CN107988124A - One plant of 2,4-DNT sulfonate efficient degrading bacterial strain Brucella sp.X2 and its application - Google Patents
One plant of 2,4-DNT sulfonate efficient degrading bacterial strain Brucella sp.X2 and its application Download PDFInfo
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Abstract
The invention discloses one plant 2,4 dinitrotoluene (DNT) sulfonate efficient degrading bacterial strain Brucella sp.X2 and its application.The bacterial strain is preserved in China General Microbiological culture presevation administrative center on the 15th in September in 2017, and preserving number is CGMCC NO.14588.Above-mentioned bacterial strains have extraordinary degradation effect to 2,4 DNT, 3 SA and 2,4 DNT, 5 SA, have application well in the reparation of nitro compound species organic polluted soil.
Description
Technical field
The invention belongs to environmental organism field, and in particular to major pollutants 2 in the red water pollution soil of one plant of TNT, 4- bis-
The separation and application of nitrobenzyl tosylate degradation bacteria strains Brucella sp.X2.
Background technology
TNT is one of most important explosive in the world, in its manufacturing process, in order to remove asymmetric TNT generally uses
The method for adding sodium sulfite is refined, and produces 2,4- dinitrotoluene (DNT)s sulfonate (DNTs), such as 2,4- dinitrotoluene (DNT)s -3-
Sulfonate (2,4-DNT-3-SA) and 2,4- dinitrotoluene (DNT) -5- sulfonate (2,4-DNT-5-SA) are simultaneously dissolved in water, warp
After crossing phase separation, waste water is known as " TNT " red water, is mainly made of dinitrotoluene (DNT) sulfonate, it is also incomplete comprising some
Nitrify metabolin and complicated harmful by-products.U.S. environment protection mechanism (EPA) is classified as the dangerous substance of RCRA specifications.
The red water of undressed TNT is directly drained into water or soil will seriously pollute surrounding environment.The red water of TNT and the red water pollutions of TNT
The processing of soil is the environmental problem that must be solved in TNT productions.
Geobiont repair technology is risen in the 1980s, compared with physics and chemical method, and bioremediation technology has
Have the advantages that advantage of lower cost, treatment effect it is preferable, it is easy to operate, do not easily cause secondary pollution, in-situ treatment can be implemented, into
For a kind of efficient, economy and the recovery technique of Eco-friendly, it is the forward position of current soil recovery technique research field, and have
Actual application value.
Many scholars have isolated the bacterial strain that can apply to explosive wastewater contaminant degradation.Duque etc. is first from TNT Polluted Soils
Bacterium-Pseudomonas alba the C1S1 for the TNT that can degrade is isolated in earth, which can be by TNT, 2,4-DNT, 2-MNT as only
One nitrogen source.Oh et al. isolates Pseudomonas aeruginosa category from TNT contaminated soils, this, which belongs to, can produce nitroreductase promotion TNT's
Degraded.Nyanhongo et al. isolates pseudomonas GG04 and bacillus SF from TNT polluted-waters and soil, can
Know degraded TNT in Yangpu.Gumuscu et al. has isolated achromobacter STE 11, can be main to turn using TNT as unique nitrogen source
Turn to DNT, AMNT.Khan et al. isolates new thermophilic Methylobacillus from TNT contaminated sites, and the flora is starch strengthened in addition
In the case of can not produce the open loop of TNT phenyl ring and other harmful by-products.
But there has been no research to point out to isolate the microorganism for the dinitrotoluene (DNT) sulfonate that can degrade in the soil.This
Invention isolates the engineered strain of the one plant of dinitrotoluene (DNT) sulfonate that can degrade from soil and is made into bacterium solution, is added to
Soil remediation is carried out in the red water pollution soil of TNT.
The content of the invention
The defects of it is an object of the invention to overcome the red water of existing TNT and TNT red water pollution soil restoring technologies and deficiency,
One plant of 2,4-DNT sulfonate efficient degrading bacteria and its application are provided.
