown single colony to screen out the single colony taking tetracycline as a unique carbon source.
Step two: primary screen for degrading bacterial strain
And (3) selecting a single colony on the plate, inoculating the single colony on a screening solid culture medium containing 100mg/L tetracycline by adopting a continuous streaking method, culturing for 24 hours, selecting the single colony, storing the single colony by using a slant, and continuously purifying until the single colony is observed as a pure strain under a microscope.
Step three: rescreening of degrading strains
Inoculating the preliminarily screened strain into an LB liquid culture medium, inoculating the strain serving as a seed solution into a basic culture medium containing 50mg/L tetracycline after culturing for 24 hours, placing the basic culture medium in a shaking table at the temperature of 30 ℃ and at the speed of 140r/min for culturing for 2d, using the strain not inoculated as a control group, measuring the content of the tetracycline by using a High Performance Liquid Chromatography (HPLC), eliminating the strain with poor degradation capability, and storing the strain with high tetracycline degradation capability by using a glycerin pipe and a slope for later use.
Step four: identification of degrading strains
And selecting a single colony which is subjected to constant temperature culture for 24 hours at the temperature of 30 ℃ after streaking of the plate, and observing the characteristics of the colony such as shape, color, transparency and the like. The physical and chemical identification is mainly carried out by analyzing indexes of the strain such as carbon source utilization, starch hydrolysis, cellulose utilization, gelatin liquefaction, hydrogen sulfide generation, oxidase, catalase and the like, and the physiological and chemical test of the strain is carried out according to Bergey's bacteria identification manual, general bacteria common identification method and the like.
In a second aspect, the invention provides the use of an alcaligenes strain according to any one of the embodiments described above for the degradation of tetracycline.
As an optional embodiment, in the application of the Alcaligenes to tetracycline degradation, the pH value of the degradation solution is 5-9, and the degradation temperature is 20-40 ℃ in the tetracycline degradation process of the Alcaligenes.
As an alternative embodiment, in the application of the Alcaligenes degrading tetracycline provided by the invention, the concentration of the Alcaligenes degrading tetracycline is 50-250mg/L.
In a third aspect, the present invention provides an antibiotic bacterial agent for degrading tetracycline, wherein the antibiotic bacterial agent comprises the alcaligenes described in any one of the above embodiments.
As an optional embodiment, in the antibiotic bacterial agent for degrading tetracycline provided by the invention, the preparation method of the antibiotic bacterial agent comprises the steps of performing activated culture on the Alcaligenes by using a liquid culture medium, and collecting a culture solution.
The invention has the following beneficial effects:
the strain is identified to Alcaligenes sp by physicochemical identification and 16S rRNA sequence analysis, and is named as Alcaligenes sp.R3. The preservation number is: CCTCC NO: m2020882. The alcaligenes can reach 80.16 percent of the highest degradation rate of the tetracycline, shows good degradation effect, can be applied to removing or reducing the residue of the tetracycline in the environment, and provides a safe and environment-friendly microbial degradation method for the problem of tetracycline residue.
Drawings
FIG. 1 is a colony morphology map of the strain Alcaligenes sp.R3 in example 1;
FIG. 2 is an electrophoretogram of the strain Alcaligenes sp.R3 in example 1;
FIG. 3 is a phylogenetic tree of the strain Alcaligenes sp.R3 in example 2;
FIG. 4 is a graph showing the results of the tetracycline degradation by the strain Alcaligenes sp.R3 at pH in example 4;
FIG. 5 is a graph showing the results of the tetracycline degradation by the strain Alcaligenes sp.R3 under temperature in example 4;
FIG. 6 is a graph showing the results of the tetracycline degradation by the strain Alcaligenes sp.R3 according to the inoculation amount in example 4;
FIG. 7 is a graph showing the results of the tetracycline degradation by the strain Alcaligenes sp.R3 at the rotation speed in example 4;
FIG. 8 is a graph showing the results of experiments on tetracycline degradation by the starting substrate concentration versus the strain Alcaligenes sp.R3 in example 4.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the description herein, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Reagents and equipment required in the experimental process
The reagents used in the following examples are, unless otherwise indicated, conventional in the art, and the equipment and methods are conventional in the art.
