CN115141782A - Sulfamethazine degrading microbial inoculum and preparation method and application thereof - Google Patents
Sulfamethazine degrading microbial inoculum and preparation method and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention relates to the field of environmental protection. A mixed sulfadimethoxine bactericide contains Arthrobacter protoformiae (Arthrobacter protophorae) and Acinetobacter sp. The sulfadimethoxine mixed bacterial agent is derived from livestock and poultry manure and has good compatibility with native microbial flora of manure compost.
Description
Technical Field
The invention relates to the field of environmental protection, and in particular relates to a sulfadimidine degrading microbial inoculum, a preparation method thereof and application thereof in removing livestock and poultry manure antibiotics.
Background
The aquaculture industry is a major field of antibiotic use. Antibiotic residues in the breeding excrement are common, and if the antibiotic residues are not completely treated, the antibiotic residues are discharged into the environment along with wastewater and excrement, so that harm is caused to human bodies and the ecological environment. How to effectively remove antibiotic residues in livestock and poultry feces becomes an important problem to be solved urgently.
Biodegradation is an effective means for solving the problem of antibiotic environmental residue, and has attracted wide attention of scholars at home and abroad. Currently, many sulfonamide antibiotics-degrading bacteria have been reported, such as microbacterium, rhodococcus, achromobacter, ralstonia, brevundimonas, pseudomonas, acinetobacter, variovorax, alcaligenes, ochrobacterum, paenibacillus, pseudomonas, aeromonas, and alcaligenes.
In addition, compared with a single microorganism, the mixed microorganism community has great advantages, has stronger adaptability to the environment, and can better meet the current situation of compound pollution caused by the simultaneous existence of multiple antibiotics due to the mutual matching of multiple microorganism communities, so that the research on mixed bacteria degradation of antibiotics is also concerned. In particular, the mixed bacteria agent manually screened and constructed in a laboratory has obvious advantages in biodegradation of pollutants compared with natural mixed bacteria and single strains with incompletely-defined components. In addition, because the strain components of the artificially constructed mixed microbial inoculum are definite, the optimal and most reasonable production process is easier to find, the product quality is more stable, and the microbial inoculum has stronger pertinence and is safer and more reasonable in practical application. However, few reports on antibiotic-degrading mixed bacteria with definite strains are reported at present.
From the above, in order to meet the increasingly expanding requirements for microbial remediation of antibiotic pollution, different antibiotic-degrading strains can be selected according to specific application conditions such as main antibiotic residue types and compatibility with medium native microbial flora, so that more strain resources are provided for the preparation of mixed bacteria, more different strains and degrading bacteria aiming at different antibiotics are screened, and the requirement for expanding an antibiotic-degrading microbial strain library is indispensable.
Disclosure of Invention
The invention aims to provide a sulfadimethoxine mixed bacterial agent which is derived from livestock and poultry manure, has good compatibility with native microbial flora of manure compost and high sulfadimethoxine degradation rate. The invention also aims to provide a domestication and screening method of the dimethoxypyrimidine mixed fungicide and application of the domestication and screening method in livestock and poultry manure antibiotic removal.
The specific technical scheme of the invention is as follows: a sulfadimethoxine mixed bacterial preparation comprises Arthrobacter protoformiae (Arthrobacter protophorae) and Acinetobacter sp; arthrobacter protozogenes (Arthrobacter protophorae) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.25224, the preservation date is 2022 years, 7 months and 4 days, and the preservation unit address is No. 3 Beijing of No. 1 Beijing West Lu of the sunward district of Beijing; acinetobacter (Acinetobacter sp.) is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.25225, the preservation date is 2022 years, 7 months and 4 days, and the preservation unit address is No. 3 of Xilu No. 1 of Beijing Kogyo area, chaoyang district.
Further, the sulfadimethoxine mixed bacterial agent consists of Arthrobacter protoformis (Arthrobacter protophorae), acinetobacter sp, bacillus lactis (Lysinibacillus fusiformis) and Microbacterium mitrae, and is obtained by domesticating and screening livestock and poultry manure; the mixed bacterial agent is prepared by mixing various strains in equal proportion.
