CN101016529A - Agrobacterium tumefaciens capable of metabolizing nicotine and application thereof - Google Patents
Agrobacterium tumefaciens capable of metabolizing nicotine and application thereof Download PDFInfo
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Abstract
The invention discloses a pseudomonas putida to metabolite nicotine, which is characterized by the following: preserving in China Typical Culture Preservation Center (Wuhan university, china Wuhan) with keeping number as CCTCC NO.M 206131; possessing stronger nicotine metabolic ability and nicotine toxin immunity ability; growing highest at 5g/L nicotine density; culturing a great deal of a strain; using intact cell as biological activator; co-suspending in phosphoric acid buffer with solid waste matter from tobacco industry; oscillating; degrading nicotine completely. This bacterium possesses simple operation, which can be used to dispose tobacco waste matter widely.
Description
Technical field
The invention belongs to biological technical field, but relate to the Agrobacterium tumefaciens and the application thereof of a strain metabolizing nicotine specifically.
Background technology
Nicotine (nicotine) is the main alkaloid in many tobaccos, and content reaches 0.5～12%.It is playing the part of important role in people suck the behavior of tobacco, make people produce very strong dependency to tobacco.Yet Nicotine also is a kind of " deleterious Hazardous substances " simultaneously, and disposable soakage reaches the 40ml/ man-hour, can suppress nervus centralis, causes the heart paralysis, and fatal danger is arranged; Other alkaloid in Nicotine and the tobacco in tobacco processing course and smoking moment can be generated distinctive N-nitrosamine (tobacco-specific nitrosamines in the tobacco by nitrosification, TSNA), as nitrosamine4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), they are intensive lung cancer carcinogens.
Tobacco processing course has produced a large amount of solid waste, and these refuses wherein contain the toxic substance based on Nicotine because its physical properties can't be used again, becomes a kind of " dangerous Toxic waste ".They since be difficult for being degraded and long-term existence in environment, brought very big threat to human survival.Nicotine average content in these refuses reaches the 18g/Kg dry weight, and when nicotine content was higher than the 500mg/Kg dry weight, European Union's law was decided to be them " dangerous Toxic waste ".1994, EPA Nicotine has been listed in toxic substance discharge catalogue (Toxics Release Inventory, TRI) in.Even more serious is, and that Nicotine has is well water-soluble, very easily causes phreatic pollution.Therefore press for to take some measures and effectively handle the problems such as refuse that tobacco processing produces.
Nearly decades, some microorganisms are owing to the ability with metabolizing nicotine gets more and more people's extensive concerning, and some microorganisms are also reported in succession.Strain oxidation Arthrobacter (Arthrobacter oxidans) P-34 who separates carclazyte earth as American scholar Rittenberg seminar (1959).Uchida etc. (1983) have investigated the ability of 11 fungal strains that comprise the tobacco pathogenic bacterium and 6 strain actinomycetes degraded Nicotine, find to have only Pellicularia filamentosa (Pellicularia filamentosa) JTS-208 and the little Ke Yinhan of sour jujube spore mould (Cuninghamella echinulata) IFO-4444 to have the ability of degraded Nicotine.The separation from soil of Yuan Yongjun etc. (2005) report obtains a strain Ochrobactrum intermedium, and this bacterial strain can Nicotine be the sole carbon source growth, under its optimal conditions, the Nicotine of 0.5g/L can be degraded 97.6% in 36h.The separation from the soil of tobacco waste pollution of Ruan etc. (2005) report obtains a pseudomonas (Pseudomonas sp.) HF-1, this bacterial strain can Nicotine be sole carbon source and nitrogenous source growth, under its optimal conditions, the Nicotine of 1.3g/L can be degraded 99.6% in 25h.But though these microorganism metabolizing nicotines, its metabolic capacity all a little less than, to the toxic resistance of Nicotine a little less than, these have all limited their application in actual production.
Summary of the invention
But shortcoming at above-mentioned existing metabolizing nicotine microorganism, the invention provides the Agrobacterium tumefaciens (Agrobacterium tumefaciens) that a strain has stronger nicotine metabolite ability and Nicotine toxicity resistance, the intact cell that reaches with this bacterial strain is the application that biological catalyst biological degradation tobacco is processed Nicotine in the solid waste that produces.It is short, simple to operate that this method has the reaction times, the characteristics that security is good.
But the bacterial strain of the metabolizing nicotine that the present invention relates to is Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4.Be preserved in Chinese typical culture collection center (Wuhan University, China, Wuhan) on November 22nd, 2006, its deposit number is: CCTCC NO.M206131.
Colonial morphology and the cell morphological characteristic of above-mentioned Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 be, colony diameter 0.5-1.0mm, and smooth surface, glossy, the edge is translucent, central oyster white; Cell is an elongated rod shape, microbend, and the blunt circle in two ends, length is 1.60～1.84 μ m, diameter is 0.32～0.41 μ m (accompanying drawing 1); Gram-negative, semi-solid puncture is mobility.
The physiological and biochemical property of above-mentioned Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 sees table 1 for details.
The physiological and biochemical property of table 1 Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131
| Physiological and biochemical test | The result | Physiological and biochemical test | The result |
| 35 ℃ of growths | + | Catalase | + |
| The 2%NaCl growth | + | Gelatine liquefication | - |
| Glucose fermentation produces acid | + | Methyl red test | - |
| The litmus milk test | Produce alkali | The V-P test | + |
| The demand of the organic growth factor | - | Produce indole test | - |
| N 2As only nitrogen source | - | Produce H 2S | - |
| Nitrate reduction | + | Produce 3-ketone group lactose | + |
| Nitrate is as only nitrogen source | + | Arg and Lys decarboxylase | - |
| The starch hydrolysis | - | Ferrous citrate hydrogen ammonium | + |
Annotate :+the positive ,-feminine gender.
The utilization of carbon source situation of above-mentioned Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 sees table 2 for details.
