CN102433272A - Xanthobacter flavus DT8 and the use thereof for degrading cyclic ethers - Google Patents
Xanthobacter flavus DT8 and the use thereof for degrading cyclic ethers Download PDFInfo
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Abstract
The invention discloses xanthobacter flavus DT8 and use thereof for degrading cyclic ethers. In the invention, a bacterial agent containing xanthobacter flavus DT8 is used as an enzyme source, cyclic ether compounds are used as a substrate, and the xanthobacter flavus DT8 is cultured in an organic salt culture medium at 30 to 37 DEG C on a shake at a shaking speed of 120 to 160r/min for 40 to 48 hours to degrade the cyclic ether compounds; and the cyclic ether compounds are tetrahydrofuran and 1,4-dioxane. The enlarged culture of the xanthobacter flavus DT8 disclosed by the invention is easy to implement, the xanthobacter flavus DT8 has high cyclic ether degrading capacity, is suitable for biodegradation of waste water or gas containing cyclic ether compounds, is environment-friendly and has very high engineering value and economic value.
Description
(1) technical field
The present invention relates to a kind of cyclo other compounds degraded bacillus and application thereof, particularly a kind of yellow bacillus flavus DT8 and the application in the microbiological deterioration cyclo other compounds thereof.
(2) background technology
Cyclo other compounds is that application organic solvent is extremely widely gone up in industry, comprises THF (THF) 、 diox 、 trioxane etc.Cyclo other compounds can pass through respiratory tract, digestive tube, skin intrusion body, during lower concentration skin and mucous membrane is had hormesis; Anesthetic action, hepatotoxicity and lethal effect are arranged during high density; Secular toxicity test shows that cyclo other compounds also has stronger carinogenicity to animal.In recent years, especially THF is with the consumption of diox increases cyclic ethers class material year by year, and problem of environmental pollution is on the rise.The present a plurality of developed country such as the U.S., Japan, Canada etc. have detected THF and 1, the pollution of 4-diox in underground water well and atmosphere; Discovery China pharmaceutical factory output waters such as Li Haiyan are drunk the pollution that all there is THF in surface water with certain.Therefore, cause the control of environmental pollution very urgent by cyclic ethers class material.
Cyclic ethers class material is an oxygen heterocyclic ring class material, is difficult to its degraded through general advanced oxidation such as ozone method.The pollution that biological process is removed cyclic ethers class material has obtained both domestic and externally consistently approving.Screen at present have the degraded THF and 1, the bacterial strain of 4-diox has Rhodococcus ruber 219, actinomycetes CB1190, Pseudonocardia sp.ENV478 and Cordyceps sinesis etc.But the substrate broad spectrum of these bacterial strains remains to be further expanded, and the enlarged culturing of thalline is also difficult.Therefore, filter out the bacterial strain of multiple cyclic ethers class material of to degrade, and carry out large scale culturing and have extremely important construction value and economic worth with the improvement that is applied to cyclic ethers class material and pollutes.
(3) summary of the invention
The object of the invention provides a kind of yellow bacillus flavus DT8 and application in the microbiological deterioration cyclo other compounds thereof that is obtained by screening in the environment; And thalline spread cultivation method and fungicide preparation mode are provided, can be used for containing in the environment improvement of the waste water and the waste gas of cyclo other compounds.
The technical scheme that the present invention adopts is:
Yellow bacillus flavus DT8 (Xanthobacter flavus DT8) is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, preservation date: on April 29th, 2011, deposit number: CCTCC NO:M 2011148.
The screening of yellow bacillus flavus DT8 bacterial strain according to the invention:
1) bacterial classification source: the active sludge of the medical factory in Shanghai sewage plant Aerobic Pond.
2) bacterial strain screening: take from the active sludge of the medical factory in Shanghai sewage plant Aerobic Pond, and process mixed culture medium after minimal medium mixes by 1: 1 (v/v) and place wide-necked bottle, TV is 4L; With 1, the 4-diox be substrate as the sole carbon source and the energy, 25 ℃～35 ℃ acclimation shaking culture; And concentration of substrate reached 500mg/L (substrate adds preceding sampling detection residual substrate concentration) after 2d added a feasible adding of substrate at interval; 7d changes mixed culture medium at interval, and after 7～September, the mud of domestication can be stablized degraded 200mg/L diox every day; This sample is transferred into shaking a bottle further enrichment, obtains the domestication sample;
3) will tame sample and be seeded to the 50mL minimal medium by 10% access amount, with 1, the 4-diox is a substrate, detects residual substrate concentration after cultivating 24h on 30 ℃, 160r/min shaking table; Treat that the degradation of substrates rate reaches about 80%, be transferred to 10% access amount again and contain 1 of equal concentrations, in the fresh minimal medium of 4-diox (medium component is identical with the acclimation shaking culture base), after the enrichment of repeatedly going down to posterity; Dilute coating, picking list bacterium colony, again with 1, the 4-diox is a substrate; Carry out degrading activity and measure, separation and purification obtains a strain and has 1; The bacterial strain DT8 of 4-diox degrading activity, said 1,4-diox concentration is 100mg/L.