To realize the purpose of foregoing invention, the technical scheme is that：
One plant of 2,4- dinitrotoluene (DNT) sulfonate efficient degrading bacterial strain brucella Brucella sp.X2, in 2017
September is preserved in China General Microbiological culture presevation administrative center on the 1st, and preserving number is CGMCC NO.14588, preservation address：North
No. 3 Institute of Microorganism, Academia Sinica of institute of Jing Shi Chaoyang Districts North Star West Road 1.
Efficient degrading bacterial strain Brucella sp.X2 provided by the present invention are from the red water pollution soil of Gansu Province silver TNT
It is enriched with, tames, isolates and purifies to obtain.Bacterium colony is creamy white, rounded protuberance, the smooth moistening in surface, easy picking, and Gram's staining is cloudy
Property.Cell is rod-shaped under the microscope.The 16S rRNA gene sequence characteristics of above-mentioned bacterial strains, using analytic approach by sequence and database
It is compared, it is found that the bacterial strain belongs to Brucella (Brucella sp.), its DNA sequence dna table is as shown in sequence table.
Above-mentioned 2,4-DNT sulfonate degradation bacteria strains Brucella sp.X2 or its bacteria suspension are in degraded nitro virtue
Application in terms of fragrant race's explosive (TNT, DNT, MNT etc.) is also within protection scope of the present invention.
2,4- dinitrotoluene (DNT) sulfonate degradation bacteria Brucella sp.X2 can be grown very well under the conditions of 15-40 DEG C,
2,4- dinitrotoluene (DNT) -3- sulfonate and 2,4- dinitrotoluene (DNT) -5- sulfonate to high concentration are respectively provided with good tolerance,
It is wider to temperature, the accommodation of pH value, it can survive in the presence of a harsh environment, and can effectively degrade within a short period of time two kinds
Dinitrotoluene (DNT) sulfonate.There is good application prospect in the red water of TNT and contaminated soil remediation.
The specific method of above application is：Using it is preceding by degradation bacteria strains Brucella sp.X2 in LB fluid nutrient mediums,
It is about 10 12h to be activated under the conditions of 30 DEG C cell concentration is made9The bacteria suspension of CFU/mL, bacterium solution is inoculated with the inoculum concentration of 5-10%
In the red water of TNT or contaminated soil.
Preferably, the optimum condition of the degraded is：10% inoculum concentration, liquid soil ratio 2:5, pH 9, temperature 35
℃。
Brief description of the drawings
Fig. 1 is group's form of bacterial strain Brucella sp.X2
Fig. 2 is the scanning electron microscope (SEM) photograph of bacterial strain Brucella sp.X2
Fig. 3 is growth curves of the bacterial strain Brucella sp.X2 at 30 DEG C
Fig. 4 is degradation curves of the Brucella sp.X2 to two kinds of 2,4- dinitrotoluene (DNT) sulfonate.
Fig. 5 for Brucella sp.X2 under different pH to the degradation rate of two kinds of 2,4- dinitrotoluene (DNT) sulfonate.
Fig. 6 is Brucella sp.X2 at different temperatures to the degradation rate of two kinds of 2,4- dinitrotoluene (DNT) sulfonate.
Embodiment
For a better understanding of the present invention, present invention is further elaborated with reference to embodiment, but
Present disclosure is not limited solely to the following examples.
Following embodiments agents useful for same is as follows：
(1) the inorganic salts solid medium containing pollutant：2,4-DNT-3-SA 0.5g, 2,4-DNT-5-SA 0.5g,
NaCl30g, NH4NO33g, KH2PO4 1g,K2HPO4 1g,CaCl2 0.02g,MgSO40.5g, agar powder 20g；Deionized water
1L；Trace element solution 10ml.Taken out after autoclaving and be inverted tablet, it is spare after condensation.