The HPLC chromatographic determination conditions of the invention include: high Performance Liquid Chromatography (HPLC) was used to quantify the concentration of tetracycline. The high-efficiency liquid phase is Agilent-1260, a C18 column (5 mu m,4.6mm multiplied by 250 mm), and the mobile phase: phosphate acetonitrile =80 (v/v). The column temperature was: 30 ℃, flow rate: 1mL/min, sample size: 20 mu L, and the wavelength of an ultraviolet detector is 360nm.
Standard substance of tetracycline hydrochloride in the present invention (C) 22 H 24 N 2 O 8 HCl), molecular weight 480.9, purity ≥ 98%; acetonitrile and methanol were both chromatographically pure, peptone, LB broth were purchased from changshuding national biotechnology limited.
The method for configuring the culture medium used by the invention comprises the following steps:
1. basic culture medium: na (Na) 2 HPO 4 ，KH 2 PO 4 NaCl, peptone, 1L of ultrapure water, starting pH 7.0, sterilized at 121 ℃ for 25min (2% agar added in solid medium).
LB broth: 5g of beef extract, 10g of peptone, 5g of NaCl, 1L of pure water, pH7.4-7.6, and sterilizing at 121 ℃ for 15min.
3. Screening a culture medium: adding tetracycline hydrochloride into the sterilized basic culture medium, and adding tetracycline mother liquor filtered by a microporous membrane with the diameter of phi 0.45 mu m according to different concentration requirements.
Standard stock solutions of tetracycline hydrochloride: 0.02g of tetracycline hydrochloride is accurately weighed, and is dissolved in a 100mL brown volumetric flask with ultrapure water to prepare a tetracycline standard solution with the concentration of 200mg/L, and the tetracycline standard solution is stored at 4 ℃ for later use.
Example 1
The application finds an antibiotic degrading bacterium capable of degrading tetracycline, the tetracycline degrading bacterium is Alcaligenes sp, and the Alcaligenes sp is delivered to China Center for Type Culture Collection (CCTCC) for collection at 12 months and 10 days in 2020 with the name: r3, the preservation number is CCTCC NO: m2020882 address: wuhan university in Wuhan city, hubei province, china.
The bacillus caldarius (Alcaligenes sp.R 3) is obtained by enriching, separating and purifying pig manure and comprises the following specific steps:
the method comprises the following steps: enrichment, separation and purification of bacterial strain
Taking a pig manure sample from a certain pig farm, and storing the pig manure sample in a refrigerator at the temperature of-20 ℃ by using a self-sealing bag device. Taking 10g of the sample, placing the sample into a triangular flask containing glass beads, adding 90mL of sterile water, 1Shaking at 30 deg.C for 20min at 70 r/min. Taking the treated sample, sucking 2mL of supernatant, and inoculating the supernatant into a liquid culture medium for culture for 2d; then inoculating to screening culture medium containing 20mg/L tetracycline, culturing for 2d, sequentially inoculating to screening culture medium containing 40, 80, 120, 160, 200, 240, 280, and 320mg/L tetracycline, culturing for 2d under constant conditions, enriching, diluting by 10-fold dilution method, and collecting 10 -4 、10 -5 、10 -6 、10 -7 And (3) coating the diluent, inverting the coated plate into a constant-temperature incubator at 30 ℃ for culturing for 24h, and carrying out plate streaking on the grown single colony to screen out the single colony taking tetracycline as a unique carbon source.
Step two: primary screen for degrading bacterial strain
And (3) selecting a single colony on the plate, inoculating the single colony on a screening solid culture medium containing 100mg/L tetracycline by adopting a continuous streaking method, culturing for 24 hours, selecting the single colony, storing the single colony by using a slant, and continuously purifying until the single colony is observed as a pure strain under a microscope.
Step three: rescreening of degrading strains
Inoculating the preliminarily screened strain into an LB liquid culture medium, inoculating the strain serving as a seed solution into a basic culture medium containing 50mg/L tetracycline after culturing for 24 hours, placing the basic culture medium in a shaking table at the temperature of 30 ℃ and at the speed of 140r/min for culturing for 2d, using the strain not inoculated as a control group, measuring the content of the tetracycline by using a High Performance Liquid Chromatography (HPLC), eliminating the strain with poor degradation capability, and storing the strain with high tetracycline degradation capability by using a glycerin pipe and a slope for later use.