The strain identification of purpose bacterial strain DT8 according to the invention:
1) described bacterial strain DT8 morphological specificity and physiological and biochemical property are: observation of cell is shaped as shaft-like or arcuation under the opticmicroscope, and size is 0.2～0.5 μ m * 1～2.5 μ m, amphitrichous; No gemma, Gram-negative is after 30 ℃ of solid plate substratum are cultivated 48h; Single bacterium colony is yellow, and circle is opaque; Neat in edge is prone to provoke; The physiological and biochemical test result is: oxydase and catalase are positive, and nitrate reduction test and citrate test are positive; Indoles, methyl red test feminine gender, amylolytic enzyme is negative, and the gelatin test is negative.
2) the 16S rRNA sequential analysis of described bacterial strain DT8
At first by extracting the full genome that the genome test kit extracts the purpose bacterial strain; DNA to this bacterial strain carries out pcr amplification; Obtain the 16S rRNA amplified production of about 1500bp, amplimer is universal primer F8 (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and the R1541 (5 '-AGAAAGGAGGTGATCCAGCC-3 ') of bacterium conserved sequence.This sequence is carried out the homology comparison with the gene order among the GenBank after order-checking, find that the similarity of bacterial strain DT8 and yellow bacillus flavus (Xanthobacter flavus) reaches 99%.
Through 16S rRNA homology analysis and combine physiological and biochemical property, be yellow bacillus flavus with identification of strains of the present invention, the yellow bacillus flavus DT8 (Xanthobacter flavus DT8) of called after.
The application of yellow bacillus flavus DT8 of the present invention in the degraded cyclo other compounds.
Described cyclo other compounds is preferably THF (THF) or 1,4-diox.
Yellow bacillus flavus DT8 of the present invention being applied as in the degraded cyclo other compounds: with the microbial inoculum that contains yellow bacillus flavus DT8 is the enzyme source; With the cyclo other compounds is substrate; In minimal medium; At 25～35 ℃, 120～160r/min shaking table is cultivated 40～80h, makes the cyclo other compounds degraded; Said cyclo other compounds is THF or 1, the 4-diox; Said substrate starting point concentration is 50～1200mg/L; Said minimal medium final concentration consists of: Na
2HPO
412H
2O 4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
22H
2O 0.023g/L, micro-mother liquor 1mL/L, pH 7.0, and solvent is a water, and said micro-mother liquor final concentration is formed: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O 0.10g/L, ZnSO
47H2O 0.10g/L, Na
2MoO
42H
2O 0.02g/L, CoCl
26H
2O 0.02g/L.
Further, the said microbial inoculum that contains the cell of yellow bacillus flavus DT8 bacterium is the cell fermentation liquid that yellow bacillus flavus DT8 contains the DT8 bacterium.
Further; The said microbial inoculum that contains yellow bacillus flavus DT8 is the solid-state microbial inoculum that contains yellow bacillus flavus DT8, and the said solid-state microbial inoculum that contains yellow bacillus flavus DT8 prepares as follows: the cell fermentation liquid that yellow bacillus flavus DT8 is contained the DT8 bacterium is in peat composed of rotten mosses carrier and minimal medium, behind the stirring and evenly mixing; In 25～35 ℃ of following solid state fermentation 24～48h; 30～37 ℃ of perseverances are surely dry, dry back grind into powder, and acquisition contains the solid-state microbial inoculum of cell of DT8 bacterium; Said peat composed of rotten mosses carrier is that the peat composed of rotten mosses and water are with 1: 2 mixture of mass ratio; Dry mycelium concentration is 0.77～1.54g/L in the cell fermentation liquid that contains the DT8 bacterium of said yellow bacillus flavus DT8; Number of cells is 0.1 * 10 in the said solid-state microbial inoculum that contains yellow bacillus flavus DT8
10～5 * 10
10CFU/g; The consumption of said peat composed of rotten mosses carrier and the consumption of minimal medium are to not influence of the present invention, and the solid-state microbial inoculum that the present invention contains yellow bacillus flavus DT8 measures with the number of cells of DT8, and the cell fermentation liquid-solid state fermentation that contains the DT8 bacterium is got final product.
