CN113817648B - Bordetella adss-6 and application thereof - Google Patents
Bordetella adss-6 and application thereof Download PDFInfo
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- CN113817648B CN113817648B CN202111196261.1A CN202111196261A CN113817648B CN 113817648 B CN113817648 B CN 113817648B CN 202111196261 A CN202111196261 A CN 202111196261A CN 113817648 B CN113817648 B CN 113817648B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
Abstract
The invention belongs to the technical field of environmental microbiology. The invention provides a Bossella (Bosea sp.) Ads-6, which is preserved in China general microbiological culture Collection center (CGMCC) at 9-13.2021, wherein the accession number of the strain Ads-6 is CGMCC No.23391, the collection center is CGMCC for short, and the collection center is No. 3 of West Lu 1 of the sunward district in Beijing. The Bos' Ads-6 can use the antibiotic amoxicillin as the only carbon source, nitrogen source and energy source substance for growth and metabolism, and compared with simple modification and transformation, chemical bond breakage and the like, the degradation mode is more thorough and more environment-friendly.
Description
Technical Field
The invention belongs to the technical field of environmental microbiology.
Background
Amoxicillin, which is the most widely used antibiotic, plays an important role in human anti-infection history, and is widely applied to the fields of livestock breeding, fishery and agriculture in recent years. However, the amoxicillin can not be completely absorbed or degraded in vivo, and about 55% -90% of the amoxicillin is discharged out of the body in the form of original drug or primary metabolite along with urine or feces, and finally is collected in sewage environment and soil environment. Not only can direct ecological damage be brought to the water and soil environment, but also more importantly, long-term amoxicillin accumulation can bring selective pressure to original microorganisms in the environment, the growth of the microorganisms sensitive to amoxicillin is inhibited, and drug-resistant microorganisms quickly become dominant populations, so that the original ecological structure is changed. At the same time, the resistance gene is more easily formed and propagated at this time. Creates good conditions for the appearance of multiple drug-resistant bacteria and super bacteria. The antibiotics, the drug-resistant bacteria and the drug-resistant genes form a novel pollution system, and challenge is provided for the treatment of the ecological environment.
Therefore, how to effectively degrade or remove residual antibiotics in sewage environment is an urgent problem to be solved. In fact, in recent years, a lot of researches on amoxicillin degradation are carried out, but most of the amoxicillin in the sewage is treated by physical or chemical methods, such as photocatalysis, fenton oxidation, nano material treatment and the like. The method is not only high in cost and inconvenient and difficult to industrially use, but also more important is that the physical and chemical treatment methods are used for converting amoxicillin raw medicines into other compounds with relatively low toxicity by means of modification conversion or chemical bond breakage, and the long-term influence on the ecological environment is unknown. In contrast, the microbial degradation of environmental pollutants is a method which is widely used and has a long history, and compost, landfill, activated sludge and the like are all based on the degradation and utilization of organic pollutants by microorganisms, and play an irreplaceable role in the aspect of environmental management. However, these traditional microbial degradation methods use complex microbial flora as functional units, and usually require long-term acclimation culture for specific pollutants to exhibit degradation performance, and cannot ensure high degradation efficiency.
The traditional microbial degradation method has low degradation efficiency on amoxicillin and longer period. At present, few single strains which have degradation effects on amoxicillin are separated, many single strains cannot grow and metabolize by taking amoxicillin as a unique carbon and nitrogen source, degradation products cannot enter a definite substance metabolism cycle, and the specific way of amoxicillin in the bacterial degradation process is rarely known.
Disclosure of Invention
In view of the above, the invention provides a Bossella (Bosea sp.) Ads-6, wherein the Bossella Ads-6 has been deposited in China general microbiological culture Collection center (CGMCC) at 9.13.2021, the accession number of the collection center is CGMCC No.23391, the collection center is CGMCC for short, and the collection center is No. 3 of North West Lu No.1 of the sunward area in Beijing.
On the other hand, the bordetella sp Ads-6 provided by the invention can effectively degrade amoxicillin in sludge and sewage.
