CN115960783A - Anaerobic microorganism combination with sulfonamide antibiotic degradation function and application thereof - Google Patents
Anaerobic microorganism combination with sulfonamide antibiotic degradation function and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of antibiotic degradation treatment, and particularly relates to an anaerobic microorganism combination with a sulfonamide antibiotic degradation function and application thereof. The present invention provides two anaerobic microorganisms, namely Paracistriumbifermentans J2 and Clostridium sp.J2, for sulfonamide antibiotic treatment. The two anaerobic microorganisms can degrade the sulfonamide antibiotics, and the removal effect of the sulfonamide antibiotics can be enhanced through the combination of the two anaerobic microorganisms. In addition, after the Paracistriumbifermentans J2 and/or Clostridium sp.J2, sodium alginate and charcoal are prepared into the microbial immobilized bacteria balls, the microbial immobilized bacteria balls can still be quickly started in a short time, and the sulfonamide antibiotics can be efficiently removed, so that a new way is provided for the degradation of the sulfonamide antibiotics and the treatment of pharmaceutical wastewater of the sulfonamide antibiotics.
Description
Technical Field
The invention belongs to the technical field of antibiotic degradation treatment, and particularly relates to an anaerobic microorganism combination with a sulfonamide antibiotic degradation function and application thereof.
Background
Antibiotics have been widely used as antibacterial agents for preventing and treating diseases of human beings and animals for a long time, and also have been used as feed additives for promoting the growth of animals in the process of animal breeding, and are one of the most widely used drugs in the world. Statistically, the yield and sales of sulfa antibiotic drugs are increasing year by year, with sulfamethoxazole being the most common sulfa antibiotic. However, sulfamethoxazole is not completely absorbed by human/animals, and more than 85% of the antibiotics are discharged into the environment in the form of raw or metabolites via the feces and urine of patients and livestock and poultry, and pollute soil and water through different ways. Meanwhile, because the sulfonamide antibiotics have high solubility and fluidity and are easy to migrate and distribute in water environment, the sulfonamide antibiotics are easier to exist in the environment for a long time than other antibiotics due to higher stability and weaker adsorbability.
At present, the method for treating antibiotics in wastewater mainly comprises a physical method, a chemical method and a biological method. Among them, the conventional physical and chemical methods mainly include precipitation, ion exchange, adsorption, etc., but their use is limited due to their high cost. The microbiological method has the advantages of good effect, low cost and the like, and is widely concerned by students. At present, sewage treatment plant often uses aerobic microorganism to handle waste water, but this process can produce a large amount of excess sludge, and the running cost is too high, and anaerobic microorganism has the energy consumption low, and excess sludge output low grade advantage has obtained extensive concern in waste water treatment. Therefore, the method has wide application prospect in treating the wastewater by adopting the anaerobic microorganisms.
In addition, anaerobic microorganisms are immobilized by a microorganism immobilization method, and pollutants in an environmental medium are adsorbed and biologically converted by virtue of the metabolic process of the microorganisms. The method has the outstanding advantages of quick start, toxicity resistance, strong adaptability, easy solid-liquid separation, quick precipitation, small and light treatment process equipment, small occupied area and the like, and has attracted wide attention of domestic and foreign scholars in recent years.
Therefore, the discovery of the anaerobic bacteria capable of efficiently degrading the sulfonamide antibiotics and the application thereof have important significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides two anaerobic microorganisms for treating the sulfonamide antibiotics, namely Clostridium paratungensis J2 (Clostridium bifermentans J2) and bacillus cereus J2 (Clostridium sp.j2), and the two strains or the combination of the two strains have the remarkable advantages of high degradation efficiency, short treatment period and the like.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the invention provides the use of paracrystalline bifermentates j2 and/or Clostridium sp.j2 for degrading antibiotics of the sulphonamide type.
In a second aspect, the invention provides the use of parastryiumbifermentans j2 and/or Clostridium sp.j2 in the treatment of pharmaceutical wastewater containing a sulfonamide antibiotic.
