CN103952365B - One strain has the polyP bacteria of higher phosphor-removing effect and screening method thereof and application at low temperatures - Google Patents
One strain has the polyP bacteria of higher phosphor-removing effect and screening method thereof and application at low temperatures Download PDFInfo
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Abstract
The invention discloses the polyP bacteria that a strain has higher phosphor-removing effect at low temperatures, it is characterized in that, does is the preserving number of described polyP bacteria CCTCC? NO:M2014140.Bacterial strain of the present invention is that utilize low temperature enrichment culture and dilution mixture flat band method to screen from the bed mud of Chaohu to obtain a kind of at low temperatures can the polyP bacteria of efficient dephosphorization, still the carbon source effectively absorbed in waste water poly-beta-hydroxy alkyl salt can be converted into when anaerobism at 8 DEG C, discharge the polyphosphoric acid salt stored in born of the same parents simultaneously, therefrom obtain energy; When aerobic, be oxidized the poly-beta-hydroxy alkyl salt generate energy in born of the same parents, and from waste water, take the photograph phosphorus in large quantities, accumulated with the form of polyphosphoric acid salt.Bacterial strain of the present invention has thermal adaptability widely, has poly-phosphorus effect preferably, can be used for the wastewater treatment under winter or cold condition in 8-30 DEG C.
Description
Technical field
The present invention relates to a strain and still there is the polyP bacteria of higher phosphor-removing effect and screening method thereof and application at low temperatures, belong to field of environment microorganism.
Background technology
Water pollution problems is the major issue that countries in the world generally face, and this problem is still serious in such developing country of China.In recent decades, China's population sharp increase, urbanization paces are accelerated, and urban wastewater discharge is increasing, and water pollution present situation is very serious, and has the trend obviously worsened.Along with increasingly sharpening of water pollutions, the eutrophication problem of water body is more and more outstanding.Since nearly ten years, the ratio of China's eutrophication water rises to 55% from 50%, and oligotrophication's water body ratio drops to 0.53% from 3.2% simultaneously.Body eutrophication can cause the amount reproduction of algae, causes the generation of red tide or wawter bloom phenomenon, reduces the transparency of water body, affects water quality; Algal bloom also can consume the dissolved oxygen in water body, affects other hydrobiological existence, destroys aquatic ecological balance; Part algae can discharge toxic substance in water body simultaneously, harm other biological and HUMAN HEALTH.The major cause of natural water eutrophication is containing a large amount of nutritive substances, and particularly the waste water of the element such as nitrogen phosphorus is injected in rivers and lakes.In numerous element, exceeding standard of phosphorus content is considered to one of key factor causing body eutrophication.Therefore, how efficiently, economic dephosphorization has become the important research direction in current water prevention and cure of pollution field.
Biological phosphate-eliminating technology is the phosphorus removing method of widespread use in the world at present, and its advantage is a large amount of chemical sludges can avoiding producing in chemical phosphorus removal method, and reduce sludge bulking phenomenon, save energy, working cost are lower, and are conducive to the recovery of phosphorus.Biological phosphate-eliminating is mainly called as by a class that the microorganism of polyP bacteria (Poly-phosphateAccumulatingOrganisms, PAOs) come, and this quasi-microorganism all belongs to heterotrophic bacterium.Its core be utilize polyP bacteria under anaerobic to discharge phosphorus and under aerobic condition to the feature of the excessive consumption of phosphorus, and the phosphorus absorbed under aerobic condition is more than the phosphorus discharged under anaerobic condition, thus the phosphorus in waste water is removed.Phosphorus content in general bacterial body only has about 2% of dry cell weight, and polyP bacteria under aerobic condition can excess by the phosphorus suction body in waste water, make phosphorus content more than 10%, even can reach 30%.In recent years, people conduct in-depth research microorganism dephosphorization technique, and devise multiple dephosphorization process.