The utilization of carbon source situation of table 2.Agrobacterium tumefaciens wsn1-4CCTCCNO.M206131
| Carbon source | The result | Carbon source | The result |
| Glucose | + | Histidine | + |
| Xylitol | + | Ala | + |
| Pectinose | + | Fructose | + |
| Trisodium Citrate | + | Dextrin | + |
| Semi-lactosi | + | Seminose | + |
| Lactose | + | Glycogen | - |
| Sucrose | + | Trehalose | + |
| Rhamnosyl | + | Raffinose | + |
| Glycerine | + | Inositol | + |
| Propanedioic acid | + (weak) | 2, the 3-butyleneglycol | + (weak) |
Annotate :+can utilize ,-do not utilize.
The 16S rDNA sequence of above-mentioned Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 has 100% homology with the 16S rDNA sequence of other bacteriums of other Agrobacterium tumefaciens reported and Agrobacterium, but different is, other bacterial strain of having reported all can not metabolizing nicotine, sees table 3 for details.
The 16S rDNA sequence alignment result of table 3 Agrobacterium tumefaciens wsn1-4 CCTCC NO.M206131
| Data source | Bacterial strain | Homology |
| gb|AY850392.1 | Agrobacterium tumefaciens B8S | 100％ |
| gb|AY513492.1 | Agrobacterium tumefaciens 2001025242 | 100％ |
| gb|AY513491.1 | Agrobacterium tumefaciens 2003015367 | 100％ |
| gb|AY513490.1 | Agrobacterium tumefaciens 2003018195 | 100％ |
| gb|AY513489.1 | Agrobacterium tumefaciens 2002000903 | 100％ |
| gb|AY174112.1 | Agrobacterium sp.JS71 | 100％ |
| dbj|AB116668.1 | Agrobacterium tumefaciens | 100％ |
| emb|AJ130719.1 | Agrobacterium radiobacter LMG 383 | 100％ |
| gb|DQ202333.1 | | Agrobacterium sp.CCBAU 31104 | 100％ |
| emb|AJ389908.1 | Agrobacterium tumefaciens O363 | 100％ |
| emb|AJ389907.1 | Agrobacterium tumefaciens O362 | 100％ |
| emb|AJ389900.1 | Agrobacterium tumefaciens CIP43-76 | 100％ |
| emb|AJ389899.1 | Agrobacterium tumefaciens CIP28-75 | 100％ |
| emb|AJ389898.1 | Agrobacterium tumefaciens CIP127-76 | 100％ |
| emb|AJ389894.1 | Agrobacterium tumefaciens CFBP2884 | 100％ |
| gb|DQ267109.1 | Agrobacterium tumefaciens CCBAU 01041 | 100％ |
The 16S rDNA sequence length of above-mentioned Agrobacterium tumefaciens wsn1-4 CCTCC NO.M206131 is 1371bp, and its nucleotide sequence is shown in SEQ ID NO.1.
Above-mentioned Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 can be sole carbon source, nitrogenous source and energy growth with the Nicotine, growth temperature range is 20～37 ℃, the pH scope is 6.0～8.0, but the concentration of metabolizing nicotine can reach 5g/L (accompanying drawing 2).
The application that utilizes the intact cell of the Agrobacterium tumefaciens that a strain has stronger nicotine metabolite ability and Nicotine toxicity resistance for the Nicotine in the biological catalyst biological degradation tobacco processing solid waste of the present invention, the method steps that this application relates to is as follows:
(1) microorganism strains: select Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in contains on the solid slant culture base that mass volume ratio is 0.02～0.3% Nicotine, under 20 ℃～37 ℃ conditions, static cultivation 24～36 hours;
(3) seed culture: with the bacterial strain of step (2) cultivation, encircle with inoculation articulating 1～2 under aseptic condition that to contain mass volume ratio in 20～100mL be in 0.02～0.3% the Nicotine liquid nutrient medium, under 20 ℃～37 ℃ conditions, shaking culture is 6～30 hours on shaking table, makes first order seed;
(4) enlarged culturing: the inoculum size of volume ratio with 5～10%, connecing first order seed, to contain mass volume ratio in 200～1000mL be in 0.02～0.3% the Nicotine liquid nutrient medium, under 20 ℃～37 ℃ conditions, shaking culture is 6～30 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: the inoculum size of volume ratio with 5～10%, connecing secondary seed, to contain mass volume ratio in 5～20L be in 0.1～0.5% the Nicotine liquid nutrient medium, cultivate under 25 ℃～37 ℃ conditions, the period detecting cell concn, OD value under 620nm reaches 1.2～3.9, stops fermentation culture;
(6) collecting cell: the nutrient solution 5 of getting step (5), under 000 rev/min of condition centrifugal 10～15 minutes, collect the cell of fermentation culture, and with the washing of 20～100mmol/L pH7.0 phosphoric acid buffer, centrifugal with identical condition again, repeat 2～3 times, the cell of collecting precipitation, this cell is biological catalyst, and is 4 ℃ of storages, standby;
(7) removal of Nicotine in the tobacco processing solid waste: the biological catalyst that step (6) is made is suspended in 20～100mmol/L pH7.0 phosphoric acid buffer, mixing, making the OD value of biological catalyst under 620nm in the mixture is 2～10, be 1: 8～20 ratio again according to the volume ratio of tobacco processing refuse quality and biological catalyst suspension, in g/mL, in the biological catalyst suspension, add tobacco processing solid waste, under 20 ℃～40 ℃, 180～350 rev/mins conditions, vibrate; Take a sample during the processing, detect the degraded situation of Nicotine with vapor-phase chromatography, when Nicotine in the mixture detect fully less than after, continue vibration termination after 1～4 hour;
(8) with the mixture after step (7) termination, with 6,000～12,000 rev/min 10～30 minutes centrifugal, remove the supernatant liquor do not contain Nicotine, obtain the biological catalyst and the residual waste that are added in the step (7), remove the residual waste on upper strata, collect the biological catalyst of lower floor;
(9) recycling of biological catalyst: with step (8) collect biological catalyst according to the recycling of the described method of step (7), repeat this step 3～7 time, discard the centrifugal precipitation at last;
(10) supernatant liquor that step (8) and step (9) are obtained mixes, and further handles together in company with general waste water, and step (8) obtains the precipitation that residual waste and step (9) obtain and then can handle with general refuse.
