CN116769668B - Rhodococcus sp.NJF-7 and application thereof in degradation of low-fluorine alkane - Google Patents
Rhodococcus sp.NJF-7 and application thereof in degradation of low-fluorine alkane Download PDFInfo
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- 239000011737 fluorine Substances 0.000 title abstract description 17
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- 230000015556 catabolic process Effects 0.000 title description 13
- 238000006731 degradation reaction Methods 0.000 title description 13
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 15
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 8
- 238000006065 biodegradation reaction Methods 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- LHLRHWJTTUCDQA-UHFFFAOYSA-N 1-fluorodecane Chemical compound CCCCCCCCCCF LHLRHWJTTUCDQA-UHFFFAOYSA-N 0.000 claims 3
- 229910052500 inorganic mineral Inorganic materials 0.000 claims 1
- 239000011707 mineral Substances 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000000593 degrading effect Effects 0.000 abstract description 5
- 238000009629 microbiological culture Methods 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 238000005067 remediation Methods 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 13
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- 230000000694 effects Effects 0.000 description 6
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- 239000002689 soil Substances 0.000 description 4
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006115 defluorination reaction Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
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- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
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- 238000012408 PCR amplification Methods 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 241000168435 Rhodococcus wratislaviensis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
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- DIOQZVSQGTUSAI-NJFSPNSNSA-N decane Chemical compound CCCCCCCCC[14CH3] DIOQZVSQGTUSAI-NJFSPNSNSA-N 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
- 150000002222 fluorine compounds Chemical class 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- DIOQZVSQGTUSAI-UHFFFAOYSA-N n-butylhexane Natural products CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
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- 239000012137 tryptone Substances 0.000 description 1
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The invention discloses Rhodococcus sp.NJF-7 which is preserved in China general microbiological culture Collection center, with the preservation number of 2023, 5 months and 19 days: CGMCC No.1.19448. Also discloses the application of the catalyst in degrading low-fluorine alkane. The strain NJF-7 obtained by the invention can biologically crack C-F bonds and degrade 1-FD in different pH ranges, so that the strain can be applied to the remediation of polluted sites in different background environments.
Description
Technical Field
The invention belongs to the technical field of environmental microbial engineering, and particularly relates to Rhodococcus sp.NJF-7 and application thereof in degradation of low-fluorine alkane.
Background
Fluorine (F) is the most electronegative and reactive element of the periodic table, and also the 13 th abundant element of the crust, and is widely used in the synthesis of industrial preparations, agricultural chemicals and pharmaceuticals. Many fluoroorganic chemicals have environmental durability and can affect the environment and human health. The recalcitrance of fluorochemicals is generally believed to be due to the presence of C-F bonds, affecting the overall properties of the compound, making it difficult for microorganisms to biodegrade it. The C-F bond becomes one of the single bonds which are recognized as the most difficult to break due to the higher bond energy, and the fluorine atom outer electron layer contains three pairs of non-bonded electrons, so that the C-F in the fluorine-containing compound can be effectively protected to be subjected to strong acid, alkali, light, heat, biodegradation and the like. In addition, since fluorine is the element with the strongest electronegativity, F tends to be generated after C-F bond cleavage - While fluorine (F) exists in the form of ions - ) Has strong toxic action on bacterial cells even at micromolar concentration, so that the biodegradation of fluorine-containing compounds by microorganisms is overcome - Toxicity challenges. Microorganism disruption C-FThe functional research of the bond has important scientific significance for researching the biodegradation of the polyfluoro compound in the future.
