CN115433701A - Proteobacterium and microbial inoculum thereof and application of proteobacterium in degrading cephalosporin antibiotics - Google Patents
Proteobacterium and microbial inoculum thereof and application of proteobacterium in degrading cephalosporin antibiotics Download PDFInfo
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Abstract
The invention discloses a Proteus and a microbial inoculum thereof and application in degrading cephalosporin antibiotics, wherein the strain is Proteus (Proteus sp.), the strain is named as CW-1, and is preserved in China general microbiological culture Collection Center (CCM) at 9 months and 7 days in 2022, the strain preservation number is as follows: CGMCC No.25658. The strain CW-1 has good degradation effect, is a strain capable of degrading cephalosporin antibiotics and having strong antibiotic tolerance, has strong adaptability to cephalosporin antibiotics, and has good application prospect in bioremediation of environment polluted by cephalosporin antibiotics.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a proteus and a microbial inoculum thereof, and application of the proteus in degrading cephalosporin antibiotics.
Background
Antibiotics are now available for clinical treatment of humans and animals, or are added directly to animal feed, and their rapid development brings great benefits to humans. Antibiotics are drugs which can inhibit normal life activities of other organisms and destroy ecological balance at extremely low concentration, and can be synthesized biologically or chemically. The global antibiotic consumption is estimated to be between 10 and 20 million tons/year, because the investment of antibiotics in the cultivation process is an indispensable link in China as a large cultivation country, the annual antibiotic consumption of China exceeds 2.5 million tons, and the large antibiotic consumption country is formed, and the rapid emergence of ARB and ARG is stimulated by the use and improper discharge of antibiotics, so that the large antibiotic consumption poses a great potential threat to the environmental health and brings about no small environmental problem.
Antibiotics can be classified into sulfonamides, β -lactams, fluoroquinolones, chloramphenicol, macrolides, tetracyclines, and the like according to their chemical structures. The investigation found that the production of Chinese antibiotics in 2013 was 24.8 ten thousand tons, the total amount of consumed antibiotics was about 16.2 ten thousand tons, and the human consumption was about 48%, wherein the consumption proportion of beta-lactams was 24% at the highest. Beta-lactams are further classified into penicillins, cephalosporins and the like, and cephalosporins are currently widely used antibiotics.
Several bacteria have been reported to have a degrading effect on cephalosporin antibiotics in the literature, including Pseudomonas, sphingomonas, and Ochrobactrum. Wherein, the pseudomonads CE21 and CE22 are cultured for 24 hours under the condition that the mass concentration of the cephalexin is 1mg/L, and the degradation rates of the cephalexin can respectively reach 90 percent and 46.7 percent; the ochrobactrum is cultured for 36 hours under the condition that the mass concentration of the cefalexin is 10mg/L, and the degradation rate can reach 100 percent. The invention provides a microorganism which can resist high-concentration cephalosporin antibiotics and can rapidly degrade the cephalosporin antibiotics.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a degrading strain which can not only tolerate high-concentration cephalosporin antibiotics but also efficiently degrade cephalosporin antibiotics.
In order to achieve the above object, the present invention adopts the following technical solutions:
a strain of Proteus, the strain is Proteus sp, the strain is named as CW-1, and is preserved in China general microbiological culture Collection center (CGMCC) at 9 month and 7 days 2022, and the strain preservation number is as follows: CGMCC No.25658.
The application of the proteobacterium in degrading the cephalosporin antibiotics is disclosed, wherein the cephalosporin antibiotics are one or more of cefalexin, cefotaxime or cefaclor.
Preferably, the concentration of the cephalosporin antibiotics is 25 to 500mg/L, and the addition amount of the proteus is 0.5 to 1.5 percent; the degradation temperature is 15 to 45 ℃, and the pH is 6 to 10.
A microbial inoculum prepared by the proteus.
The application of the microbial inoculum in degrading cephalosporin antibiotics, wherein the cephalosporin antibiotics are one or more of cephalexin, cefotaxime, cephamycin or cefaclor.
Preferably, the concentration of the cephalosporin antibiotics is 25 to 500mg/L, and the addition amount of the fungicide is 0.5 to 1.5 percent; the degradation temperature is 15 to 45 ℃, and the pH value is 6 to 10.
The preparation method of the microbial inoculum comprises the following specific steps:
s1, preparing an LB liquid culture medium;
s2, inoculating the proteus to an LB liquid culture medium for culture to obtain the microbial inoculum.