The Bos' Ads-6 can use the antibiotic amoxicillin as the only carbon source, nitrogen source and energy source substance for growth and metabolism, and compared with simple modification and transformation, chemical bond breakage and the like, the degradation mode is more thorough and more environment-friendly.
Drawings
FIG. 1 is a dendrogram of strain Ads-6 based on the 16S rRNA gene sequence.
FIG. 2 is a colony and cell morphology diagram of the strain Ads-6, wherein A is an electron micrograph and B is a colony map.
Detailed Description
Examples
The invention takes sewage treatment activated sludge of pharmaceutical factories as a separation source to separate a strain Ads-6 which can perform growth and metabolism by taking amoxicillin as a unique carbon source, nitrogen source and energy substance. Compared with the traditional degradation mode, the bacterial strain does not simply convert amoxicillin into a certain intermediate product, but converts amoxicillin into substances required by self growth by taking the amoxicillin as a substrate and further converts the substances into inorganic substances through respiration, thereby realizing the mineralization of amoxicillin and the cycle of substance metabolism, and being more environment-friendly. On one hand, provides excellent strain resources for the research of the next degradation path and degradation mechanism. On the other hand, the strain is a member of the bradyrhizobiaceae, belongs to a strain resource common in the environment, and can play a role in bioaugmentation by adding the strain in the amoxicillin-containing sewage treatment or the bioremediation of polluted soil, so that the degradation efficiency of amoxicillin is greatly improved.
In the present invention, amoxicillin is abbreviated as AMX.
1. Screening of Strain Ads-6
The amoxicillin degrading strain Ads-6 is obtained by collecting Gaobacil from 2019 in 3 months, domesticating and separating the strain in activated sludge of pharmaceutical factory in Tonghua City of Jilin province, and the specific method comprises the following steps:
a Minor Salt Medium (MSM) liquid culture medium is used as an acclimatization culture medium, and the configuration method of the MSM culture medium comprises the following steps: KH (Perkin Elmer) 2 PO 4 (0.5g/L),Na 2 HPO 4 ·12H 2 O(1.5g/L),NaCl(1.0g/L),MgSO 4 ·7H 2 O (0.2 g/L), a trace element stock solution (1 mL/L), and the formula of the trace element stock solution is as follows: mnCl 2 ·4H 2 O(0.13g/L)，ZnCl 2 (0.23g/L),CuSO4·5H 2 O(0.03g/L),CoCl 2 ·6H 2 O(0.42g/L),Na 2 MoO 4 ·2H 2 O(0.15g/L)，Al 2 (SO 4 ) 3 (0.05g/L)，CaCl 2 (0.23g/L)，FeCl 3 (0.2g/L)，NiCl 2 ·6H 2 O (0.2 g/L), adjusting pH to 7.0, packaging into conical flask, and sterilizing at 121 deg.C under high temperature and high pressure for 20min.
Inoculating the uniformly mixed activated sludge into MSM according to the volume ratio of 10%, adding 20mg/L amoxicillin as a carbon source and a nitrogen source, additionally adding 50mg/L yeast extract to provide a growth factor at the initial stage of enrichment and domestication, and carrying out enrichment culture in a shaking table at 160rpm and 30 ℃ for 2 weeks to obtain an enrichment culture solution.
Then, the enriched culture solution obtained above was inoculated into a new MSM medium at an inoculation amount of 1%, the initial concentration of amoxicillin was adjusted to 50mg/L, and the culture was carried out under the same conditions for 2 weeks without adding yeast extract.
Then sequentially transferring the amoxicillin to an MSM culture medium with the initial concentration of 80mg/L and 100mg/L for domestication and enrichment culture in the same way.
And scribing or coating the obtained enrichment solution on an LB flat plate for multiple times, and separating single bacteria to obtain each strain resource surviving in the enrichment solution.
Then inoculating the obtained strains into LB liquid culture medium subpackaged in test tubes respectively for pure culture, and after about 1-2 days of culture, the growth of the strains enters logarithm or is stableAt this time, the cells were collected, the supernatant was removed and washed with ultrapure water 2 to 3 times to completely remove the residual medium, and OD was prepared 600 Inoculating 1.0 bacterial suspension into MSM according to 1% inoculation amount, adding amoxicillin 100mg/L, culturing in a shaking table at 160rpm and 30 deg.C for one week, sampling at regular intervals, and detecting OD 600 And variation in amoxicillin content.
