CN104560778A - Degrading bacterium taking melamine as substrate as well as screening method and application of degrading bacterium - Google Patents
Degrading bacterium taking melamine as substrate as well as screening method and application of degrading bacterium Download PDFInfo
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- CN104560778A CN104560778A CN201410653049.7A CN201410653049A CN104560778A CN 104560778 A CN104560778 A CN 104560778A CN 201410653049 A CN201410653049 A CN 201410653049A CN 104560778 A CN104560778 A CN 104560778A
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Abstract
The invention discloses a degrading bacterium taking melamine as a substrate as well as a screening method and application of the degrading bacterium. The degrading bacterium is onion Burkholderia cepacia CGMCC No.9843. The screening method comprises the following steps: enriching the degrading bacteria; performing domesticated incubation on the degrading bacteria; performing plate streaking on the degrading bacterium to separate a single colony; and performing transparent zone screening on the degrading bacteria. The application specifically is that the degrading bacterium taking melamine as the substrate is used for repairing melamine polluted soil. A use method comprises a step of adding a bacterium solution of the degrading bacterium or charcoal adsorbed with the degrading bacterium into the melamine polluted soil. The degrading bacterium screened by the method disclosed by the invention can use melamine as an only carbon source and can effectively degrade melamine, thereby providing a quick and convenient means for developing a new bioremediation microbial resource.
Description
Technical field
The present invention relates to biological degradation organic pollutant technical field, a specifically strain take trimeric cyanamide as the degradation bacteria of substrate and screening thereof and application.
Background technology
Trimeric cyanamide (Melamine), also known as melamine, melon, melamine, molecular formula is C
3n
6h
6, be a kind of triazines nitrogen heterocyclic ring organic compound, be widely used in the organic synthesis monomer of chemical industry.In industry, a large amount of trimeric cyanamide used finally can cause soil and water environmental pollution, and then indirect pollution animals and plants product.In addition, studies have reported that, because trimeric cyanamide nitrogen content is up to 66.67%, a large amount of melamine waste residue produced in industrial production can be made into incorporation of fertilizerin the soil.These trimeric cyanamides residual in environment can cause the accumulation of trimeric cyanamide in plant, and then affect the edible safety of vegetalitas agricultural-food, and therefore, in recent years, in soil, the degraded of trimeric cyanamide causes extensive concern both domestic and external.
Utilize microorganism to the organic pollutant of all kinds of synthetic of degrading, make it degradable for inorganics is one of effective ways of current most potentiality.The microbiological deterioration research starting of trimeric cyanamide is late, achievement is few, only has several sections of reports both at home and abroad now.Therefore, be separated, screen the microorganism with efficient degradation trimeric cyanamide, to environment purification, ensuring food safety has important practical significance.
Summary of the invention
For defect of the prior art, the object of this invention is to provide degradation bacteria and screening thereof and application that a strain take trimeric cyanamide as substrate, be specially degradation bacteria and screening method, purposes, using method and the composition containing this bacterium that a strain take trimeric cyanamide as substrate.
Involved by the present invention, bacterial strain Burkholderia Burkholderia cepacia is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on October 27th, 2014, deposit number is CGMCC No.9843, and depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the invention provides a kind of take trimeric cyanamide as the degradation bacteria of substrate, and described degradation bacteria is Burkholderia cepacia (Burkholderia cepacia) CGMCC No.9843.
Second aspect, the invention provides a kind of take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and described screening method comprises the steps:
The enrichment culture of step one, degradation bacteria: take by trimeric cyanamide pollute soil sample in containing trimeric cyanamide enrichment medium in, 20-37 DEG C, cultivate 7-10 days under 140-210r/min condition, be forwarded to the fresh enrichment medium containing trimeric cyanamide and cultivate, switching for several times, obtains enrichment bacterium liquid;
The domestication of step 2, degradation bacteria is cultivated: get enrichment bacterium liquid, be forwarded in basic medium, adds trimeric cyanamide and carries out domestication and cultivate, 20-37 DEG C, cultivate 7-10 days under 140-210r/min condition, and switching several, must tame bacterium liquid;
Step 3, be separated single bacterium colony: dilution domestication bacterium liquid, is seeded to LB solid medium dull and stereotyped, is inverted and cultivates 3-5 days, obtain single bacterium colony of degradation bacteria in 20-37 DEG C of constant temperature;
Step 4, bacterial isolation: the single bacterium colony getting degradation bacteria, transfer containing in the screening culture medium of trimeric cyanamide, 20-37 DEG C of constant temperature is inverted and is cultivated 7-10 days, selects and can grow and have the bacterial strain of obvious transparent circle, be namely able to the degradation bacteria that trimeric cyanamide is substrate.