Preferably, in step S1, the LB liquid medium comprises: 5.0g of NaCl5, 10.0g of peptone, 5.0g of yeast powder and deionized water to 1000mL, and sterilizing at 121 ℃ for 20 min.
Preferably, in step S2, the culture conditions are: the temperature is 20 to 40 ℃, the time is 24 to 48h, and the pH is 6 to 10.
The invention has the advantages that: the strain obtained by separation can take cephalosporin antibiotics as a unique carbon source, and the degradation rate can reach more than 90% after the cephalosporin antibiotics are cultured in an inorganic salt culture medium with the initial concentration of 200mg/L for 26 hours; after the cephalosporin antibiotics are cultured in an inorganic salt culture medium with the initial concentration of 500mg/L for 48 hours, the degradation rate can reach more than 90 percent; the strain can resist alkali growth, the pH is within the range of 6 to 10, and the degradation rate of the strain CW-1 to cephalexin can reach more than 80 percent; the strain has a good degradation effect, is a strain capable of degrading cephalosporin antibiotics and having strong antibiotic tolerance, has strong adaptability to cephalosporin antibiotics, and has a good application prospect in bioremediation of environment polluted by cephalosporin antibiotics.
Drawings
FIG. 1 is the colony morphology of the growth of strain CW-1 of the present invention;
FIG. 2 is a growth curve of the strain CW-1 of the present invention;
FIG. 3 is a graph showing the degradation profile of the strain CW-1 of the present invention for various cephalosporin antibiotics;
FIG. 4 is the effect of pH on the degradation of cefalexin by strain CW-1;
FIG. 5 is a graph showing the effect of different initial concentration conditions on CW-1 degradation of cephalexin;
FIG. 6 is the effect of temperature on the degradation of cefalexin by strain CW-1;
FIG. 7 shows the degradation effect of the microbial inoculum of the invention on actual domestic sewage.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
Separation, purification and identification of the strains:
collecting sewage of a Nanjing Tokyo domestic sewage treatment plant, transferring 1mL of the sewage into 100mL of LB liquid culture medium in a super clean bench, oscillating the sewage for 2d in a constant-temperature oscillation incubator at 30 ℃ and 150r/min, setting cephalosporin antibiotics with different mass concentration gradients in the LB liquid culture medium, and sequentially transferring and culturing the cephalosporin antibiotics every 2d until the mass concentration of the cephalosporin antibiotics in the culture medium is 50mg/L to obtain the final bacterial liquid culture solution. And (3) coating and separating the obtained final bacterial liquid culture solution by using a dilution plate method, culturing a sample at the temperature of 30 ℃ by using an LB solid culture medium, after obvious single colonies are formed on the surface, selecting a plurality of different single colonies according to the characteristics of the colony, such as shape, size, color, transparency and the like, and performing streak purification and culture. And finally determining a strain capable of efficiently degrading the cephalosporin antibiotics according to the respective degradation effects of the cephalosporin antibiotics on the cephalosporin antibiotics.
The specific components of the LB liquid medium are as follows:
5.0g of NaCl, 10.0g of peptone, 5.0g of yeast powder and deionized water to be supplemented to 1000ml, thus obtaining the LB liquid culture medium. On the basis, 1.5-2% of agar powder is additionally added to obtain the LB solid culture medium, and the LB solid culture medium is sterilized at 121 ℃ for 20 min.
After the strain is activated, the strain can grow on a flat plate made of a solid nutrient medium for 24 hours at 30 ℃ under the aerobic condition of 150r/min, a yellowish, slightly upwards-convex and opaque colony with the diameter of 0.3 to 0.5cm can be formed, and the colony map is shown in figure 1.
At present, there is no report about the degradation of beta-lactam antibiotics by Proteus at home and abroad. The strain CW-1 is a new strain with the function of degrading beta-lactam antibiotics.