The experiment confirms that the strain OD is generated in the process 600 And the amoxicillin content is reduced at the same time of increasing, and the repeated test results are consistent. This strain was named Ads-6.
TABLE 1 degradation of Amoxicillin by Ads-6
2. Identification of Strain Ads-6
The universal primers 27F and 1492R of the 16S rRNA gene of the bacteria are used as upstream and downstream primers, the 16S rRNA gene of the strain is subjected to PCR amplification by a colony PCR method, and an amplification product is sent to a company for sequencing to obtain a sequence shown in a sequence table SEQ ID NO. 1.
According to the whole genome sequencing result of the strain Ads-6, the 16S rRNA gene sequence of the strain Ads-6 is completely consistent with the sequence obtained by the colony PCR, and the obtained sequence results are compared on line by using NCBI-blast and EzBioCloud to find that the 16S rRNA gene sequence of the strain Ads-6 is matched with Bosea robiniee DSM 26672 T Most closely, the similarity reaches 99.22%. This sequence was also clustered with the strain of Bosea sp on the phylogenetic clade (fig. 1).
Colony characteristics of Strain Ads-6 on LB plates (see FIG. 2): round, wet, milky-white, convex. Scanning electron micrographs show that the cells of the strain Ads-6 are short rods and grow polar hairs singly. These characteristics all correspond to those of the genus Bos of the family bradyrhizobiaceae.
By combining the information, the strain Ads-6 is identified to belong to the bordetella (Bosea sp.), the strain Ads-6 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms at 9.13.2021, the accession number of the preservation center is CGMCC No.23391, the preservation center is CGMCC for short, and the preservation center address is No. 3 of West Lu No.1 of the sunward district, beijing.
3. Characteristic of bacterial strain Ads-6 in degradation of amoxicillin
Using MSM culture medium as culture medium, 0.25mmol/L amoxicillin as unique carbon and nitrogen source, inoculating 1% Ads-6 bacterial suspension (OD) 600 = 1.0) and placed in a shaking table at 160rpm and 30 ℃ and samples are taken every 24h to detect the biomass and amoxicillin content (detection by HPLC). The experiment was set up in triplicate, the results were averaged and the error was the standard deviation of the results for the triplicate experiments.
TABLE 2 degradation of Ads-6 on AMX at a concentration of 0.25mM
The result shows that the amoxicillin is completely transformed within 24 hours and completely degraded within 4 days under the condition, the biomass is increased from 0.013 +/-0 at 0 time to 0.043 +/-0.003 at 4 days, and the Ads-6 strain has excellent degradation capability on the amoxicillin and can utilize the amoxicillin to grow and metabolize so as to realize the complete degradation of the amoxicillin.
The bacterial strain Ads-6 realizes growth and metabolism by taking amoxicillin as the only carbon and nitrogen source in the growth process, which shows that the amoxicillin can be assimilated by the bacterial strain Ads-6 into nucleic acid, protein and other substances necessary for self-survival, and then converted into inorganic substances through self-respiration to form substance closed circulation. Compared with other degradation modes such as co-matrix metabolism or other incomplete modification and transformation, chemical bond breakage and the like, the process not only saves the consumption of other energy materials, but also avoids the secondary pollution to the environment caused by simple material transformation.
4. The strain Ads-6 degrades amoxicillin with different initial concentrations
Using LB liquid mediumCulturing Ads-6 to index or stationary phase, collecting thallus, washing with ultrapure water for 2-3 times to completely remove residual culture medium, and making into OD 600 About 1 percent of the bacterial suspension is inoculated into an MSM culture medium (without any carbon and nitrogen source) according to the inoculation amount of 1 percent, the amoxicillin concentrations are respectively 2mM,1mM,0.5mM,0.25mM and 0 five gradients, each gradient is three biological repetitions, and the variation of the amoxicillin in the MSM culture medium under the condition of not inoculating the strain Ads-6 is set as a control.
