CN110591948B - Compound microbial agent for degrading quinolone antibiotics and preparation method and application thereof - Google Patents
Compound microbial agent for degrading quinolone antibiotics and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of soil organic pollutant degradation, and discloses a compound microbial agent for degrading quinolone antibiotics, and a preparation method and application thereof. The quinolone antibiotic degradation bacterial liquid, the energy supply bacterial liquid, the magnetic medium bacterial liquid, the surfactant and the fermentation medium are mixed to obtain a mixed liquid; fermenting to obtain the compound microbial agent for degrading quinolone antibiotics. The energy supply bacteria adopted by the invention enable the compound microbial agent to have high colonization and proliferation capacity in the in-situ soil environment; the adopted surfactant and magnetic medium bacteria enable the compound microbial agent to have low water tension and antibiotic polarity. The prepared compound microbial agent has high activity and strong environmental adaptability. The compound microbial agent is used for degrading quinolone antibiotics in soil, and has wide application range and high degradation efficiency.
Description
Technical Field
The invention belongs to the technical field of soil organic pollutant degradation, and particularly relates to a compound microbial agent for degrading quinolone antibiotics, and a preparation method and application thereof.
Background
Quinolone antibiotics (Quinolones, QNs) are artificially synthesized antibacterial drugs containing 4-quinolone basic structures, have the characteristics of wide antibacterial spectrum, strong antibacterial activity, no cross resistance with other antibacterial drugs, small toxic and side effects and the like, and are human and animal universal anti-infective drugs with the clinical prescription rate second to that of beta-lactams. China is a large consumer of quinolone antibiotics. A large amount of quinolone antibiotics finally enter a soil system through the ways of human or animal body discharge, pharmaceutical sewage discharge, solid waste stacking and the like, so that the detection content of the quinolone antibiotics in farmland soil in China reaches the level of mg/kg. Because the quinolone antibiotics have the characteristics of stronger biological activity and slow biodegradation, the high-content quinolone antibiotics in the soil form direct or potential pollution, and finally cause adverse effects on the health and survival of human beings.
The method for removing quinolone antibiotics in soil mainly comprises photolysis, hydrolysis and biodegradation. Wherein, the photolysis efficiency is limited by the influence of environmental light intensity, frequency and environmental factors (such as soil water content, pH, organic matter content, cation exchange capacity and the like), and the intermediate product of the hydrolysis method has biotoxicity and unobvious antibiotic removal effect, so that the photolysis and the hydrolysis are difficult to realize large-scale application. In contrast, biodegradation is favored because of its safety and ease of use. However, the efficiency of the existing microbial agent is not high, so that the degradation efficiency of the biodegradable quinolone antibiotics is low, and the technical application is difficult to realize.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of a compound microbial agent for degrading quinolone antibiotics.
The invention also aims to provide the compound microbial agent for degrading the quinolone antibiotics, which is prepared by the method.
The invention also aims to provide application of the compound microbial agent for degrading quinolone antibiotics in soil remediation.
The purpose of the invention is realized by the following technical scheme: a preparation method of a compound microbial agent for degrading quinolone antibiotics comprises the following steps: mixing quinolone antibiotic degrading bacteria liquid, energy supply bacteria liquid, magnetic medium bacteria liquid, surfactant, fermentation medium and water to obtain mixed liquid; fermenting to obtain the compound microbial agent for degrading quinolone antibiotics.
The quinolone antibiotic degrading bacteria are preferably one or more of Staphylococcus caprae (NOR-36), Candida parapsilosis (CICC 1973), Rhodococcus rhodochrous (Rhodococcus sp.) RP-11 (with the preservation number of CGMCC No.13024, and the China center for microbiological culture Collection, general microbiological culture Collection center, China institute for microbiology, institute of sciences, 3, located in the North Dynasty, west Lot 1, Nyoho, Beijing at 2016 (9.21.2016), Pseudomonas (Pseudomonas sp.) ACCC 11715, Acinetobacter (Acinetobacter sp.) ACCC 11710, and Sphingomonas (Sphingomonas sp.) ACCC 11703.
The energy supplying bacteria are preferably one or more of Corynebacterium glutamicum ACCC 04261, Bacillus pumilus ACCC 10387, Brevibacterium ammoniagenes SCTCC 100664, Corynebacterium stasis CGMCC1.844 and Corynebacterium ammoniagenes ATCC 19183.
The magnetic medium bacteria are preferably one or more than two of Pseudomonas fluorescens (CICC 23919), Sphingomonas (Sphingomonas yabuuchiae) ACCC 19937 and Burkholderia cepacia (Burkholderia cepacia) CICC 10828.
The fermentation medium comprises the following components: 22-27 g/L of molasses powder, 3-7 g/L of 45% amino acid powder, 1-3 g/L of bone meal, 1-3 g/L of peptone, 1-3 g/L of wheat bran, 2-4 g/L of potato extract, 1-3 g/L of sweet corn juice, 0.3-0.7 g/L of malt extract powder, 1-3 g/L of glucose, 1-3 g/L of dipotassium hydrogen phosphate, 0.5-1.5 g/L of potassium dihydrogen phosphate, 0.3-0.7 g/L of diammonium hydrogen phosphate, 0.2-0.4 g/L of magnesium sulfate heptahydrate, 0.05-0.15 g/L of ferric chloride, 0.05-0.15 g/L of manganese sulfate monohydrate, L, Fe g/L of manganese sulfate monohydrate3O40.5-1.5 g/L, Fe g of magnetic nanoparticles3O4@SiO21-3 g/L of magnetic nano composite particles, water as a solvent and natural pH; the following are preferred: 24.5g/L of molasses powder, 5g/L of 45% amino acid raw powder, 2g/L of bone meal (crushed crude bone meal), 2g/L of peptone, 2g/L of wheat bran, 3g/L of potato extract, 2g/L of sweet corn juice, 0.5g/L of malt extract powder, 2g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 1g/L of potassium dihydrogen phosphate, 0.5g/L of diammonium hydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate, 0.1g/L of ferric chloride, 0.1g/L, Fe g/L of manganese sulfate monohydrate3O4Magnetic nanoparticles 1g/L, Fe3O4@SiO22g/L of magnetic nano composite particles, water as a solvent and natural pH.