Example 2
Growth curve of strain CW-1:
the strain CW-1 to be tested is inoculated into the culture solution of a large test tube, the test tube is taken out at regular time under the conditions of proper culture temperature and good ventilation state, the concentration (OD value) of bacterial liquid is measured at the wavelength of 600 nanometers, and the concentration of the bacterial liquid is in linear relation with the optical density value in a certain range. Therefore, the growth and propagation process of the bacteria can be deduced according to the OD value of the bacterial liquid; the growth curve of the strain can be drawn by plotting a set of measured OD values with their corresponding culture times, as shown in FIG. 2. The method comprises the following specific steps:
(1) Pre-culturing strains: taking 1 strain, selecting 1-ring bacterial lawn by aseptic technique, inoculating into LB liquid culture medium, and standing for 12 hr to obtain thallus culture solution; the LB liquid medium comprises the following components: 5.0g of NaCl5, 10.0g of peptone, 5.0g of yeast powder and deionized water are added to 1000mL, and sterilization is carried out for 20 min at 121 ℃;
(2) Inoculation: sucking 1ml of thallus culture solution, transferring the thallus culture solution into a 250ml triangular flask containing 100ml of sterilized LB liquid culture medium, and fully shaking to uniformly mix the thallus culture solution;
(3) Culturing: and (3) placing the triangular flask inoculated with the bacterium liquid into a shaking table for shaking culture at 30 ℃ and 200rmp, respectively culturing for 0, 2, 4, 6, 8, 10, 12, 14, 16 and 20 hours, taking 2ml of the bacterium liquid at a corresponding time, placing the bacterium liquid into a 5ml sterile centrifuge tube, immediately storing the bacterium liquid in a refrigerator, and carrying out turbidimetry on the bacterium liquid at the last moment.
As can be seen from FIG. 2, strain CW-1 reached the logarithmic growth phase at 20 h.
Example 3
The strain CW-1 has the degradation effect on cephalosporin antibiotics:
inoculating the strain CW-1 identified as Proteobacteria into inorganic salt degradation culture medium containing different cephalosporin antibiotic pollutants (cefalexin, cefaclor, cefotaxime and cefamycin) with an inoculation amount of 1%, wherein the concentration of each pollutant in the culture medium is 200mg/L, and the components of the culture medium are NaCl 1.0g/L, (NH) 4 ) 2 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，Na 2 HPO 4 •12H 2 O 3.0862g/L，MgSO 4 •7H 2 O0.2 g/L, deionized water (ultrapure water) is supplemented to 1000mL, pH is 7.0, sterilization is carried out for 20 min at 121 ℃, then culture media containing pollutants are respectively placed under the conditions of the same temperature of 30 ℃ and the same rotating speed of 150r/min for 24h, the content of each pollutant is measured, and the degradation rate is calculated, and the result is shown in figure 3.
As can be seen from FIG. 3, the degradation rate of the proteus bacteria on cefalexin, cefaclor, cefotaxime and cefamycin can reach more than 80% within 24 hours, and the degradation effect is excellent.
Example 4
Effect of pH on the degradation Effect of the strain CW-1:
inoculating bacterial suspension of the strain CW-1 into a degradation medium containing 200mg/L of cephalexin according to the inoculation amount of 1% (v/v) under the condition of different pH values, wherein the component of the degradation medium is 1.0g/L of NaCl, (NH) 4 ) 2 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，Na 2 HPO 4 •12H 2 O 3.0862g/L，MgSO 4 •7H 2 O0.2 g/L, adding deionized water (ultrapure water) to 1000ml, sterilizing at 121 deg.C for 20 min, and adjusting pH to 7.0; culturing at the same temperature of 30 deg.C and the same rotation speed of 150r/min for 24 hr, and screening out the optimum pH range, with the results shown in figure 4.
As can be seen from FIG. 4, the degradation rate of the strain CW-1 to cefalexin with a concentration of 200mg/L can reach more than 80% within a pH range of 6 to 10, the effect is remarkable, and the strain is alkali-resistant, and has a degradation rate of more than 30% under a condition of pH 11.
Example 5
Effect of different initial concentrations on the degradation effect of strain CW-1:
under the condition of different initial concentrations of the cefalexin pollutants, respectively inoculating bacterial suspensions of the strain CW-1 into a degradation culture medium according to the inoculation amount of 1% (v/v), wherein the components of the degradation culture medium are NaCl 1.0g/L, (NH) 4 ) 2 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，Na 2 HPO 4 •12H 2 O 3.0862g/L，MgSO 4 •7H 2 O0.2 g/L, adding deionized water (ultrapure water) to 1000ml, sterilizing at 121 deg.C for 20 min, and adjusting pH to 7.0; culturing at the same temperature of 30 deg.C and the same rotation speed of 150r/min for 24h, and observing the influence of different initial concentrations of cephalexin pollutant on its degradation, the results are shown in figure 5.
As can be seen from FIG. 5, the degradation rate of the strain CW-1 to cephalexin with the concentration of 200mg/L can reach more than 85% in 24 hours and can reach 100% in 48 hours, and the degradation rate to cephalexin with the concentration of 500mg/L can reach more than 90% after 48 hours and can reach 100% in 60 hours, which can show that the strain CW-1 can tolerate cephalexin with higher concentration and can degrade cephalexin with high concentration.