Existing studies indicate that small amounts of monofluorochemicals, such as monofluoroacetate, exist in nature, and thus microorganisms have the potential to evolve defluorinated enzymes to defluorinate fluorides. Although there are many studies on biodegradation of fluorochemicals, the effect of microorganisms on breaking C-F bonds is not disclosed; still other studies have been carried out mostly in complex flora, which do not localize the critical flora for breaking the C-F bonds, and are not conducive to further research on the mechanism of microbial degradation and functional development. Therefore, it is very important to find functional bacteria capable of degrading fluorine-containing compounds and grasp more strain resources, only Pseudomonas sp.strain 273 which can degrade fluorine-containing alkane in garden soil in 2020 is found by Xie and the like through literature search, and other functional strains which take fluorine-containing alkane as a single carbon source and can break C-F bonds are not found.
Disclosure of Invention
1. Object of the invention.
The Rhodococcus sp.NJF-7 is screened out, can biologically crack C-F bonds, and can effectively degrade low-fluorine alkane.
2. The technical scheme adopted by the invention is as follows.
Rhodococcus sp.NJF-7 of the invention is preserved in China general microbiological culture Collection center (CGMCC) of 5 months and 19 days in 2023, and has an address of 1 st Xielu No. 3 of North Chen in the Korean region of Beijing, and a classification name of Rhodococcus (Rhodococcus) as a strain of the microorganism institute of China academy of sciences, post code 100101, and the preservation number: CGMCC No.1.19448.
The Rhodococcus sp.NJF-7 has the basic biological characteristics that:
(1) Colony morphology: early white, light orange red when ripe, smooth surface and light-proof;
(2) Physiological and biochemical characteristics: gram positive;
(3) Culturing characteristics: LB medium at 28 ℃.
The Rhodococcus sp.NJF-7 provided by the invention can biologically cleave C-F bonds, so that the Rhodococcus sp.NJF-7 can be applied to degradation of low-fluorine alkane, especially 1-fluorine decane (1-FD).
Specifically, rhodococcus sp.NJF-7 is inoculated into an inorganic salt culture medium containing low-fluorine alkane, and the culture is carried out at 28 ℃ and 160r/min to realize the biodegradation of the low-fluorine alkane. The pH of the inorganic salt medium is controlled to be 5.5-8.5, preferably 7.2.
Inorganic salt medium (MM): mgSO (MgSO) 4 ·2H 2 O 0.2g、CaCl 2 ·2H 2 O 20mg、FeSO 4 ·7H 2 O10mg、KH 2 PO 4 0.4g、Na 2 HPO 4 0.6g、MnSO 4 20mg、NaNO 3 1g、NH 4 Cl 0.6g, deionized water 1000mL, natural pH, and sterilizing at 121deg.C for 20min.
The Rhodococcus sp.NJF-7 is added to the inorganic salt culture medium in the form of resting cells, and the addition amount of the resting cells is calculated as the percentage of the culture system, preferably 10%.
Resting cells were prepared as follows:
(1) Plate culture: rhodococcus sp.NJF-7 is inoculated on an LB solid plate and cultured for 24-48 hours at 28 ℃ to obtain a single colony.
(2) And (3) performing expansion culture: inoculating the single colony obtained in the step (1) into LB liquid medium by using an inoculating loop, and culturing at 28 ℃ and 160r/min for 24-36 h to obtain OD 600 Bacterial liquid with the concentration of 0.6-0.8 is centrifuged, bacterial cells are collected, sterilized inorganic salt culture medium is washed for 3 times, and resting cell liquid is obtained after equal volume resuspension.
LB (Luria-Bertani) liquid medium: 10g of NaCl, 10g of tryptone, 5g of yeast extract, 1000mL of deionized water, natural pH value and sterilization at 121 ℃ for 20min. The LB solid culture medium is prepared by adding 1.5% agar powder on the basis of the culture medium.
3. The invention has the technical effects.
(1) The rhodococcus NJF-7 can break C-F bond to remove fluoride ion from fluorinated alkane to degrade, and the related report of the genus rhodococcus for degrading fluorinated alkane is not known at present.
(2) The strain NJF-7 obtained by the invention can degrade 1-FD in different pH ranges, so that the strain can be applied to the remediation of polluted sites in different background environments.