The cell fermentation liquid that contains the DT8 bacterium of above-mentioned said yellow bacillus flavus DT8 can prepare as follows: 1) slant culture: yellow bacillus flavus DT8 is seeded to the R2A solid medium; 30～32 ℃ of slant culture 40～48h; Obtain the inclined-plane thalline, said R2A solid medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water; Agar 15～18g/L; 2) seed culture: the inclined-plane thalline of picking step 1) preparation is seeded to the R2A liquid nutrient medium; Cultivate 24～48h for 30～37 ℃; Obtain seed liquor, said R2A liquid nutrient medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water; 3) fermentation culture: with step 2) seed liquor of preparation is seeded to fermention medium with 5～10% inoculum sizes, interior 30～37 ℃ of fermentor tank, and pH value 6.5～7.3, dissolved oxygen (DO) maintains 2.0～3.0, cultivates 40～48h, obtains to contain the cell fermentation liquid of DT8 bacterium; Said fermention medium final concentration consists of: yeast powder 0.5g/L, Na
2HPO
412H
2O 4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
20.023g/L micro-mother liquor 1mL/L, solvent are water, original ph is 6.7～7.2, and micro-mother liquor final concentration consists of: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O 0.10g/L, ZnSO
47H
2O 0.10g/L, Na
2MoO
42H
2O 0.02g/L, CoCl
26H
2O 0.02g/L.
In addition; The cell fermentation liquid that contains the DT8 bacterium of described yellow bacillus flavus DT8 also can prepare as follows: a) slant culture: yellow bacillus flavus DT8 is seeded to the R2A solid medium; 30～32 ℃ of slant culture 40～48h; Obtain the inclined-plane thalline, said R2A solid medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L, agar 15～18g/L, pH7.2, solvent are water; B) seed culture: the inclined-plane thalline of picking step a) preparation is seeded to the R2A liquid nutrient medium, cultivates 24～48h for 30～37 ℃, obtains seed liquor; Said R2A liquid nutrient medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water; C) fermentation culture: the seed liquor of step b) preparation is seeded to R2A liquid nutrient medium or broth culture with 5～10% inoculum sizes; Add the glycerine skimmer in the fermentor tank; 30～37 ℃, pH value 6.5～7.5, dissolved oxygen (DO) maintains 2.0～3.0; Cultivate 40～48h, obtain to contain the cell fermentation liquid of DT8 bacterium; Said R2A liquid nutrient medium final concentration is formed same step b); Said broth culture final concentration consists of: yeast powder 10g/L, peptone 10g/L, NaCl 5g/L; Solvent is a water; PH7.2～7.4, the consumption of said glycerine skimmer can reach the froth breaking purpose usually and get final product not influence of the present invention.
Further; Yellow bacillus flavus DT8 of the present invention being applied as in the degraded cyclo other compounds: with the microbial inoculum that contains yellow bacillus flavus DT8 is the enzyme source, and the described microbial inoculum that contains yellow bacillus flavus DT8 is the solid-state microbial inoculum that contains yellow bacillus flavus DT8, is the sole carbon source and the energy with the cyclo other compounds; In minimal medium; 30～37 ℃, 120～160r/min shaking table is cultivated 40～48h, makes the cyclo other compounds degraded; Number of cells is 1 * 10 in the said solid-state microbial inoculum that contains yellow bacillus flavus DT8
10CFU/g, said cyclo other compounds are THF or 1, the 4-diox, and the starting point concentration of THF Huo diox is preferably 50～1200mg/L.
The cell fermentation liquid that yellow bacillus flavus DT8 according to the invention contains the DT8 bacterium can directly be used for the degraded of cyclo other compounds, and (stem cell concentration is 0.77～1.54g/mL) or gets solid-state microbial inoculum 0.5g (number of cells is 10 to get the cell fermentation liquid 0.5mL that yellow bacillus flavus DT8 contains the DT8 bacterium
10CFU/g) be seeded to and contain 100mg/L 1, in the minimal medium of 4-diox, detect 1, the degraded situation of 4-diox; Wherein, the cell fermentation liquid that contains the DT8 bacterium can be with 1 in 30～40h, and the 4-diox is degraded fully; Solid-state microbial inoculum can degrade 1 fully in 35～50h, the 4-diox.
Appropriate pH value and temperature that the present invention contains the microbial inoculum degraded cyclo other compounds of yellow bacillus flavus DT8 are respectively 6.5～7.5 and 30～37 ℃; Through the degraded situation of research microbial inoculum to the different concns substrate, show 1, the degradation rate of 4-diox receives the influence of substrate starting point concentration height less, do not have obvious lag phase at the degraded initial stage, but high density (1.2g/L) substrate can not be degraded fully.When concentration of substrate was 100mg/L, the degraded of THF only needed 28h, and 1, the 4-diox then needs 48h, and visible DT8 is different to the degradation capability of different substrates.
Dry mycelium method for measurement of concentration according to the invention is: get a certain amount of fermented liquid, measure OD
600Value, again that fermented liquid is centrifugal, deposition is dried to constant weight, measures dry cell weight in the fermented liquid, with OD
600Value is X-coordinate, and dry cell weight is an ordinate zou, draws concentration map, according to OD in the fermented liquid
600Value is calculated dry mycelium concentration.
