CN1410528A - Construction method of bacillus phosphorus relieving engineering bacterial strain - Google Patents
Construction method of bacillus phosphorus relieving engineering bacterial strain Download PDFInfo
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- CN1410528A CN1410528A CN 02145369 CN02145369A CN1410528A CN 1410528 A CN1410528 A CN 1410528A CN 02145369 CN02145369 CN 02145369 CN 02145369 A CN02145369 A CN 02145369A CN 1410528 A CN1410528 A CN 1410528A
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- 229910052698 phosphorus Inorganic materials 0.000 title claims description 68
- 239000011574 phosphorus Substances 0.000 title claims description 68
- 230000001580 bacterial effect Effects 0.000 title claims description 27
- 238000010276 construction Methods 0.000 title claims description 16
- 241000894006 Bacteria Species 0.000 claims abstract description 51
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Abstract
An engineered dephosphorylating strain of bacillus is configured through slant culture in beef paste-pepton culture medium, eluting the activated bacteria with physiological saline or buffering phosphoric acid solution to obtain the bacillus suspension, centrifugal separation to collect the spores, diluting with physiological saline or buffering phosphoric acid solution, coating on aseptic glass, drying, implanting ion beams in the bacillus, and eluting.
Description
Technical field
The present invention relates to a kind of construction process of genus bacillus phosphorus decomposing engineering strain.
Background technology
In the present age, the development sustainable agriculture has become the universe's common recognition.Yet chemistry agricultural, livestock industry and industrial expansion have caused the serious environmental pollution for a long time, and wherein organophosphorus is one of important pollution sources.Phytate phosphorus in the agriculture production in organophosphorus pesticide, the herding movement and the multiple organophosphorus in trade effluent and the sanitary sewage etc. cause huge load to environment, have restricted the development of industries such as agricultural and aquatic products.Therefore, eliminate the problem that organophosphorus pollutes becomes the countries in the world concern.
Microbiological deterioration is to eliminate the important channel that organophosphorus pollutes.Action of microorganisms is depended in the decomposition of occurring in nature organophosphorus to a great extent, another kind of main phosphorus in the soil---insoluble inorganic phosphorus also must just dissociate under action of microorganisms and for crop absorbs, so microorganism plays an important role in the effectuation of the removing of soil phosphorus pollution and phosphorus nutrition.But under the natural condition, action of microorganisms is extremely slow.Utilize modern technologies that microorganism is transformed, make up the phosphorus decomposing engineering bacteria, strengthen or induce the capacity of decomposition of organophosphorus and inorganic phosphorus, eliminate phosphorus and pollute, simultaneously for crop provides phosphorus nutrition, this is the desirable means that solve phosphorus pollution and phosphorus nutrition at present.Transform microbial strains, making up engineering bacteria has two kinds of approach, i.e. mutagenesis and hybridization generally speaking.
1. make up the principle and the method for microbial engineering bacteria
Microorganism modern breeding cardinal principle is hybridization and sudden change, produces two kinds of microbial engineering bacteria construction processs thus: selection by mutation and cross-breeding.Each division is as follows:
Its theoretical basis of selection by mutation is to bring out sudden change.Mutagenic compound commonly used have physical mutagen (as ray, laser), chemical mutagen (as HNO
2, the 5-bromo pyrimi piperidine) and biological induced-mutation agent (as mutator gene and transposon) etc., they mostly by change DNA base pairing situation or insertion, one section base of disappearance causes the change of gene structure, thereby change biological character.Selection by mutation can be divided into mutagenesis in vivo and vitro mutagenesis again.Vitro mutagenesis is also named positional mutation, refers to produce sudden change effectively in the external specific region of gene that makes.Mutant strain can obtain the various engineering bacterium by seed selection.
Mutagenesis in vivo breeding mutation rate is higher, and the amplitude of improved properties is bigger, and application is convenient, rapid, economical, so be widely adopted, at present a lot of strain excellents all obtain with this method.But it needs the effect of intensive physical chemical factor, is easy to the genetic background of badly damaged bacterial strain, and directional property almost do not have, and blindness is bigger, also can cause certain injury to human body sometimes.Vitro mutagenesis but has fabulous directional property, and mutation rate is easy to artificial control up to percentum, so be applied to industrial production at present again and again.This shows, along with the develop rapidly of biotechnology and raising day by day that breeding is required, the vitro mutagenesis technology will further obtain breeding work persons' favor.
The theoretical basis of cross-breeding cross-breeding is hybridization technique.Several cross breeding methods commonly used at present have:
(1) protoplastis merges: be mainly used in the cell that can not or be difficult to hybridize under the natural situation, can break through the boundary of planting.
(2) cell hybridization: this is the hybridization mode that just can carry out under a kind of natural condition.