The invention provides two sulfonamide antibiotic degrading bacteria, wherein one is a Clostridium parahaemolyticum strain J2, and the other is a bacillus cereus strain Clostridia sp.J2. The two strains are obtained by carrying out long-term domestication on sulfate-reduced sludge collected in a sulfur-mediated bioreactor and then carrying out screening, separation and purification for multiple times. The deposit information of ParacistriumbifertinansJ 2 is: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No:62845, deposit address: the microbiological research institute of Guangdong province, no. 59 building, no. 5 building, guangdong province, of the Fuli Zhonglu 100, guangzhou city, the preservation date: 9/28/2022. Deposit information for Clostridium sp.j2 is: the preservation unit: guangdong province microorganism culture Collection (GDMCC), preservation number: GDMCC No:62934, deposit address: the microbiological research institute of Guangdong province, no. 59 building, no. 5 building, guangdong province, of the Fuli Zhonglu 100, guangzhou city, the preservation date: 10/28/2022.
Preferably, the sulfa antibiotic comprises sulfamethoxazole.
More preferably, the concentration of sulfamethoxazole is not more than 10mg/L.
The removal rate of 1mg/L, 2mg/L and 5mg/L sulfamethoxazole in 72 hours by Paracistriumbifermentans J2 is respectively 53.0%, 52.6% and 44.25% through a sulfamethoxazole removal experiment; the removal rates of 1mg/L, 2mg/L and 5mg/L sulfamethoxazole in 72 hours by Clostridia sp.J2 are 34.7%, 31.2% and 24.92%, respectively, and the removal rates of 1mg/L, 2mg/L and 5mg/L sulfamethoxazole in 24 hours are 68.7%, 69.5% and 64.8% when the two microorganisms are treated in combination (the total inoculation amount is unchanged), which proves that Parastrium bifermentans J2 and Clostridia sp.J2 have better tolerance and degradability to sulfamethoxazole.
Preferably, the paracoccidentalis bifermentations and j2 and/or Clostridium sp.j2 are made into immobilized bacteria pellets before use.
More preferably, bacterial suspension and bacterial suspension of Parastrikiumbifermentans J2Adding rice straw biochar into Clostridium sp.J2 bacterial solution, mixing the biochar-microorganism solution with sodium alginate solution after shaking culture, and dripping the obtained mixed solution into CaCl which is continuously stirred 2 In the solution, gel beads are formed after cross-linking.
Sulfamethoxazole removal experiments show that the immobilized microbial sphere can be quickly started in a short time and efficiently removed, wherein the removal rate of the sodium alginate-charcoal-combined microbial sphere on the sulfamethoxazole in 24 hours is as high as 26.61%, and the immobilized microbial sphere is proved to have better tolerance and removal effect on the sulfamethoxazole.
More preferably, the mass concentration of the sodium alginate solution is 3 percent, and the CaCl is added 2 The mass concentration of the solution is 5%, and the concentration of the bacterial liquid is OD 600 1.5-2; the using amount ratio of the straw biochar to the bacterial liquid is 0.4-0.6g/50mL, the volume ratio of the sodium alginate solution to the bacterial liquid is 1 2 The volume ratio of the solution is 1.
More preferably, the shaking culture time is 1-3h; the time for crosslinking does not exceed 1 hour.
More preferably, the liquid medium used for cultivating the bacterial Parastribiumpormenzans J2 broth and/or the bacterial Clostridium sp.J2 broth comprises Na 2 SO 4 ，NH 4 Cl，CaCl 2 ·6H 2 O，MgSO 4 ·7H 2 O，KH 2 PO 4 Yeast extract, CH 3 COONa，FeSO 4 ·7H 2 O，C 3 H 8 ClNO 2 S·H 2 O (L-cysteine hydrochloride hydrate), C 6 H 7 O 6 Na (ascorbic acid).