But in the winter time or under the cold condition such as cold district, the Be very effective of biological phosphate-eliminating declines.This is mainly because warm polyP bacteria (phosphorusaccumulationorganism in playing a major role in biological phosphate-eliminating, PAOs) growth metabolism is under cryogenic in holddown, and low temperature PAOs is fewer than middle temperature PAOs at a normal temperature, metabolic rate is slow, is difficult to form dominant microflora in its natural state.Therefore, effectively dephosphorization under cold condition be realized, can not only from the research and development aspect such as technique and combustion adjustment research, also must from the feasibility of microbiological angle research and inquirement low-temperature biological dephosphorization.
In recent years, people have carried out certain research to low temperature polyP bacteria, are mainly tamed by thermograde, therefrom screen the bacterial strain still at low temperature with poly-phosphorus usefulness in warm polyP bacteria.But the research of this bacterioid and dephosphorization characteristics thereof is all in the junior stage, and does not cultivate acclimation efficiently, do not have systematic authentication method yet.Verify kind and the characteristic of low temperature polyP bacteria, specify their dephosphorization condition, will research, the development and application of waste water dephosphorization technique be contributed to.
Summary of the invention
For above problem, the object of this invention is to provide the polyP bacteria that a strain still has higher phosphor-removing effect at low temperatures.This bacterium has thermal adaptability widely, can effectively utilize various carbon source simultaneously.Another object of the present invention is to provide screening method and the application of this bacterium.
Bacterial strain of the present invention is that utilize low temperature enrichment culture and dilution mixture flat band method to screen from the bed mud of Chaohu to obtain a kind of at low temperatures can the polyP bacteria of efficient dephosphorization, still the carbon source effectively absorbed in waste water poly-beta-hydroxy alkyl salt can be converted into when anaerobism at 8 DEG C, discharge the polyphosphoric acid salt stored in born of the same parents simultaneously, therefrom obtain energy; When aerobic, be oxidized the poly-beta-hydroxy alkyl salt generate energy in born of the same parents, and from waste water, take the photograph phosphorus in large quantities, accumulated with the form of polyphosphoric acid salt.
The present invention adopts following technical scheme:
One of the object of the invention is to provide a strain to have the polyP bacteria of higher phosphor-removing effect at low temperatures, and the preserving number of described polyP bacteria is CCTCCNO:M2014140.
Another object of the present invention is to provide a kind of screening to have the method for higher phosphor-removing effect polyP bacteria at low temperatures, and its feature is, described method comprises the steps:
(1) culture medium A, B and C is prepared respectively:
Culture medium A is strains separation, purifying, storage medium, and it consists of: often liter of culture medium A comprises peptone 10g, extractum carnis 5g, sodium-chlor 5g, agar 19g, and all the other are water, regulates pH to 7.0-7.2,
Substratum B is pre-culture medium, and it consists of: often liter of substratum B comprises sodium acetate 2g, Na
2hPO
446mg, NH
4cl152.8mg, CaCl
28.3mg, MgSO
482mg, K
2sO
417.83mg, HEPES7g, micro-2mL, all the other are water, regulate pH to 7.0-7.2;
Culture medium C is rich phosphorus substratum, and it consists of: often liter of culture medium C comprises sodium acetate 1g, MgSO
482mg, FeSO
43.7mg, CaCl
260mg, (NH
4)
2sO
40.2g, extractum carnis 0.22g, K
2hPO
474mg, micro-2mL, all the other are water, regulate pH to 7.0-7.2;
Consisting of of described trace element: often liter of trace element comprises FeCl
30.9g, H
3bO
3150mg, CuSO
430mg, KI180mg, MnCl
260mg, ZnSO
4120mg, CoCl
2140mg, Na
2moO
460mg, ethylenediamine tetraacetic acid (EDTA) 10g, all the other are water;
(2) enrichment culture