(3) genetically engineered: i.e. DNA recombinant technology is the cross-breeding of molecular level.Its process is, recombinates at external dna molecular with different sources, changes in the appropriate host and expresses.
Cross-breeding can be combined the good character of different sources, learns from other's strong points to offset one's weaknesses, and more becomes perfect.The obstacle of overcoming property of Protoplast Fusion Technique and kind wherein, recombination frequency is very high, and recon has panoramic kind, also can be used with other breeding method simultaneously.But this method operating process is more loaded down with trivial details, and difficulty is bigger, so non-direct, effective means.Hybridoma technique is more convenient, but directivity and artificial controlled still not ideal enough, and can't overcoming property and the obstacle of kind, so neither the ideal approach.The modern genetic engineering technology not only breaks through the constraint of kind and sibship fully, also can carry out directed mutagenesis, even start brand-new species, obtains the range gene engineering bacteria.But genetic engineering technique is used for breeding, and its prerequisite is that the genetic background of bacterial strain must be clear, and therefore for most production bacterium, because the genetic background complexity, genetic engineering technique is used in original production bacterium improvement and be few.For microbial fertilizer because be the thalline that will live directly be manured into soil and environment in, the therefore application of clear and definite restriction gene engineering bacteria in the bacterial manure standard of China.
More than various selection by mutation approach can obtain the various engineering bacterium.Compare two kinds of breeding approach, selection by mutation is still the main mode that makes up engineering bacteria.Ray (β, gamma-rays), nuclear radiation, chemomorphosis etc. are the breeding techniques that generally uses at present, but life-time service is with a kind of mutagenic compound, and bacterial classification often produces resistance, seek out bigger variation and just must adopt new mutation source.Ionic fluid, laser, aviation are the species mutafacient system that latest developments are got up, and wherein ionic fluid is the new technology of the structure engineering strain of China's original creation.2. the principle of ionic fluid effect and principal feature
It is that China scientific and technical personnel found the eighties that ionic fluid injects biological effect, and drops into the Breeding Application field rapidly, has developed into a kind of brand-new breeding technique so far.
The principle of ionic fluid effect is in ion source, and gas (or steam) discharge generation plasma body, plasma body obtain the ionic fluid of different-energy again by the corresponding identical charges ion of mass analyzer separation acquisition through accelerator, and promptly carrying can ionic fluid.At present, N has been arranged
+, Ar
+, He
+, H
+Be used for breeding etc. the different kinds of ions bundle.Carry and to act on organism by ionic fluid (target chamber is necessary for vacuum, to avoid carrying the gas molecule collision of energy ion and air power loss or scattering takes place) in target chamber, get final product induce variation.
Carry and can ion to bring out variation and will experience a plurality of processes.At first, after energetic ion enters organism, with multiple atom and interaction of electrons wherein, gradually kinetic energy is passed to target atom and electronics, scatter and disappear fully and stop until ionic kinetic energy, this process is called ionic " slowing down ", and this is incident ion generation transmission ofenergy and deposition process.To cross potential barrier and leave original position if target atom obtains enough energy in collision, be shifted.Slowing down ion, displaced atom can produce room, interstitial impurity atom and substitutional impurity atom, and the rearrangement of room, displaced atom, slowing down ion and target atom and compound can form new molecule and group, produce abundant mutant.Secondly, the charge-exchange effect, incident ion in target after the slowing down, all can lose certain electronics with the collision each time of target atom or from target trapped electron, thereby charge-exchange takes place.Charge-exchange often causes the variation that organism surface ionizing group quantity, dissociation constant and topology are arranged, and causes the effect between cell and the cell, Ca
2+With the interaction of cytolemma and the change of cytolemma ion permeability, thereby cause a series of physiological change.In addition, ion implantationly also can cause radical damage.When energetic ion injects organism, because the exciting of target molecule, ionization will produce free radical in the collision scope.Active free radical easily and target molecule generation addition, take out reactions such as hydrogen and transfer transport, thereby cause organism recurring structure and changing function, mutagenesis.As seen, ion (H
+, N
+, Ar
+Deng) inject cell after, acting in conjunction by electricity, energy, matter, not only influence the cells physiological biochemical function, the more important thing is chromosome aberration, DNA and the damage of RNA chain, the fracture that make in the cell, cause the sudden change or the activation of enzyme, produce various mutations, thereby provide the foundation for the acquisition of engineering bacteria.