The solid-state microbial inoculum that contains yellow bacillus flavus DT8 according to the invention adopts number of cells CFU/g to represent; Its method for expressing is: get the solid-state microbial inoculum of 0.5g and be dissolved in the saline water of 5mL; Drip to blood counting chamber behind the dilution different concns and carry out the thalline counting, obtain number of cells through unit conversion again in microscopically.
Employing R2A liquid nutrient medium of the present invention or broth culture are as fermention medium; During fermentation need in fermentor tank, not add all the other materials; Reduce the microbiological contamination probability; And shorten the generation time of cell growth, thereby in the identical time, obtain more thalline, the thalline that this substratum obtains does not influence its degrading activity to cyclo other compounds.
Compared with prior art; Beneficial effect of the present invention is mainly reflected in: the yellow bacillus flavus DT8 of the present invention bacterial strain is easy to enlarged culturing; Performance with efficient degradation cyclo other compounds; Be applicable to the waste water that contains cyclo other compounds or the biological degradation of waste gas, environmental friendliness has extremely important construction value and economic worth.
(4) description of drawings
Fig. 1 contains the degraded situation of yellow bacillus flavus DT8 microbial inoculum to cyclo other compounds
Fig. 2 contains the solid-state fungicide preparation schema of yellow bacillus flavus DT8
The performance of the yellow bacillus flavus DT8 degraded of Fig. 3 cyclo other compounds
Fig. 4 pH value is to the influence of the solid-state microbial inoculum degraded cyclo other compounds that contains yellow bacillus flavus DT8
Fig. 5 temperature is to the influence of the solid-state microbial inoculum degraded cyclo other compounds that contains yellow bacillus flavus DT8
Fig. 6 contains the performance of the solid-state microbial inoculum of yellow bacillus flavus DT8 to the degraded of different concns cyclo other compounds; A is that the solid-state microbial inoculum of yellow bacillus flavus DT8 is to different concns 1; The performance of 4-diox degraded, B is the performance of the solid-state microbial inoculum of yellow bacillus flavus DT8 to different concns THF degraded.
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Said minimal medium final concentration consists of: Na
2HPO
412H
2O 4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
22H
2O 0.023g/L, micro-mother liquor 1mL/L, pH 7.0, and solvent is a water, and said micro-mother liquor is formed: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O0.10g/L, ZnSO
47H
2O 0.10g/L, Na
2MoO
42H
2O 0.02g/L, CoCl
26H
2O 0.02g/L.
Said R2A liquid nutrient medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water.
Said fermention medium final concentration consists of: yeast powder 0.5g/L, Na
2HPO
412H
2O4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
20.023g/L micro-mother liquor 1mL/L, solvent are water, original ph is 6.7～7.2, and micro-mother liquor final concentration consists of: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O 0.10g/L, ZnSO
47H
2O 0.10g/L, Na
2MoO
42H
2O0.02g/L, CoCl
26H
2O 0.02g/L.
The final concentration of said broth culture consists of: yeast powder 10g/L, peptone 10g/L, NaCl5g/L, solvent are water, pH7.2～7.4.
Said R2A solid medium final concentration consists of: add 1.5%～1.8% agar in the R2A liquid nutrient medium.
The screening and the evaluation of embodiment 1 yellow bacillus flavus DT8 bacterial classification
(1) bacterial strain screening
A, primary dcreening operation: take from the active sludge of the medical factory in Shanghai sewage plant Aerobic Pond, and process mixed culture medium after minimal medium mixes by 1: 1 (v/v) and place wide-necked bottle, with 1; The 4-diox be substrate as the carbon source and the energy, 30 ℃ of following aeration acclimation shaking culture of bath temperature, and at interval 2d adds substrate one time; Sampling added an amount of substrate after detecting residual substrate concentration again before substrate added, and made to add back 1, and 4-diox concentration reaches 500mg/L; At interval 7d changes mixed-culture medium, after 7～9 months, and the mud of domestication about 200mg/L diox of can on average degrading every day; Explain that domestication produces effect, this sample is transferred into shaking a bottle further enrichment;
B, multiple sieve and separation and purification: will tame sample and be seeded in the 50mL minimal medium (identical with the acclimation shaking culture based component) by 10% access amount, with 1 of 100mg/L, the 4-diox is the sole carbon source and the energy, carries out the screening of purpose bacterial strain; Cultivate on 30 ℃, 160r/min shaking table, after 24h detects the substrate residual concentration, treats that the concentration of substrate degradation rate reaches more than 80%; Be forwarded to 10% inoculum size again and contain 1 of 100mg/L, in the fresh minimal medium of 4-diox, by that analogy; The enrichment of repeatedly going down to posterity is diluted coating, picking list bacterium colony; With 1, the 4-diox is a substrate again, carries out degrading activity and measures; Separation and purification obtains a strain and has 1, the bacterial strain DT8 of 4-diox degrading activity.