The prescription of above-mentioned steps (3), the described liquid nutrient medium in (4) (5) is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L.
The prescription of the described solid slant culture base of above-mentioned steps (2) is that the preferred mass volume ratio that adds is 1.5～2.0% agar in described liquid nutrient medium.
Above-mentioned steps (2), (3), the described nicotine concentration in (4) (5) preferred 0.1～0.3%.
Preferred 29～32 ℃ of above-mentioned steps (2), (3), the described yeast culture temperature in (4) (5).
Preferred 26～30 hours of described yeast culture time of above-mentioned steps (2).
Above-mentioned steps (3), preferred 12～24 hours of (4) described yeast culture time.
The preferred 50mmol/L of concentration of the described phosphoric acid buffer of above-mentioned steps (6).
The described biological catalyst of above-mentioned steps (7) is meant the intact cell of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131.
Preferred 40～the 80mmol/L of the described phosphoric acid buffer of above-mentioned steps (7).
The OD value preferred 5～7 of biological catalyst in the described mixture of above-mentioned steps (7) under 620nm.
The volume ratio preferred 1: 10～15 of described tobacco processing refuse quality of above-mentioned steps (7) and biological catalyst suspension.
Preferred 28 ℃～32 ℃ of the described conversion reaction temperature of above-mentioned steps (7).
Preferred 2～3 hours of the time of the described continuation vibration of above-mentioned steps (7).
The described vapor-phase chromatography of above-mentioned steps (7) adopts VARIAN CP3380 gas chromatograph, chromatographic column is SPB-5 capillary column (internal diameter 0.32mm, long 30m, thickness 0.25 μ m, U.S. Supelcol company), nitrogen is as carrier gas, testing conditions is 260 ℃ of sampler temperature, 140 ℃ of column temperatures, 280 ℃ of detector temperatures, sample size 1 μ l.
Institute's sample thief utilizes the gas chromatography determination Nicotine after needing to handle in accordance with the following methods during the described processing of above-mentioned steps (7): sample is in 6,000～12,000 rev/min 10～30 minutes centrifugal, getting the 0.2ml supernatant has in the bottle of sealing cover in one, add 1.8ml distilled water, add 50 μ l 1M aqueous sodium carbonates again, with 30 ℃ of oscillation extraction 30min of 2ml chloroform, chloroform phase solution is used for gas chromatography determination.
But the Agrobacterium tumefaciens of a strain metabolizing nicotine provided by the invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131, have stronger nicotine metabolite ability and the toxic ability of anti-Nicotine, but the concentration of metabolizing nicotine can reach 5g/L, and suitable concentration is 1.0～3.0g/L.This has solved bacterial strain nicotine metabolite ability and the low problem of toxin immunity reported at present.
But the intact cell of the Agrobacterium tumefaciens of utilization metabolizing nicotine provided by the invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 is cooked biological catalyst, handles the method for tobacco processing solid waste, has following characteristics:
(1) intact cell of using Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 is as biological catalyst, and waste treatment speed is fast, saves time and the energy.
(2) the required substratum of the bacterial strain that adopts of preparation biological catalyst is simple, cost is low.
(3) the cell permeability of this bacterial strain is good, does not need fragmentation, can be directly with complete cell degradation Nicotine, and easy to operate.
(4) biological catalyst can be removed with centrifuging, and can reclaim and reuse 3～7 times.
(5) treating processes is simple, and mild condition is easy to operate, and is easy to control.
(6) Nicotine in the refuse can thoroughly be removed.
Therefore, but the Agrobacterium tumefaciens of a strain metabolizing nicotine provided by the invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 and the method for handling tobacco processing solid waste thereof have important application value.
Description of drawings
But the Agrobacterium tumefaciens of a strain metabolizing nicotine that the present invention relates to (Agrobacterium tumefaciens), name is called Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4, be preserved in Chinese typical culture collection center on November 22nd, 2006, its deposit number is: CCTCC NO.M206131.
Fig. 1 is that Agrobacterium tumefaciens of the present invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 cell amplifies 15000 times stereoscan photograph.
Fig. 2 is that Agrobacterium tumefaciens of the present invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 grows in the liquid nutrient medium that contains the 5g/L Nicotine and the situation of the Nicotine of degrading.
Wherein, symbol ■ represents the concentration (g/L) of Nicotine in the substratum, symbol ● the dry cell weight (g/L) behind the expression bacterial growth.
Fig. 3 is that 6 cell is made biological catalyst and handled nicotine content and be about the degraded situation that the tobacco of 30mg/g dry weight is processed Nicotine in the refuse process for utilizing Agrobacterium tumefaciens of the present invention (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 OD value under 620nm.
Wherein, the volume (mL) of tobacco processing refuse quality (g) and biological catalyst suspension is than being 1: 10.
Embodiment
The screening of embodiment 1:Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 bacterial strain
Gather tobacco plant root soil sample from the big Tanaka of the long-term planting tobacco on ground such as Weifang, Shandong, getting 2～4g joins in the liquid nutrient medium that contains the 1g/L Nicotine, under 30 ℃ of conditions on shaking table shaking culture 2～4 days, the 5mL that transfers then cultivates in identical substratum, method is transferred 3～5 times continuously like this, to be applied on the solid plate substratum that contains the 1g/L Nicotine after the nutrient solution dilution that obtain, cultivated 1～3 day under 30 ℃ of conditions, after waiting to grow bacterium colony, the picking macrocolony is line continuously on the solid plate substratum of same Nicotine, confirm that this bacterial strain has the ability of degraded Nicotine, and be pure culture, picking list bacterium colony is to the solid slant culture base that contains the 1g/L Nicotine then, cultivated 1～3 day under 30 ℃ of conditions, obtain Agrobacterium tumefaciens of the present invention (Agrobacterium tumefaciens) wsn1-4, oneself is preserved in Chinese typical culture collection center (Wuhan University this bacterial strain on November 22nd, 2006, China, Wuhan), its deposit number is: CCTCC NO.M206131.