The principal feature of ionic fluid effect is because the synergy of matter, energy, lotus three factors, ionic fluid injects the variation that causes a large amount of acceptor atom displacements, reorganization and biomolecules, form new molecular structure and group, produce abundant transgenation, thereby ionic fluid injects and can obtain higher mutation rate under the light situation of damage.Particularly, ion beam mutagenesis has following characteristics: at first similar with ray mutagenesis, ionic fluid injects and has the mutagenic effect that energy deposition causes; Different with ray mutagenesis, ionic fluid injects the cascade damage that also exists momentum exchange to produce, and shows as genetic material atomic displacement, rearrangement or genetically deficient, produces transgenation; Secondly, similar chemomorphosis, ionic fluid injects the slowing down ion, displaced atom and the background elements compounding that produce can cause chemical variation; In addition, the charge-exchange of ionic fluid injection process can cause biomolecules transfer transport and free radical reaction, causes biological induced-mutation.As seen, ionic fluid injects the characteristic that has physics, chemistry and biological induced-mutation concurrently, this complex mutation has significantly improved mutagenic frequency and efficient, as under identical survival rate, ionic fluid injects typhoid fever class salmonella, and its auxotrophic mutation frequency and efficiency of inducing mutation improve 14～45% and 16～35% respectively than gamma-rays.In a word, ion beam mutagenesis has multiple superiority, shows as high excitability, measures and concentrate and controllability, and cell injury is light, mutation rate is high, mutation spectrum is wide, can carry out directed mutagenesis, has become the important new tool of contemporary germplasm improvement and breeding.
Ionic fluid use the present situation ionic fluid on genetic improvement, use the earliest be selection by mutation, wherein mainly be the improvement of plant germplasm.In the Research for Industrial Microbial Germ improvement, use ionic fluid and inject the also many efficient engineering bacterias of structure, and in industrial production, brought into play vital role.Conventional production strain often shows in various degree resistance to physical chemistry mutafacient system commonly used in the past, and ionic fluid shows good effect as a kind of new mutation source.Hefei Inst. of Plasma Physics, Chinese Academy of Sciences and the cooperation of domestic dozens of unit are the bacterium that sets out to produce the superior strain that uses, and have increased substantially the output of multiple industrial microorganism tunning, and the microbial strains of having put into production has 3.By ion beam mutagenesis, research of agricultural science institute in Zhejiang brings up to 20,000 units with the enzyme work of saccharification enzyme-producing bacteria from 1.5 ten thousand fermentation units; Yao Jianming etc. have improved 40% with the fermentation level of rifomycin bacterial strain; The production of vitamin C bacterium that this method of usefulness such as Xu An obtains is created domestic and international two step method fermentation glucose acid invert ratio new peak, and molar yield is up to 97.5%.As seen, ionic fluid is very remarkable to the mutagenesis effect of microorganism.
Although ion beam mutagenesis has obtained many high yield engineering bacterias on industrial micro breeding, almost also do not involving in aspect agricultural and the environmental microorganism.Ionic fluid is injected the structure that induced-mutation technique is applied to agricultural and environmental microorganism engineering bacteria, be expected to break through effectively the proterties of existing bacterial strain, obtain brand-new bioengineered strain, improve microorganism and improve fertilizer provision from soil and remove the ability of environmental pollution, thereby start the new way of agricultural and environmental microorganism strain improvement and breeding.
Summary of the invention
The construction process that the purpose of this invention is to provide a kind of genus bacillus phosphorus decomposing engineering strain.
The step that makes up the method for genus bacillus phosphorus decomposing engineering strain is:
1) activation culture genus bacillus on the inclined-plane, used substratum is a beef-protein medium, the bacterium after the activation directly becomes bacteria suspension with physiological saline or phosphoric acid buffer wash-out;
2) bacteria suspension is centrifugal, collect spore, with physiological saline or phosphoric acid buffer with the spore dilution and coat on the aseptic blank plate or slide glass dry 0.5～2h;
3) with N
+, H
+, Ar
+Or He
+Ionic fluid injects genus bacillus, and the ion energy that is injected is that 10～30KeV, dosage are 5～300 * 10
14Ions/cm
2, 2 * 10
-2～6 * 10
-3Carry out intermittent pulse type under Pa vacuum, the aseptic condition and inject, each 50～100 seconds recurrent intervals;
4) with physiological saline or phosphoric acid buffer wash-out spore, and be diluted to finite concentration,,, obtain the efficient engineering strain that decomposes organophosphorus then through identifying at the enterprising row filter of NBRIY substratum.
The another kind of step that makes up the method for genus bacillus phosphorus decomposing engineering strain is:
1) activation culture genus bacillus on the nitrogen-free agar inclined-plane, the bacterium after the activation is inoculated in the liquid nutrient medium that produces no pod membrane gemma, and 25～30 ℃, 150～250rpm/min are cultivated 36～72h, centrifugal collection spore;
2) with spore with the dilution of physiological saline or phosphoric acid buffer and coat on the aseptic blank plate or slide glass dry 0.5～2h;
3) with N
+, H
+, Ar
+Or He
+Ionic fluid injects genus bacillus, and the ion energy that is injected is that 10～30KeV, dosage are 5～300 * 10
14Ions/cm
2, 2 * 10
-2～6 * 10
-3Carry out intermittent pulse type under Pa vacuum, the aseptic condition and inject, each 50～100 seconds recurrent intervals;
4) with physiological saline or phosphoric acid buffer wash-out spore, and be diluted to finite concentration, after process screening on the NBRIY substratum, on the NBRIP substratum, carry out multiple sieve, be tested and appraised at last, obtain the efficient engineering strain that decomposes organophosphorus, inorganic phosphorus or inorganic potassium.