(2) identification of strains
A, strain morphology characteristic and physiological and biochemical property:
Observation of cell is shaped as shaft-like or arcuation under the opticmicroscope, and size is 0.2～0.5 μ m * 1～2.5 μ m, amphitrichous, no gemma; Gram-negative, after 30 ℃ of solid plate substratum were cultivated 48h, it is yellow that single bacterium colony is, circle; Opaque, neat in edge is prone to provoke;
Part physiological and biochemical test result is: oxydase and catalase are positive, and nitrate reduction test and citrate test are positive; Indoles, methyl red test feminine gender, amylolytic enzyme is negative, and the gelatin test is negative, and sugar fermentating test is negative.
B, 16S rDNA sequencing
At first by extracting the full genome that genome test kit (3S DNA Isolution Kit V2.2 Shen ability lottery industry bio tech ltd) extracts the purpose bacterial strain; DNA to this bacterial strain carries out pcr amplification; Obtain the 16S rRNA amplified production of about 1500bp, amplimer is universal primer F8 (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and the R1541 (5 '-AGAAAGGAGGTGATCCAGCC-3 ') of bacterium conserved sequence.This amplified production carries out the homology comparison with the gene order among the GenBank after order-checking, find that the similarity of bacterial strain DT8 and yellow bacillus flavus (Xanthobacter flavus) reaches 99%.Through 16S rDNA homology analysis and combine physiological and biochemical property, be yellow bacillus flavus (Xanthobacter flavus) with identification of strains of the present invention, yellow bacillus flavus (Xanthobacter flavus) DT8 of called after.
Embodiment 2 yellow bacillus flavus DT8 contain the preparation of the cell fermentation liquid of DT8 bacterium
1) slant culture: yellow bacillus flavus DT8 is seeded to the R2A solid medium, cultivates 48h for 30 ℃, obtain the inclined-plane thalline.
2) seed culture: the inclined-plane thalline of picking step 1) preparation is seeded to the R2A liquid nutrient medium, cultivates 24h for 30 ℃, obtains seed liquor;
3) fermentation culture: with step 2) seed liquor of preparation is seeded to fermention medium with inoculum size 10%, interior 30 ℃ of fermentor tank, and pH value 7.0, dissolved oxygen (DO) maintains 2.0～3.0, cultivates 48h, obtains to contain the cell fermentation liquid of DT8 bacterium; The cell fermentation liquid of the said DT8 of containing bacterium finally records thalline OD
600Reach 2.0 approximately, dry mycelium concentration reaches 0.77g/L.
Embodiment 3 yellow bacillus flavus DT8 contain the preparation of the cell fermentation liquid of DT8 bacterium
1) slant culture and seed culture method are with embodiment 2.
2) fermentation culture: the seed liquor of preparation is seeded to the R2A liquid nutrient medium of 3.5L with inoculum size 10%, adds 0.5g glycerine in the fermentor tank as skimmer, 30 ℃; PH value 7.0; Dissolved oxygen (DO) keeps 2.0～3.0, cultivates 48h, obtains to contain the cell fermentation liquid of DT8 bacterium; The cell fermentation liquid of the said DT8 of containing bacterium finally records thalline OD
600Reach 4.0, the cell fermentation liquid dry mycelium concentration that contains the DT8 bacterium reaches 1.54g/L.
Embodiment 4 yellow bacillus flavus DT8 contain the preparation of the cell fermentation liquid of DT8 bacterium
1) slant culture and seed culture method are with embodiment 2.
2) fermentation culture: the seed liquor of preparation is seeded to the broth culture of 3.5L with inoculum size 10%, adds 0.5g glycerine in the fermentor tank as skimmer, 30 ℃; PH value 7.0; Dissolved oxygen (DO) keeps 2.0～3.0, cultivates 48h, obtains to contain the cell fermentation liquid of DT8 bacterium; The cell fermentation liquid of the said DT8 of containing bacterium finally records thalline OD
600Reach 3.0, the cell fermentation liquid dry mycelium concentration that contains the DT8 bacterium reaches 1.2g/L.
1) contain the preparation of the cell fermentation liquid microbial inoculum of DT8 bacterium: the plastic barrel that the fermented liquid that embodiment 3 preparing methods are obtained goes out jar directly with 10L encapsulates as liquid microbial inoculum, and this liquid state microbial inoculum can directly apply to and contain 1, the improvement of 4-diox sewage.Get liquid microbial inoculum 0.5mL and be seeded to and contain 100mg/L 1, in the 50mL minimal medium of 4-diox, 4h at interval, sampling detects 1, the residual concentration of 4-diox.The liquid microbial inoculum of result such as Fig. 1 can be with 1 in 36h, and the 4-diox is degraded fully.Therefore, prove that liquid microbial inoculum has higher degrading activity.