Further, liquid medium used (per 1000 mL): 1000mg Na 2 SO 4 ，1300mg NH 4 Cl，100mg CaCl 2 ·6H 2 O，2000mg MgSO 4 ·7H 2 O，550mg KH 2 PO 4 1000mg of yeast extract, 1300mg of CH 3 COONa，200mg FeSO 4 ·7H 2 O，500mg C 3 H 8 ClNO 2 S·H 2 O (L-cysteine)Hydrochloride hydrate of hydrochloric acid), 100mg of C 6 H 7 O 6 Na (ascorbic acid) and the balance ultrapure water.
More preferably, the culture environment of the paracoccidentalis bifermentans j2 and/or Clostridium sp.j2 is: standing at constant temperature of 35 deg.C under anaerobic condition, and initial pH value is 7.0-7.5.
In a third aspect of the present invention, a microbial agent for degrading a sulfonamide antibiotic, wherein the microbial agent comprises paracssiumbifermentations j2 and/or Clostridium sp.j2 as a main active ingredient.
Preferably, the microbial inoculum further comprises auxiliary materials acceptable in the microbial inoculum field, such as liquid culture medium and other fillers or carriers.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains two anaerobic bacteria after carrying out long-term domestication on sulfate reduction sludge collected in a sulfur-mediated bioreactor and carrying out multiple screening, separation and purification, wherein one anaerobic bacterium is a Clostridium parahaemolyticum strain named as Paraclostridium bifermentans J2, and the other anaerobic bacterium is a bacillus cereus named as Clostridium sp.J2. The two anaerobic microorganisms can degrade the sulfonamide antibiotics, can enhance the removal effect of the sulfonamide antibiotics through the combination of the two anaerobic microorganisms, and can be applied to the pharmaceutical wastewater of the sulfonamide antibiotics treated by the biological anaerobic method. In addition, after the Paracistriumbifermentations J2 and/or Clostridium sp.J2, sodium alginate and charcoal are prepared into the microbial immobilized bacteria balls, the microbial immobilized bacteria balls can still be quickly started in a short time, and the sulfonamide antibiotics can be efficiently removed. The invention provides a new way for the degradation of the sulfonamide antibiotics and the treatment of the pharmaceutical wastewater of the sulfonamide antibiotics.
Drawings
FIG. 1 shows the effect of different microbial treatments on the removal of sulfamethoxazole antibiotics;
FIG. 2 shows the effect of rice straw charcoal on the removal of sulfamethoxazole antibiotic;
FIG. 3 is a diagram of sodium alginate-biochar-microorganism immobilized bacteria beads;
FIG. 4 shows the effect of different immobilized beads on the removal of sulfamethoxazole antibiotic (a, b, c, d are used to distinguish different experimental groups).
Detailed Description
The following further describes embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
EXAMPLE 1 acquisition of anaerobic microorganisms
The composition of the liquid medium used in this example is as follows:
every 1000mL of medium consisted of: 1000mg Na 2 SO 4 ，1300mg NH 4 Cl，100mg CaCl 2 ·6H 2 O，2000mg MgSO 4 ·7H 2 O，550mg KH 2 PO 4 1000mg of yeast extract, 1300mg of CH 3 COONa，200mg FeSO 4 ·7H 2 O，500mg C 3 H 8 ClNO 2 S·H 2 O (L-cysteine hydrochloride hydrate), 100mg C 6 H 7 O 6 Na (ascorbic acid) and the balance ultrapure water.