Step four: identification of degrading strains
And selecting a single colony which is subjected to constant temperature culture for 24 hours at the temperature of 30 ℃ after streaking of the plate, and observing the characteristics of the colony such as shape, color, transparency and the like. The physical and chemical identification is mainly carried out by analyzing indexes of the strain such as carbon source utilization, starch hydrolysis, cellulose utilization, gelatin liquefaction, hydrogen sulfide generation, oxidase, catalase and the like, and the physiological and chemical test of the strain is carried out according to Bergey's bacteria identification manual, general bacteria common identification method and the like.
The high performance liquid chromatography determination method comprises the following steps:
high Performance Liquid Chromatography (HPLC) was used to quantify the concentration of tetracycline. The high-efficiency liquid phase is Agilent-1260, a C18 column (5 mu m,4.6mm multiplied by 250 mm), and the mobile phase: phosphate acetonitrile =80 (v/v). The column temperature is: 30 ℃, flow rate: 1mL/min, sample size: 20 mu L, and the wavelength of an ultraviolet detector is 360nm.
Standard substance of tetracycline hydrochloride in the present invention (C) 22 H 24 N 2 O 8 HCl), molecular weight 480.9, purity ≥ 98%; acetonitrile and methanol are both chromatographically pure and purchased from Changshuding national biotechnology Limited.
Inoculating the purified Alcaligenes sp.R3 on a screening culture medium, and culturing at 30 ℃ for 24 hours, wherein colonies are milky and round, the surface is smooth and moist, the colonies are not tightly combined with the culture medium, and the edges are tidy; the form of the cells was short rods, as shown in FIG. 1.
And (3) taking purified Alcaligenes sp.R3 to perform physiological and biochemical measurement such as starch hydrolysis, and performing physiological and biochemical test of the strain by referring to Bojie's Manual of identification of bacteria (general identification method of bacteria) and the like. In particular, see the following table:
TABLE 1
Note: "+" indicates positive; "-" indicates negative.
Example 2
R3 and its phylogenetic analysis.
(1) The DNA of the strain R3 is used as a template, and the universal primer is used for carrying out PCR amplification on the DNA. The results of agarose gel electrophoresis are shown in FIG. 2.
(2) The obtained gene sequences were subjected to BLAST alignment using NCBI and phylogenetic trees were made using the software MEGA7.0 as shown in FIG. 3, the results of the BLAST analysis showing: the strain had the closest relationship to Alcaligenes sp. (KU 986676) and the homology was 99%, and the strain was determined to be Alcaligenes sp. The nucleotide sequence of Alcaligenes sp.R3 is shown in SEQ ID NO. 1.
Example 3
And (3) verifying the tetracycline degradation effect of Alcaligenes sp.
R3 from the plate picking a loop into LB broth, after 24h culture as seed liquid into the base medium containing 50mg/L tetracycline. The cells were then incubated at 30 ℃ for 48h in a shaker at 140r/min, and the supernatant was centrifuged and filtered through a 0.45 μm microfiltration membrane, and the tetracycline content of the filtered supernatant was determined by High Performance Liquid Chromatography (HPLC), and the experiment was repeated three times with Alcaligenes sp.R.3 not inoculated as a control. The results of HPLC measurements are shown in Table 2, which shows that the tetracycline degradation rate was 9.34% in the medium without addition of Alcaligenes sp.R3, and 56.70% in the medium with addition of Alcaligenes sp.R3, and the contribution rate of the strain was 70% or more.
TABLE 2
Example 4
Optimal conditions for the degradation of tetracycline by Alcaligenes sp.R3.
Preparing an inoculation solution: inoculating an Alcaligenes sp.R3 strain obtained by separation and purification by using an inoculating loop into an LB broth culture medium, culturing in a constant-temperature shaking incubator at 30 ℃ and 140r/min, after culturing for 12h, taking a certain amount of culture solution in a centrifuge tube at 8000r/min, centrifuging for 10min, pouring out the supernatant, repeatedly washing for 1-2 times by using 0.9% physiological saline, diluting by using the physiological saline, measuring the absorbance of the sample at 600nm of an ultraviolet spectrophotometer by using the physiological saline as a reference, taking a sample with OD600 nm =1.0 as an inoculation solution, and placing the sample in a refrigerator at 4 ℃ for later use.