Advantage of the present invention is:
At the with serious pollution problem of present organophosphorus, application efficiency of inducing mutation height, the novel induced-mutation technique that the sudden change amplitude is big---ionic fluid injects and makes up engineering bacteria, strengthen the ability that genus bacillus is decomposed organophosphorus and inorganic phosphorus, realize the dual-use function of soil self-purification and nutrition self-sufficiency.Silicate bacteria is to be widely used in the genus bacillus that microorganism potash fertilizer is produced at present, injects by ionic fluid, makes up new engineering strain, can strengthen its decomposition to soil phosphorus, improves the effect that this bacterium provides available phosphorus and potassium.Inject constructed engineering strain by ionic fluid the capacity of decomposition of organophosphorus or inorganic phosphorus is improved 15～45% and 10%～25% respectively.
Description of drawings
Accompanying drawing is the schema of the construction process of genus bacillus phosphorus decomposing engineering strain.
Embodiment
Ion beam mutagenesis is advanced in the world at present induced-mutation technique, utilize this technology that genus bacillus is transformed, structure can decompose the engineering bacteria of organophosphorus or inorganic phosphorus, provide elite seed for producing the novel microorganism phosphate fertilizer or the phosphorus potassium complex fertilizer that can efficiently decompose organophosphorus or inorganic phosphorus, the quality of promotion microorganism phosphate fertilizer or phosphorus potassium complex fertilizer improves and applies, quicken the ecological agriculture development process, improvement and the breeding for microbial fertilizer production bacterium simultaneously provides novel method.
Selection by mutation has two key problem in technology: one, create mass efficient muton its two, set up fast, accurate and easily efficient screening.The present invention injects by specific ionic fluid and carries out mutagenesis, obtains the variant of a large amount of high mutation rates, screens by effective screening culture medium then, obtains engineering strain.Concrete steps are as follows:
1, the cultivation of microbial spores and pre-treatment
The existence of pod membrane has a strong impact on the mutagenesis effect.Do not produce the spore of the genus bacillus of pod membrane and directly on the beef extract-peptone slant medium, cultivate acquisition.Producing the genus bacillus such as the silicate bacteria of pod membrane then must cultivate on specific substratum.
1.1 not producing the spore of the genus bacillus of pod membrane cultivates
1) the extractum carnis egg is from the peptone substratum: extractum carnis 5.0g; Peptone 10.0g; NaCl 5.0g; Agar powder 18～20g; Distilled water 1000; PH7.0～7.5.
2) on the beef-protein medium inclined-plane, inoculate genus bacillus, cultivate 24～48h for 25～30 ℃;
3) with the bacterium colony on physiological saline or the phosphoric acid buffer wash-out inclined-plane, the centrifugal 3～5min of 5000～8000rpm/min collects spore, washes 1～3 time with physiological saline, and the dilution spore is to finite concentration; Get this spore suspension 0.3～0.5mL and coat on the aseptic empty plate, after fully smoothening, air seasoning 0.5～1h on Bechtop carries out ionic fluid immediately and injects processing.
1.2 the spore of genus bacillus is cultivated
1) actication of culture is inoculated kind of a daughter bacteria on nitrogen-free agar, cultivates 24～36h for 28～30 ℃;
Nitrogen-free agar: potassium aluminum silicate 1.2～2.0g; Sucrose 5.0～10g; Na
2HPO
41.0～2.0g; FeCl
30.003～0.005g; MgSO
47H
2O 0.3～0.5g; Agar 18～20g; Distilled water 1000ml; PH7.0～7.5.
2) produce the liquid production substratum that gemma does not have the pod membrane gemma, the bottled nutrient solution 50ml of 500ml triangle, the kind daughter bacteria liquid of inoculation inclined-plane wash-out, 28～30 ℃ of joltings are cultivated, and rotating speed 200～250 commentaries on classics/min cultivate 48～72h;
The production substratum of no pod membrane gemma: starch 10～20g; (NH
4)
2SO
41.0～2.0g; MgSO
47H
2O 0.5～1.0g; K
2HPO
40.5～1.0g; FeCl
30.005～0.008g; Distilled water 1000ml; PH7.0～7.5.