The domestication and screening method of the sulfadimethoxine mixed bacterial agent comprises the following steps:
1) A refrigerated pig manure sample, 50g, was weighed into a 1L triangular flask, and 450ml of sterile water (manure: water =1:9, v: v), 180rpm, shaking at room temperature for 2 hours, filter paper filtering to obtain a pig manure wastewater suspension 1, and sterilizing at 121 ℃ for 30min.
2) Mixing the refrigerated pig manure with water (100g + 100ml) according to 1:1, oscillating at room temperature for 2 hours at 180rpm, standing for 2 hours, and taking the supernatant to obtain a pig manure suspension 2.
3) 49ml of pig manure wastewater suspension 1 subjected to high-temperature sterilization treatment is added with sulfadimidine to the final concentration of 10mg/L, and 1ml of pig manure suspension 2 is added, and the mixture is subjected to shake culture at 25 ℃ and 180rpm in the dark.
4) After the completion of the culture, 100. Mu.l of the culture was spread on an LB plate containing sulfadimidine, and single colonies were randomly picked and dropped on an LB medium containing 10mg/L sulfadimidine, cultured for 5 days, and the antibiotic degradation rate was measured.
5) Selecting a strain capable of degrading antibiotics, extracting total DNA of bacteria, performing PCR amplification by using a 16SrDNA universal primer, and then performing sequencing and homology comparison to determine the species of the degrading bacteria.
6) Selecting and screening the obtained sulfadimidine degrading bacteria, and formulating different bacterium agent combination schemes; inoculating the microbial inoculum combination into the pig manure wastewater suspension according to 1 percent, adding sulfadimidine to the final concentration of 10mg/L, and performing shake culture at 180rpm and 25 ℃ in the dark; after culturing for 2 and 5 days, determining the degradation rate of the sulfadimidine; by comparing the degradation rate of the sulfadimidine, an optimal microbial inoculum combination scheme is obtained.
Compared with the prior art, the invention has the beneficial effects that:
1) The Arthrobacter protoformis (Arthrobacter protophorae), acinetobacter sp, bacillus lactis (Lysinibacillus fusiformis) and Microbacterium mitis (Microbacterium mirae) of the present invention are derived from livestock manure and have good compatibility with the native microbial flora of manure compost. The application of the mixed microbial inoculum to the treatment of fecal sewage and fecal wastewater relative to the mixed microbial inoculum of other sources such as sludge in biochemical treatment tanks in pharmaceutical factories can cause the situations of poor environmental compatibility and poor adaptation, which leads to the failure of better executing degradation function.
2) The degrading performance of the sulfonamides antibiotics of Arthrobacter protozormiae and Acinetobacter sp in the strain is not reported, and a new microbial strain resource is provided for repairing antibiotic pollution.
3) In the process of microbial inoculum mutual matching, the composite microbial inoculum prepared by mixing all the antibiotic degrading bacteria from excrement has poor removal effect on sulfadimidine in excrement, and the removal effect is only 17.7%; without optimization, the degradation rate of Arthrobacter protothecoides (Arthrobacter protophorae) and Acinetobacter (Acinetobacter sp.) on sulfadimidine in raw pig manure wastewater was 11.3% and 13.9%. But after optimization, the removal rate of the composite microbial inoculum to sulfadimidine in the excrement can be improved to 60% under the same conditions. Compared with a control without adding the microbial inoculum, the removal efficiency of the sulfadimidine in the livestock and poultry manure inoculated with the microbial inoculum is improved from 20-35 percent to 43-60 percent.
Detailed Description
The present invention will be further described with reference to the following examples.
Example (b):
1. sulfamethazine degrading strain domestication, screening and identification test
1. Test procedure
1) A refrigerated pig manure sample, 50g, was weighed into a 1L triangular flask, and 450ml of sterile water (manure: water =1, 9,v), 180rpm, shaking at room temperature for 2 hours, filter paper filtering to obtain a pig manure wastewater suspension 1, and sterilizing at 121 ℃ for 30min.
2) Mixing the refrigerated pig manure with water (100g + 100ml) according to 1:1, oscillating at room temperature for 2 hours at 180rpm, standing for 2 hours, and taking the supernatant to obtain a pig manure suspension 2.