The mixture comprises the following components in percentage by mass: the concentration of the bacteria is (0.8-1.2) × 10118-12% cfu/mL carbostyril antibiotic degrading bacteria liquid, with the bacteria concentration of (0.8-1.2) × 10116-12% of cfu/mL energy supply bacterial liquid with a bacterial concentration of (0.8-1.2) × 10115-10% of cfu/mL magnetic medium bacterium liquid, 1-6% of surfactant, 8-15% of fermentation medium and the balance of water; the following are preferred: the concentration of the bacteria is (0.8-1.2) × 10119-12% cfu/mL carbostyril antibiotic degrading bacteria liquid with the concentration of (0.8-1.2) x 10116-10% of cfu/mL energy supply bacteria liquid, 0.8-1.2 bacteria concentration and production10115-10% of cfu/mL magnetic medium bacterium liquid, 1-6% of surfactant, 8-15% of fermentation medium and the balance of water.
More preferably, the concentration of the bacterial cells of the quinolone antibiotic-degrading bacterial solution is 1.2X 1011cfu/mL。
The bacterial concentration of the energy supply bacterial liquid is 1 multiplied by 1011cfu/mL。
The concentration of the magnetic medium bacterium liquid is 1 multiplied by 1011cfu/mL。
The surfactant is dodecyl polyoxyethylene ether (C)12EnWherein n is 4,10,23), triton X-100, coconut fatty acid diethanolamide (CDEA, formula C)11H23CON(CH2CH2OH)2) Sodium dodecyl sulfate (C)12H25NaO4S, CAS 151-21-3), sodium dodecyl sulfate (molecular formula C)12H25NaO3S，CAS:2386-53-0)、NεOne or more of-lauroyl-L-lysine sodium (LL), Tween 20 and Tween 80.
When the surfactant is a mixture formed by more than two kinds of surfactants, the components are the same in parts by weight.
The quinolone antibiotic degrading bacteria liquid is preferably obtained by a method comprising the following steps:
oscillating and culturing the quinolone antibiotic degrading bacteria to a logarithmic growth period;
secondly, continuously culturing for 4-6 days at the temperature of 30-37 ℃ and at the speed of 120-150 r/min;
or obtained by a method comprising the following steps:
A. performing shake culture on the quinolone antibiotic degrading bacteria until the logarithmic growth period is reached;
B. then carrying out acclimatization culture by using a culture medium containing quinolone antibiotics;
C. domesticating the obtained bacteria to logarithmic growth period;
D. and then continuously culturing for 4-6 days at the temperature of 30-37 ℃ and the speed of 120-150 r/min.
When the quinolone antibiotic degrading bacteria are mixed bacteria, culturing each bacteria in the quinolone antibiotic degrading bacteria to a logarithmic growth period by oscillation culture, such as the steps I, A and C; when the acclimation step is available, respectively acclimating each bacterium, as in step B; then mixing according to the equal volume ratio and continuously culturing, such as the step II and the step D.
The culture conditions in the first step and the second step are preferably culture at 28-30 ℃ and 120-150 r/min; more preferably, the culture is carried out at 28-30 ℃ and 130-150 r/min.
The culture medium in the culture in the step I and the step A is an LB liquid culture medium or an YPD liquid culture medium. LB liquid medium is suitable for the cultivation of bacteria, YPD liquid medium is suitable for the cultivation of yeast.
The composition of the culture medium containing the quinolone antibiotics in the step B is as follows: 240-440 mg/L quinolone antibiotic, 5.0g/L ammonium sulfate, 1.0g/L monopotassium phosphate, 0.5g/L magnesium sulfate and 0.2g/L yeast extract; preferably 240-440 mg/L of quinolone antibiotics, 5.0g/L of ammonium sulfate, 1.0g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and 0.2g/L of yeast extract.
The quinolone antibiotics comprise norfloxacin, ciprofloxacin and ofloxacin.
The condition of the domestication culture is preferably culture for 5-8 days at 28-30 ℃ under the condition of 120-150 r/min; more preferably, the culture is carried out for 5-8 days at 28-30 ℃ and 130-150 r/min.
The times of the domestication culture are 3-8 times; preferably 5 to 6 times.
The culture condition in the step C is preferably culture for 48-68 hours at 30-37 ℃ under the condition of 110-130 r/min; more preferably, the culture is carried out for 48-68 h under the conditions of 30-37 ℃ and 120 r/min.
The continuous culture conditions in the second step and the step D are preferably 32-37 ℃ and 130-150 r/min for 4-6 days.
The energy supplying bacterium solution is preferably obtained by the following method: culturing the energy supply bacteria by using a CM 0002 improved culture medium to a logarithmic growth period, and culturing for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min to obtain an energy supply bacteria liquid; more preferably, the culture is carried out for 5 days at the temperature of 32-37 ℃ and at the speed of 150 r/min; the composition of the CM 0002 modified medium was as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride and 10g/L, pH 7.0.0-7.5 of lactose.
When the energy supply bacteria are mixed bacteria liquid, the bacteria are respectively cultured to logarithmic growth period, then mixed according to the equal volume ratio, and cultured for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min; more preferably, the culture is carried out for 5 days at a temperature of between 32 and 37 ℃ and at a speed of 150 r/min.
The culture conditions for culturing the energy supply bacteria to the logarithmic growth phase are as follows: shake culturing at 28-30 ℃ and 130-150 r/min for 48-72 h; more preferably: shake culturing at 28-30 deg.C and 140r/min for 48-72 h.