Preferably, in step one, described enrichment medium comprises component and content:
More preferably, described enrichment medium comprises component and content:
Preferably, in step one, in described enrichment medium, the concentration of trimeric cyanamide increases with the increase of switching number of times.
More preferably, the concentration first for the trimeric cyanamide in the enrichment medium of enrichment culture is 10mg/L, in switching subsequently, often transfers once, and the melamine concentration in enrichment medium increases 10mg/L.
Preferably, in step one, the inoculum size of described switching is 10wt%, and switching number of times is 4 times.
Preferably, in step 2, described melamine concentration is 40mg/L.
Preferably, in step 2, the inoculum size of described switching is 10wt%, and the number of times of described switching is 3 times.
Preferably, in step 2, component and the content of described basic medium comprise:
More preferably, the component of described basic medium and content comprise:
Preferably, in step 3, described dilution specifically refers to by domestication bacterium liquid dilution 100-10000 doubly.
Preferably, in step 3, the mode of described inoculation comprises line, coating.
Preferably, in step 3, component and the content of described LB solid culture body comprise: Tryptones 10g/L, yeast extract 10g/L, NaCl 5g/L, pH are 6.0-8.0.
More preferably, component and the content of described LB solid medium comprise: Tryptones 10g/L, and yeast extract 10g/L, NaCl 5g/L, pH are 7.0.
Preferably, in step 4, described switching comprises to be inoculated by the mode of spot printing.
Preferably, in step 4, component and the content of described screening culture medium comprise:
More preferably, the component of described screening culture medium and content comprise:
PH is 7.0; Described per-cent is mass ratio.
Preferably, in step 4, the concentration of described trimeric cyanamide is 10 ~ 60mg/L; Be less than 10mg/L screening effect undesirable, be greater than 60mg/L bacteria growing inhibiting.
It is take trimeric cyanamide as the degradation bacteria strains of sole carbon source that the present invention screens the degradation bacteria strains obtained.
The third aspect, the invention provides a kind of is that the degradation bacteria of substrate is repairing the purposes in trimeric cyanamide contaminated soil with trimeric cyanamide.
Fourth aspect, the invention provides a kind of is that the degradation bacteria of substrate is repairing the method in trimeric cyanamide contaminated soil with trimeric cyanamide.
Preferably, described method comprise the bacterium liquid adding with trimeric cyanamide the degradation bacteria being substrate in trimeric cyanamide contaminated soil, the charcoal being adsorbed with trimeric cyanamide the degradation bacteria being substrate or be adsorbed with trimeric cyanamide be substrate degradation bacteria containing polyvinyl alcohol and alginate carrier.
More preferably, described method is in trimeric cyanamide contaminated soil, add the charcoal being adsorbed with degradation bacteria.
5th aspect, the invention provides a kind of containing with trimeric cyanamide is the degradation bacteria of substrate and the composition of charcoal.
The present invention has following beneficial effect:
(1) pass through the formula of the various substratum of design bacterial strain screening in screening method of the present invention, from ortho-water paddy soil, obtain the bacterial strain of efficient degradation trimeric cyanamide.The bacterial strain screened is obvious to trimeric cyanamide degradation effect, and the rate of clearing up reaches more than 82%.Described simple and feasible screening process, cheap economy.The bacterial strain of gained can be used for preparing the degradation bacterial agent of trimeric cyanamide, has good application potential in practice.