3) the centrifugal 3～5min of pre-treatment 5000～8000rpm/min collects spore, washes 1～3 time with physiological saline, and the dilution spore is to finite concentration; Get this spore suspension 0.3～0.5mL and coat on the aseptic empty plate, after fully smoothening, air seasoning 0.5～1h on Bechtop carries out ionic fluid immediately and injects processing;
2, ionic fluid injects
1) characteristic of ionic fluid kind, implantation dosage, starting strain all can influence efficiency of inducing mutation, therefore must determine the best ion implantation dosage according to concrete beam types, bacterial strain kind.Ionic fluid can be selected N
+, H
+, Ar
+Or He
+Plasma is as injecting ion, and the injection energy of ions is 10～30KeV, and dosage is 5～300 * 10
14Ions/cm
2Ion implantation storehouse vacuum tightness is 1 * 10
-2～4.2 * 10
-3Pa; Intermittent pulse type injects, each 55～90 seconds recurrent intervals.
2) under above-mentioned injection condition, adopt serial dosage that genus bacillus is carried out ion implantation processing,, on beef extract-peptone or nitrogen-free agar, measure cytoactive with the spore that the physiological saline wash-out was handled, carry out regression analysis, make cell viability/dosage regression curve;
3) according to cell viability/dosage regression curve, be the mutagenesis implantation dosage with the corresponding dosage of 55～95% lethality rates, under aseptic condition, carry out ion implantation processing;
4) spore after the mutagenesis is used the physiological saline wash-out immediately, and is diluted to finite concentration;
3, screening method
1) screening: the genus bacillus to mutagenesis is inoculated on the NBRIY screening culture medium, cultivate 48～72h for 25～30 ℃, organophosphorus degrading bacteria produces opaque circle or transparent circle with organic phosphorus degrading, therefore having or not and the big or small ability that just can determine bacterial strain decomposition organophosphorus according to opaque circle or transparent circle.Select the bigger bacterium colony of opaque circle or transparent circle as aimed strain.
The NBRIY culture medium prescription is: glucose 10.0～20.0g; Yelkin TTS or phytate phosphorus 2.0～5.0g or organophosphorus pesticide 100～1000mg; (NH
4)
2SO
40.5～2.0g; MgSO
47H
2O 0.3～0.5g; KCl 0.2～0.3g; NaCl 0.2～0.3g; MnSO
47H
2O 0.003～0.005g; FeSO
47H
2O0.003～0.005g; CaCO
32.0～5.0g; Agar 18.0～20.0g; Distilled water 1000mL; PH7.0～7.5.
2) multiple sieve: genus bacillus is after screening on the above-mentioned NBRIY substratum, the target bacterium colony is forwarded to multiple sieve on the NBRIP substratum, this substratum is through the inorganic phosphorus of improvement and the screening culture medium of potassium decomposer, wherein tetrabromophenol sulfonphthalein or methyl red are as acid base indicator, indication bacterium colony acid producing ability, yellow haloing appears in the acid-producing bacteria strain.Haloing is big more, and acid producing ability is strong more, and the ability of decomposing insoluble inorganic phosphorus and potassium is just strong more.The bigger bacterium colony of yellow haloing is the target bacterium colony.
The NBRIP culture medium prescription is: glucose 10.0～20.0g; Ground phosphate rock 2.0～5.0g; (NH
4)
2SO
40.1～0.5g; MgCl
26H
2O 2.0～5.0g; MgSO
47H
2O 0.25～0.5g; Agar 18.0～20.0g; Tetrabromophenol sulfonphthalein or methyl red 0.002～0.010g; Distilled water 1000mL:pH7.0～7.5;
3, identification of strains
1) the phosphorus capacity of decomposition is measured the target bacterium colony that above screening is obtained and is carried out liquid culture respectively, cultivate 96～120h after, measure free phosphorus, biomass phosphorus and total phosphorous respectively, can calculate the ability that isolated strains decomposes organophosphorus or inorganic phosphorus.The detection molybdenum antimony resistance colorimetric method of phosphorus.The remarkable enhanced bacterial strain of the capacity of decomposition of organophosphorus or inorganic phosphorus is an engineering strain.
2) genetic stability is identified engineering strain was transferred for 3～7 generations on the NBRIY solid medium continuously, to the 3rd～7 generation bacterial strain measure the ability of decomposing organophosphorus and inorganic phosphorus respectively, show to have genetic stability preferably in 3～7 generations with the interior bacterial strain of good character that can keep, can be used as the production bacterial strain.
Embodiment 1
Use N
+Ionic fluid injects induces the bacillus megaterium variation, obtains the efficient engineering strain that decomposes Yelkin TTS.On the inclined-plane of beef-protein medium, activate bacillus megaterium, 28～30 ℃ cultivate 36～48h after, use the physiological saline wash-out, centrifugal collection spore dilutes and coats on the aseptic empty plate air seasoning 0.5～1h on the super clean bench; With energy is that 15～25KeV, dosage are 40～150 * 10
14Ions/cm
2N
+(vacuum tightness is 2 * 10 in vacuum
-2～4.2 * 10
-3Pa), carrying out intermittent pulse type under the aseptic condition injects; Use physiological saline wash-out spore then, after screening on NBRIY (the containing Yelkin TTS) substratum, through identifying, obtain 1 plant height and imitate the engineering strain that decomposes Yelkin TTS, the ability that this bacterial strain decomposes Yelkin TTS improves 18～25%.