(1) Effect of initial pH on Alcaligenes sp.R3 degradation of Tetracycline
100mL of the liquid medium was added to a 250mL Erlenmeyer flask and autoclaved at 121 ℃ for 25min. Adding fresh tetracycline mother liquor filtered by a 0.45 mu m microporous membrane into a conical flask respectively to ensure that the initial concentration of tetracycline in the culture medium is 100mg/L, adding 5% of inoculation solution, setting the pH value of the culture medium to be 5, 6, 7, 8 and 9, setting blank control (only tetracycline is not added with degrading bacteria) for each treatment group, setting 3 treatment groups in parallel, placing the treatment groups in a shaking table at 30 ℃ and 140r/min, culturing for 72h, taking a certain amount of culture solution for measurement, measuring the content of tetracycline by using a High Performance Liquid Chromatography (HPLC), and calculating the degradation rate. The results of HPLC measurements are shown in FIG. 4, where the initial pH was different and the degradation rate of tetracycline was different. The highest degradation rate of tetracycline is achieved at pH8, reaching 67.17%, with pH =7 being the second degradation rate.
(2) Effect of temperature on Alcaligenes sp.R3 degradation of Tetracycline
100mL of the liquid medium was added to a 250mL conical flask and autoclaved at 121 ℃ for 25min. Adding fresh tetracycline mother liquor filtered by a 0.45 mu m microporous membrane into a conical flask to ensure that the initial concentration of tetracycline in a culture medium is 100mg/L, adding 5% of seed solution, setting a blank control for each treatment group, repeating the treatment groups for 3 times, culturing in a shaking table at the temperature of 20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃ and 140r/min respectively, taking a certain amount of culture solution for measuring after culturing for 72h, measuring the content of tetracycline by using a High Performance Liquid Chromatography (HPLC), and calculating the degradation rate. The HPLC measurement results are shown in FIG. 5, and the tetracycline degradation rate shows a first-rising and second-falling trend with the increase of the temperature. When the temperature is 30 ℃, the degradation efficiency is the highest and reaches 55.8 percent.
(3) Effect of inoculum size on the degradation of tetracycline by Alcaligenes sp.R3
100mL of the liquid medium was added to a 250mL conical flask and autoclaved at 121 ℃ for 25min. Respectively adding fresh tetracycline mother liquor filtered by a 0.45 mu m microporous filter membrane into an erlenmeyer flask to enable the initial concentration of tetracycline in a culture medium to be 100mg/L, respectively setting blank control for each treatment group, repeating the treatment groups for 3 times, adding inoculation liquid with the concentration of 1%, 2%, 3%,4% and 5% into a basic culture medium, placing the culture medium in a shaker at 30 ℃ and 140r/min for culture, taking a certain amount of culture solution for measurement after 72 hours of culture, measuring the content of tetracycline by using a High Performance Liquid Chromatography (HPLC), and calculating the degradation rate. The HPLC analysis results are shown in FIG. 6, in which the degradation rate is increased and then decreased with increasing inoculum size. When the inoculation amount is 4%, the tetracycline degradation rate is obvious and reaches 56.17%.
(4) Effect of rotational speed on Alcaligenes sp.R3 degradation of Tetracycline
100mL of the liquid medium was added to a 250mL conical flask and autoclaved at 121 ℃ for 25min. Adding fresh tetracycline mother liquor filtered by a microporous filter membrane with the diameter of 0.45 mu m into a conical flask respectively to ensure that the initial concentration of the tetracycline in a culture medium is 100mg/L, adding 4% inoculation liquid, respectively setting blank control for each treatment group, repeating each treatment group for 3 times, setting the rotating speed of a shaking table to be 100, 120, 140, 160 and 180r/min, culturing at the temperature of 30 ℃, taking a certain amount of culture solution for measuring after culturing for 72 hours, measuring the content of the tetracycline by using a High Performance Liquid Chromatography (HPLC), and calculating the degradation rate. As shown in FIG. 7, the degradation rate of tetracycline by R3 strain was 80.16% at a rotation speed of 140R/min, but the degradation rate was slightly decreased by increasing the rotation speed of the shaker.
(5) Effect of substrate concentration on Alcaligenes sp.R3 degradation of Tetracycline
100mL of the liquid medium was added to a 250mL conical flask and autoclaved at 121 ℃ for 25min. The tetracycline concentration in the culture medium is set to be 50, 100, 150, 200 and 250mg/L (filtered by a 0.45 mu m microporous membrane), 4% inoculation liquid is added, blank control is respectively arranged for each treatment group, each treatment group is repeated for 3 times, the culture is carried out in a shaking table at 30 ℃ and 140r/min, a certain amount of culture solution is taken for measurement after 72 hours of culture, the content of tetracycline is measured by High Performance Liquid Chromatography (HPLC), and the degradation rate is calculated. As shown in FIG. 8, the degradation rate of tetracycline is significant up to 72.01% when the substrate concentration is 50mg/L.