Culturing at 30 deg.C and 160rpm in shaker, and sampling daily to determine biomass (OD) 600 ) And amoxicillin and other intermediate degradation events until the biomass stabilizes and no longer grows. The results show that amoxicillin also slightly degrades in the control sample, but does not affect the results of the experimental group.
At a low initial concentration of amoxicillin (<1 mM), the growth amount of the strain Ads-6 is increased along with the increase of the concentration of amoxicillin, but in the case of high concentration (more than or equal to 1 mM), the strain Ads-6 grows to OD 600 Around 0.66, this may be due to the fact that certain degradation products inhibit the growth of Ads-6 at high concentrations. In addition, it can be seen from the results that DP1 and DP2 also achieved a sequence from none to zero during the degradation process, indicating that DP1 and DP2 are important intermediate metabolites during the degradation of amoxicillin.
TABLE 3 degradation of Ads-6 for different AMX starting concentrations
5. Degradation of amoxicillin by bacterial strain Ads-6 under different pH values
As in the 4 th experimental procedure above, three initial pH gradients of 6,7 and 8 were set, and a control experiment for spontaneous amoxicillin degradation at different pH was set, again in triplicate with an initial concentration of amoxicillin of 0.25mM at 160rpm at 30 ℃. The results show that the degradation rates of amoxicillin and DP1 and DP2 are close in the pH range, namely the strain Ads-6 has a wide pH adaptation range for the degradation of amoxicillin.
TABLE 4 degradation of AMX by Ads-6 at different pH
6. Degradation of amoxicillin by strain Ads-6 at different temperatures
The same experiment operation as the step 4, the initial concentration of amoxicillin is 0.25mM, the pH value of the culture medium is 7, the culture condition is 160rpm, three gradients of 20 ℃,30 ℃ and 40 ℃ are set, and experiments of spontaneous amoxicillin degradation at different temperatures are set for reference, and each gradient is repeated three times. The result shows that the temperature has great influence on the degradation of amoxicillin, and at the temperature of 20 ℃, ads-6 grows slowly, but can still rapidly continue to degrade amoxicillin. 40 ℃ is not the temperature at which strain Ads-6 can grow normally, at which temperature the biomass of strain Ads-6 tends to decrease, but inoculated Ads-6 can still degrade part of the amoxicillin.
TABLE 5 Ads-6 degradation of AMX at different temperatures
7. Degradation of amoxicillin by strain Ads-6 under the condition of additionally adding carbon source or nitrogen source
The same procedure as the above experiment in step 4 was carried out, where the initial concentration of amoxicillin was 0.25mM, the medium pH was 7, the culture conditions were 160rpm,30 ℃. However, 100mg/L of yeast extract, 100mg/L of ammonium sulfate or 100mg/L of glucose was additionally added to the medium, and a blank (no additional carbon or nitrogen source was added) was set, and each set of experiments was similarly repeated three times. The results show that. The additional carbon source (glucose) and additional nitrogen source (ammonium sulfate) did not promote the degradation of amoxicillin, and Ads-6 was able to utilize glucose for growth. In comparison, the yeast extract can greatly promote the degradation and metabolism of amoxicillin.
TABLE 6 Effect of exogenous carbon and nitrogen on Ads-6 degradation of AMX
The Bos' Ads-6 can use the antibiotic amoxicillin as the only carbon source, nitrogen source and energy source substance for growth and metabolism, and compared with simple modification and transformation, chemical bond breakage and the like, the degradation mode is more thorough and more environment-friendly.
Sequence listing
<110> institute for microbiology of Chinese academy of sciences
<120> bordetella Ads-6 and application thereof
<141> 2021-10-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1493
<212> DNA
<213> Bos bacterium (Bosea sp.)