(2) the present invention utilizes ortho-water paddy soil for handling object, and the degradation bacteria of gained is the indigenous bacterium in field soil, is not easily ostracised after making microbial inoculum at Field information, respond well.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the aspect graph of bacterial strain MB4 under transmission electron microscope;
Fig. 2 is provided by the invention take trimeric cyanamide as the growth curve of the degradation bacteria of substrate and the degradation effect figure to trimeric cyanamide.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of trimeric cyanamide degradation bacteria MB4, i.e. Burkholderia cepacia Burkholderia cepacia, the screening of (CGMCC No.9843).
The preparation of bacterium liquid: take the long-term soil sample by trimeric cyanamide pollution in the enrichment medium containing trimeric cyanamide, culture condition 37 DEG C, 180r/min, shaking table cultivates 7 days, the gradient inoculum size of trimeric cyanamide is 10,20,30,40mg/L, increase progressively with the gradient of 10mg/L, transfer 4 times;
The component of described enrichment medium comprises: MgSO
47H
2o (0.2g/L), K
2hPO
4(0.1g/L), (NH
4)
2sO
4(0.1g/L), CaSO
4(0.05g/L), FeSO
47H
2o (0.002g/L), glucose 10.0g/L, peptone 5.0g/L, pH are 7.0, sterilising conditions: 121 DEG C of moist heat sterilization 20min.
Get the final enrichment bacterium liquid of above-mentioned cultivation, be transferred in basic medium with the inoculum size of 5wt%, add and carry out domestication cultivation, culture condition 37 DEG C, 180r/min, shaking table cultivates 7 days, so repeats 3 domestications, obtains the bacterium liquid of degraded trimeric cyanamide.
The composition of described basic medium comprises: MgSO
47H
2o (0.4g/L), K
2hPO
4(0.2g/L), (NH
4)
2sO
4(0.2g/L), CaSO
4(0.1g/L), FeSO
47H
2o (0.002g/L), trimeric cyanamide (40mg/L), pH is 7.0.
Degradation bacteria plate streaking is separated single bacterium colony: after getting the final domestication bacterium liquid dilution of above-mentioned cultivation, line is coated on LB solid medium flat board, and 37 DEG C of constant temperature are inverted cultivation 7 days, obtain single bacterium colony of degradation bacteria.
The composition of described LB solid culture body comprises: Tryptones 10g/L, yeast extract 5g/L, NaCl (10g/L), and pH is 7.0.
Get single bacterium colony that above-mentioned separation obtains again, point to be applied in the screening culture medium being added with trimeric cyanamide 37 DEG C of constant temperature and to be inverted cultivation 7 days, selects and can grow and have the bacterial strain of obvious transparent circle, and as screening take trimeric cyanamide as the degradation bacteria strains of sole carbon source.
The composition of described screening culture medium comprises: MgSO
47H
2o (0.1g/L), K
2hPO
4(0.75g/L), (NH
4)
2sO
4(0.8g/L), KH
2pO
40.5g/L), NaCl (1.0g/L), agar 1.8-2.0%, trimeric cyanamide (40mg/L), pH is 7.0.
Final acquisition trimeric cyanamide degradation bacteria MB4.
By the form of optics and transmission electron microscope observation bacterial strain MB4, display (see Fig. 1) observed by transmission electron microscope (20000 ×), and somatic cells is elongated rod shape, without gemma, and length 3.2 ~ 5.5 μm, width 0.8 ~ 1.2 μm.
embodiment 2
The present embodiment relates to the mensuration of bacterium MB4 to trimeric cyanamide degradation capability.
Activated by bacterial strain MB4 after above-mentioned separation, purifying, described activation is namely the MB4 bacterial strain that inorganic salt solid medium is preserved, and transfer in 250mL triangular flask, often bottled have the sterilized LB substratum of 150mL.180r/min, 37 DEG C of shaking culture 1d.Be MB4 bacterium liquid.Described LB substratum: Tryptones 10g/L, yeast extract 5g/L, NaCl (10g/L), pH is 7.0.