Trace element solution：CuSO40.05g, MnSO4 0.05g,FeSO4.7H2O 0.05g, deionized water 50ml.
(2) LB fluid nutrient mediums：Tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL.High pressure
It is spare after sterilizing.
(3) 18g/L agar is added in LB solid mediums LB fluid nutrient mediums, is taken out after autoclaving and is inverted tablet, coagulated
It is spare after knot.
Embodiment 1：The screening of 2,4-DNT sulfonate degradation bacteria strains
Pedotheque picks up from Baiyin City, gansu Province (104 ° 13 ' 43.907 of east longitude ", north latitude 36 ° 30 ' 44.676 "), loaded on from
In envelope, laboratory is transported back in 4 DEG C of preservations.
Enrichment culture：Take contaminated soil 10g to be put into the conical flask equipped with 100mlLB fluid nutrient mediums, 30 DEG C,
The constant temperature of 120rpm is wanted to be shaken on bed, enrichment culture 24h,
Domestication：The above-mentioned nutrient solutions of 1ml are taken to be applied to spreading rod containing 2,4-DNT-3-SA and 2,4-DNT-5-SA's consolidates
In body minimal medium, constant incubator culture 7d is put into；
Isolate and purify：After bacterium colony is grown, colonial morphology is observed, with aseptic inoculation ring from above-mentioned solid inorganic salt culture medium
In select poor morphology away from big bacterium colony, be inoculated in using plate streaking partition method on LB solid mediums, be placed in constant incubator
In 30 DEG C be inverted culture 24h, the picking single bacterium colony after bacterium colony is grown, and be seeded to LB solid mediums in the same way.Weight
Multiple above-mentioned line separation process, until forming the single purifying bacterium colony of form.
It is prepared by bacteria suspension：By inoculation after purification into LB fluid nutrient mediums, in 30 DEG C, 24h is cultivated in 120rpm,
And cell concentration is adjusted to 109CFU/mL is spare.
Bacterial strain is identified：Extract bacterial strain DNA, and using universal primer 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and
1513R (5'-TACGGTTACCTTGTTACGACTT-3') is expanded, and amplification system, which is the positive primers of 2.5 μ L, 2.5 μ L are counter draws
Thing, 45 μ L distilled waters, 50 μ Lsupermix, make DNA profiling with the single bacterium colony of purifying.Amplification condition is 96 DEG C of pre-degeneration 10min,
96 DEG C of denaturation 1sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 2min, totally 35 circulations, 72 DEG C extend 5min.After 16SrRNA amplifications
5 μ L of DNA of bacteria sample mixed with 6 × loading buffer, 1 μ L after, using 1.0% Ago-Gel (5% nucleic acid dye) into
Row electrophoresis detection, DNA maker used are 100bp DNA Ladder (Suo Laibao).Deposition condition is as follows：1 × TAE is buffered
Liquid, voltage 100V, electrophoresis time 20min.After electrophoresis using gel imaging system (Isogen Proxima 10Phi) into
As observation.Amplified production is sent to U.S. lucky biology and carries out 16SrRNA sequencings, the 16S rDNA gene orders measured are passed through
Blast carries out similar sequences search, the gene order of bacterial strain is compared with the 16SrDNA in Genbank, utilization is most like
Sequence determines its Phylogenetic.
Embodiment 2：The degradation capability measure of 2,4-DNT sulfonate degradation bacteria strains Brucella sp.X2
Uncontaminated soil is taken, grinding is air-dried, crosses 1mm sieves, it is spare.Weigh a certain amount of 2,4-DNT-3-SA and 2,4-DNT-
5-SA is dissolved in acetone.In fume hood, the uniformly acetone soln of two kinds of sulfonate of sprinkling, and stirring equal into above-mentioned soil
It is even.It is about 2,4-DNT-3-SA and 2 to make soil concentration, and the concentration of 4-DNT-5-SA is respectively 500mg/kg；Make it in fume hood
Natural air drying 2 days.