The magnetic medium bacterium liquid is preferably obtained by the following method: culturing the magnetic medium bacteria to a logarithmic growth period by using a CM 0002 improved culture medium, and culturing for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min to obtain magnetic medium bacteria liquid; more preferably, the culture is carried out for 4-5 days at the temperature of 34-37 ℃ and at the speed of 150 r/min; the composition of the CM 0002 modified medium was as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride and 10g/L, pH 7.0.0-7.5 of lactose.
When the magnetic medium bacteria are mixed bacteria liquid, the bacteria are respectively cultured to logarithmic growth period, then mixed according to the equal volume ratio, and cultured for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min; more preferably, the culture is carried out at a temperature of between 34 and 37 ℃ and at a speed of 150r/min for 4 to 5 days.
The culture conditions for culturing the magnetic medium bacteria to the logarithmic growth phase are as follows: carrying out shake culture for 48-72 h at the temperature of 28-30 ℃ and under the condition of 130-150 r/min; more preferably: carrying out shake culture for 48-72 h at the temperature of 28-30 ℃ and the speed of 140 r/min.
The fermentation conditions are preferably that sterile air is forced to be introduced every 4 hours for 30min at the temperature of 32-37 ℃ and the speed of 100-120 r/min, the ventilation quantity is 0.5-2 times of the volume of the fermentation tank per minute, and the fermentation is continuously carried out for 5-8 days.
The fermentation medium is sterilized before adding bacteria, and the sterilization condition is preferably sterilization at 115-121 ℃ for 15-20 min.
A compound microbial agent for degrading quinolone antibiotics is prepared by the preparation method.
The compound microbial agent for degrading quinolone antibiotics is applied to soil remediation.
The soil is polluted by quinolone antibiotics.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a compound microbial agent which has high activity and strong environmental adaptability and can efficiently degrade the pollution of quinolone antibiotics.
(2) The quinolone antibiotic degrading bacteria can obviously improve the degrading, colonizing and proliferating capacities of the strains in the in-situ soil environment through energy supply bacteria companion and coupling fermentation.
(3) The addition of the surfactant and the magnetic medium bacteria is beneficial to digesting the water tension and the antibiotic polarity of the quinolone antibiotic degrading bacteria applied to soil, enhancing the affinity of the quinolone antibiotic and the degrading bacteria and accelerating the degradation of the quinolone antibiotic residue.
(4) The compound microbial agent for degrading quinolone antibiotics in the invention is used for degrading quinolone antibiotics in soil, and has wide application conditions and high degradation efficiency.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
The strain can be obtained from Sichuan province microorganism resource platform strain preservation center (SICC), China industrial microorganism strain preservation management center (CICC), American mode strain collection center (ATCC), China agricultural microorganism strain preservation management center (ACCC) and China general microorganism strain preservation management center (CGMCC); among them, the ATCC strain can be obtained from American type culture center, or from North Name.
Fe3O4Magnetic nanoparticles, Fe3O4@SiO2Magnetic nanocomposite particles are provided by sienna millennium biotechnology limited; the remaining raw materials were obtained commercially from the market.
Norfloxacin, ciprofloxacin, ofloxacin and enrofloxacin, which are purchased from Chinese pharmaceutical and biological product institute, and have the purity of 95.0 percent
LB liquid medium formula: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
YPD liquid medium formula: tryptone 20g/L, yeast powder 10g/L, glucose 10 g/L.
The formula of the fermentation initial culture medium is as follows: 24.5g/L of molasses powder, 5g/L of 45% amino acid raw powder, 2g/L of bone meal (crushed crude bone meal), 2g/L of peptone, 2g/L of wheat bran, 3g/L of potato extract, 2g/L of sweet corn juice, 0.5g/L of malt extract powder, 2g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 1g/L of potassium dihydrogen phosphate, 0.5g/L of diammonium hydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate, 0.1g/L of ferric chloride, 0.1g/L, Fe g/L of manganese sulfate monohydrate3O4Magnetic nanoparticles 1g/L, Fe3O4@SiO22g/L of magnetic nano composite particles, water as a solvent and natural pH.
Potato extract juice: peeling 200g of potatoes, removing bud eyes, cutting into small strips, putting the small strips into an aluminum pot, adding 1000mL of water, boiling for about 20-30 minutes until the potatoes are soft but not rotten, filtering by 6-8 layers of gauze, and taking filtered juice for later use.
Sweet corn juice: the juice obtained by directly squeezing sweet corn.
Liquid CM 0002 modified medium: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride and 10g/L, pH 7.0.0-7.5 of lactose.