2) contain the preparation of the solid-state microbial inoculum of cell of DT8 bacterium: the preparation process is as shown in Figure 2: at first with the water that adds the twice quality in the 20g peat composed of rotten mosses, mix and obtain peat composed of rotten mosses carrier 60g, the bacterium of going out is subsequent use; After the fermented liquid that embodiment 2 preparing methods are obtained goes out jar, get fermented liquid (OD
600About 2.0; Dry mycelium concentration is 0.77g/L) 10mL joins containing in the 60g peat composed of rotten mosses carrier container of the bacterium of going out, and adds the 10mL minimal medium again as nutritive medium, room temperature solid fermentation 48h behind the stirring and evenly mixing; 37 ℃ of freeze-day with constant temperature; The grind into powder packing of dry back is preserved, and obtains to contain the solid-state microbial inoculum of yellow bacillus flavus DT8 bacterium, and its number of cells is stabilized in 1 * 10
10About CFU/g, get solid-state microbial inoculum 0.5g and be seeded to and contain 100mg/L 1, in the 50mL minimal medium of 4-diox, 4h at interval, sampling detects 1, the residual concentration of 4-diox.Solid-state microbial inoculum as shown in Figure 1 can be with 1 in 40h, and the 4-diox is degraded fully.Therefore, prove that solid-state microbial inoculum also has higher degrading activity.
The characteristic of embodiment 6 yellow bacillus flavus DT8 degraded different rings ether compounds
Liquid microbial inoculum (cell concentration OD with the preparation of embodiment 5 methods
600Reach 2.0) the centrifugal enrichment thalline; Process the cell bacteria suspension that contains the DT8 bacterium for twice with the phosphoric acid buffer rinse of 0.5mol/L; Be seeded to respectively with 1,4-diox and THF are the 50mL minimal medium of the sole carbon source and the energy, make the initial cell concentration (OD of the cell bacteria suspension that contains the DT8 bacterium
600) be 0.01 (dry weight 4.4mg/L), 1,4-diox and THF substrate starting point concentration are respectively 100mg/L, and put into temperature respectively and be 30 ℃, revolution and be the shaking table of 160r/min and cultivate, the not timing sampling, the degraded situation of observing substrate, the result sees Fig. 3.Along with the prolongation of time, concentration of substrate is reduced to degraded fully gradually.The THF degraded only needs 28h in the substrate of same concentrations, and 1, the 4-diox then needs 48h, and visible X.flavus DT8 is different to the degradation capability of different substrates.
Embodiment 7pH value is to the influence of yellow bacillus flavus DT8 degraded cyclo other compounds
(solid-state microbial inoculum number of cells is 1 * 10 to take by weighing the solid-state microbial inoculum 0.5g that contains yellow bacillus flavus DT8 of case study on implementation 5 methods preparations respectively
10CFU/g), be dissolved in the minimal medium of different initial pH enzyme source respectively as the degraded cyclo other compounds.Use the 1mol/LNaOH aqueous solution or the 1mol/LHCl aqueous solution to regulate minimal medium and be different pH values (4.0,5.0,6.0,6.5,7.0,7.5,8.0,9.0,10.0), experiment divides two groups (1,4-diox group and THF group); Each pH value establish 3 parallel, 1 blank adds 1 respectively; 4-diox and THF are as the unique carbon source and the energy, and the substrate starting point concentration is respectively under the condition of 100mg/L, do not connect the enzyme source as blank; Place shaking culture in 30 ℃, 160r/min constant temperature shaking table respectively, take a sample after cultivating 36h, record in the reaction solution remaining 1 respectively; The concentration of 4-diox and THF, the result sees Fig. 4.
Visible by Fig. 4, in pH4.0～10.0, mikrobe all can degrade THF and 1,4-diox; Along with pH increases to 10.0 from 4.0, the degradation rate of substrate all increases afterwards earlier and reduces, solid-state microbial inoculum degraded 1, and the pH value that suits of 4-diox and THF is about 7.0.The solid-state microbial inoculum substrate of in the neutral wider pH range, better must degrading is described, for its application at different pH environment provides assurance.
Embodiment 8 temperature are to the influence of yellow bacillus flavus DT8 degraded cyclo other compounds
(solid-state microbial inoculum number of cells is 1 * 10 to take by weighing the solid formulation 0.5g that contains yellow bacillus flavus DT8 of case study on implementation 5 methods preparations respectively
10CFU/g), be dissolved in the enzyme source of conduct degraded cyclo other compounds in the minimal medium respectively.Substrate is divided into 1, two groups of 4-diox and THF, each temperature establish 3 parallel, 1 blank, blank does not add solid-state microbial inoculum, the substrate starting point concentration is the 100mg/L minimal medium.It is 25 ℃, 30 ℃, 37 ℃, 40 ℃ shaking table constant-temperature shaking culture (the shaking table revolution is 160r/min) that each sample is placed temperature respectively, the concentration of residual substrate in the measured reaction liquid behind the cultivation 36h.Can know that by Fig. 5 in 25 ℃～40 ℃ TRs, solid-state microbial inoculum all can degrade 1,4-diox and THF, and when being the sole carbon source and the energy with THF its degradation rate all greater than 1, the 4-diox.But solid-state microbial inoculum degraded 1, the optimal temperature of 4-diox is 37 ℃, and the optimal temperature of degraded THF is 30 ℃.Therefore infer that optimum temperuture is between 30～37 ℃.