The method for screening and obtaining the anaerobic microorganisms comprises the following steps:
(1) Screening enrichment
Pouring 100mL of a sterile liquid culture medium with sulfamethoxazole concentration of 10mg/L into a serum bottle, taking 10mL of uniformly mixed sludge (sulfate-reduced sludge) from a sulfur-mediated bioreactor for processing sulfamethoxazole, inoculating the sludge into the 100mL serum bottle, fully mixing and shaking the sludge, placing the sludge in a constant-temperature biological incubator at 35 ℃, and standing and culturing the sludge for 10 days to obtain the bacterial liquid after the first screening and enrichment. And then taking 10mL of supernatant from the bacteria liquid enriched by the first screening, transferring the supernatant into a 100mL serum bottle, filling 100mL of sterilization liquid culture medium with sulfamethoxazole concentration of 10mg/L in the serum bottle in advance, placing the mixture into a constant-temperature biological incubator at 35 ℃ after fully mixing and shaking, and then standing and culturing for 10 days to obtain the bacteria liquid enriched by the second screening. Then, the concentration of sulfamethoxazole in the liquid medium is increased by a concentration gradient of 10mg/L, the above operations are repeated three times until the concentration of sulfamethoxazole in the medium is 50mg/L, and the mixture is statically cultured in an incubator at 35 ℃ for 10 days, so that the final mixed bacterial liquid is obtained.
(2) Gradient dilution
Taking 1mL of the mixed bacterial liquid obtained in the step (1), using 9 anaerobic tubes according to the proportion of 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 The gradient of (c) is subjected to a gradient dilution.
(3) Purification and enrichment
And (3) respectively arranging three parallel samples on the 9 mixed bacteria liquid subjected to gradient dilution obtained in the step (2) for separation and purification: 0.1mL of the mixed bacterial solution was plated to uniformly coat the bacterial solution on a sterilized solid medium (1.5% agar was added to the liquid medium, sulfamethoxazole concentration was 20mg/L, and the remaining components were the same as those in the liquid medium), and the mixture was subjected to inverted culture in a constant temperature anaerobic incubator at 35 ℃ for 7 days.
A dilution gradient (10) with a moderate number of colonies and with the colonies clearly visible was selected 4 ) And (3) purifying and separating the corresponding plate culture medium: selecting dominant bacterial colonies with obvious growth difference characteristics on a culture medium, inoculating the dominant bacterial colonies to 100mL of sterile liquid culture medium with the sulfamethoxazole concentration of 20mg/L, fully mixing, shaking uniformly, placing the mixture in a constant-temperature incubator at 35 ℃ for static culture, and culturing for 10 days to show that black flocculent bacteria exist in a serum bottle.
(4) Further purifying and enriching
Taking the bacterial liquid obtained in the step (3), spreading on a sterilized solid culture medium (added with 1.8% agar, the sulfamethoxazole concentration is 20mg/L, and the other components are the same as the liquid culture medium), and inversely culturing in a constant-temperature anaerobic incubator at 35 ℃ for 7 days.
Then, the bacterial colony with better growth state on the plate culture medium is inoculated into a 100mL serum bottle for enrichment culture, the serum bottle is filled with 100mL of sterilization culture medium (the concentration of sulfamethoxazole is 20 mg/L) in advance, and the bacterial colony is placed in a constant temperature incubator at 35 ℃ for static culture for 7 days after being fully mixed, shaken uniformly. Repeating the operation for three times until the growth forms of the colonies on the plates are basically consistent, and judging that the bacteria can tolerate sulfamethoxazole.
And (3) carrying out 16S rRNA sequencing detection and NCBI database pairing on the two purified sulfamethoxazole degrading pure bacteria. The purified strains were highly similar to the strains in the table below.
| Chinese name of genus | Genus of highest degree of similarity | Degree of similarity (%) |
| Clostridium paradoxum | Paraclostridiumbifermentans | 99.66 |
| Bacillus cereus | Clostridium sp. | 100 |
Finally, the two strains are named as Clostridium parahaemolyticum J2 (Clostridium J2) and Bacillus cereus J2 (Clostridium J2) respectively, and are sent to a strain collection center for preservation.
The deposit information of Clostridium parapsilosis J2 is as follows:
the generic name: paracistriumbifertinans J2;
the preservation number is: GDMCC No:62845;
the preservation date is as follows: 9/28/2022;
the name of the depository: guangdong province microbial strain preservation center;
the address of the depository: building No. 59, building No. 5 of the prefecture midroad No. 100 yard in Guangzhou city.