Example 1
1. Preparation of quinolone antibiotic degrading flora
Taking degradation bacteria: staphylococcus capriae (Staphylococcus caprae) -NOR-36 (in the literature, "Paraflomine, Chenliwei, Caitamin, et al. norfloxacin degrading bacterium NOR-36 separation screening and degradation characteristics research [ J ]]Published 576-584, and Candida parapsilosis (Candid), proceedings of environmental science, 2017,37(2)a parapsilosis) CICC 1973, Rhodococcus (Rhodococcus sp.) CGMCC 13024, Pseudomonas (Pseudomonas sp.) ACCC 11715, Acinetobacter (Acinetobacter sp.) ACCC 11710 and Sphingomonas (Sphingomonas sp.) ACCC 11703, wherein the single bacteria are respectively cultured by LB liquid medium, the single yeasts are respectively cultured by YPD liquid medium to logarithmic phase, and the culture is carried out under the shaking condition of 29 ℃ and 150 r/min. Adjusting the concentration of each obtained degrading bacteria seed stock solution to make the concentration of each bacteria liquid about 1 × 1011cfu/mL, then uniformly mixing according to the equal volume ratio, placing on a shaking bed, and carrying out liquid fermentation for 6 days at 36 ℃ and 130r/min to prepare the quinolone antibiotic degradation flora with the thallus concentration of 1.2 multiplied by 1011cfu/mL。
2. Preparation of energy-supplying bacterium solution
Corynebacterium glutamicum ACCC 04261, Bacillus pumilus ACCC 10387, Brevibacterium ammoniagenes SCTCC 100664, Corynebacterium stasis CGMCC1.844 and Corynebacterium ammoniagenes ATCC 19183 which grow well on LB agar slant culture medium are respectively added into a liquid CM 0002 modified culture medium (pH7.0) with the same inoculation amount and are shake-cultured for 48h under the conditions of 28 ℃ and 140 r/min. Mixing the obtained strain seed stock solutions at equal volume ratio, placing on a shaking table, performing liquid fermentation at 37 deg.C and 150r/min for 5 days to obtain energy supplying strain solution with a thallus concentration of about 1 × 1011cfu/mL。
3. Preparation of magnetic medium bacterium liquid
Adding single strains of Pseudomonas fluorescens (Pseudomonas fluorescens) CICC 23919, Sphingomonas yanbuuchiae (Sphingomonas yabuuchiae) ACCC 19937 and Burkholderia cepacia CICC 10828 which grow well on LB agar slant culture medium into a liquid CM 0002 modified culture medium (pH7.0) with the same inoculation amount respectively, performing shake culture for 48h under the conditions of 30 ℃ and 140r/min, uniformly mixing the obtained strain seed stock solutions in an isometric ratio, placing the mixed strain seed stock solutions on a shaking bed, and performing liquid culture at the conditions of 37 ℃ and 150r/minFermenting for 4 days to obtain magnetic medium bacteria solution with a bacteria concentration of about 1 × 1011cfu/mL。
4. Preparation of compound microbial agent for degrading quinolone antibiotics
15% (w/w) of the fermentation starting medium and 50% (w/w) of tap water were added to a 3000L microbial fermenter, followed by sterilization at 121 ℃ for 20 min. When the temperature of the fermentation medium in the fermentation tank is reduced to the culture temperature, respectively inoculating 12% (w/w) of the quinolone antibiotic degradation bacteria, 10% (w/w) of the energy supply bacteria, 7% (w/w) of the magnetic medium bacteria and 6% (w/w) of a surfactant under aseptic conditions, wherein the surfactant comprises dodecyl polyoxyethylene ether (C) with equal mass fraction12EnWhere n is 4,10,23) and triton X-100, to obtain 2800L of fermentation broth. Then, the fermentation was continued for 5 days by forcibly introducing sterile air at 32 ℃ at 120r/min for 30min every 4 hours at an aeration rate of 2 times the volume of the culture broth in the fermenter. The fermentation product meets the standard of GB20287-2006 agricultural microbial inoculum through detection, and the compound microbial inoculum for degrading quinolone antibiotics is prepared.
Example 2
1. Preparation of quinolone antibiotic degrading flora
The single degrading bacteria of Staphylococcus caprae (Staphylococcus caprae) -NOR-36 and Pseudomonas (Pseudomonas sp.) ACCC 11715 were cultured in LB liquid medium to logarithmic phase respectively, and the culturing was performed at 30 ℃ under 130r/min shaking condition. Then, acclimatization: A. respectively putting 200 mu L of the cultured degradation bacterium suspension into 250mL triangular flasks, adding 100mL of sterilized liquid selective medium (440 mg/L of norfloxacin, 5.0g/L of ammonium sulfate, 1.0g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate and 0.2g/L of yeast extract) into the triangular flasks, and carrying out wild domestication on the degradation bacterium for 5 days (the domestication condition is carried out under 30 ℃ and 130r/min shaking conditions); B. repeating the step A6 times with 200 μ L wild domestication solution, shake culturing in LB liquid culture medium at 37 deg.C and 120r/min for 48h to obtain single degrading bacteria seed stock solution with thallus concentration of about 1 × 1011cfu/mL; C. mixing the seed stock solutions of the single degrading bacteriaMixing at equal volume ratio, placing in shaking table, performing liquid fermentation at 32 deg.C and 150r/min for 4 days to obtain norfloxacin degrading flora with thallus concentration of about 1.2 × 1011cfu/mL。
2. Preparation of energy-supplying bacterium solution
Adding Corynebacterium glutamicum ACCC 04261 (Corynebacterium glutamicum) with good growth on LB agar slant culture medium into CM 0002 modified culture medium (pH7.0), shake culturing at 30 deg.C and 140r/min for 72h, placing the obtained strain seed stock solution on a shaking bed, and liquid fermenting at 32 deg.C and 150r/min for 5 days to prepare energy supply strain solution with the strain concentration of about 1 × 1011cfu/mL。
3. Preparation of magnetic medium bacterium liquid
Adding Burkholderia cepacia (Burkholderia cepacia) CICC 10828 which grows well on LB agar slant culture medium into liquid CM 0002 modified culture medium (pH7.0), performing shake culture at 28 ℃ and 140r/min for 72h, placing the obtained strain seed stock solution on a shaking bed, performing liquid fermentation at 37 ℃ and 150r/min for 5 days to prepare magnetic medium strain solution with the strain concentration of about 1 × 1011cfu/mL。
4. Preparation of compound microbial agent for degrading quinolone antibiotics
8% (w/w) of the fermentation starting medium and 70% (w/w) of tap water were added to a 5000L microbial fermenter, followed by sterilization at 121 ℃ for 20 min. When the temperature of the culture medium in the fermentation tank is reduced to the required culture temperature, 10% (w/w) of the norfloxacin degrading bacteria, 6% (w/w) of the energy supply bacteria liquid, 5% (w/w) of the magnetic medium bacteria liquid and 1% (w/w) of the surfactant are respectively inoculated under aseptic conditions to obtain a fermentation liquid 4600L. Wherein the surfactant is composed of sodium dodecyl sulfate (molecular formula C) with equal mass fraction12H25NaO3S，CAS:2386-53-0)、NεSodium lauroyl-L-lysine (LL) and Tween 80. Then, the fermentation was continued for 8 days by forcing sterile air at 36 ℃ for 30min every 4h at 100r/min at an aeration rate of 1.5 times the volume of the culture broth in the fermentor. The detection of the fermentation product meets GB20287-2006 agricultural microbial agent, namely preparing the compound microbial agent for degrading norfloxacin.