Picking single bacterium is fallen within the LB fluid nutrient mediums of sterilizing, is sealed with breathable sealing film, and conical flask is placed in shaking table
In 120rpm, 30 DEG C of activation 24h.Above-mentioned soil 20g is weighed, the Brucella of OD600=1 is added according to 5% inoculum concentration
The bacterium solution of sp.X2 activation, and it is 2 to adjust soil ratio with inorganic salt liquid culture medium:5, sealed with breathable sealing film.Will be certain dense
The bacteria suspension of degree is seeded in above-mentioned soil according to 5% inoculum concentration.As for being cultivated in 30 DEG C of constant incubators, each 24h takes
Sample, 2,4-DNT-3-SA and 2,4-DNT-5-SA concentration are measured using high performance liquid chromatography.Degradation curve is as shown in Figure 4.By
Figure understands that the bacterial strain has preferable degradation effect for two kinds of sulfonate that concentration is respectively 500mg/kg.To 2,4-DNT-3-
The degraded of SA reached 100% at the 12nd day, and the degradation rate to 2,4-DNT-5-SA was comparatively fast close to 100% at the 8th day.
Embodiment 3：The degradation effect of Brucella sp.X2 p-sulfonic acid salt at various ph values
Uncontaminated soil is taken, grinding is air-dried, crosses 1mm sieves, it is spare.Weigh a certain amount of 2,4-DNT-3-SA and 2,4-DNT-
5-SA is dissolved in acetone.In fume hood, the uniformly acetone soln of two kinds of sulfonate of sprinkling, and stirring equal into above-mentioned soil
It is even.It is about 2,4-DNT-3-SA and 2 to make soil concentration, and the concentration of 4-DNT-5-SA is respectively 500mg/kg；Make it in fume hood
Natural air drying 2 days.Above-mentioned soil 20g is weighed in conical flask, with dilute H2SO4With NaOH adjust soil pH be respectively 3,5,7,
9th, 11, the bacterium solution that the Brucella sp.X2 of OD600=1 activate is added according to 5% inoculum concentration, and with inorganic salt liquid culture
Keynote section soil ratio is 2:5, sealed with breathable sealing film.All conical flasks are positioned in 30 DEG C of constant incubator and stand training
Support a period of time and sampled after 6 days, measure the concentration of two kinds of sulfonate.
Bacterial strain Brucella sp.X2 are as shown in Figure 5 to the degradation effect of two kinds of sulfonate under condition of different pH.By Fig. 5
Understand, bacterial strain of the present invention has wider accommodation to pH, to 500mg/ in scope under faintly acid to alkaline condition
2, the 4-DNT-5-SA of 2, the 4-DNT-3-SA and 500mg/kg of kg are respectively provided with certain removal effect, more have under the conditions of pH7-11
Beneficial to Brucella sp.X2 two kinds of sulfonate of degraded, degradation effect is optimal under the conditions of wherein pH9, to two kinds of sulfonate in 6 days
Degradation rate be respectively 63.72% and 100%.
Embodiment 4：The degradation effect of Brucella sp.X2 p-sulfonic acid salt at different temperatures
Uncontaminated soil is taken, grinding is air-dried, crosses 1mm sieves, it is spare.Weigh a certain amount of 2,4-DNT-3-SA and 2,4-DNT-
5-SA is dissolved in acetone.In fume hood, the uniformly acetone soln of two kinds of sulfonate of sprinkling, and stirring equal into above-mentioned soil
It is even.It is about 2,4-DNT-3-SA and 2 to make soil concentration, and the concentration of 4-DNT-5-SA is respectively 500mg/kg；Make it in fume hood
Natural air drying 2 days.Above-mentioned soil 20g is weighed in conical flask, the bacterial strain of OD600=1 is added according to 10% inoculum concentration
The bacterium solution of Brucella sp.X2 activation, and it is 2 to adjust soil ratio with inorganic salt liquid culture medium:5, sealed with breathable sealing film
Mouthful.Conical flask is respectively placed in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, one section of quiescent culture in 40 DEG C of constant incubator
Time simultaneously sampled after 6 days, measured the concentration of two kinds of sulfonate.