The performance of embodiment 9 yellow bacillus flavus DT8 degraded different concns cyclo other compounds
(solid-state microbial inoculum number of cells is 1 * 10 to take by weighing the solid-state microbial inoculum 0.5g that contains yellow bacillus flavus DT8 of case study on implementation 5 methods preparations respectively
10CFU/g), be substrate with the cyclo other compounds of different starting point concentrations, than suitable culture condition (pH7.0; 37 ℃ of temperature) under, research is the degradation property of the microbial inoculum of dominant strain to different concentration of substrate cyclic ethers class materials with yellow bacillus flavus, and experiment is divided into two groups (1; 4-diox group and THF group); Each concentration of substrate establish 3 parallel, 1 blank, blank does not add solid-state microbial inoculum.The substrate that in minimal medium, adds different concns; PH7.0, temperature is reacted 135h down for 37 ℃, at interval the concentration of substrate in 6h or the 12h measured reaction liquid; Wherein 1, the substrate starting point concentration of 4-diox is respectively 50,100,200,300,400,500,650,850 and 1200mg/L; The substrate starting point concentration of THF is respectively 50,100,200,500,800,1000 and 1500mg/L.The result is as shown in Figure 6, can be known by figure A, solid-state microbial inoculum degraded 1, and the degradation rate of 4-diox receives the influence of substrate starting point concentration less, and visible diox is less to the toxicity of solid-state microbial inoculum, and can be with its mineralising of degrading fully under high density.Slightly different with it is solid-state microbial inoculum is significantly higher than 1 to the degradation rate of THF, 4-diox (figure B), but this bacterial strain THF of degrading high concentration fully promptly degrades and to finite concentration, can not THF be degraded again.
Claims (10)
1. yellow bacillus flavus DT8 (Xanthobacter flavus DT8) is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, preservation date: on April 29th, 2011, deposit number: CCTCC NO:M 2011148.
2. yellow bacillus flavus DT8 as claimed in claim 1 is characterized in that described yellow bacillus flavus DT8 is shaft-like or arcuation, and size is 0.2～0.5 μ m * 1～2.5 μ m, amphitrichous, no gemma, Gram-negative; After 30 ℃ of solid plate substratum were cultivated 48h, it is yellow that single bacterium colony is, circle, and opaque, neat in edge is prone to provoke; Oxydase and catalase are positive, and nitrate reduction test and citrate test are positive; Indoles, methyl red test feminine gender, amylolytic enzyme is negative, and the gelatin test is negative.
3. the application of yellow bacillus flavus DT8 as claimed in claim 1 in the degraded cyclo other compounds.
4. the application of yellow bacillus flavus DT8 as claimed in claim 3 in the degraded cyclo other compounds is characterized in that described cyclo other compounds is: THF or 1,4-diox.
5. the application of yellow bacillus flavus DT8 as claimed in claim 3 in the degraded cyclo other compounds; It is characterized in that described being applied as: with the microbial inoculum that contains yellow bacillus flavus DT8 is the enzyme source; With the cyclo other compounds is substrate, in minimal medium, at 25～35 ℃; 120～160r/min shaking table is cultivated 40～80h, makes the cyclo other compounds degraded; Said cyclo other compounds is THF or 1, the 4-diox; Said substrate starting point concentration is 50～1500mg/L; Said minimal medium final concentration consists of: Na
2HPO
412H
2O 4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
22H
2O 0.023g/L, micro-mother liquor 1mL/L, pH 7.0, and solvent is a water, and said micro-mother liquor final concentration is formed: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O 0.10g/L, ZnSO
47H
2O 0.10g/L, Na
2MoO
42H
2O 0.02g/L, CoCl
26H
2O 0.02g/L.
6. the application of yellow bacillus flavus DT8 as claimed in claim 5 in the degraded cyclo other compounds is characterized in that the said microbial inoculum that contains yellow bacillus flavus DT8 is the cell fermentation liquid that yellow bacillus flavus DT8 contains the DT8 bacterium.