The deposit information for Bacillus cereus J2 is as follows:
the name of the genus: js 2;
the preservation number is as follows: GDMCC No:62934;
the preservation date is as follows: 10 months and 28 days 2022;
the name of the depository: guangdong province microbial strain preservation center;
the address of the depository: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
The 16S rRNA sequence of Paracistriumbifermentans J2 (SEQ ID NO. 1) is shown below:
AGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGATCTCTTCGGAGAGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCTGTACACACGGATAACATACCGAAAGGTATACTAATACGGGATAACATATAAAAGTCGCATGGCTTTTGTATCAAAGCTCCGGCGGTACAGGATGGACCCGCGTCTGATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGAATTACTGGGCGTAAAGGGTGAGTAGGTGGTTTTTTAAGTCAGAAGTGAAAGGCTACGGCTCAACCGTAGTAAGCTTTTGAAACTAGAGAACTTGAGTGCAGGAGAGGAGAGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAATACCAGTAGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACTAGTGTCGGGGGTTGCCCCCCTCGGTGCCGCAGCTAACGCATTAAGTACTCCGCCTGGGAAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCCATTGACCTCTCCCTAATCGGAGATTTCCCTTCGGGGACAGTGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCTTTAGTTGCCAGCATTAAGTTGGGCACTCTAGAGGGACTGCCGAGGATAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGGGTGGTACAGAGAGTTGCCAAGCCGCGAGGTGGAGCTAATCCCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTACTAGTAATCGCAGATCAGAATGCTGCGGTGAATGCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTTGGGGGCGCCCGAAGCCGGTTAGCTAACCTTTTAGGAAGCGGCCGTCGAAGGTGAAACCAATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAG。
the 16S rRNA sequence of clostridium sp.j2 (SEQ ID No. 2) is shown below:
AGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTTACGGCTACCTTGTTACGACTTCACCCCAGTCATTGGTTTCACCTTCGACGGCCGCTTCCTAAAAGGTTAGCTAACCGGCTTCGGGCGCCCCCAACTTCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGCATTCACCGCAGCATTCTGATCTGCGATTACTAGTAACTCCAGCTTCATGTAGGCGAGTTTCAGCCTACAATCCGAACTGAGAATGGCTTTAAGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCACCCATTGTAGCACGTGTGTAGCCCTAAGCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGAGTTATCCTCGGCAGTCCCTCTAGAGTGCCCAACTTAATGCTGGCAACTAAAGGCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCACTGTCCCCGAAGGGAAATCTCCGATTAGGGAGAGGTCAGTGGGATGTCAAGCTTAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCGCTACTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCGTACTTCCCAGGCGGAGTACTTAATGCGTTAGCTGCGGCACCGAGGGGGGTAACCCCCGACACCTAGTACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTTACAGTCCAGAGAGCCGCCTTCGCTACTGGTATTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCTACTCTCCTCTCCTGCACTCAAGTTCTCTAGTTTCAAAAGCTTACTACGGTTGAGCCGTAGCCTTTCACTTCTGACTTAAAAAACCACCTACGCACCCTTTACGCCCAGTAATTCCGGATAACGCTAGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCCTCCTCAAGTACCGTCATTATCTTCCTTGAGGACAGAGCTTTACGACCCGAAGGCCTTCATCGCTCACGCGGCGTTGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACTGATCGTTGCCTTGGTAAGCCATTACCTTACCAACTAGCTAATCAGACGCGGGTCCATCCTGTACCGCCGGAGCTTTGATACAAAAGCCATGCGACTTTTATATGTTATCCCGTATTAGTATACCTTTCGGTATGTTATCCGTGTGTACAGGGCAGGTTACCCACGCGTTACTCACCCGTCCGCCGCTCTCTCCGAAGAGATCGCTCGACTTGCATGTGTTAGGCACGCCGCCAGCGTTCATCCTGAGCCAGGATCAAACTCTAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGA。
example 2 Parastridium bifermentatans J2 and Clostridium sp.J2 for sulfamethoxazole removal
To investigate the removal effect of paracrostiniumbifermentans j2 and Clostridium sp.j2 on Sulfamethoxazole (SMX), the following experiments were performed:
paraclostridiambiferners J2 and Clostridium sp.J2 are respectively inoculated into a 100mL serum bottle for enrichment culture, 100mL of sterilized culture medium liquid (the components are the same as the culture medium in the example 1, and the initial pH is 7.0-7.5) is filled in the serum bottle in advance, the mixture is fully shaken and uniformly mixed and then placed in a 35 ℃ incubator for standing culture for 1 day, and the bacterial liquid in the serum bottle is observed to be flocculent black.