Get active sludge 0.5g to be added in 100mL sterilized water in 6 DEG C of-10 DEG C of shaking culture 24h, it is in the culture medium A of 5mg/L that the mixed solution got after 10mL shaking culture is transferred to 100mL phosphorus concentration, 5-9 DEG C of enrichment culture, often cultivate 2d switching once, the phosphorus concentration of often transferring in a culture medium A improves 5mg/L, corotation connects 4 times, enrichment culture 8d, obtains enrichment culture liquid;
(3) separation and purification
Described enrichment culture liquid is reached 10 to extent of dilution
-6, draw 0.5ml diluent in separation and purification flat board, mix with culture medium A at 40 DEG C-42 DEG C, solidify rear inversion, go out bacterium colony in 8 DEG C of constant temperature culture; Single bacterium colony on flat board described in picking is repeatedly rule purifying on flat board, to microscopic examination display without miscellaneous bacteria, namely obtains purifying bacterial strain;
(3) screen
By described purifying inoculation in the Erlenmeyer flask that 100mL substratum B is housed, at 130r/min, shaking culture 24h at 8 DEG C, obtain pre-culture solution, described pre-culture solution is transferred in the Erlenmeyer flask that 100mL culture medium C is housed by 10%, at 130r/min, shaking culture 2d at 8 DEG C; Wherein first Anaerobic culturel 24h aerobic cultivation 24h again; Get the centrifugal 10min of nutrient solution 10000r/min after cultivation completes and get supernatant liquor, measure the total phosphorus concentration of supernatant liquor, investigate bacterial strain to the clearance of total phosphorus, thus the polyP bacteria at low temperatures with higher phosphor-removing effect filtered out.
Screening method of the present invention can filter out the bacterial strain of preservation of the present invention, but is not limited to, only for screening bacterial strain of the present invention, also can be used for the screening that other has higher phosphor-removing effect polyP bacteria at low temperatures.
Another object of the present invention is to polyP bacteria application in the treatment of waste water.
PolyP bacteria 16SrDNA sequence of the present invention is as shown in SEQIDNO:1.
Bacterial strain of the present invention has the following advantages:
(1) bacterial strain of the present invention has thermal adaptability widely, has poly-phosphorus effect preferably, can be used for the wastewater treatment under winter or cold condition in 8-30 DEG C.
(2) bacterial strain of the present invention has very high dephosphorization activity, and this bacterial strain is after cultivation 48h, and dephosphorizing rate reaches 94.5, can be used for natural water body ecological reconstruction or in-situ immobilization.
Bacterial strain of the present invention is screened from the bed mud of Chaohu by contriver in October, 2012 to obtain, through qualification, belong to Rhodopseudomonas, it is a fluorescent pseudomonads, called after pseudomonas D6PseudomonasSP.D6, in China typical culture collection center preservation, preservation address: Wuhan, China Wuhan University; Preservation date is on April 20th, 2014; Deposit number is CCTCCNO:M2014140.
Accompanying drawing explanation
Fig. 1 is after bacterial strain of the present invention cultivates 48h at different temperatures, the OD value of substratum and dephosphorizing rate.
Fig. 2 is after bacterial strain of the present invention cultivates 48h in different carbon source substratum, the OD value of substratum and dephosphorizing rate.
Fig. 3 is after bacterial strain of the present invention cultivates 48h in different pH value substratum, the OD value of substratum and dephosphorizing rate.
Fig. 4 is OD value and the real-time detected result figure of dephosphorizing rate of bacterial strain of the present invention substratum in culturing process.
Embodiment
Below by the drawings and specific embodiments, the invention will be further described.
The separation screening of embodiment 1 bacterial strain of the present invention and qualification:
(1) substratum
Culture medium A: often liter of substratum is containing peptone 10g, extractum carnis 5g, sodium-chlor 5g, agar 19g; Surplus is water; PH7.0-7.2.
Culture medium A is used for strains separation, purifying, preservation.
Substratum B: often liter substratum is containing sodium acetate (carbon source) 2g, Na
2hPO
446mg, NH
4cl152.8mg, CaCl
28.3mg, MgSO
482mg, K
2sO
417.83mg, HEPES7g, micro-2mL; Surplus is water, pH7.0-7.2.