Bacterial strain Brucella sp.X2 are as shown in Figure 6 to the degradation effect of two kinds of sulfonate at different temperatures.Can by Fig. 6
Know, the temperature range that bacterial strain of the present invention uses is wider.In the range of 15-40 DEG C to the 2,4-DNT-3-SA of 500mg/kg and
2, the 4-DNT-5-SA of 500mg/kg is respectively provided with certain degradation effect, and degradation rate after the first rise of temperature with reducing, 35 DEG C
Under the conditions of degradation effect it is optimal, 55.28% is respectively reached to 2,4-DNT-3-SA and 2, the degradation rate of 4-DNT-5-SA in 6 days
With 97.35%.Repair time should be appropriately extended when the temperature is low.
Embodiment 5：Brucella sp.X2 repair actual contaminated soil
On the basis of above example, further improve make Brucella sp.X2 can be used in repairing actual TNT it is red
Water pollution soil, experiment comprise the following steps that：
(1) prepared by the bacteria suspension of bacterial strain Brucella sp.X2：The single bacterium of picking strain X 2 is fallen within LB fluid nutrient mediums,
It is positioned in shaking table, in 35 DEG C, 1d is cultivated under the conditions of 120rpm.Fermented and cultured is carried out using fermentation tank, by the kind of above-mentioned culture
Sub- liquid is inoculated in LB culture mediums by 5% inoculum concentration, is aerated using air compressor, speed of agitator 200r/min,
Timing sampling detects OD600 values, grows to spare after exponential phase.
(2) for examination soil：Silver somewhere is picked up from by the red water pollution soil of TNT, through natural air drying, crosses 8 mesh sieves (3mm sieves).
Soil sample is fitted into plexiglass box, starts to spray above-mentioned X2 bacteria suspensions with 10% inoculum concentration, and with addition inorganic salt liquid
Body culture medium is to liquid soil than being 2:5.To reach optimal degradation effect, it is 9 to adjust soil pH, and container is covered with heating and thermal insulation and is wrapped
Wrap up in, maintain temperature at 35 DEG C.
Soil is detected after the processing of 10 days, finds the degraded to 2,4-DNT-3-SA, 2,4-DNT-5-SA
Rate is 100%, and corresponding pollutant is can't detect in soil after repair.
Sequence table
<110>Beijing collaborative innovation research institute
<120>One plant of 2,4- dinitrotoluene (DNT)s sulfonate efficient degrading bacterial strain Brucella sp. X2 and its application
<130> 2010
<141> 2018-01-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1360
<212> DNA
<213>Brucella (Brucella sp.)