The prescription of above-mentioned described liquid nutrient medium is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L.
The prescription of above-mentioned described solid plate substratum and solid slant culture base is that to add mass volume ratio in described liquid nutrient medium be 1.5% agar.
Embodiment 2: above-mentioned Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 bacterial strain 16S rDNA obtains
Collection grows into the culture 2mL of Agrobacterium tumefaciens (Agrobacterium tumefaciens) the wsn1-4CCTCC NO.M206131 of stationary phase, 6,000 rev/mins centrifugal 5 minutes, remove supernatant; Add 565 μ L TE solution in the throw out, the prescription of TE solution is: the ethylenediamine tetraacetic acid (EDTA) (EDTA) of the Tutofusin tris of 10mmol/L (Tris), 1mmol/L, to adjust pH with hydrochloric acid be 8.0; Blow and beat repeatedly with suction pipe throw out is suspended, add 30 μ L mass volume ratio concentration again and be 10% sodium laurylsulfonate (SDS) and the Proteinase K (Merck company, the U.S.) of 5 μ L 20mg/mL, mixing was in 37 ℃ of following water-baths 1 hour; The NaCl that adds 100 μ L 5mol/L, abundant mixing, add the CTAB/NaCl solution of 80 μ L again, the prescription of CTAB/NaCl solution is: with mass volume ratio concentration is that 10% cetyltriethylammonium bromide (CTAB) is dissolved in the NaCl solution of 0.7mol/L; Behind the mixing in 65 ℃ of water-baths 10 minutes; Add isopyknic chloroform/primary isoamyl alcohol, mixing, 12,000 rev/mins centrifugal 5 minutes, supernatant is changed in the new pipe; Add isopyknic phenol/chloroform/primary isoamyl alcohol, mixing, 12,000 rev/mins centrifugal 5 minutes, supernatant is changed in the new pipe; Add the Virahol of 0.6 times of volume, mixing precipitates until DNA gently, 12,000 rev/mins centrifugal 5 minutes, abandon supernatant, use 70% washing with alcohol; 12,000 rev/min centrifugal 5 minutes, abandon supernatant, be deposited under the room temperature dry 10 minutes, it is heavy molten to add 100 μ l TE damping fluids, and this solution is the genomic dna solution of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131.Genomic dna with extraction is a template, utilizes eubacterium universal primer 27f and 1492r, carries out pcr amplification, adds following reagent successively in the reaction system of 50 μ L: 38 μ LH
2O, the PCR reaction buffer of 5 μ L10 times concentration, the PCR reaction buffer of 10 times of concentration prescription is as follows: the KCl of 500mmol/L, the dithiothreitol (DTT) of 30mmol/L (DTT), 100 mmol/L to adjust pH with hydrochloric acid be 8.8 Tris damping fluid, the bovine serum albumin of 1mg/mL, 4 μ l 25mmol/L MgCl
2The mixture of 1 μ L4 kind dNTP, 0.5 μ L upstream primer, 0.5 μ L downstream primer, 0.5 μ L template DNA is the genomic dna solution of above-mentioned Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131,0.5 μ LTaq archaeal dna polymerase in centrifugal 5 seconds behind the mixing, heats mixture 5 minutes at 94 ℃.According to 94 ℃ of sex change 1 minute, to anneal 1 minute for 50 ℃, 30 circulations are carried out in 72 ℃ of programs reactions of extending 2 minutes altogether.After last circulation, be incubated 10 minutes down in 72 ℃, the mixture that obtains is the pcr amplification product of the 16S rDNA of Agrobacterium tumefaciens (Agrobacteriumtumefaciens) wsn1-4CCTCC NO.M206131, and product is checked order.