<400> 1
attcaatctg agagtttgat cctggctcag agcgaacgct ggcggcaggc ttaacacatg 60
caagtcgaac gggcacttcg gtgctagtgg cagacgggtg agtaacgcgt gggaacgtgc 120
ctttcggttc ggaataattc agggaaactt ggactaatac cggatacgcc cttcggggga 180
aagatttatc gccgaaagat cggcccgcgt ctgattagct agttggtgag gtaatggctc 240
accaaggcga cgatcagtag ctggtctgag aggatgatca gccacattgg gactgagaca 300
cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggc gcaagcctga 360
tccagccatg ccgcgtgagt gatgaaggcc ttagggttgt aaagctcttt tgtccgggaa 420
gataatgact gtaccggaag aataagcccc ggctaacttc gtgccagcag ccgcggtaat 480
acgaaggggg ctagcgttgc tcggaatcac tgggcgtaaa gggcgcgtag gcggactctt 540
aagtcggggg tgaaagccca gggctcaacc ctggaattgc cttcgatact gggagtcttg 600
agttcggaag aggttggtgg aactgcgagt gtagaggtga aattcgtaga tattcgcaag 660
aacaccggtg gcgaaggcgg ccaactggtc cgaaactgac gctgaggcgc gaaagcgtgg 720
ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat gccagccgtt 780
ggggagcttg ctcttcagtg gcgcagctaa cgctttaagc attccgcctg gggagtacgg 840
tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 900
ttaattcgaa gcaacgcgca gaaccttacc agcttttgac atgtccggtt tgatcggcag 960
agatgccttt cttcagttcg gctggccgga acacaggtgc tgcatggctg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa ccctcgcccc tagttgccat 1080
cattcagttg ggaactctag ggggactgcc ggtgataagc cgcgaggaag gtggggatga 1140
cgtcaagtcc tcatggccct tacaggctgg gctacacacg tgctacaatg gcggtgacaa 1200
tgggcagcga aggggcgacc tggagctaat cccaaaaagc cgtctcagtt cagattgcac 1260
tctgcaactc gagtgcatga aggtggaatc gctagtaatc gtggatcagc atgccacggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagttg ggtttacccg 1380
aaggcgtcgc gctaaccgca aggaggcagg cgaccacggt aggctcagcg actggggtga 1440
agtcgtaaca aggtagccgt aggggaacct gcggctggat cacctccttt cta 1493
Claims (7)
1. Bordetella bacteria (B), (B)Boseasp.) Ads-6 with the preservation number of CGMCC No.23391.
2. The bordetella of claim 1 (b), (b)Boseasp.) the application of Ads-6 in the degradation of amoxicillin.
3. The bacterium Bordetella (B) according to claim 2Boseasp.) application of Ads-6 in degradation of amoxicillin, which is characterized in that the amoxicillin is amoxicillin in polluted soil, sludge or sewage.
4. The method of claim 2 Bordetella bacteria (B), (B)Boseasp.) the use of Ads-6 for the degradation of amoxicillin, characterized in that the conditions of said degradation are: the concentration of amoxicillin is not higher than 1 mM.
5. The bacterium Bordetella (B) according to claim 2Boseasp.) the use of Ads-6 for the degradation of amoxicillin, characterized in that the conditions of said degradation are: the pH is 6-8.
6. The method of claim 2 Bordetella bacteria (B), (B)Boseasp.) the use of Ads-6 for the degradation of amoxicillin, characterized in that the conditions of said degradation are: the temperature is 20-40 ℃.
7. The method of claim 2 Bordetella bacteria (B), (B)Boseasp.) Ads-6 for degrading amoxicillin, characterized in that said Bordetella Ads-6 is used in the form of a composition of Bordetella Ads-6 and a yeast extract.
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| JP2004248618A (en) * | 2003-02-21 | 2004-09-09 | Hoomaa Clean Kk | Bacterial group symbiotically living with fungus used for treating organic material and its application |
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|---|---|---|---|---|
| CN101031637A (en) * | 2005-04-21 | 2007-09-05 | 揖斐电株式会社 | Method of treating wastewater containing organic compound |
| KR20170068259A (en) * | 2015-12-09 | 2017-06-19 | 대한민국(관리부서:국립수산과학원) | Microbiological strain having nitrous acid decomposition ability and process of decomposing nitrous acid |
| CN111378601A (en) * | 2020-02-25 | 2020-07-07 | 南京农业大学 | Halogenated phenol degradation strain and microbial inoculum produced by same |
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