Substratum B is used for the pre-culture medium of bacterial strain.
Culture medium C: often liter of substratum is containing sodium acetate (carbon source) 1g, MgSO
482mg, FeSO
43.7mg, CaCl
260mg, (NH
4)
2sO
40.2g, extractum carnis 0.22g, K
2hPO
474mg, micro-2mL; Surplus is water; PH7.0-7.2.
The rich phosphorus that culture medium C is used for bacterial strain is cultivated.
D, trace element composition (g/L): often liter of substratum is containing FeCl
30.9g, H
3bO
3150mg, CuSO
430mg, KI180mg, MnCl
260mg, ZnSO
4120mg, CoCl
2140mg, Na
2moO
460mg, ethylenediamine tetraacetic acid (EDTA) 10g.
Before above-mentioned substratum uses, 121 DEG C, sterilizing 20 minutes.
(2) separation of Pseudomonas fluorescens D6 bacterial strain, purifying and screening:
Carry out enrichment culture under cryogenic.Getting Chaohu bed mud 0.5g is added in 100mL sterilized water, shaking culture 24h at 8 DEG C, gets 10mL mixed solution and is transferred to 100mL and adds phosphorus to phosphorus concentration and reach low temperature enrichment culture in 5mg/L beef-protein medium.Often cultivate 2d switching once, the phosphorus concentration of often transferring in a beef-protein medium improves 5mg/L, and corotation connects 4 times, enrichment culture 8d, and enrichment obtains the stronger bacterial strain of poly-phosphorus ability.
Mixed diluting flat band method and plate streak is adopted to carry out separation and purification.Draw 0.5ml enrichment culture liquid in the test tube that 4.5ml sterilized water is housed with liquid-transfering gun, mixing, the rest may be inferred.The extent of dilution of enrichment culture liquid is finally made to reach 10
-6.Draw the diluent after dilution, with the amount of the dull and stereotyped 0.5ml of every block, mix with about 40 DEG C isolation mediums, be positioned over Bechtop and make it solidify.Afterwards flat board is inverted, puts into 8 DEG C of constant incubators and cultivate.
Picking list bacterium colony, and the single strain of picking is repeatedly rule purifying on flat board, to microscopic examination display without miscellaneous bacteria, now can think that bacterial strain purifying is complete.
By the purifying inoculation of gained in the Erlenmeyer flask that 100mL pre-culture medium is housed, at 130r/min, shaking culture 24h at 8 DEG C.Pre-culture solution is transferred in the Erlenmeyer flask that the rich phosphorus substratum of 100mL is housed by 10%, at 130r/min, shaking culture 2d at 8 DEG C.In culturing process, front 24h Anaerobic culturel, anaerobic condition is sealed by rubber plug, extracts the high pure nitrogen that reinjects of air in bottle reach with sterilized syringe.The aerobic cultivation of rear 24h of cultivating, is, in Bechtop, rubber plug is changed to air-permeable envelope, oxygen is entered.Get the centrifugal 10min of nutrient solution 10000r/min after cultivation completes and get supernatant liquor, measure total phosphorus concentration, investigate bacterial strain to the clearance of total phosphorus, the Pseudomonas fluorescens D6 in the efficient polyP bacteria filtered out and the present invention, be seeded to slant medium and preserve.
(3) colony morphology characteristic of Pseudomonas fluorescens D6:
Culture medium A is cultivated bacterium colony oyster white after 2 days, in regular circle shapes, intermediate projections, edge be opaque, glossy.
(4) Molecular Identification of 16SrDNA
Experimental technique: picking thalline from the inclined-plane of bacterial strain D6, adds in the centrifuge tube containing 100 μ L distilled waters, and vortex mixes, and thermo-cracking bacteria suspension take genomic dna as template amplification 16SrDNA.
Forward primer is 7F:5 '-CAGAGTTTGATCCTGGCT-3 ';
Reverse primer is 1540R:5 '-AGGAGGTGATCCAGCCGCA-3 '.