Sequencing result: the 16S rDNA sequence length of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 is 1371bp, and nucleotide sequence is as follows:
gcttaacaca tgcaagtcga acgccccgca aggggagtgg cagacgggtg agtaacgcgt 60
gggaacatac cctttcctgc ggaatagctc cgggaaactg gaattaatac cgcatacgcc 120
ctacggggga aagatttatc ggggaaggat tggcccgcgt tggattagct agttggtggg 180
gtaaaggcct accaaggcga cgatccatag ctggtctgag aggatgatca gccacattgg 240
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
gcaagcctga tccagccatg ccgcgtgagt gatgaaggcc ttagggttgt aaagctcttt 360
caccgatgaa gataatgacg gtagtcggag aagaagcccc ggctaacttc gtgccagcag 420
ccgcggtaat acgaaggggg ctagcgttgt tcggaattac tgggcgtaaa gcgcacgtag 480
gcggatattt aagtcagggg tgaaatcccg cagctcaact gcggaactgc ctttgatact 540
gggtatcttg agtatggaag aggtaagtgg aattccgagt gtagaggtga aattcgtaga 600
tattcggagg aacaccagtg gcgaaggcgg cttactggtc cattactgac gctgaggtgc 660
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 720
gttagccgtc gggcagtata ctgttcggtg gcgcagctaa cgcattaaac attccgcctg 780
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 840
agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc agctcttgac attcggggta 900
tgggcattgg agacgatgtc cttcagttag gctggcccca gaacaggtgc tgcatggctg 960
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa ccctcgccct 1020
tagttgccag catttagttg ggcactctaa ggggactgcc ggtgataagc cgagaggaag 1080
gtggggatga cgtcaagtcc tcatggccct tacgggctgg gctacacacg tgctacaatg 1140
gtggtgacag tgggcagcga gacagcgatg tcgagctaat ctccaaaagc catctcagtt 1200
cggattgcac tctgcaactc gagtgcatga agttggaatc gctagtaatc gcagatcagc 1260
atgctgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagttg 1320
gttttacccg aaggtagtgc gctaaccgca aggaggcagc taaccacgta g 1371
Embodiment 3: utilize the intact cell of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 to handle the method that tobacco is processed solid waste, wherein the volume of tobacco processing solid waste quality and biological catalyst suspension is 1: 8 in g/mL ratio
(1) microorganism strains: select Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in contains on the solid slant culture base that mass volume ratio is 0.02% Nicotine, under 20 ℃ of conditions, static cultivation 28 hours;
(3) seed culture: the bacterial strain that step (2) is cultivated, encircle with inoculating articulating 1 that to contain mass volume ratio in 20mL be that under 20 ℃ of conditions, shaking culture is 6 hours on shaking table, makes first order seed in 0.02% the Nicotine liquid nutrient medium under aseptic condition;
(4) enlarged culturing: the inoculum size of volume ratio with 5%, connecing first order seed, to contain mass volume ratio in 200mL be that under 20 ℃ of conditions, shaking culture is 6 hours on shaking table, makes secondary seed in 0.02% the Nicotine liquid nutrient medium;
(5) fermentor cultivation: the inoculum size of volume ratio with 5%, connecing secondary seed, to contain mass volume ratio in 5L be in 0.1% the Nicotine liquid nutrient medium, cultivates the period detecting cell concn under 25 ℃ of conditions, OD value under 620nm reaches 1.2, stops fermentation culture;
(6) collecting cell: the nutrient solution 5 of getting step (5), under 000 rev/min of condition centrifugal 10 minutes, collect the cell of fermentation culture, and with the washing of 20mmol/L pH7.0 phosphoric acid buffer, centrifugal with identical condition again, repeat 2 times, the cell of collecting precipitation, this cell is biological catalyst, and is 4 ℃ of storages, standby;
(7) removal of Nicotine in the tobacco processing solid waste: the biological catalyst that step (6) is made is suspended in the 20mmol/L pH7.0 phosphoric acid buffer, mixing, making the OD value of biological catalyst under 620nm in the mixture is 2, be 1: 8 ratio again according to the volume ratio of tobacco processing refuse quality and biological catalyst suspension, in g/mL, in the biological catalyst suspension, add tobacco processing solid waste, under 20 ℃, 180 rev/mins conditions, vibrate; Take a sample during the processing, detect the degraded situation of Nicotine with vapor-phase chromatography, when Nicotine in the mixture detect fully less than after, continue vibration termination after 4 hours;
(8) with the mixture after step (7) termination, with 6,000 rev/mins 30 minutes centrifugal, remove the supernatant liquor that does not contain Nicotine, obtain the biological catalyst and the residual waste that are added in the step (7), remove the residual waste on upper strata, collect the biological catalyst of lower floor;
(9) recycling of biological catalyst: with step (8) collect biological catalyst according to the recycling of the described method of step (7), repeat this step 3 time, discard the centrifugal precipitation at last;
(10) supernatant liquor that step (8) and step (9) are obtained mixes, and further handles together in company with general waste water, and step (8) obtains the precipitation that residual waste and step (9) obtain and then can handle with general refuse.
The prescription of above-mentioned steps (3), the described liquid nutrient medium in (4) (5) is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L.
The prescription of the described solid slant culture base of above-mentioned steps (2) is that the preferred mass volume ratio that adds is 1.6% agar in described liquid nutrient medium.
The described vapor-phase chromatography of above-mentioned steps (7) adopts VARIAN CP3380 gas chromatograph, chromatographic column is SPB-5 capillary column (internal diameter 0.32mm, long 30m, thickness 0.25 μ m, U.S. Supelcol company), nitrogen is as carrier gas, testing conditions is 260 ℃ of sampler temperature, 140 ℃ of column temperatures, 280 ℃ of detector temperatures, sample size 1 μ l.
Institute's sample thief utilizes the gas chromatography determination Nicotine after needing to handle in accordance with the following methods during the described processing of above-mentioned steps (7): sample is in 8,000 rev/min 20 minutes centrifugal, getting the 0.2ml supernatant has in the bottle of sealing cover in one, add 1.8ml distilled water, add 50 μ l 1M aqueous sodium carbonates again, with 30 ℃ of oscillation extraction 30min of 2ml chloroform, chloroform phase solution is used for gas chromatography determination.