3) 49ml of pig manure wastewater suspension 1 subjected to high-temperature sterilization treatment is added with sulfadimidine to the final concentration of 10mg/L, and 1ml of non-sterilized pig manure suspension 2 is added, and the mixture is subjected to shake culture at 25 ℃ and 180rpm in the dark.
4) Samples were taken at 2 days of culture and the sulfadimidine residual concentration was determined. And calculating the degradation rate of the sulfadimidine.
5) Taking 100 mul of pig manure treated culture (2 days), spreading the culture on an LB plate containing sulfadimidine, and culturing for 36-38 hours;
6) From the drug-resistant LB plate, 11 single strains were randomly picked and dropped into LB medium containing 10mg/L sulfadimidine, cultured for 5 days, and the antibiotic degradation rate was determined as shown in Table 1.
7) Selecting a strain capable of degrading antibiotics, extracting total DNA of bacteria, performing PCR amplification by using a 16SrDNA universal primer, performing sequencing and homology comparison, and determining the species of the degrading bacteria by combining physiological biochemistry and strain forms.
2. Test results
From the 2-day degradation rate of the sulfadimidine, the pig manure extracting solution contains flora capable of degrading the sulfadimidine, and the 2-day degradation rate is 29.6%. Further from the culture, we screened 11 sulfadimidine-resistant bacteria and identified them, respectively, arthrobacter protoformis (Arthrobacter prototrophiae), acinetobacter sp, bacillus amyloliquefaciens (Lysinibacillus fusiformis), microbacterium mitis (Microbacterium mitie), bacillus psychrophilus (Psychrobacter sp), bacillus cereus (Bacillus cereus), bacillus thuringiensis (Bacillus thuringiensis), kurthia kurthi (Kurthia populi), enterobacter shensuensis (Enterobacter kobei), bacillus butyricum (Peribacillus butyrophilus), acinetobacter loensis (Acinetobacter loffii). Wherein, the degrading performance of the Arthrobacter protoformiae (Arthrobacter protophorae) and the Acinetobacter sp (sulfadimidine) is higher than that of other strains.
TABLE 1 Sulfadimidine-degrading bacteria identification result and sulfadimidine degradation efficiency%
| Bacterial strain | SMZ degradation Rate (%) |
| Arthrobacter protophormiae | 13.90 |
| Lysinibacillus fusiformis | 7.80 |
| Microbacterium mitrae | 4.90 |
| Psychrobacillus sp. | 5.91 |
| Bacillus cereus | 5.67 |
| Bacillus thuringiensis | 7.74 |
| Kurthia populi | 10.80 |
| Enterobacter kobei | 5.78 |
| Peribacillus butanolivorans | 5.85 |
| Acinetobacter sp. | 11.25 |
| Acinetobacter lwoffii | 0 |
Arthrobacter protophoriae, namely Arthrobacter protophorus 4-11 with the preservation number of CGMCC No.25224; acinetobacter sp.SM2-1 with the preservation number of CGMCC No. 25224.
2. Sulfamethazine compound microorganism application effect test
1. Test method
1) Preparing a seed solution: transferring all strains in the table 1 into an LB liquid culture medium according to the inoculation amount of 0.5%, carrying out shaking culture at 25 ℃ and 180rpm until the OD600 value is about 0.6-1, and collecting bacterial liquid as seed liquid for later use after the culture is finished.
2) Preparing a complex microbial inoculum: mixing the strains according to equal proportion, washing the mixed strains by using sterile normal saline before use, and concentrating the mixed strains by 10 times to obtain the compound microbial inoculum for later use.
3) Pig manure wastewater: mixing pig manure and water according to a ratio of 1:9, oscillating at 180rpm and 25 ℃ for 2h, filtering with gauze, collecting filtrate, subpackaging, sterilizing with high pressure steam at 121 ℃ for 1h to obtain sterile leaching solution, and cooling for later use.
4) Inoculating the microbial inoculum to the pig manure wastewater according to the inoculation amount of 1%, culturing for 5 days, sampling, and determining the residual concentration of sulfadimidine. And calculating the degradation rate of the sulfadimidine. The inoculation concentration of all the microbial inoculum treatments is consistent.