10mL of each of the two types of bacterial solutions (exponential growth phase) was taken out to a 100mL anaerobic flask (filled with nitrogen gas to maintain anaerobic conditions), and then 100mL of a sterilized liquid medium (v: v =10 100) containing sulfamethoxazole at different concentrations (1 mg/L, 2mg/L, 5 mg/L). After fully shaking and mixing evenly, placing the mixture in a constant temperature incubator with the temperature of 35 ℃ for static culture for 3 days, and finally using a high performance liquid chromatograph to measure the removal condition of sulfamethoxazole. As a result, parastridium bifermentations J2 showed that the removal rates of sulfamethoxazole at 1mg/L, 2mg/L and 5mg/L were 53.0%, 52.6% and 44.25% respectively within 72 hours; the removal rates of 1mg/L, 2mg/L and 5mg/L sulfamethoxazole by Clostridium sp.J2 were 34.7%, 31.2% and 24.92%, respectively.
Furthermore, 5mL of each of the two types of bacterial solutions were taken out to 100mL of the same anaerobic culture flask, and 100mL of a sterilized liquid medium (v: v =10: 100) containing sulfamethoxazole at different concentrations (1 mg/L, 2mg/L, 5 mg/L) was put into the anaerobic culture flask. After fully shaking and mixing evenly, standing and culturing for 3 days in a constant temperature incubator at 35 ℃, and finally determining the removal condition of sulfamethoxazole by using a high performance liquid chromatograph. As a result, the microbial combination of Parastridium bifermentations J2 and Clostridium sp.J2 (FIG. 1) was found to have the removal rate of 68.7%, 69.5% and 64.8% of sulfamethoxazole at 1mg/L, 2mg/L and 5mg/L within 72h, which proves that the two selected anaerobic microorganisms can degrade sulfamethoxazole and the removal effect of sulfamethoxazole can be enhanced by the combination of the two microorganisms.
Example 3 immobilization of Parastribiumemetanans J2 and Clostridium sp.J2 and removal of sulfamethoxazole by immobilized microorganisms
1. Removing effect of straw biochar on sulfamethoxazole
(1) Obtaining straw biochar
Putting agricultural waste rice straw in an oven at 80 ℃, drying water, crushing the material by a crusher, compacting raw material powder, filling the compacted raw material powder into a crucible with a cover, transferring the crucible into a muffle furnace, heating the crucible for 2 hours at 700 ℃ under constant temperature and oxygen deficiency conditions, cooling the crucible to room temperature, taking out the crucible, crushing and grinding the material by the crusher, sieving the crushed material by a 100-mesh sieve, and filling the crushed material into a white transparent wide-mouth bottle for later use.
(2) Sulfamethoxazole removal experiment
To investigate the removal effect of biochar on sulfamethoxazole, the following experiments were performed:
5g of rice straw biochar is placed in a 1000mL serum bottle, a bioreactor with sulfamethoxazole concentrations of 0.1, 0.5, 1, 3 and 5mg/L is filled in the serum bottle in advance for manual water distribution, the mixture is sufficiently shaken at 150rpm and 30 ℃ for 24 hours, and then a high performance liquid chromatograph is used for measuring the removal condition of the sulfamethoxazole. As shown in FIG. 2, the removal rates of the straw biochar to sulfamethoxazole with different concentrations in 24h are 77.94%, 56.33%, 34.75%, 23.47% and 16.63%, respectively, and therefore the straw biochar can be applied to the treatment of the wastewater containing sulfamethoxazole.