<400> 1
ccctaccgtg gtcgcctgcc tccttgcggt tagcacagcg ccttcgggta aaaccaactc 60
ccatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120
ccgcgattac tagcgattcc aacttcatgc actcgagttg cagagtgcaa tccgaactga 180
gatggctttt ggagattagc tcacactcgc gtgctcgctg cccactgtca ccaccattgt 240
agcacgtgtg tagcccagcc cgtaagggcc atgaggactt gacgtcatcc ccaccttcct 300
ctcggcttat caccggcagt ccccttagag tgcccaactg aatgctggca actaagggcg 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca 420
tgcagcacct gtctccgatc cagccgaact gaaggatagt gtctccacta accgcgatcg 480
ggatgtcaag ggctggtaag gttctgcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcctttgag ttttaatctt gcgaccgtac tccccaggcg 600
gaatgtttaa tgcgttagct gcgccaccga agagtaaact ccccaacggc taacattcat 660
cgtttacggc gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcacct 720
cagcgtcagt aatggaccag tgagccgcct tcgccactgg tgttcctccg aatatctacg 780
aatttcacct ctacactcgg aattccactc acctcttcca tactcaagac ttccagtatc 840
aaaggcagtt ccggggttga gccccgggat ttcacccctg acttaaaagt ccgcctacgt 900
gcgctttacg cccagtaaat ccgaacaacg ctagccccct tcgtattacc gcggctgctg 960
gcacgaagtt agccggggct tcttctccgg ttaccgtcat tatcttcacc ggtgaaagag 1020
ctttacaacc ctagggcctt catcactcac gcggcatggc tggatcaggc ttgcgcccat 1080
tgtccaatat tccccactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg 1140
tggctgatca tcctctcaga ccagctatgg atcgtcgcct tggtaggcct ttaccccacc 1200
aactagctaa tccaacgcgg gctcatcatt tgccgataaa tctttccccg ttagggcaca 1260
tacggtatta gcagtcgttt ccaactgttg ttccgtagca aatggtagat tcccacgcgt 1320
tactcacccg tctgccgctc cccttgcggg gcgctcgact 1360
Claims (5)
1. one plant of 2,4- dinitrotoluene (DNT) sulfonate degradation bacteria strains Brucella sp.X2, it is characterised in that the bacterial strain preservation
In China General Microbiological culture presevation administrative center, deposit number is CGMCC NO.14588, preservation address：Beijing's southern exposure
The institute 3 of area North Star West Road 1.
2. one plant of 2,4-DNT sulfonate degradation bacteria strains Brucella sp.X2 as claimed in claim 1 are red in TNT
Application in water and the red water pollution soil remediations of TNT.
3. application according to claim 2, it is characterised in that：Major pollutants are 2,4-DNT -3- sulfonate
(2,4-DNT-3-SA) and 2,4- dinitrotoluene (DNT) -5- sulfonate (2,4-DNT-5-SA).
4. apply according to claim 3, it is characterised in that the cell concentration in Brucella sp.X2 bacterial suspensions is
109CFU/mL, the inoculum concentration of the bacteria suspension is 10%.
5. application as claimed in claim 2, the application are specially：Picking Brucella sp.X2 single bacteriums are fallen within cultivates containing LB
In the conical flask of base, in 35 DEG C, 1d is cultivated under the conditions of 120rpm, seed culture fluid is made.The seed liquor of above-mentioned culture is pressed 5%
Inoculum concentration be inoculated in culture medium fermentation tank containing LB and cultivated, be aerated using air compressor, speed of agitator 200r/
Min, is cultivated to exponential phase.Above-mentioned X2 bacteria suspensions are sprayed with 10% inoculum concentration, and with adding inorganic salt liquid culture medium
To liquid soil than being 2:5.To reach optimal degradation effect, it is 9 to adjust soil pH, and maintains temperature at 35 DEG C,
Wherein, inorganic salt liquid culture medium：NaCl 30g, NH4NO33g, KH2PO4 1g,K2HPO4 1g,CaCl2 0.02g,
MgSO40.5g；Deionized water 1L；Trace element solution 10ml.
Trace element solution：CuSO40.05g, MnSO4 0.05g,FeSO4.7H2O 0.05g, deionized water 50ml.
LB fluid nutrient mediums：Tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL.Autoclaving standby
With.
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| CN116790407A (en) * | 2023-03-09 | 2023-09-22 | 中国科学院青岛生物能源与过程研究所 | Geobacillus D47 for degrading 2,4-DNT and 2,4-DNT-3-SA and application thereof |
| CN116790407B (en) * | 2023-03-09 | 2024-01-02 | 中国科学院青岛生物能源与过程研究所 | Geobacillus D47 for degrading 2,4-DNT and 2,4-DNT-3-SA and application thereof |
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