Drawings
FIG. 1 shows a phylogenetic tree (a) and a colony cell morphology (b) of 1-FD-degrading bacteria NJF-7;
FIG. 2 is a degradation curve of strain NJF-7 for 1-FD;
FIG. 3 shows the degradation effect of strain NJF-7 on 1-FD at different pH values.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention will be further illustrated with reference to specific examples, which are not intended to limit the invention. The technical means used in the examples are conventional means well known to those skilled in the art, if specifically indicated.
Example 1: separation, purification and identification of 1-FD degradation bacteria
The strain NJF-7 is obtained by screening from soil of Suzhou chemical plant, and comprises the following specific steps:
(1) Enrichment culture: 20g of Suzhou chemical plant soil and 0.1% (v/v) of 1-FD were placed in a Erlenmeyer flask containing 50mL of an inorganic salt medium, and cultured at 28℃for 4 weeks at 160 r/min. After enrichment, taking the soil suspension, centrifuging at 1400r/min for 3min, taking supernatant, passing through a 0.22 mu m water-based filter membrane, detecting F-concentration in the solution by using an Ion Chromatograph (IC), and judging the biological defluorination effect by the presence or absence of F-release.
(2) And (3) separating and purifying: and (3) separating and purifying the enriched liquid with the biological defluorination effect in the step (1). The specific implementation is as follows: firstly, preparing a double-layer flat plate, wherein the lower layer of the flat plate is an inorganic salt solid culture medium (containing 1.5% of agar), then preparing a solution of 1-FD with the concentration of 20mM (the solvent is acetone), mixing the solution with the inorganic salt solid culture medium (containing 1.0% of agar) according to the ratio of 1:20 (v/v) while the solution is hot, and taking 4-5 mL of the solution to be spread on the lower layer flat plate before solidification. Taking the steps(1) The concentrated solution in the sample was subjected to gradient dilution (10) -3 ，10 -4 ，10 -5 ) Coating on a double-layer plate, inversely culturing at 28 ℃ until colony is generated on the plate, and picking single colony on an LB plate to continuously streak for separation and purification.
(3) And (3) re-screening: culturing the single colony obtained in the step (2) in LB liquid medium until OD 600 After centrifugation at 5000rpm for 4min, the supernatant was removed, washed three times with inorganic salt medium and resuspended in equal volume of inorganic salt medium. 1% (v/v) of the resuspended strain was inoculated into 5mL of an inorganic salt medium containing 0.1% of 1-FD (v/v), cultured for one week at 28℃and then 1mL of the culture medium was filtered through a 0.22 μm aqueous filter membrane, and the F-concentration was detected by using an Ion Chromatograph (IC), whereby the strain obtained in the step (2) was verified to be defluorinated.
(4) And (3) identification: the strain verified in the step (3) was subjected to PCR amplification using the universal primers 27F and 1492R and then subjected to 16S sequencing by the company (Major Bio, shanghai, china), the obtained sequences were subjected to BLAST alignment, 100% similarity with Rhodococcus wratislaviensis strain DLC-cam was found, and the obtained strain was identified as Rhodococcus genus and designated NJF-7 by combining the morphological characteristics of the colony with the gram staining results (FIG. 1 b) using the MEGA11 software building block phylogenetic tree (FIG. 1 a).
Example 2: degradation of 1-FD by degrading bacteria NJF-7
Inoculating 1-FD degrading bacteria NJF-7 into LB liquid culture medium, culturing at 28deg.C at 160r/min to logarithmic phase, centrifuging at 5000rpm for 5min, collecting thallus, and inoculating at 10% (v/v) ratio in degradation experiment. Degradation experiments were performed in 20mL sterile flasks containing 5mL of inorganic salt medium with a sole carbon source of 1-FD (0.1%, i.e., 5 mmol/L) and incubated for 3d at 28℃in a 160r/min incubator. The experiment was set up with a total of 6 sampling time points, 6 replicates for each time point. In sampling, 1mL of the sample was passed through a 0.22 μm aqueous filter for detection of F-concentration in any 3 replicates, then double the volume of n-hexane was added to the remaining 3 replicates to extract residual 1-FD, 1mL of the organic phase was sucked through a 0.22 μm organic filter, and the residual amount was measured by a gas chromatograph (GC, agilent 7890A, shimadzu).