Example 3
1. Preparation of quinolone antibiotic degrading flora
Single degrading bacteria of Staphylococcus caprae (Staphylococcus caprae) -NOR-36, Pseudomonas sp (Pseudomonas sp.) ACCC 11715 and Sphingomonas sp (ACCC 11703) are respectively cultured in LB liquid medium to logarithmic phase and cultured under the shaking condition of 150r/min at 29 ℃. Then, acclimatization: A. respectively placing 250 mu L of the degradation bacterium suspension subjected to enrichment culture into a 250mL triangular flask, adding 100mL of sterilized liquid selective culture medium (ciprofloxacin 345mg/L, ammonium sulfate 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L and yeast extract 0.2g/L) into the triangular flask, and carrying out wild domestication on the degradation bacterium for 8 days (the domestication condition is carried out under 29 ℃ and 150r/min shaking conditions); B. repeating the step A6 times with 250 μ L of wild domestication solution, shake culturing in LB liquid culture medium at 30 deg.C and 120r/min for 60 hr to obtain single degrading bacteria seed stock solution with thallus concentration of about 1 × 1011cfu/mL; C. mixing the seed stock solutions of the single degrading bacteria at equal volume ratio, placing in a shaking table, and performing liquid fermentation at 37 deg.C and 140r/min for 5 days to obtain ciprofloxacin degrading flora with thallus concentration of about 1.2 × 1011cfu/mL。
2. Preparation of energy-supplying bacterium solution
Respectively adding Bacillus pumilus (ACCC 10387) and Corynebacterium stasis (CGMCC 1.844) which grow well on LB agar slant culture medium into CM 0002 modified culture medium (pH7.5) with the same volume in the same inoculation amount, shake-culturing at 29 deg.C and 140r/min for 60h, mixing the obtained strain seed stock solutions uniformly in equal volume ratio, placing on a shaking bed, and performing liquid fermentation at 35 deg.C and 150r/min for 5 days to prepare energy supply strain solution with the concentration of about 1 × 1011cfu/mL。
3. Preparation of magnetic medium bacterium liquid
Good growth on LB agar slant culture mediumAdding Sphingomonas yabuuchiae ACCC 19937 and Burkholderia cepacia CICC 10828 into CM 0002 modified culture medium (pH7.5) with the same volume in the same inoculation amount respectively, performing shake culture at 29 ℃ and 140r/min for 60h, mixing the obtained strain seed stock solutions uniformly in an equal volume ratio, placing on a shaking bed, performing liquid fermentation at 34 ℃ and 150r/min for 5 days to prepare magnetic medium bacterium solution, wherein the bacterium concentration is about 1 × 1011cfu/mL。
4. Preparation of compound microbial agent for degrading quinolone antibiotics
10% (w/w) of the fermentation medium components and 57% (w/w) of tap water were added to a 3000L microbial fermenter, followed by sterilization at 121 ℃ for 20 min. When the temperature of the fermenter medium was lowered to the desired culture temperature, 9% (w/w) of the ciprofloxacin-degrading bacterial population, 8% (w/w) of the energy-supplying bacterial liquid, 10% (w/w) of the magnetic medium bacterial liquid, and 6% of a surfactant were inoculated under aseptic conditions, respectively, to obtain 2800L of a fermentation liquid. Wherein the surfactant is coconut oil fatty acid diethanolamide (CDEA, molecular formula C) with equal mass fraction11H23CON(CH2CH2OH)2) Sodium dodecyl sulfate (C)12H25NaO4S, CAS:151-21-3) and Tween 20. Then, the fermentation was continued for 7 days at 37 ℃ at 110r/min with forced ventilation of sterile air for 30min every 4h at a rate of 1 volume/min of the volume of the fermentor. The fermentation product meets the standard of GB20287-2006 agricultural microbial inoculum through detection, and the compound microbial inoculum for degrading ciprofloxacin is prepared.
Example 4
1. Preparation of quinolone antibiotic degrading flora
Taking degradation bacteria: staphylococcus caprae (Staphylococcus caprae) -NOR-36, Candida parapsilosis CICC 1973, Acinetobacter Acinetobacter sp ACCC 11710, Sphingomonas sp ACCC 11703, respectively, culturing the bacteria in LB liquid medium, culturing the yeast in YPD liquid medium to logarithmic phase, culturing at 28 deg.C, 140r/min, shakingAnd (4) performing under oscillating conditions. Then, acclimatization: A. respectively putting 200 mu L of the degradation bacteria suspension subjected to enrichment culture into 250mL triangular flasks, adding 100mL of sterilized liquid selective culture medium (240 mg/L of ofloxacin, 5.0g/L of ammonium sulfate, 1.0g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate and 0.2g/L of yeast extract) into the triangular flasks, and performing wild domestication on the degradation bacteria for 6 days (the domestication condition is carried out under the shaking condition of 28 ℃ and 140 r/min); B. repeating the step A5 times with 300 μ L wild domestication solution, shake culturing in LB liquid culture medium or YPD liquid culture medium (bacteria are cultured in LB liquid culture medium, yeast are cultured in YPD liquid culture medium) at 34 deg.C and 120r/min for 68 hr to obtain single degrading bacteria seed stock solution with thallus concentration of 1 × 1011cfu/mL; C. mixing the seed stock solutions of the single degrading bacteria at an equal volume ratio, placing on a shaking bed, and performing liquid fermentation at 36 deg.C and 140r/min for 5 days to obtain ofloxacin degrading flora with a thallus concentration of about 1.2 × 1011cfu/mL。