Amplification reaction system is: DNA profiling 0.5 μ L, 5 × Buffer damping fluid 2.5 μ L, dNTP1 μ L, each 0.5 μ L of positive anti-primer, enzyme 0.2 μ L, distilled water 19.8 μ L.
Reaction conditions is: 98 DEG C of denaturation 3min, 98 DEG C of 25sec, 55 DEG C of 25sec, 72 DEG C of 1min, 30 circulations, 4 DEG C of preservations after 72 DEG C of prolongation 10min.
Do electrophoresis detection with the 16SrDNA amplified production of 1% sepharose to bacterial strain, after checking, cut adhesive tape, adopt DNA gel to reclaim kits PCR primer.Pcr amplification product entrusts Shanghai Sheng Gong biotechnology company limited to check order.
(5) 16SrDNA sequential analysis and Phylogenetic Analysis:
The 16SrDNA length obtaining D6 bacterial strain after order-checking is the sequence of 1451bp, be submitted to NCBIGenbank database and carry out Blast comparison, find that the evolutionary distance of bacterial strain D6 and Pseudomonasfluorescens is the most close, determine that it is Rhodopseudomonas, preliminary identification its be Pseudomonas fluorescens, called after PseudomonasfluorescensD6.
D6 bacterial strain 16SrDNA sequence is as shown in SEQIDNO:1.
The best dephosphorization condition of embodiment 2 bacterial strain of the present invention
1, the seed liquor obtained by the strain culturing of preservation of the present invention by volume 1:10 is inoculated in culture medium C, shaking culture 48h at 8 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C respectively, wherein first Anaerobic culturel 24h, aerobic cultivation 24h again, detect OD value and the dephosphorizing rate of culture medium C after 48h, detected result as shown in Figure 1.From Fig. 1 result, bacterial strain of the present invention has thermal adaptability widely, has poly-phosphorus effect preferably, can be used for the wastewater treatment under winter or cold condition in 8-30 DEG C.
2, the seed liquor obtained by the strain culturing of preservation of the present invention by volume 1:10 is inoculated in the rich phosphorus substratum of different carbon source, at 20 DEG C of shaking culture 48h, wherein first Anaerobic culturel 24h, more aerobic cultivation 24h, detect OD value and the dephosphorizing rate of culture medium C after 48h, detected result as shown in Figure 2.The rich phosphorus substratum of above-mentioned different carbon source refers to culture medium C, with etc. the culture medium C 1 that sodium acetate obtains in carbon content glucose substitutive medium C, with etc. the culture medium C 2 that sodium acetate obtains in carbon content fructose substitutive medium C, with etc. the culture medium C 3 that sodium acetate obtains in carbon content cane sugar substitution culture medium C, with etc. the culture medium C 4 that sodium acetate obtains in carbon content lactose substitutive medium C; With etc. the culture medium C 5 that sodium acetate obtains in carbon content starch in replace culture medium C.From Fig. 2 result, bacterial strain of the present invention can well utilize various carbon source, and the suitableeest carbon source is sodium acetate.
3, the seed liquor obtained by the strain culturing of preservation of the present invention by volume 1:10 is inoculated in the substratum of different pH value, shaking culture 48h at 20 DEG C, wherein first Anaerobic culturel 24h, more aerobic cultivation 24h, detect OD value and the dephosphorizing rate of culture medium C after 48h, detected result as shown in Figure 3.The substratum of the different pH value of above-mentioned difference refers to respectively: culture medium C; Culture medium C 6: composition is identical with culture medium C, pH5.0; Culture medium C 7: composition is identical with culture medium C, pH6.0; Culture medium C 8: composition is identical with culture medium C, pH8.0; Culture medium C 9: composition is identical with culture medium C, pH9.0; Culture medium C 10: composition is identical with culture medium C, pH10.0; Culture medium C 11: composition is identical with culture medium C, pH11.0; From Fig. 3 result, bacterial strain of the present invention, in the scope of pH7-9, has very strong dephosphorization active.