The result shows that: strain NJF-7 was able to degrade 43% of 1-FD within 36h and release an equivalent amount of F-, indicating that NJF-7 achieved biodegradation of 1-FD by cleavage of the C-F bond, as shown in FIG. 2.
Example 3: NJF-7 degradation of 1-FD under different pH conditions
The pH of the inorganic salt medium was adjusted to 5.5, 6.5, 7.2, 8.5 (1 mol/LNaOH or 1mol/L HCl aqueous solution adjustment), and 10% (v/v) of the resting cell fluid NJF-7 obtained in the same procedure as in example 2 was inoculated at a 1-FD application concentration of 0.05% (v/v), 3 replicates per treatment. After incubating the sample in a 160r/min incubator at 28℃for 3 days, 1mL of the solution was taken to determine the F-concentration.
The result shows that: strain NJF-7 can degrade 1-FD under various pH conditions, and has better degradation effect under neutral condition with pH of 7.2, and F-release rate reaches 75%, as shown in figure 3.
The above examples are given solely for the purpose of better illustration of the present invention and are not intended to limit the invention thereto, and modifications, improvements and substitutions made within the principles of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. Rhodococcus spRhodococcussp.) NJF-7, accession number: CGMCC No.1.19448.
2. The rhodococcus of claim 1Rhodococcussp.) NJF-7 for cleaving a C-F bond containing compound, said C-F bond containing compound being 1-fluorodecane.
3. The use according to claim 2, characterized in that the rhodococcus is treated withRhodococcus sp.) NJF-7 was inoculated into an inorganic salt medium containing 1-fluorodecane for cultivation, thereby effecting biodegradation of 1-fluorodecane.
4. Use according to claim 3, characterized in that the culture conditions are 28 ℃, 160 rpm.
5. Use according to claim 3, characterized in that the pH of the mineral salt medium is 5.5-8.5.
6. The use according to claim 3, characterized in that the ratio of the inorganic salt medium is: mgSO (MgSO) 4 ·2H 2 O 0.2g、CaCl 2 ·2H 2 O 20mg、FeSO 4 ·7H 2 O 10mg、KH 2 PO 4 0.4g、Na 2 HPO 4 0.6g、MnSO 4 20mg、NaNO 3 1g、NH 4 Cl 0.6g was dissolved in deionized water 1000mL and sterilized at 121℃for 20min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965309A (en) * | 2012-11-14 | 2013-03-13 | 浙江工业大学 | Rhodococcus sp. and application thereof to micro-biologically degrading 4-fluorocinnamic acid |
| CN103589659A (en) * | 2013-09-18 | 2014-02-19 | 中国科学院南京土壤研究所 | Rhodococcus globerulus WJ4 and application thereof to remediation of phthalic acid ester (DEHP) polluted soil |
| CN116004459A (en) * | 2022-12-29 | 2023-04-25 | 浙江工业大学 | Rhodococcus YZ-1 and application thereof in degrading organic pollutants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965309A (en) * | 2012-11-14 | 2013-03-13 | 浙江工业大学 | Rhodococcus sp. and application thereof to micro-biologically degrading 4-fluorocinnamic acid |
| CN103589659A (en) * | 2013-09-18 | 2014-02-19 | 中国科学院南京土壤研究所 | Rhodococcus globerulus WJ4 and application thereof to remediation of phthalic acid ester (DEHP) polluted soil |
| CN116004459A (en) * | 2022-12-29 | 2023-04-25 | 浙江工业大学 | Rhodococcus YZ-1 and application thereof in degrading organic pollutants |
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