The composition of the manual water distribution of the bioreactor used in this example is as follows:
every 1000mL of the composition comprises the following components: 1232mg Na 2 SO 4 ，640.63mg CH 3 COONa，26mg CaCl 2 ，38.95mg MgCl 2 ·6H 2 O，95.5mg NH 4 Cl，0.72mg KH 2 PO 4 ，2.52mg K 2 HPO 4 ，0.2mg KI，5mg FeCl 3 ·6H 2 O，0.5mg H 3 BO 3 ，0.5mg CuSO 4 ，0.63mg MnSO 4 ·H 2 O，0.83mg ZnSO 4 ·7H 2 O，0.5mg CoCl 2 ·6H 2 O。
2. Acquisition of immobilized microbial spheres
In order to further improve the removal effect of clostridium subtotae J2 and bacillus cereus J2 on sulfamethoxazole, the following embedding and curing treatments are carried out on two anaerobic microorganisms:
(1) 10mL of the bacterial solutions (OD) of the two strains were taken separately 600 =1.5 to 2), each of which was inoculated into 100mL serum bottles previously filled with 100mL of a sterilized liquid medium (the composition was the same as that of the liquid medium in example 1). After fully mixing and shaking evenly, the mixture is placed in a constant temperature incubator with the temperature of 35 ℃ for standing culture for 1 day, and the thalli which are black floccules in a serum bottle can be obviously observed.
(2) Preparing sodium alginate-biochar-microorganism immobilized bacteria balls: sterilizing the rice straw biochar at 121 deg.C for 20min under wet heat and high pressure, cooling the biochar, and respectively sterilizing 50ml of bacteria liquid (OD) 600 = 1.5-2), clostridium sp.J2 bacterial fluid (OD) 600 = 1.5-2), and coculture broth (OD) 600 =1.5-2, the mixing volume ratio of the two bacterial liquids is 1) transferring to a 50mL serum bottle, then adding 0.5g sterilized straw biochar, shaking and culturing for 2h at 35 ℃ of a shaking table and 150 rpm. Then mixing the biochar-microorganism solution with 3% (w/v) sodium alginate solution in equal volume, and dripping the mixed solution into 500mL of 5% (w/v) CaCl which is continuously stirred at constant speed 2 In solution, crosslinking was carried out for 1h. After the gel beads are formed, the gel beads are washed by deionized water for at least three times, and are placed in 0.75% NaCl solution to be stored at 4 ℃ for later use, so that the appearance of the sodium alginate-biochar-microbial microspheres is obviously observed to be black spherical (figure 3).
(3) Preparing sodium alginate-microorganism immobilized bacteria balls: take 50mL 3% (w)V) sodium alginate solution and 500mL of 5% (w/v) CaCl 2 The solution was further prepared from 50mL of a co-culture broth (OD) 600 = 1.5-2) transferring to 50mL serum bottle, shaking table 35 ℃, 120rpm shaking culture for 2h. Then the combined microorganism bacterium liquid and the sodium alginate solution are mixed in equal volume, and the mixed liquid is dripped into CaCl which is continuously stirred at constant speed 2 In solution, crosslinking was carried out for 1h. After the gel beads had formed, the beads were rinsed at least three times with deionized water and stored in 0.75% NaCl solution at 4 ℃ for further use.
3. Removing effect of immobilized microbial sphere on sulfamethoxazole
The research on the removal effect of the immobilized microbial spheres on sulfamethoxazole mainly comprises the following steps:
(1) The microbial cells were placed in 100mL of a sterilized liquid medium (the composition of which was the same as that of the liquid medium in example 1), and cultured again at 35 ℃ for 24 hours, whereby black flocculent cells were observed in the medium.