Embodiment 2
Use H
+Ionic fluid injects induces the variation of silicate bacteria KNP414 bacterial strain, obtains the efficient engineering strain that decomposes inorganic phosphorus, inorganic potassium and Yelkin TTS.
On nitrogen-free agar, activate KNP414, behind 28～30 ℃ of cultivation 30～36h, be seeded in the liquid nutrient medium of no pod membrane gemma, 28～30 ℃, 200～250 commentaries on classics/min are cultivated 48～56h, centrifugal collection spore dilutes and coats on the aseptic empty plate seasoning 0.5～1h; With energy is that 15～25KeV, dosage are 60～200 * 10
14Ions/cm
2H
+(vacuum tightness is 3 * 10 in vacuum
-2～5 * 10
-3Pa), intermittent pulse type injects under the aseptic condition; Use physiological saline wash-out spore then, at the enterprising row filter of NBRIY substratum (containing Yelkin TTS), multiple sieve on the NBRIP substratum through identifying, obtains 2 plant heights and imitates engineering strain KNP41401 and the KNP41402 that decomposes inorganic potassium, inorganic phosphorus and organophosphorus immediately.The ability that KNP41401 decomposes inorganic potassium, inorganic phosphorus and organophosphorus improves 15～20%, 18～25% and 20～40% respectively, and KNP41402 improves 18～20%, 20～25% and 25～45% respectively.
Embodiment 3
Use He
+Ionic fluid injects induces the subtilis variation, obtains the efficient engineering strain that decomposes phytate phosphorus.On the inclined-plane of beef-protein medium, activate subtilis, 28～30 ℃ cultivate 36～48h after, use the phosphoric acid buffer wash-out, centrifugal collection spore dilutes and coats on the aseptic slide glass air seasoning 0.5～1h on the super clean bench; With energy is that 10～20KeV, dosage are 40～150 * 10
14Ions/cm
2He
+(vacuum tightness is 2 * 10 in vacuum
-2～5 * 10
-3Pa), intermittent pulse type injects under the aseptic condition; Use phosphoric acid buffer wash-out spore then, screen on NBRIY (phytate phosphorus) substratum, through identifying, obtain 1 plant height and imitate the engineering strain that decomposes phytate phosphorus, the ability that this bacterial strain decomposes phytate phosphorus improves 25～35%.
Embodiment 4
Use Ar
+Ionic fluid injects induces the variation of silicate bacteria KNP411 bacterial strain, obtains the efficient engineering strain that decomposes inorganic phosphorus and inorganic potassium and effectively decompose triazophos.On the inclined-plane of nitrogen-free agar, activate KNP411, behind 28～30 ℃ of cultivation 30～36h, be seeded in the liquid nutrient medium of no pod membrane gemma, 28～30 ℃, 200～250 commentaries on classics/min are cultivated 48～72h, centrifugal collection spore, dilution is also coated on the aseptic empty plate air seasoning 0.5～1h on super clean bench; With energy is that 10～20KeV, dosage are 80～160 * 10
14Ions/cm
2Ar
+(vacuum tightness is 2 * 10 in vacuum
-2～5 * 10
-3Pa), intermittent pulse type injects under the aseptic condition; Use physiological saline wash-out spore then, on NBRIY (containing triazophos) substratum, screen, on the NBRIP substratum, sieve again immediately, through identifying, obtain 1 plant height and imitate the engineering strain that decomposes inorganic potassium, inorganic phosphorus and triazophos, the ability that this bacterial strain decomposes inorganic potassium, inorganic phosphorus and triazophos improves 15～20%, 18～25% and 20～45% respectively.
Claims (10)
1. the construction process of a genus bacillus phosphorus decomposing engineering strain is characterized in that its step is:
1) activation culture genus bacillus on the inclined-plane, used substratum is a beef-protein medium, the genus bacillus after the activation directly becomes bacteria suspension with physiological saline or phosphoric acid buffer wash-out;
2) bacteria suspension is centrifugal, collect spore, dilute spore and coat on the aseptic blank plate or slide glass dry 0.5～2h with physiological saline or phosphoric acid buffer;
3) with N
+, H
+, Ar
+Or He
+Ionic fluid injects genus bacillus, and the ion energy that is injected is that 10～30KeV, dosage are 5～300 * 10
14Ions/cm
2, 2 * 10
-2～6 * 10
-3Carry out intermittent pulse type under Pa vacuum, the aseptic condition and inject, each 50～100 seconds recurrent intervals;
4) inject the spore of handling with physiological saline or phosphoric acid buffer wash-out through ionic fluid, and be diluted to finite concentration,,, obtain the efficient engineering strain that decomposes organophosphorus then through identifying at the enterprising row filter of NBRIY substratum.
2. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 1, it is characterized in that the pre-treating process that said ionic fluid injects is: at first clean spore 1～3 time with physiological saline or phosphoric acid buffer, centrifugal, suspend with physiological saline or phosphoric acid buffer again, and be diluted to 10
3～10
6Individual L
-1, coating diameter then is on the blank flat board or slide glass of 9cm, air seasoning 0.5～2h on super clean bench.
3. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 1 is characterized in that the screening method step of said engineering strain is:
1) preparation screening culture medium, its prescription is: glucose 10～20g; Yelkin TTS or phytate phosphorus 2.0～5.0g or organophosphorus pesticide 100～1000mg; (NH
4)
2SO
40.5～2.0g; MgSO
47H
2O 0.3～0.5g; KCl 0.1～0.3g; NaCl 0.1～0.3g; MnSO
47H
2O 0.001～0.005g; FeSO
47H
2O0.001～0.005g; CaCO
31.0～5.0g; Agar powder 18～20g; Distilled water 1000mL; PH7.0～7.5.
2) will inject the spore inoculating handled on screening culture medium through ionic fluid, cultivate 48～96h for 25～30 ℃.Opaque halo or transparent circle occurring and enclosing bigger bacterium colony is the target bacterium colony.
4. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 1 is characterized in that the step of said authentication method is:
1) will screen single colony inoculation of acquisition in the NBRIY liquid nutrient medium, 25～30 ℃, 150～250 commentaries on classics/min cultivate 96～120h, and are centrifugal, measure free phosphorus, biomass phosphorus and total phosphorous respectively, the degradation capability enhancing 5～15% or the above bacterial strain of organophosphorus are engineering strain.
2) above engineering strain was transferred for 3～7 generations on the NBRIY solid medium continuously, ability to the 3rd～7 generation bacterial strain mensuration decomposition organophosphorus, can keep the bacterial strain of higher organic phosphorus degrading ability to show to have genetic stability preferably, can be used as the production bacterial strain.
5. the construction process of a genus bacillus phosphorus decomposing engineering strain is characterized in that its step is:
1) activation culture genus bacillus on the nitrogen-free agar inclined-plane, the bacterium after the activation is inoculated in the liquid nutrient medium that produces no pod membrane gemma, and 25～30 ℃, 150～250 commentaries on classics/min are cultivated 36～72h, centrifugal collection spore;
2) with spore with the dilution of physiological saline or phosphoric acid buffer and coat on the aseptic blank plate or slide glass dry 0.5～2h;
3) with N
+, H
+, Ar
+Or He
+Ionic fluid injects genus bacillus, and the ion energy that is injected is that 10～30KeV, dosage are 5～300 * 10
14Ions/cm
2, 2 * 10
-2～6 * 10
-3Carry out intermittent pulse type under Pa vacuum, the aseptic condition and inject, each 50～100 seconds recurrent intervals;
4) with physiological saline or phosphoric acid buffer wash-out spore, and be diluted to finite concentration, after process screening on the NBRIY substratum, on the NBRIP substratum, carry out multiple sieve, be tested and appraised at last, obtain the efficient engineering strain that decomposes organophosphorus, inorganic phosphorus and potassium.
6. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 5 is characterized in that the liquid culture based formulas of said nitrogen-free agar and the no pod membrane gemma of product is respectively:
Nitrogen-free agar: potassium aluminum silicate 1.2～2.0g; Sucrose 5.0～10g; Na
2HPO
41.0～2.0g; FeCl
30.003～0.005g; MgSO
47H
2O 0.3～0.5g; Agar 18～20g; Distilled water 1000ml; PH7.0～7.5.
The liquid nutrient medium of no pod membrane gemma: starch 10～20g; (NH
4)
2SO
40.5～2.0g; MgSO
47H
2O0.3～1.0g; K
2HPO
40.5～20g; FeCl
30.005～0.010g; Agar powder 18～20g; Water 1000ml; PH7.0～7.5.
7. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 5, it is characterized in that the pre-treating process that said ionic fluid injects is: at first clean spore 1～3 time with physiological saline or phosphoric acid buffer, centrifugal, suspend with physiological saline or phosphoric acid buffer again, and be diluted to 10
3～10
6Individual L
-1, coating diameter then is on the blank flat board or slide glass of 9cm, air seasoning 0.5～2h on super clean bench.
8. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 5 is characterized in that the screening method step of said engineering strain is:
1) preparation screening culture medium, its prescription is: glucose 10～20g; Yelkin TTS or phytate phosphorus 2.0～5.0g or organophosphorus pesticide 100～1000mg; (NH
4)
28O
40.5～2.0g; MgSO
47H
2O 0.3～0.5g; KCl 0.1～0.3g; NaCl 0.1～0.3g; MnSO
47H
2O 0.001～0.005g; FeSO
47H
2O0.001～0.005g; CaCO
31.0～5.0g; Agar powder 18～20g; Distilled water 1000mL; PH7.0～7.5.
2) will inject the spore inoculating handled on screening culture medium through ionic fluid, 25～30 ℃ of cultivation 48～96h.Opaque halo or transparent circle occurring and enclosing bigger bacterium colony is the target bacterium colony.
9. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 5 is characterized in that the step of said multiple screen method is:
1) substratum is sieved in preparation again, and its prescription is: glucose 10～20g; Potassium breeze, ground phosphate rock or Ca
3(PO4)
22.0～5.0g; (NH
4)
2SO
40.1～1.0g; MgCl
26H
2O 2.0～5.0g; MgSO
47H
2O0.25～0.5g; Agar powder 18～20.0g; Tetrabromophenol sulfonphthalein or methyl red indicator 0.002～0.005gL
-1Distilled water 1000mL:pH7.0～7.5.
2) colony inoculation that above screening culture medium screening is obtained is cultivated 48～96h for 28～30 ℃ in sieving again on the substratum.The bigger bacterium colony of yellow haloing and haloing occurring is the target bacterium colony.
10. the construction process of a kind of genus bacillus phosphorus decomposing engineering strain according to claim 5 is characterized in that the step of said authentication method is:
1) will sieve single colony inoculation of acquisition again in the NBRIY liquid nutrient medium, 25～30 ℃, 150～250 commentaries on classics/min cultivate 96～120h, and are centrifugal, measure free phosphorus, biomass phosphorus and total phosphorous respectively, the degradation capability enhancing 5～15% or the above bacterial strain of organophosphorus are engineering strain.
2) inoculation that above evaluation is obtained is in the NBRIP liquid nutrient medium, 25～30 ℃, 150～250 commentaries on classics/min are cultivated 96～120h, centrifugal, the ability of detect decomposing inorganic phosphorus and potassium respectively, the capacity of decomposition of inorganic phosphorus and potassium than starting strain increase respectively 5～15% or above bacterial strain be engineering strain.
3) above engineering strain was transferred for 3～7 generations on NBRIP and NBRIY solid medium respectively continuously, to the 3rd～7 generation bacterial strain measure the ability of decomposing inorganic potassium, inorganic phosphorus and organophosphorus respectively, can keep the bacterial strain of higher inorganic phosphorus, inorganic potassium and organophosphorus capacity of decomposition to show to have genetic stability preferably, can be used as the production bacterial strain.
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|---|---|---|---|
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|---|---|---|---|
| CN 02145369 CN1233817C (en) | 2002-11-21 | 2002-11-21 | Construction method of bacillus phosphorus relieving engineering bacterial strain |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250784A (en) * | 2011-04-25 | 2011-11-23 | 南京中医药大学 | Angelica root rhizosphere efficient phosphate-solubilizing bacterium and microbial inoculum prepared from same and application thereof |
| CN102399713A (en) * | 2011-09-22 | 2012-04-04 | 华南农业大学 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
| CN111924982A (en) * | 2020-08-12 | 2020-11-13 | 中国科学院武汉岩土力学研究所 | Biological permeable reactive barrier for composite contaminated site and preparation method thereof |
| CN114181003A (en) * | 2021-09-24 | 2022-03-15 | 中农新科(苏州)有机循环研究院有限公司 | Preparation method of efficient phosphate fertilizer from kitchen waste |
-
2002
- 2002-11-21 CN CN 02145369 patent/CN1233817C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250784A (en) * | 2011-04-25 | 2011-11-23 | 南京中医药大学 | Angelica root rhizosphere efficient phosphate-solubilizing bacterium and microbial inoculum prepared from same and application thereof |
| CN102250784B (en) * | 2011-04-25 | 2013-06-19 | 南京中医药大学 | Angelica root rhizosphere efficient phosphate-solubilizing bacterium and microbial inoculum prepared from same and application thereof |
| CN102399713A (en) * | 2011-09-22 | 2012-04-04 | 华南农业大学 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
| CN102399713B (en) * | 2011-09-22 | 2013-01-30 | 华南农业大学 | Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving |
| CN111924982A (en) * | 2020-08-12 | 2020-11-13 | 中国科学院武汉岩土力学研究所 | Biological permeable reactive barrier for composite contaminated site and preparation method thereof |
| CN114181003A (en) * | 2021-09-24 | 2022-03-15 | 中农新科(苏州)有机循环研究院有限公司 | Preparation method of efficient phosphate fertilizer from kitchen waste |
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|---|---|
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