2. Test results
After 5 days of culture, the degradation rate of sulfadimidine in the control culture wastewater is 0.1%, and the degradation rate of sulfadimidine in the microbial inoculum inoculation treatment is 17.7%.
3. Sulfadimidine composite microbial agent combination optimization test
1. Test method
1) Preparing a seed solution: transferring all strains in the table 2 into LB liquid culture medium according to the inoculation amount of 0.5%, carrying out shaking culture at 25 ℃ and 180rpm until the OD600 value is about 0.6-1, and collecting bacterial liquid as seed liquid for later use after the culture is finished.
2) Preparing a complex microbial inoculum: the strains are mixed according to the equal proportion shown in the table 2, washed by sterile normal saline before use, and then concentrated by 10 times to be used as a compound microbial inoculum for later use.
3) Pig manure wastewater: mixing pig manure with water according to a ratio of 1. Standing at room temperature for 1 day, and sterilizing with high pressure steam at 121 deg.C for 1 hr. The sterile leach solution is then cooled for use.
4) According to the inoculation amount of 1%, the microbial inoculum combination is inoculated into the pig manure wastewater, the sampling is carried out after 5 days of culture, and the residual concentration of sulfadimidine is determined. And calculating the degradation rate of the sulfadimidine. Each treatment was 3 replicates.
TABLE 2 Mixed Strain combinations
Bacillus megaterium and Bacillus proteoliticus were purchased from the market, and the other strains were derived from Table 1 and screened from pig manure.
2. Test results
As shown in Table 3, the sulfadimidine degradation rate of the microbial inoculum 2 reaches 42.8 percent, which is obviously higher than that of other microbial inoculants and single-bacterium treatment. The microbial inoculum 3 and 4 has one more strain than the microbial inoculum 2, but the degradation rate of the sulfadimidine is reduced, which is probably caused by competitive antagonism among strains.
TABLE 3 degradation rates of different combinations of agents Sulfadimidine (SMZ)%
3. Application effect evaluation test of sulfadimidine composite microbial agent combination (case 3)
1. Test method
1) Preparing a seed solution: transferring all strains in the table 1 into an LB liquid culture medium according to the inoculation amount of 0.5%, carrying out shaking culture at 25 ℃ and 180rpm until the OD600 value is about 0.6-1, and collecting bacterial liquid as seed liquid for later use after the culture is finished.
2) Preparing a complex microbial inoculum: mixing the strains according to equal proportion, washing the mixed strains by using sterile normal saline before use, and concentrating the mixed strains by 10 times to obtain the compound microbial inoculum for later use.
3) The mixed strain combinations are shown in Table 2 (same as example 1).
4) Pig manure wastewater: mixing pig manure and water according to a ratio of 1. Compared with example 2, the pig manure has different storage time in the refrigerator, and the extract is slightly different.
5) And (3) inoculating the microbial inoculum combination into the pig manure wastewater according to the inoculation amount of 1%, culturing for 2 days, sampling, and determining the residual concentration of sulfadimidine. And calculating the degradation rate of the sulfadimidine. Each treatment was 3 replicates.
2. Test results
As shown in Table 4, the 2-day sulfadimidine degradation rate of the microbial inoculum 2 treatment is 60.1%, which is still significantly higher than that of other microbial inoculum combinations.
TABLE 3 degradation rates% of different sulfadimidine-degrading bacterial agents combined with Sulfadimidine (SMZ)
| Combination of | 2 day SMZ degradation (%) |
| CK | 20.2 |
| Bacterial agent 1 | 38.0 |
| Bacterial preparation 2 | 60.1 |
| Bacterial agent 3 | 58.7 |
| Bacterial agent 4 | 32.7 |
Compared with the microbial inoculum 2, the antibiotic degradation strains in the microbial inoculants 1, 3 and 4 are more, but the sulfadimidine degradation rate of the microbial inoculants 1, 3 and 4 is greatly lower than that of the microbial inoculants 2, and further shows that unreasonable strain interworking can cause mutual influence among the strains due to competitive antagonism, and finally inhibit the application effect.