(2) The pellets were added to a 100mL serum bottle (previously filled with nitrogen to remove air) containing 100mL of sterilized liquid medium containing 5mg/L sulfamethoxazole (the composition of the medium was the same as that described in example 1) at a volume ratio of 10% (v/v). Fully shaking to mix the liquid evenly, placing the liquid in a constant temperature incubator with the temperature of 35 ℃ for standing culture for 1 day, and finally determining the removal condition of the sulfamethoxazole by using a high performance liquid chromatograph. As shown in FIG. 4, the immobilized microbial community can rapidly remove sulfamethoxazole in the water body within 24 hours, wherein the sodium alginate-charcoal-combined culture microbial community is optimal, and the removal rate can reach 26.61%.
According to the results of the examples 2, 4 and 5, the combined microorganism and straw biochar has higher removal efficiency on sulfamethoxazole, can be applied to the biological anaerobic method for treating pharmaceutical wastewater of sulfonamide antibiotics, and provides a practical method for treating wastewater containing sulfonamide antibiotics.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (10)
- Use of Paracistridium bifermentations J2 and/or Clostridium sp.J2 for degrading antibiotics of the sulphonamide type, characterised in that the deposit number of Paracistriumbifermentations J2 is GDMCC No:62845, clostridium sp.j2 with accession number GDMCC No:62934.
- use of Paracistridium bifermentations J2 and/or Clostridium sp.J2 for the treatment of pharmaceutical waste water containing sulphonamide antibiotics, characterised in that the deposit number of Paracistriumbifermentations J2 is GDMCC No:62845, clostridium sp.j2 with accession number GDMCC No:62934.
- 3. use according to claim 1 or 2, characterized in that said sulfonamide antibiotic comprises sulfamethoxazole.
- 4. Use according to claim 3, characterized in that the sulfamethoxazole concentration is not more than 10mg/L.
- 5. Use according to claim 1 or 2, wherein the paracoccidiumbifermentatans j2 and/or Clostridium sp.j2 are made into immobilized bactereulus prior to use.
- 6. The use according to claim 5, wherein the biochar is added to the bacterial paracystiniformens J2 and/or Clostridium sp.J2, the biochar-microorganism solution is mixed with the sodium alginate solution after the shaking culture, and the resulting mixture is added dropwise to the CaCl solution under stirring 2 In the solution, gel beads are formed after cross-linking.
- 7. Use as claimed in claim 5, characterized in that said sodium alginate is solubleThe mass concentration of the liquid is 3 percent, and the CaCl is 2 The mass concentration of the solution is 5%, and the concentration of the bacterial liquid is OD 600 1.5-2; the using amount ratio of the straw biochar to the bacterial liquid is 0.4-0.6g/50mL, the volume ratio of the sodium alginate solution to the bacterial liquid is 1 2 The volume ratio of the solution is 1.
- 8. The use according to claim 5, wherein the shaking culture is carried out for a period of 1 to 3 hours; the time for crosslinking does not exceed 1 hour.
- 9. Use according to claim 5, characterized in that the liquid medium used for culturing the bacterial Parastridium bifermentations J2 and/or Clostridium sp.J2 bacteria comprises Na 2 SO 4 ，NH 4 Cl，CaCl 2 ·6H 2 O，MgSO 4 ·7H 2 O，KH 2 PO 4 Yeast extract, CH 3 COONa，FeSO 4 ·7H 2 O，C 3 H 8 ClNO 2 S·H 2 O (L-cysteine hydrochloride hydrate), C 6 H 7 O 6 Na (ascorbic acid).
- 10. A bacterial agent for degrading sulfonamide antibiotics, which comprises paracstridiumbifermentans j2 and/or Clostridium sp.j2 as a main active ingredient.
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| CN117286065A (en) * | 2023-09-27 | 2023-12-26 | 华中农业大学 | Double-fermentation clostridium parahaemolyticum B1251a and application thereof |
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| CN117286065A (en) * | 2023-09-27 | 2023-12-26 | 华中农业大学 | Double-fermentation clostridium parahaemolyticum B1251a and application thereof |
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