The degradation capability of the strain Alcaligenes sp.R3 on tetracycline is influenced by pH, temperature, rotating speed, substrate concentration and the like. The results of the above examples show that when the pH is 8, the temperature is 30 ℃, the rotating speed is 140r/min, the initial concentration of tetracycline is 50mg/L, the inoculation amount is 4%, the tetracycline degradation rate can reach 80.16% after shaking culture for 72 h.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
SEQUENCE LISTING
<110> Hunan agriculture university
<120> Alcaligenes and product and application thereof
<130> 20211130
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1375
<212> DNA
<213> Alcaligenes sp
<400> 1
cctgcagtcg acggcagcgc gagagagctt gctctcttgg cggcgagtgg cggacgggtg 60
agtaatatat cggaacgtgc ccagtagcgg gggataacta ctcgaaagag tggctaatac 120
cgcatacgcc ctacggggga aaggggggga tcgcaagacc tctcactatt ggagcggccg 180
atatcggatt agctagttgg tggggtaaag gctcaccaag gcaacgatcc gtagctggtt 240
tgagaggacg accagccaca ctgggactga gacacggccc agactcctac gggaggcagc 300
agtggggaat tttggacaat gggggaaacc ctgatccagc catcccgcgt gtatgatgaa 360
ggccttcggg ttgtaaagta cttttggcag agaagaaaag gtacctccta atacgaggta 420
ctgctgacgg tatctgcaga ataagcaccg gctaactacg tgccagcagc cgcggtaata 480
cgtagggtgc aagcgttaat cggaattact gggcgtaaag cgtgtgtagg cggttcggaa 540
agaaagatgt gaaatcccag ggctcaacct tggaactgca tttttaactg ccgagctaga 600
gtatgtcaga ggggggtaga attccacgtg tagcagtgaa atgcgtagat atgtggagga 660
ataccgatgg cgaaggcagc cccctgggat aatactgacg ctcagacacg aaagcgagca 720
aacaggatta gataccctgg tagtccacgc cctaaacgat gtcaactagc tgttggggcc 780
gttaggcctt agtagcgcag ctaacgcgtg aagttgaccg cctggggagt acggtcgcaa 840
gattaaaact caaaggaatt gacggggacc cgcacaagcg gtggatgatg tggattaatt 900
cgatgcaacg cgaaaaacct tacctaccct tgacatgtct ggaaagccga agagatttgg 960
ccgtgctcgc aagagaaccg gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg caacgagcgc aacccttgtc attagttgct acgcaagagc 1080
actctaatga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat 1140
ggcccttatg ggtagggctt cacacgtcat acaatggtcg ggacagaggg tcgccaaccc 1200
gcgaggggga gccaatctca gaaacccgat cgtagtccgg atcgcagtct gcaactcgac 1260
tgcgtgaagt cggaatcgct agtaatcgcg gatcagaatg tcgcggtgaa tacgttcccg 1320
ggtcttgtac acaccgcccg tcacaccatg ggagtgggtt tcaccagaag taggt 1375
Claims (6)
1. An Alcaligenes sp R3 strain, which is characterized in that the preservation number of the Alcaligenes sp R3 in China center for type culture Collection is CCTCC NO: m2020882.
2. Use of the alcaligenes according to claim 1 for the degradation of tetracycline.
3. The use of Alcaligenes for degrading tetracycline according to claim 2, wherein the pH of the degradation solution is 5-9 and the degradation temperature is 20-40 ℃ during the degradation of tetracycline by Alcaligenes.
4. The use of an alcaligenes bacterium for degrading tetracycline according to claim 1, wherein the concentration of the alcaligenes bacterium for degrading tetracycline is 50-250mg/L.
5. An antibiotic agent for degrading tetracycline, comprising the alcaligenes according to claim 1.
6. The antibiotic bacterial agent for degrading tetracycline according to claim 5, wherein the preparation method comprises activating the Alcaligenes with a liquid medium, and collecting the culture solution.
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