Embodiment 4: utilize the intact cell of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 to handle the method that tobacco is processed solid waste, wherein the volume of tobacco processing solid waste quality and biological catalyst suspension is 1: 15 in g/mL ratio
(1) microorganism strains: select Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in contains on the solid slant culture base that mass volume ratio is 0.1% Nicotine, under 30 ℃ of conditions, static cultivation 24 hours;
(3) seed culture: the bacterial strain that step (2) is cultivated, encircle with inoculating articulating 1 that to contain mass volume ratio in 50mL be that under 30 ℃ of conditions, shaking culture is 12 hours on shaking table, makes first order seed in 0.1% the Nicotine liquid nutrient medium under aseptic condition;
(4) enlarged culturing: the inoculum size of volume ratio with 8%, connecing first order seed, to contain mass volume ratio in 500mL be that under 30 ℃ of conditions, shaking culture is 18 hours on shaking table, makes secondary seed in 0.2% the Nicotine liquid nutrient medium;
(5) fermentor cultivation: the inoculum size of volume ratio with 8%, connecing secondary seed, to contain mass volume ratio in 10L be in 0.3% the Nicotine liquid nutrient medium, cultivates the period detecting cell concn under 30 ℃ of conditions, OD value under 620nm reaches 2.9, stops fermentation culture;
(6) collecting cell: the nutrient solution 5 of getting step (5), under 000 rev/min of condition centrifugal 12 minutes, collect the cell of fermentation culture, and with the washing of 50mmol/L pH7.0 phosphoric acid buffer, centrifugal with identical condition again, repeat 3 times, the cell of collecting precipitation, this cell is biological catalyst, and is 4 ℃ of storages, standby;
(7) removal of Nicotine in the tobacco processing solid waste: the biological catalyst that step (6) is made is suspended in the 50mmol/L pH7.0 phosphoric acid buffer, mixing, making the OD value of biological catalyst under 620nm in the mixture is 5, be 1: 15 ratio again according to the volume ratio of tobacco processing refuse quality and biological catalyst suspension, in g/mL, in the biological catalyst suspension, add tobacco processing solid waste, under 30 ℃, 250 rev/mins conditions, vibrate; Take a sample during the processing, detect the degraded situation of Nicotine with vapor-phase chromatography, when Nicotine in the mixture detect fully less than after, continue vibration termination after 2 hours;
(8) with the mixture after step (7) termination, with 8000 rev/mins 15 minutes centrifugal, remove the supernatant liquor do not contain Nicotine, obtain the biological catalyst and the residual waste that are added in the step (7), remove the residual waste on upper strata, collect the biological catalyst of lower floor;
(9) recycling of biological catalyst: with step (8) collect biological catalyst according to the recycling of the described method of step (7), repeat this step 5 time, discard the centrifugal precipitation at last;
(10) supernatant liquor that step (8) and step (9) are obtained mixes, and further handles together in company with general waste water, and step (8) obtains the precipitation that residual waste and step (9) obtain and then can handle with general refuse.
The prescription of above-mentioned steps (3), the described liquid nutrient medium in (4) (5) is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L.
The prescription of the described solid slant culture base of above-mentioned steps (2) is that the preferred mass volume ratio that adds is 1.8% agar in described liquid nutrient medium.
The described vapor-phase chromatography of above-mentioned steps (7) adopts VARIAN CP3380 gas chromatograph, chromatographic column is SPB-5 capillary column (internal diameter 0.32mm, long 30m, thickness 0.25 μ m, U.S. Supelcol company), nitrogen is as carrier gas, testing conditions is 260 ℃ of sampler temperature, 140 ℃ of column temperatures, 280 ℃ of detector temperatures, sample size 1 μ l.
Institute's sample thief utilizes the gas chromatography determination Nicotine after needing to handle in accordance with the following methods during the described processing of above-mentioned steps (7): sample is in 8,000 rev/min 20 minutes centrifugal, getting the 0.2ml supernatant has in the bottle of sealing cover in one, add 1.8ml distilled water, add 50 μ l 1M aqueous sodium carbonates again, with 30 ℃ of oscillation extraction 30min of 2ml chloroform, chloroform phase solution is used for gas chromatography determination.
Embodiment 5: utilize the intact cell of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 to handle the method that tobacco is processed solid waste, wherein the volume of tobacco processing solid waste quality and biological catalyst suspension is 1: 20 in g/mL ratio
(1) microorganism strains: select Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in contains on the solid slant culture base that mass volume ratio is 0.3% Nicotine, under 37 ℃ of conditions, static cultivation 36 hours;
(3) seed culture: the bacterial strain that step (2) is cultivated, encircle with inoculating articulating 2 that to contain mass volume ratio in 100mL be that under 37 ℃ of conditions, shaking culture is 30 hours on shaking table, makes first order seed in 0.3% the Nicotine liquid nutrient medium under aseptic condition;
(4) enlarged culturing: the inoculum size of volume ratio with 10%, connecing first order seed, to contain mass volume ratio in 1000mL be that under 37 ℃ of conditions, shaking culture is 30 hours on shaking table, makes secondary seed in 0.3% the Nicotine liquid nutrient medium;
(5) fermentor cultivation: the inoculum size of volume ratio with 10%, connecing secondary seed, to contain mass volume ratio in 20L be in 0.5% the Nicotine liquid nutrient medium, cultivates the period detecting cell concn under 37 ℃ of conditions, OD value under 620nm reaches 3.9, stops fermentation culture;
(6) collecting cell: the nutrient solution 5 of getting step (5), under 000 rev/min of condition centrifugal 15 minutes, collect the cell of fermentation culture, and with the washing of 100mmol/L pH7.0 phosphoric acid buffer, centrifugal with identical condition again, repeat 3 times, the cell of collecting precipitation, this cell is biological catalyst, and is 4 ℃ of storages, standby;
(7) removal of Nicotine in the tobacco processing solid waste: the biological catalyst that step (6) is made is suspended in the 100mmol/L pH7.0 phosphoric acid buffer, mixing, making the OD value of biological catalyst under 620nm in the mixture is 10, be 1: 20 ratio again according to the volume ratio of tobacco processing refuse quality and biological catalyst suspension, in g/mL, in the biological catalyst suspension, add tobacco processing solid waste, under 40 ℃, 350 rev/mins conditions, vibrate; Take a sample during the processing, detect the degraded situation of Nicotine with vapor-phase chromatography, when Nicotine in the mixture detect fully less than after, continue vibration termination after 1 hour;
(8) with the mixture after step (7) termination, with 12,000 rev/mins 10 minutes centrifugal, remove the supernatant liquor that does not contain Nicotine, obtain the biological catalyst and the residual waste that are added in the step (7), remove the residual waste on upper strata, collect the biological catalyst of lower floor;
(9) recycling of biological catalyst: with step (8) collect biological catalyst according to the recycling of the described method of step (7), repeat this step 7 time, discard the centrifugal precipitation at last;
(10) supernatant liquor that step (8) and step (9) are obtained mixes, and further handles together in company with general waste water, and step (8) obtains the precipitation that residual waste and step (9) obtain and then can handle with general refuse.
The prescription of above-mentioned steps (3), the described liquid nutrient medium in (4) (5) is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L.