Bacillus megaterium and Bacillus proteoliticus were found to have some sulfadimidine degradation function in the early stage, but it was found by optimization that adding Bacillus megaterium or Bacillus proteoliticus to the bacterium agent 2 (Arthrobacter protophoriae, lysinibacillus fusiformis, microbacterium mitrae, acinetobacter sp.) reduces the sulfadimidine degradation function of the complex bacterium agent.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.
Claims (9)
1. A sulfadimethoxine mixed bacterial agent, which is characterized by comprising Arthrobacter protoformiae (Arthrobacter protophorae) and Acinetobacter (Acinetobacter sp.); arthrobacter protoformiae (Arthrobacter protophorae) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.25224; acinetobacter (Acinetobacter sp.) is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.25225.
2. The sulfadimethoxine mixed bacterial preparation according to claim 1, characterized by consisting of Arthrobacter protoformis (Arthrobacter protophorae), acinetobacter sp, bacillus lysimachiae (Lysinibacillus fusiformis) and Microbacterium mitis (Microbacterium mirae).
3. The sulfadimethoxine mixed bacterial preparation as claimed in claim 1, wherein the bacterial preparation is obtained by domesticating and screening Arthrobacter protoformis (Arthrobacter protophorae), acinetobacter sp, bacillus lysimachiae (Lysinibacillus fusiformis) and Microbacterium mirae (Microbacterium mirae) from livestock and poultry feces.
4. The sulfadimethoxine mixed bacterial preparation according to claim 2, wherein Arthrobacter protoformiae (Arthrobacter protophorae), acinetobacter sp., bacillus Lysinibacillus fusiformis (Lysinibacter fusiformis) and Microbacterium mirae (Microbacterium mirae) are domesticated and screened from pig manure.
5. The sulfadimethoxine mixed bacterial preparation according to claim 2, wherein the mixed bacterial preparation is prepared by mixing various strains in equal proportion.
6. The domestication and screening method of sulfadimethoxine mixed bacterial agent as claimed in claim 1, which comprises the following steps:
1) A refrigerated pig manure sample was weighed into a container, and sterile water (manure: water =1, 9,v), shaking at room temperature, filtering with filter paper to obtain a pig manure wastewater suspension 1, and sterilizing;
2) Mixing the refrigerated pig manure with water according to 1:1, oscillating at room temperature, standing, and taking the supernatant to obtain a pig manure suspension 2;
3) Taking the sterilized pig manure wastewater suspension 1, adding sulfadimidine, adding the pig manure suspension 2, and performing shake culture in a dark place;
4) After the culture is finished, coating the culture on an LB plate containing sulfadimidine, selecting a single colony to an LB culture medium containing sulfadimidine, and measuring the degradation rate of the antibiotic after culturing for a plurality of days;
5) Selecting a strain capable of degrading antibiotics, extracting total DNA of bacteria, performing PCR amplification by using a universal primer, and then performing sequencing and homology comparison to determine the species of the degrading bacteria.
7. The domestication and screening method of sulfadimethoxine mixed bacterial agent according to claim 6, further comprising a step 6) of screening the obtained sulfadimethoxine degrading bacteria, and formulating a bacterial agent combination scheme; inoculating the microbial inoculum combination into the pig manure wastewater suspension, and measuring the degradation rate of sulfadimidine after light-resistant shake culture; the sulfadimethoxine mixed bacterial preparation as claimed in claim 2 is obtained by comparing the degradation rate of sulfadimethoxine.
8. The domestication and screening method of sulfadimethoxine mixed bacterial agent according to claim 6 or 7, wherein in step 1), feces: water = 1; the sterilization temperature is not lower than 120 ℃; in the step 2), mixing the refrigerated pig manure with water 1:1; in the step 6), the microbial inoculum combination is inoculated into the pig manure wastewater suspension according to 1%.
9. The application of the sulfadimethoxine mixed bacterial agent as claimed in claim 2 in the removal of livestock and poultry feces antibiotics.
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2022
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| CN101935625A (en) * | 2010-06-12 | 2011-01-05 | 南京农业大学 | Triethylamine degrading bacterium and microbial agent produced therefrom |
| CN104328068A (en) * | 2014-09-30 | 2015-02-04 | 中南民族大学 | Arthrobacter protophormiae strain, screening method and uses thereof |
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