Example 6
Influence of temperature on the degradation effect of strain CW-1:
the strain CW-1 identified as proteobacteria is inoculated into an inorganic salt culture medium containing cefalexin in an inoculation amount of 1%, the initial concentration is 200mg/L, the pH value is 7, the strain is respectively cultured in a shaker at 15 ℃,20 ℃,25 ℃,30 ℃,35 ℃,40 ℃,45 ℃ and 150r/min, the pollutant content is measured after 24 hours, the degradation rate is calculated, and the result is shown in figure 6.
As can be seen from FIG. 6, the degradation rate of the strain CW-1 to cephalexin can reach more than 75% when the pH is 7.0 and the temperature is 15 to 45 ℃; the degradation rate can reach more than 80 percent within the temperature range of 20 to 45 ℃ and the pH value of 7.0. Therefore, the strain can still maintain excellent degradation effect at 15 ℃ or 45 ℃.
Example 7
Degradation of the microbial inoculum on actual domestic sewage:
the water quality source of the treated water is the inlet water of a cephalosporin antibiotic production sewage treatment plant with simulated water quality, the treatment capacity is 20L, the microbial inoculum prepared by proteus vulgaris is added into the sewage according to the adding amount of 1% volume ratio, the pH value of the sewage is 7-8, the activated sludge is inoculated into a control group, sampling is carried out after 24 hours, the degradation rate is measured and calculated, and the result is shown in figure 7.
Simulating the cefalexin-containing wastewater: 200mg/L of cefalexin, 20g/L of sodium chloride and NH 4 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，MgSO4 . 7H 2 O 0.2g/L。
Simulating wastewater containing cefaclor: cefaclor 200mg/L, sodium chloride 20g/L, NH 4 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，MgSO4 . 7H 2 O 0.2g/L。
Simulating wastewater containing cefotaxime: 200mg/L of cefotaxime, 20g/L of sodium chloride and NH 4 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，MgSO4 . 7H 2 O 0.2g/L。
Simulating the wastewater containing the cefuroxime: 200mg/L of cefuroxime axetil, 20g/L of sodium chloride and NH 4 SO 4 1.0g/L，KH 2 PO 4 0.5g/L，MgSO4 . 7H 2 O 0.2g/L。
As shown in figure 7, the degradation effect of the activated sludge on pollutants in the wastewater is not obvious, the removal rate is only about 10%, and the addition of the microbial inoculum produced by CW-1 can effectively remove the cephalosporin antibiotics in the wastewater, and the degradation rate is over 80% after 24 hours. Therefore, the strain CW-1 can be applied to the treatment of industrial wastewater containing cephalosporin antibiotics.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (7)
1. A strain of Proteus, which is characterized in that the Proteus (Proteus sp.) is named as CW-1 and is preserved in China general microbiological culture Collection center (CGMCC) at 9 month and 7 days 2022, and the strain preservation number is as follows: CGMCC No.25658.
2. The application of the proteus vulgaris in degrading cephalosporin antibiotics according to claim 1, wherein the cephalosporin antibiotics are one or more of cephalexin, cefotaxime, cephamycin or cefaclor, the concentration of the cephalosporin antibiotics is 25-500 mg/L, and the addition amount of the proteus vulgaris is 0.5-1.5%; the degradation temperature is 15 to 45 ℃, and the pH value is 6 to 10.
3. A microbial preparation produced by using the Bacillus proteus according to claim 1.
4. The application of the microbial inoculum of claim 3 in degrading cephalosporin antibiotics, wherein the cephalosporin antibiotics are one or more of cefalexin, cefotaxime, cephamycin or cefaclor, the concentration of the cephalosporin antibiotics is 25-500 mg/L, and the addition amount of the microbial inoculum is 0.5-1.5%; the degradation temperature is 15 to 45 ℃, and the pH is 6 to 10.
5. A preparation method of the microbial inoculum according to claim 3, which comprises the following specific steps:
s1, preparing an LB liquid culture medium;
s2, inoculating the proteus to an LB liquid culture medium for culture to obtain the microbial inoculum.
6. The method for preparing microbial inoculum according to claim 5, wherein in the step S1, the LB liquid culture medium comprises the following components: 5.0g of NaCl5, 10.0g of peptone, 5.0g of yeast powder and deionized water to make up to 1000mL.
7. The method for preparing a bacterial agent according to claim 5, wherein in step S2, the culture conditions are as follows: the temperature is 20 to 40 ℃, the time is 24 to 48h, and the pH is 6 to 10.
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