The prescription of the described solid slant culture base of above-mentioned steps (2) is that the preferred mass volume ratio that adds is 2.0% agar in described liquid nutrient medium.
The described vapor-phase chromatography of above-mentioned steps (7) adopts VARIAN CP3380 gas chromatograph, chromatographic column is SPB-5 capillary column (internal diameter 0.32mm, long 30m, thickness 0.25 μ m, U.S. Supelcol company), nitrogen is as carrier gas, testing conditions is 260 ℃ of sampler temperature, 140 ℃ of column temperatures, 280 ℃ of detector temperatures, sample size 1 μ l.
Institute's sample thief utilizes the gas chromatography determination Nicotine after needing to handle in accordance with the following methods during the described processing of above-mentioned steps (7): sample is in 8,000 rev/min 20 minutes centrifugal, getting the 0.2ml supernatant has in the bottle of sealing cover in one, add 1.8ml distilled water, add 50 μ l 1M aqueous sodium carbonates again, with 30 ℃ of oscillation extraction 30min of 2ml chloroform, chloroform phase solution is used for gas chromatography determination.
Sequence table
＜110〉Shandong University
＜120〉but the Agrobacterium tumefaciens of a strain metabolizing nicotine and application thereof
<141>2007-01-16
<160>1
<210>1
<211>1371
<212>DNA
＜213〉Agrobacterium tumefaciens (Agrobacterfum tumefaciens)
＜221〉Agrobacterium tumefaciens (Agrobacterium tumefacfens) CCTCC NO.M206131 16S rDNA
<222>(1)...(1371)
<400>1
gcttaacaca tgcaagtcga acgccccgca aggggagtgg cagacgggtg agtaacgcgt 60
gggaacatac cctttcctgc ggaatagctc cgggaaactg gaattaatac cgcatacgcc 120
ctacggggga aagatttatc ggggaaggat tggcccgcgt tggattagct agttggtggg 180
gtaaaggcct accaaggcga cgatccatag ctggtctgag aggatgatca gccacattgg 240
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
gcaagcctga tccagccatg ccgcgtgagt gatgaaggcc ttagggttgt aaagctcttt 360
caccgatgaa gataatgacg gtagtcggag aagaagcccc ggctaacttc gtgccagcag 420
ccgcggtaat acgaaggggg ctagcgttgt tcggaattac tgggcgtaaa gcgcacgtag 480
gcggatattt aagtcagggg tgaaatcccg cagctcaact gcggaactgc ctttgatact 540
gggtatcttg agtatggaag aggtaagtgg aattccgagt gtagaggtga aattcgtaga 600
tattcggagg aacaccagtg gcgaaggcgg cttactggtc cattactgac gctgaggtgc 660
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 720
gttagccgtc gggcagtata ctgttcggtg gcgcagctaa cgcattaaac attccgcctg 780
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 840
agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc agctcttgac attcggggta 900
tgggcattgg agacgatgtc cttcagttag gctggcccca gaacaggtgc tgcatggctg 960
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa ccctcgccct 1020
tagttgccag catttagttg ggcactctaa ggggactgcc ggtgataagc cgagaggaag 1080
gtggggatga cgtcaagtcc tcatggccct tacgggctgg gctacacacg tgctacaatg 1140
gtggtgacag tgggcagcga gacagcgatg tcgagctaat ctccaaaagc catctcagtt 1200
cggattgcac tctgcaactc gagtgcatga agttggaatc gctagtaatc gcagatcagc 1260
atgctgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagttg 1320
gttttacccg aaggtagtgc gctaaccgca aggaggcagc taaccacgta g 1371
Claims (10)
1. but the Agrobacterium tumefaciens of a strain metabolizing nicotine, it is characterized in that, this bacterial strain is called Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4, be preserved in Chinese typical culture collection center on November 22nd, 2006, its deposit number is: CCTCC NO.M 206131.
2. but the Agrobacterium tumefaciens of metabolizing nicotine as claimed in claim 1 is characterized in that, the bacterium colony smooth surface, and glossy, the edge is translucent, central oyster white; Cell is an elongated rod shape, microbend, and the blunt circle in two ends, length is 1.60～1.84 μ m, diameter is 0.32～0.41 μ m; Gram-negative; Semi-solid puncture is mobility; All can grow down and under the 2%NaCl for 35 ℃; Do not need organic somatomedin; The hydrogen peroxide enzyme positive; Methyl red test is negative; The V-P test is positive; Nitrate can be used as only nitrogen source; Alkali is produced in the litmus milk test; Do not produce indoles and H
2S; The 16S rDNA sequence length of this bacterium is 1371bp, and its nucleotide sequence is shown in SEQ ID NO.1;
Above-mentioned Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCC NO.M 206131 can be sole carbon source, nitrogenous source and energy growth with the Nicotine, growth temperature range is 20～37 ℃, the pH scope is 6.0～8.0, but metabolite concentration is up to the Nicotine of 5g/L.
3. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCC NO.M 206131 is the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste.