7. the application of yellow bacillus flavus DT8 as claimed in claim 5 in the degraded cyclo other compounds; It is characterized in that the said microbial inoculum that contains yellow bacillus flavus DT8 is the solid-state microbial inoculum that contains yellow bacillus flavus DT8; The said solid-state microbial inoculum that contains yellow bacillus flavus DT8 bacterium prepares as follows: the cell fermentation liquid that yellow bacillus flavus DT8 is contained the DT8 bacterium is in peat composed of rotten mosses carrier and described minimal medium; Behind the stirring and evenly mixing, in 25～35 ℃ of following solid state fermentation 24～48h, 30～37 ℃ of perseverances are surely dry; Dry back grind into powder, acquisition contains the solid-state microbial inoculum of cell of DT8 bacterium; Said peat composed of rotten mosses carrier is that the peat composed of rotten mosses and water are with 1: 2 mixture of mass ratio; Said yellow bacillus flavus DT8 contains that dry mycelium concentration is 0.77～1.54g/L in the cell fermentation liquid of DT8 bacterium, and number of cells is 0.1 * 10 in the said solid-state microbial inoculum that contains yellow bacillus flavus DT8
10～5 * 10
10CFU/g.
8. like claim 6 or the application of 7 described yellow bacillus flavus DT8 in the degraded cyclo other compounds; The cell fermentation liquid that contains the DT8 bacterium that it is characterized in that described yellow bacillus flavus DT8 prepares as follows: 1) slant culture: yellow bacillus flavus DT8 is seeded to the R2A solid medium; 30～32 ℃ of slant culture 40～48h obtain the inclined-plane thalline; Said R2A solid medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L, agar 15～18g/L, pH7.2, solvent are water; 2) seed culture: the inclined-plane thalline that step 1) is obtained is seeded to the R2A liquid nutrient medium, cultivates 24～48h for 30～37 ℃, obtains seed liquor; Said R2A liquid nutrient medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water; 3) seed liquor that fermentation culture: with step 2) obtains is seeded to fermention medium with 5～10% inoculum sizes, interior 30～37 ℃ of fermentor tank, and pH value 6.5～7.3, the DO value keeps 2.0～3.0, cultivates 40～48h, obtains to contain the cell fermentation liquid of DT8 bacterium; Said fermention medium final concentration consists of: yeast powder 0.5g/L, Na
2HPO
412H
2O 4.5g/L, KH
2PO
41.0g/L, NH
4Cl 1.5g/L, MgSO
47H
2O 0.2g/L, CaCl
20.023g/L micro-mother liquor 1mL/L, solvent are water, original ph is 6.7～7.2, and micro-mother liquor final concentration consists of: FeSO
47H
2O 1.0g/L, CuSO
45H
2O 0.02g/L, H
3BO
30.014g/L, MnSO
44H
2O 0.10g/L, ZnSO
47H
2O 0.10g/L, Na
2MoO
42H
2O 0.02g/L, CoCl
26H
2O 0.02g/L.
9. like claim 6 or the application of 7 described yellow bacillus flavus DT8 in the degraded cyclo other compounds; The cell fermentation liquid that contains the DT8 bacterium that it is characterized in that described yellow bacillus flavus DT8 prepares as follows: a) slant culture: yellow bacillus flavus DT8 is seeded to the R2A solid medium; 30～32 ℃ of slant culture 40～48h obtain the inclined-plane thalline; Said R2A solid medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L, agar 15～18g/L, pH7.2, solvent are water; B) seed culture: the inclined-plane thalline that step a) is obtained is seeded to the R2A liquid nutrient medium, cultivates 24～48h for 30～37 ℃, obtains seed liquor; Said R2A liquid nutrient medium final concentration consists of: yeast powder 0.50g/L, casein food grade 0.50g/L, Zulkovsky starch 0.50g/L, MgSO
47H
2O 0.05g/L, Tryptones 0.50g/L, glucose 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH
2PO
40.45g/L pH7.2, solvent are water; C) fermentation culture: the seed liquor that step b) is obtained is seeded to R2A liquid nutrient medium or broth culture with 5～10% inoculum sizes, adds glycerine skimmer, 30～37 ℃ in the fermentor tank; PH value 6.5～7.3; The DO value keeps 2.0～3.0, cultivates 40～48h, obtains to contain the cell fermentation liquid of DT8 bacterium; Said R2A liquid nutrient medium final concentration is formed same step b), and the final concentration of said broth culture consists of: yeast powder 10g/L, peptone 10g/L, NaCl 5g/L, solvent are water, pH7.2～7.4.
10. the application of yellow bacillus flavus DT8 as claimed in claim 1 in the degraded cyclo other compounds; It is characterized in that described being applied as: with the microbial inoculum that contains yellow bacillus flavus DT8 is the enzyme source, and the described microbial inoculum that contains yellow bacillus flavus DT8 is the solid-state microbial inoculum that contains yellow bacillus flavus DT8, is the sole carbon source and the energy with the cyclo other compounds; In minimal medium; 30～37 ℃, 120～160r/min shaking table is cultivated 40～48h, makes the cyclo other compounds degraded; Number of cells is 1 * 10 in the said solid-state microbial inoculum that contains yellow bacillus flavus DT8
10CFU/g, said cyclo other compounds are THF or 1, the 4-diox; Said cyclo other compounds starting point concentration is 50～1200mg/L.
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