Degradation effect experiment adopts shaking flask liquid culture method.3 repetitions are established in each process.Degradation bacteria strains is made A
600the bacteria suspension of=1.0, with 5% access containing in the minimal medium of 30mg/L trimeric cyanamide, 37 DEG C, 180r/min shakes training, and arrange and do not connect bacterium for contrast, each process arranges 3 repetitions, interval sampling in 1 day, continuous 2 weeks, the Simultaneously test A of every sub-sampling
600value.
Nutrient solution ammoniacal liquor methanol solution of getting (5/95, v/v) wash-out, collects elutriant N
2dry up, with 20% methanol solution constant volume, cross 0.45 μm of organic filter membrane, detect it with high performance liquid chromatography and remain.
Chromatographic condition: chromatographic column: SPHERI-5RP-18 (5 μm, 250mm × 4.6mm); Column temperature: 30 DEG C; Moving phase: acetonitrile/sodium heptanesulfonate and citrate buffer solution (15/85, V/V); Determined wavelength: 240nm; Flow velocity: 1.0mL/min; Sample size: 10 μ L.
Calculate the residual quantity of trimeric cyanamide in sample according to typical curve, obtain its degradation rate according to formula.Trimeric cyanamide degradation rate (%)=[1-(process actual measurement residual quantity/contrast actual measurement residual quantity)] × 100%
Draw through efficient liquid phase chromatographic analysis, this bacterial strain cultivates 10d in the trimeric cyanamide liquid inorganic salt culture medium of 30mg/L, and degradation efficiency can reach 81.67%, learns that this strains for degrading circulation ratio is good through repeatedly verifying.Growth and the trimeric cyanamide degradation curve of bacterial strain MB4 are shown in Fig. 2.
embodiment 3
Efficient degrading bacteria MB4 measures the degradation effect of trimeric cyanamide in two kinds of soil by after charcoal immobilization process.
The making of charcoal immobilization bacterial strain MB4 bead: take 1.0g charcoal, add containing 10mL bacteria suspension (A
600=1.0), in Erlenmeyer flask, 8h cultivated by shaking table.5000r/min eccentric cleaning obtains absorption carrier, and adding sterilized water, to be settled to 20mL for subsequent use.Embedding: polyvinyl alcohol (PVA) and sodium alginate (SA) are made into the 50mL aqueous solution by 10%+0.5% mixing, heats in water-bath (90 DEG C) and continuously stirring is dissolved, and after cooling, (40 DEG C) obtain colloid mixture; Crosslinked: to add the above-mentioned absorption carrier of 20mL, be settled to after 100mL fully mixes with sterilized water, instill with syringe and contain 4%CaCl
2saturated boric acid solution in (NaOH adjust pH to 6.7), at 4 DEG C leave standstill 24h, solidification balling-up after for subsequent use.
Adopt black earth and rice soil two kinds of soil, pedotheque, after high-pressure sterilizing pot 120 DEG C of sterilizing 20min, accurately takes 500g sample, is placed in 1000mL beaker.Add 20% methanol solution containing trimeric cyanamide, form the pedotheque of 50mg/kg.Two kinds of soil arrange charcoal immobilized bacterium, bacteria suspension, sterilized bio charcoal immobilized spherule and blank four process respectively, often process repetition 3 times.Pedotheque after process is placed in 30 DEG C, humidity is the constant incubator of 40%, cultured continuously 35d, and sampling detects melamine residual amount week about, and research charcoal immobilization degradation bacteria is to the degradation effect of trimeric cyanamide in soil.
Table 1 is the Degradation of charcoal immobilization degradation bacteria to trimeric cyanamide in rice soil and black earth, from data in table, no matter is rice soil or black earth, in 35d incubation period:
In rice soil blank (PS-CK), black earth blank (BS-CK), almost occur without trimeric cyanamide Degradation; The degradation rate of the rice soil (PS-B) of bacterium liquid process and the middle trimeric cyanamide of black earth (BS-B) is respectively 14.67% and 21.86%; After charcoal process, in rice soil and black earth, the content of trimeric cyanamide have dropped 13.6% and 14.4% respectively in one week.This is mainly caused by the adsorption of charcoal to trimeric cyanamide; And after adding charcoal immobilization degradation bacteria, in soil, the concentration of trimeric cyanamide significantly declines, between 35 days incubation periods, in rice soil and black earth, the concentration of trimeric cyanamide reduces 67.34% and 70.66% respectively.