2. Preparation of energy-supplying bacterium solution
Adding Brevibacterium ammoniagenes SCTCC 100664, Corynebacterium stasis CGMCC1.844 and Corynebacterium ammoniagenes ATCC 19183 which grow well on LB agar slant culture medium into CM 0002 modified culture medium (pH7.5) with the same inoculation amount respectively, shake culturing at 30 deg.C and 140r/min for 72h, mixing the obtained strain seed stock solution uniformly at the same volume ratio, placing on a shaking bed, and carrying out liquid fermentation at 37 deg.C and 150r/min for 5 days to prepare energy supply strain solution with the strain concentration of about 1 × 1011cfu/mL。
3. Preparation of magnetic medium bacterium liquid
Adding Pseudomonas fluorescens (Pseudomonas fluorescens) CICC 23919, Sphingomonas yabuuchiae (Sphingomonas yabuuchae) ACCC 19937 and Burkholderia cepacia CICC 10828 which grow well on LB agar slant culture medium into CM 0002 modified culture medium (pH7.5) with the same inoculation amount respectively, performing shake culture for 72h at 30 ℃ and 140r/min, and performing shake culture on the obtained strain seed stock solutionMixing at equal volume ratio, placing on a shaking bed, fermenting at 37 deg.C and 150r/min for 5 days to obtain magnetic medium bacteria solution with thallus concentration of about 1 × 1011cfu/mL。
4. Preparation of compound microbial agent for degrading quinolone antibiotics
10% (w/w) of the fermentation medium components and 56% (w/w) of tap water were added to a 5000L microbial fermenter, followed by sterilization at 121 ℃ for 20 min. When the temperature of the culture medium in the fermentation tank is reduced to the required culture temperature, 11% (w/w) of the ofloxacin degradation flora, 10% (w/w) of the energy supply bacterium liquid, 8% (w/w) of the magnetic medium bacterium liquid and 5% (w/w) of the surfactant are respectively inoculated under aseptic conditions to obtain a fermentation liquid 4600L. Wherein the surfactant is Tween 80. And (3) forcibly introducing sterile air every 4h for 30min at 32 ℃ and 120r/min, wherein the ventilation rate is 0.5 volume/min of the volume of the fermentation tank, and continuously fermenting for 8 days. The fermentation product meets the standard of GB20287-2006 agricultural microbial agent, and the compound microbial agent for degrading ofloxacin is prepared.
Example 5
1. Preparation of quinolone antibiotic degrading flora
Staphylococcus caprae (Staphylococcus caprae) -NOR-36 was cultured in LB liquid medium to logarithmic phase, and the culture was performed at 30 ℃ under 130r/min shaking. Then, acclimatization: A. putting 200 mu L of the cultured degradation bacterium suspension into a 250mL triangular flask, adding 100mL of sterilized liquid selective medium (440 mg/L of norfloxacin, 5.0g/L of ammonium sulfate, 1.0g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate and 0.2g/L of yeast extract) into the triangular flask, and carrying out wild domestication on the degradation bacterium for 5 days (the domestication condition is carried out under 30 ℃ and 130r/min shaking conditions); B. repeating the step A6 times with 200 μ L wild domestication solution, shake culturing in LB liquid culture medium at 37 deg.C and 120r/min for 48h, and liquid fermenting at 32 deg.C and 150r/min for 4 days to obtain norfloxacin degrading bacteria with thallus concentration of 1.2 × 1011cfu/mL。
2. Preparation of energy-supplying bacterium solution
Well grown on LB agar slant culture mediumAdding good Corynebacterium glutamicum (Corynebacterium glutamicum) ACCC 04261 into CM 0002 modified medium (pH7.0), shake culturing at 30 deg.C and 140r/min for 72h, placing the obtained strain seed stock solution on a shaking table, and performing liquid fermentation at 32 deg.C and 150r/min for 5 days to obtain energy supply strain solution with a strain concentration of about 1 × 1011cfu/mL。
3. Preparation of magnetic medium bacterium liquid
Adding Burkholderia cepacia (Burkholderia cepacia) CICC 10828 which grows well on LB agar slant culture medium into liquid CM 0002 modified culture medium (pH7.0), performing shake culture at 28 ℃ and 140r/min for 72h, placing the obtained strain seed stock solution on a shaking bed, performing liquid fermentation at 37 ℃ and 150r/min for 5 days to prepare magnetic medium strain solution with the strain concentration of about 1 × 1011cfu/mL。
4. Preparation of compound microbial agent for degrading quinolone antibiotics
8% (w/w) of the fermentation starting medium and 70% (w/w) of tap water were added to a 5000L microbial fermenter, followed by sterilization at 121 ℃ for 20 min. When the temperature of the culture medium in the fermentation tank is reduced to the required culture temperature, 10% (w/w) of the norfloxacin degrading bacteria, 6% (w/w) of the energy supply bacteria liquid, 5% (w/w) of the magnetic medium bacteria liquid and 1% (w/w) of the surfactant are respectively inoculated under aseptic conditions to obtain a fermentation liquid 4600L. Wherein the surfactant is composed of sodium dodecyl sulfate (molecular formula C) with equal mass fraction12H25NaO3S，CAS:2386-53-0)、NεSodium lauroyl-L-lysine (LL) and Tween 80. Then, the fermentation was continued for 8 days by forcing sterile air at 36 ℃ for 30min every 4h at 100r/min at an aeration rate of 1.5 times the volume of the culture broth in the fermentor. The fermentation product meets the standard of GB20287-2006 agricultural microbial inoculum through detection, and the compound microbial inoculum for degrading norfloxacin is prepared.