4, the seed liquor obtained by the strain culturing of preservation of the present invention by volume 1:10 is inoculated in culture medium C, shaking culture 48h at 20 DEG C, wherein first Anaerobic culturel 36h, aerobic cultivation 12h again, as shown in Figure 4, after 48h, dephosphorizing rate reaches 94.5% for real-time OD value in whole culturing process and dephosphorizing rate.
Claims (3)
1. a strain has the polyP bacteria of higher phosphor-removing effect at low temperatures, it is characterized in that, described polyP bacteria belongs to Rhodopseudomonas, it is a fluorescent pseudomonads, called after pseudomonas (PseudomonasSP.) D6, described polyP bacteria is preserved in China typical culture collection center, and preserving number is CCTCCNO:M2014140.
2. screening has a method for higher phosphor-removing effect polyP bacteria at low temperatures, and it is characterized in that, described method comprises the steps:
(1) culture medium A, B and C is prepared respectively:
Culture medium A is strains separation, purifying, storage medium, and it consists of: often liter of culture medium A comprises peptone 10g, extractum carnis 5g, sodium-chlor 5g, agar 19g, and all the other are water, regulates pH to 7.0-7.2,
Substratum B is pre-culture medium, and it consists of: often liter of substratum B comprises sodium acetate 2g, Na
2hPO
446mg, NH
4cl152.8mg, CaCl
28.3mg, MgSO
482mg, K
2sO
417.83mg, HEPES7g, micro-2mL, all the other are water, regulate pH to 7.0-7.2;
Culture medium C is rich phosphorus substratum, and it consists of: often liter of culture medium C comprises sodium acetate 1g, MgSO
482mg, FeSO
43.7mg, CaCl
260mg, (NH
4)
2sO
40.2g, extractum carnis 0.22g, K
2hPO
474mg, micro-2mL, all the other are water, regulate pH to 7.0-7.2;
Consisting of of described trace element: often liter of trace element comprises FeCl
30.9g, H
3bO
3150mg, CuSO
430mg, KI180mg, MnCl
260mg, ZnSO
4120mg, CoCl
2140mg, Na
2moO
460mg, ethylenediamine tetraacetic acid (EDTA) 10g, all the other are water;
(2) enrichment culture
Get active sludge 0.5g to be added in 100mL sterilized water in 6 DEG C of-10 DEG C of shaking culture 24h, it is in the culture medium A of 5mg/L that the mixed solution got after 10mL shaking culture is transferred to 100mL phosphorus concentration, 5-9 DEG C of enrichment culture, often cultivate 2d switching once, the phosphorus concentration of often transferring in a culture medium A improves 5mg/L, corotation connects 4 times, enrichment culture 8d, obtains enrichment culture liquid;
(3) separation and purification
Described enrichment culture liquid is reached 10 to extent of dilution
-6, draw 0.5ml diluent in separation and purification flat board, mix with culture medium A at 40 DEG C-42 DEG C, solidify rear inversion, go out bacterium colony in 8 DEG C of constant temperature culture; Single bacterium colony on flat board described in picking is repeatedly rule purifying on flat board, to microscopic examination display without miscellaneous bacteria, namely obtains purifying bacterial strain;
(4) screen
By described purifying inoculation in the Erlenmeyer flask that 100mL substratum B is housed, at 130r/min, shaking culture 24h at 8 DEG C, obtain pre-culture solution, described pre-culture solution is transferred in the Erlenmeyer flask that 100mL culture medium C is housed by 10%, at 130r/min, shaking culture 2d at 8 DEG C; Wherein first Anaerobic culturel 24h aerobic cultivation 24h again; Get the centrifugal 10min of nutrient solution 10000r/min after cultivation completes and get supernatant liquor, measure the total phosphorus concentration of supernatant liquor, investigate bacterial strain to the clearance of total phosphorus, thus the polyP bacteria at low temperatures with higher phosphor-removing effect filtered out.
3. the application in the treatment of waste water of polyP bacteria described in claim 1.
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