Table 1
The degradation bacteria strains that the present invention filters out has obvious trimeric cyanamide degradation effect, in use, itself and charcoal is composite, then the degradation effect of trimeric cyanamide significantly improves, illustrate in trimeric cyanamide contaminated soil, the use of charcoal and degradation bacteria strains has obvious synergy, and this discovery is significant to environment remediation, so the present invention is creative.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a strain take trimeric cyanamide as the degradation bacteria of substrate, it is characterized in that, described degradation bacteria is Burkholderia cepacia Burkholderia cepacia CGMCC No.9843.
2. according to claim 1 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and it is characterized in that, described screening method comprises the steps:
The enrichment culture of step one, degradation bacteria: take by trimeric cyanamide pollute soil sample in containing trimeric cyanamide enrichment medium in, 20-37 DEG C, cultivate 7-10 days under 140-210r/min condition, be forwarded to the fresh enrichment medium containing trimeric cyanamide to cultivate, switching for several times, obtains enrichment bacterium liquid;
The domestication of step 2, degradation bacteria is cultivated: get enrichment bacterium liquid, be forwarded in basic medium, adds trimeric cyanamide and carries out domestication and cultivate, 20-37 DEG C, cultivate 7-10 days under 140-210r/min condition, and switching several, must tame bacterium liquid;
Step 3, be separated single bacterium colony: dilution domestication bacterium liquid, is seeded to LB solid medium dull and stereotyped, is inverted and cultivates 3-5 days, obtain single bacterium colony of degradation bacteria in 20-37 DEG C of constant temperature;
Step 4, bacterial isolation: the single bacterium colony getting degradation bacteria, transfer containing in the screening culture medium of trimeric cyanamide, 20-37 DEG C of constant temperature is inverted and is cultivated 7-10 days, selects and can grow and have the bacterial strain of obvious transparent circle, be namely able to the degradation bacteria that trimeric cyanamide is substrate.
3. according to claim 2 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and it is characterized in that, in step one, described enrichment medium comprises component and content:
4. according to claim 2 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, it is characterized in that, in step one, in described enrichment medium, the concentration of trimeric cyanamide increases with the increase of switching number of times.
5. according to claim 2 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and it is characterized in that, in step 4, component and the content of described screening culture medium comprise:
6. according to claim 5 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and it is characterized in that, the component of described screening culture medium and content are:
7. according to claim 2 take trimeric cyanamide as the screening method of the degradation bacteria of substrate, and it is characterized in that, in step 4, the concentration of described trimeric cyanamide is 10 ~ 60mg/L.
8. one kind according to claim 1 is that the degradation bacteria of substrate is repairing the purposes in trimeric cyanamide contaminated soil with trimeric cyanamide.
9. according to claim 1 is that the degradation bacteria of substrate is repairing the using method in trimeric cyanamide contaminated soil with trimeric cyanamide, described method comprise the bacterium liquid adding with trimeric cyanamide the degradation bacteria being substrate in trimeric cyanamide contaminated soil, the charcoal being adsorbed with trimeric cyanamide the degradation bacteria being substrate or be adsorbed with trimeric cyanamide be substrate degradation bacteria containing polyvinyl alcohol and alginate carrier.
10. one kind containing according to claim 1 take trimeric cyanamide as the degradation bacteria of substrate and the composition of charcoal or containing the composition taking trimeric cyanamide as the degradation bacteria of substrate, polyvinyl alcohol and alginate carrier.
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| CN112940732A (en) * | 2021-02-18 | 2021-06-11 | 陕西省微生物研究所 | Soil organic phosphorus pesticide degradation catalyst and preparation method thereof |
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| CN112940732A (en) * | 2021-02-18 | 2021-06-11 | 陕西省微生物研究所 | Soil organic phosphorus pesticide degradation catalyst and preparation method thereof |
| CN112940732B (en) * | 2021-02-18 | 2022-02-08 | 陕西省微生物研究所 | Soil organic phosphorus pesticide degradation catalyst and preparation method thereof |
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