Effects of the embodiment
With methanol as a solvent, 200mg/L of standard norfloxacin (Nomoxacin, NOR), ciprofloxacin (CIProoxacin, CIP), Ofloxacin (FOL) and Enrofloxacin (ENR) solutions were prepared, respectively. And (3) screening a fresh soil sample by a 4mm sieve, taking 100kg of the screened soil sample, and then placing the soil sample at room temperature for 3 days. After the microorganisms are activated, the prepared four groups of solutions are respectively added into the fresh soil, so that the substrate concentrations of norfloxacin, ciprofloxacin, ofloxacin and enrofloxacin in the soil are all 50 mg/kg. After the methanol is completely volatilized, the fresh soil is respectively poured into 27 plastic barrels of 5L, and each barrel contains 3kg of fresh soil. 3 plastic barrels are used as a processing group, and 9 processing groups are arranged in the test:
treatment group 1, no microbial inoculum was added;
treating group 2, applying the degrading flora obtained in step 1 of example 1 in an inoculation amount of 5% (w/w);
treating group 3, applying the compound microbial agent obtained in step 4 in example 1, wherein the inoculation amount is 5% (w/w);
treating group 4, applying the degrading flora obtained in step 1 of example 2, with an inoculum size of 5% (w/w);
treating group 5, applying the compound microbial agent obtained in step 4 in example 2, wherein the inoculation amount is 5% (w/w);
treating group 6, applying the degrading flora obtained in step 1 of example 3, with an inoculum size of 5% (w/w);
treating group 7, and applying the compound microbial agent obtained in the step 4 in the example 3, wherein the inoculation amount is 5% (w/w);
treating group 8, applying the degrading flora obtained in step 1 of example 4, with an inoculum size of 5% (w/w);
treatment group 9 was supplemented with the complex microbial inoculum obtained in step 4 of example 4 in an amount of 5% (w/w).
Treating group 10, applying the degrading flora obtained in step 1 of example 5, with an inoculum size of 5% (w/w);
treatment group 11 was supplemented with the complex microbial inoculum obtained in step 4 of example 5 in an amount of 5% (w/w).
After fresh soil is subpackaged, the water content of the soil is adjusted to be 40% of the saturated water holding capacity, the soil surface is covered by a wet cloth, and the soil is cultured at room temperature (20-25 ℃). And collecting each treated soil sample after 5 days, and detecting the content of norfloxacin, ciprofloxacin, ofloxacin and enrofloxacin in each treated soil.
The detection method refers to the method described in Ziziphus Helianthi et al (Ziziphus Helianthi, Modiadzuvine, Liyanwen, et al. solid phase extraction-high performance liquid chromatography-fluorescence detection of quinolone antibiotics [ J ] in soil, analytical chemistry, 2009,37(12): 1733-.
As shown in table 1, the degradation rate of the quinolone antibiotics in the soil after the addition of the degrading bacteria obtained in step 1 in examples 1, 2, 3, 4, and 5 was:
norfloxacin: 63.3-80.5%; ciprofloxacin: 62.0-82.0%;
ofloxacin: 62.3 to 78.7 percent; enrofloxacin: 61.6-70.3%;
the above differences reach a very significant level (P < 0.01).
After the compound microbial agent obtained in step 4 of examples 1, 2, 3, 4 and 5 is applied, the degradation rate of the quinolone antibiotics in the soil is as follows:
norfloxacin: 80.3-95.9%; ciprofloxacin: 82.8-97.6%;
ofloxacin: 84.1-98.1%; enrofloxacin: 79.9 to 93.5 percent;
the difference reaches a very significant level (P < 0.01).
Therefore, the prepared quinolone antibiotic degrading flora has an obvious degrading effect on the quinolone antibiotic, and the addition of the energy supply flora, the magnetic medium flora and the surfactant can further improve the degrading rate of the quinolone antibiotic by 15-20%.
TABLE 1 degradation Effect (mg/kg) of the products of the different examples on 4 quinolone antibiotics
Note: the data in the table are the average of the measurements of three soil samples, with different lower case letters after the same row number indicating significant inter-treatment variability (P <0.05) and different upper case letters indicating significant inter-treatment variability (P < 0.01).
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 (9)
1. A preparation method of a compound microbial agent for degrading quinolone antibiotics is characterized by comprising the following steps: mixing quinolone antibiotic degrading bacteria liquid, energy supply bacteria liquid, magnetic medium bacteria liquid, surfactant, fermentation medium and water to obtain mixed liquid; fermenting to obtain the compound microbial agent for degrading quinolone antibiotics;
the quinolone antibiotic degrading bacteria are goat staphylococcus (Staphylococcus caprae:Staphylococcus caprae) -NOR-36; or Candida parapsilosis: (Candida parapsilosis) CICC 1973, Rhodococcus (Rhodococcus spRP-11, Pseudomonas bacteria (A), (B)Pseudomonassp.) ACCC 11715, Acinetobacter (Acinetobacter: (A)Acinetobactersp.) ACCC 11710 and Sphingomonas (Sphingomonassp.) one or more of ACCC 11703 and Staphylococcus caprae ((II)Staphylococcus caprae) -a combination of bacteria obtained by NOR-36;
said Rhodococcus (A), (B), (C) and (C)Rhodococcus spThe preservation number of RP-11 is CGMCC No.13024, and the general microorganism center of the China microorganism culture preservation management committee of the institute of microbiology, No. 3 of Ministry of China, West Lu 1, Beijing, Chaoyang, is preserved in 2016, 9 and 21 days;
the energy supply bacteria are corynebacterium glutamicum (C.) (Corynebacterium glutamicum) ACCC 04261; or Bacillus pumilus (B), (B)Bacilluspumilus) ACCC 10387, Brevibacterium ammoniagenes: (Brevibacteriumammoniagenes) SCTCC 100664, Corynebacterium parvum (A. RTM.), (B. RTM.), (Corynebacterium stationis) CGMCC1.844 and Corynebacterium ammoniagenes (C)Corynebacterium ammoniagenes) One or more than two of ATCC 19183 and Corynebacterium glutamicum ((C.))Corynebacterium glutamicum) A combination bacterium obtained by combining ACCC 04261; or Bacillus pumilus (B), (B)Bacilluspumilus) ACCC 10387 and Corynebacterium parvum (A), (B)Corynebacterium stationis) CGMCC 1.844; or Brevibacterium ammoniagenes (Brevibacteriumammoniagenes) SCTCC 100664, Corynebacterium parvum (A. RTM.), (B. RTM.), (Corynebacterium stationis) CGMCC1.844 and Corynebacterium ammoniagenes (C)Corynebacterium ammoniagenes) A combination bacterium formed by ATCC 19183;
the magnetic medium bacteria is Burkholderia cepacia (Burkholderia cepacia) (Burkholderia cepacia)Burkholderia cepacia) CICC 10828; or Pseudomonas fluorescens (Pseudomonas fluorescens) CICC 23919 and Sphingomonas bacterium (II)Sphingomonas yabuuchiae) One or more than two combined bacteria of ACCC 19937;
the fermentation medium comprises the following components: 22-27 g/L of molasses powder, 3-7 g/L of 45% amino acid powder, 1-3 g/L of bone meal, 1-3 g/L of peptone, 1-3 g/L of wheat bran, 2-4 g/L of potato extract, 1-3 g/L of sweet corn juice, 0.3-0.7 g/L of malt extract powder, 1-3 g/L of glucose, 1-3 g/L of dipotassium hydrogen phosphate, 0.5-1.5 g/L of potassium dihydrogen phosphate, 0.3-0.7 g/L of diammonium hydrogen phosphate, 0.2-0.4 g/L of magnesium sulfate heptahydrate, 0.05-0.15 g/L of ferric chloride, 0.05-0.15 g/L of manganese sulfate monohydrate, L, Fe g/L of manganese sulfate monohydrate3O40.5-1.5 g/L, Fe g of magnetic nanoparticles3O4@SiO21-3 g/L of magnetic nano composite particles, water as a solvent and natural pH.
2. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 1, wherein the complex microbial inoculant comprises: the surfactant is dodecyl polyoxyethylene ether, Triton X-100, coconut oil fatty acid diethanolamide, sodium dodecyl sulfate, Nε-dodecanoyl-LOne or more of sodium lysinate, tween 20 and tween 80.
3. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 2, wherein: when the number of the surfactants is more than two, the various surfactants are mixed according to equal parts by mass.
4. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 1, wherein the complex microbial inoculant comprises: the mixture comprises the following components in percentage by mass: the concentration of the bacteria is (0.8-1.2) × 10118-12% cfu/mL carbostyril antibiotic degrading bacteria liquid, with the bacteria concentration of (0.8-1.2) × 10116-12% of cfu/mL energy supply bacterial liquid with a bacterial concentration of (0.8-1.2) × 10115-10% of cfu/mL magnetic medium bacterium liquid, 1-6% of surfactant, 8-15% of fermentation medium and the balance of water.
5. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 1, wherein the complex microbial inoculant comprises:
the quinolone antibiotic degrading bacterium liquid is prepared by the following steps:
oscillating and culturing the quinolone antibiotic degrading bacteria to a logarithmic growth period;
secondly, continuously culturing for 4-6 days at the temperature of 30-37 ℃ and at the speed of 120-150 r/min;
or is prepared by the following steps:
A. performing shake culture on the quinolone antibiotic degrading bacteria until the logarithmic growth period is reached;
B. then carrying out acclimatization culture by using a culture medium containing quinolone antibiotics;
C. domesticating the obtained bacteria to logarithmic growth period;
D. then continuously culturing for 4-6 days at the temperature of 30-37 ℃ and under the condition of 120-150 r/min;
the composition of the culture medium containing the quinolone antibiotics in the step B is as follows: 240-440 mg/L quinolone antibiotic, 5.0g/L ammonium sulfate, 1.0g/L monopotassium phosphate, 0.5g/L magnesium sulfate and 0.2g/L yeast extract;
the energy supply bacterium liquid is obtained by the following method: culturing the energy supply bacteria by using a CM 0002 improved culture medium to a logarithmic growth period, and culturing for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min to obtain an energy supply bacteria liquid; the composition of the CM 0002 modified medium was as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride and 10g/L, pH 7.0.0-7.5 g/L of lactose;
the magnetic medium bacterium liquid is obtained by the following method: culturing the magnetic medium bacteria to a logarithmic growth period by using a CM 0002 improved culture medium, and then culturing for 4-5 days at the temperature of 32-37 ℃ and at the speed of 130-150 r/min to obtain magnetic medium bacteria liquid.
6. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 5, wherein the complex microbial inoculant comprises:
culturing for 48-72 hours at the temperature of 28-30 ℃ and at the speed of 120-150 r/min;
the culture medium in the culture in the step I and the step A is an LB liquid culture medium or an YPD liquid culture medium; LB liquid culture medium is used for culturing bacteria, YPD liquid culture medium is used for culturing yeast;
the domestication culture in the step B is carried out for 5-8 days at the temperature of 28-30 ℃ and at the speed of 120-150 r/min;
the culture condition in the step C is that the culture is carried out for 48-68 hours at the temperature of 30-37 ℃ and at the speed of 120 r/min;
step two and step D in the condition of continuous culture is to continue to culture for 4-6 days at 32-37 ℃ under the condition of 130-150 r/min;
the culture conditions for culturing the energy supply bacteria to the logarithmic growth phase are as follows: shake culturing at 28-30 ℃ and 130-150 r/min for 48-72 h;
the culture conditions for culturing the magnetic medium bacteria to the logarithmic growth phase are as follows: shake culturing at 28-30 deg.C and 130-150 r/min for 48-72 h.
7. The method for preparing a complex microbial inoculant for degrading quinolone antibiotics according to claim 1, wherein the complex microbial inoculant comprises: the fermentation conditions are that sterile air is forcibly introduced every 4 hours for 30min at the temperature of 32-37 ℃ and the speed of 100-120 r/min, the ventilation quantity is 0.5-2 times of the volume of the fermentation tank per minute, and the continuous fermentation is carried out for 5-8 days.
8. A compound microbial agent for degrading quinolone antibiotics is characterized in that: obtained by the preparation method of any one of claims 1 to 7.
9. The use of the complex microbial inoculant for degrading a quinolone antibiotic according to claim 8 in soil remediation.
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