4. application as claimed in claim 3 is characterized in that, the method steps that described application relates to is as follows:
(1) microorganism strains: select Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCC NO.M 206131 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in contains on the solid slant culture base that mass volume ratio is 0.02～0.3% Nicotine, under 20 ℃～37 ℃ conditions, static cultivation 24～36 hours;
(3) seed culture: with the bacterial strain of step (2) cultivation, encircle with inoculation articulating 1～2 under aseptic condition that to contain mass volume ratio in 20～100mL be in 0.02～0.3% the Nicotine liquid nutrient medium, under 20 ℃～37 ℃ conditions, shaking culture is 6～30 hours on shaking table, makes first order seed;
(4) enlarged culturing: the inoculum size of volume ratio with 5～10%, connecing first order seed, to contain mass volume ratio in 200～1000mL be in 0.02～0.3% the Nicotine liquid nutrient medium, under 20 ℃～37 ℃ conditions, shaking culture is 6～30 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: the inoculum size of volume ratio with 5～10%, connecing secondary seed, to contain mass volume ratio in 5～20L be in 0.1～0.5% the Nicotine liquid nutrient medium, cultivate under 25 ℃～37 ℃ conditions, the period detecting cell concn, OD value under 620nm reaches 1.2～3.9, stops fermentation culture;
(6) collecting cell: the nutrient solution 5 of getting step (5), under 000 rev/min of condition centrifugal 10～15 minutes, collect the cell of fermentation culture, and with the washing of 20～100mmol/L pH7.0 phosphoric acid buffer, centrifugal with identical condition again, repeat 2～3 times, the cell of collecting precipitation, this cell is biological catalyst, and is 4 ℃ of storages, standby;
(7) removal of Nicotine in the tobacco processing solid waste: the biological catalyst that step (6) is made is suspended in 20～100mmol/L pH7.0 phosphoric acid buffer, mixing, making the OD value of biological catalyst under 620nm in the mixture is 2～10, be 1: 8～20 ratio again according to the volume ratio of tobacco processing refuse quality and biological catalyst suspension, in g/mL, in the biological catalyst suspension, add tobacco processing solid waste, under 20 ℃～40 ℃, 180～350 rev/mins conditions, vibrate; Take a sample during the processing, detect the degraded situation of Nicotine with vapor-phase chromatography, when Nicotine in the mixture detect fully less than after, continue vibration termination after 1～4 hour;
(8) with the mixture after step (7) termination, with 6,000～12,000 rev/min 10～30 minutes centrifugal, remove the supernatant liquor do not contain Nicotine, obtain the biological catalyst and the residual waste that are added in the step (7), remove the residual waste on upper strata, collect the biological catalyst of lower floor;
(9) recycling of biological catalyst: with step (8) collect biological catalyst according to the recycling of the described method of step (7), repeat this step 3～7 time, discard the centrifugal precipitation at last;
(10) supernatant liquor that step (8) and step (9) are obtained mixes, and further handles together in company with general waste water, and step (8) obtains the precipitation that residual waste and step (9) obtain and then can handle with general refuse.
5. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that the prescription of step (3), (4), (5) described liquid nutrient medium is:
K
2HPO
43H
2O 13.3g/L, KH
2PO
44g/L, MgSO
47H
2O 0.2g/L, yeast powder 1.0g/L, metal ion mixed solution 0.5mL/L; Regulate pH7.0, sterilization is 20 minutes under 115 ℃ of conditions;
Wherein, the prescription of metal ion mixed solution is:
With the 1mol/L hydrochloric acid soln is solvent, CaCl
22H
2O 0.05g/L, CuCl
22H
2O 0.05g/L, MnSO
4H
2O0.008g/L, FeSO
47H
2O 0.004g/L, ZnSO
40.1g/L, Na
2MoO
42H
2O 0.1g/L, Na
2WO
42H
2O0.05g/L;
The prescription of the described solid slant culture base of step (2) is that to add mass volume ratio in described liquid nutrient medium be 1.5～2.0% agar.
6. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that the mass volume ratio concentration of step (2), (3), (4), (5) described Nicotine is 0.1～0.3%.
7. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that the described phosphate buffer density of step (7) is 40～80mmol/L.
8. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that the OD value of biological catalyst under 620nm in the described mixture of step (7) is 5～7.
9. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that the volume of described tobacco processing refuse quality of step (7) and biological catalyst suspension is 1: 10～15.
10. the intact cell with Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4 CCTCCNO.M 206131 as claimed in claim 4 is the method that the application of Nicotine in the biological catalyst biological degradation tobacco processing solid waste relates to, it is characterized in that, step (7) described when Nicotine in the mixture detect fully less than after, continue vibration termination after 2～3 hours.
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Cited By (5)
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| CN101928690A (en) * | 2010-07-23 | 2010-12-29 | 浙江工业大学 | Shinella sp. HZN1 capable of effectively degrading nicotine and application thereof |
| CN102816721A (en) * | 2012-08-10 | 2012-12-12 | 哈尔滨师范大学 | ACC-deaminase-producing agrobacterium tumefaciens LJL-6 and application thereof |
| CN110272848A (en) * | 2019-07-10 | 2019-09-24 | 西北农林科技大学 | One plant of rhizobium J16 and its application with potassium decomposing effect |
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2007
- 2007-01-22 CN CNB2007100130698A patent/CN100434514C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101928690A (en) * | 2010-07-23 | 2010-12-29 | 浙江工业大学 | Shinella sp. HZN1 capable of effectively degrading nicotine and application thereof |
| CN102816721A (en) * | 2012-08-10 | 2012-12-12 | 哈尔滨师范大学 | ACC-deaminase-producing agrobacterium tumefaciens LJL-6 and application thereof |
| CN102816721B (en) * | 2012-08-10 | 2013-11-20 | 哈尔滨师范大学 | ACC-deaminase-producing agrobacterium tumefaciens LJL-6 and application thereof |
| CN110272848A (en) * | 2019-07-10 | 2019-09-24 | 西北农林科技大学 | One plant of rhizobium J16 and its application with potassium decomposing effect |
| CN110272848B (en) * | 2019-07-10 | 2023-01-03 | 西北农林科技大学 | Rhizobium J16 with potassium-dissolving effect and application thereof |
| CN115484836A (en) * | 2020-03-31 | 2022-12-16 | 百欧穆斯有限公司 | Bacteria for preventing and treating smoke-induced lung injury |
| CN114540233A (en) * | 2022-02-25 | 2022-05-27 | 广东省科学院生态环境与土壤研究所 | Tailing soil sulfur oxidizing bacteria and application thereof |
| CN114540233B (en) * | 2022-02-25 | 2023-06-23 | 广东省科学院生态环境与土壤研究所 | Tailing soil sulfur oxidizing bacteria and application thereof |
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| CN100434514C (en) | 2008-11-19 |
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