CN103966129A - Screening cultivating method for denitrifying phosphorus removing bacterial strain and application thereof - Google Patents
Screening cultivating method for denitrifying phosphorus removing bacterial strain and application thereof Download PDFInfo
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- CN103966129A CN103966129A CN201410179439.5A CN201410179439A CN103966129A CN 103966129 A CN103966129 A CN 103966129A CN 201410179439 A CN201410179439 A CN 201410179439A CN 103966129 A CN103966129 A CN 103966129A
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Abstract
The invention discloses a screening cultivating method for a denitrifying phosphorus removing bacterial strain and application thereof. The bacterial strain is an amphimicrobe and can be used under anaerobic and aerobic conditions; the selected bacterial strain grows under the condition of pH 6.5-9.5 and at the temperature of 10-60 DEG, is strong in alkali-resisting capacity, and has denitrifying phosphorus removing functions under strong alkaline conditions; the growing temperature range is wide and the optimum temperature is between 25-35 DEG; the bacterial strain can revive within a short time in actual use. According to the invention, the bacterial strain is high in denitrifying phosphorus removing efficiency and can be applied to various eutrophic water; the denitrifying phosphorus removing bacteria and ecological water filtering device are placed in eutrophic water to carry out a biofilm culturing and treatment effect test for 3 months, and the result indicates that the removal rates of CODcr, BOD5, ammonia nitrogen, TP and TN in sewage are 70%, 78%, 84%, 74% and 82% respectively.
Description
Technical field
The invention belongs to biotechnology, field of environment engineering technology, be specially a kind of screening method of cultivation and application thereof of denitrification dephosphorization bacterial strain.
Background technology
In recent years, the water pollution situation of China is day by day serious, especially outstanding with the eutrophication problem of water body, and the content that is mainly manifested in nitrogen, phosphorus surpasses water body environment capacity, and then causes wawter bloom, causes water quality to worsen.Outstanding day by day along with " body eutrophication " problem, the standard of sewage discharge is also increasingly strict, how economical, remove the study hotspot that nitrogen and phosphorus pollution becomes water pollution prevention and control field efficiently.Although adopt the method for physics, chemistry and combination each other all can obtain good phosphor-removing effect, the physical methods such as electrolysis, absorption, often because its cost is compared with high, technical sophistication and cannot large-scale application; Chemistry rule can cause secondary pollution, and the sludge quantity generating is larger, and working cost is higher.Biological denitrification phosphorous removal technique is widely applied to the fields such as surface water, sanitary sewage and trade effluent because of advantages such as its economy, environmental protection.
Denitrifying phosphorus removing bacteria is bacterial strain conventional in biological carbon and phosphorous removal, it is a kind of facultative denitrifier, it can discharge phosphorus in anaerobic process, under anoxia condition, utilize nitrate for electron acceptor(EA), when completing excessive suction phosphorus, nitrate nitrogen is reduced into nitrogen, thereby biological phosphorus and nitrogen removal is organically united two into one.NICHOLLS etc. find that some bacteriums in Rhodopseudomonas, Aeromonas and acinetobacter can complete anti-nitration reaction in the oxygen-starved area of biological denitrification system in dephosphorization bacterial.The researchs such as Zhou Kangqun find that Rhodopseudomonas, paracoccus and enterobacteriaceae have poly-phosphorus effect in various degree at anoxia condition in denitrification phosphorus-collecting integral system.And the poly-phosphorus effect of Rhodopseudomonas is the strongest, utilizes nitrate comparatively thorough, the poly-phosphorus effect of enterobacteriaceae is taken second place, and the poly-phosphorus effect of secondary coccus is the most weak, and in the poly-phosphorus process of enterobacteriaceae and secondary coccus, nitrate utilization is incomplete.It is 10-45 ℃ that the people such as Ma Fang screen its growth temperature of strain bacillus cereus H-hrb01 in " a kind of Denitrifying Phosphate Accumulating Organisms bacillus cereus H-hrb01 and screening method and application " patent, is not suitable for the denitrification dephosphorization in high temperature polluted water.
Therefore, how to filter out temperature tolerance range denitrification bacterial strain wide, alkaline-resisting, that integrate denitrogenation and dephosphorization and become one of gordian technique solving body eutrophication.
Summary of the invention
The object of the invention is to filter out the bacterial strain with efficient denitrification dephosphorization usefulness from nature, for bio-denitrifying sewage dephosphorization process biological reinforced provides efficient bacteria preparation, the pollutents such as nitrogen, phosphorus are carried out to biological restoration processing.
The another one object of invention is that denitrifying phosphorus removing bacteria is combined to use with water ecological substance filtering device, effectively repairs eutrophication water, builds graceful water surrounding.
To achieve these goals, the present invention realizes by the following technical solutions:
A denitrification dephosphorization bacterial strain, selected bacterial strain is amphimicrobe, under anaerobic condition and aerobic condition, all can use.
As further scheme of the present invention, selected bacterial strain is 6.5 ~ 9.5 at pH, and temperature is to grow under 10 ~ 60 ℃ of conditions, and preferred pH is 6.7 ~ 7.3, and temperature is 25 ~ 35 ℃.
As further scheme of the present invention, selected bacterial strain can be recovered in 3d.
A screening method of cultivation for denitrification dephosphorization bacterial strain, comprises the following steps:
(1) sample collecting
The active sludge gathering from the aeration tank of dyeing waste water with aseptic bottle and waste water, the i.e. source of denitrification dephosphorization bacterial classification;
(2) separation of denitrifying phosphorus removing bacteria and purifying
Adopt dilution plate coating method and plate streak to carry out separation and purification, the bacterial classification that separation and purification is gone out is inoculated on slant medium and saves backup; Wherein, separation and purification vessel used and water are all processed through autoclaving;
(3) domestication of denitrifying phosphorus removing bacteria
Isolated bacterial classification is seeded to the waste water of pre-configured high concentration N-P, shaking culture, makes bacterial classification amount reproduction, eliminates the microorganism that is not suitable with nitrogen phosphorus environment simultaneously, progressively improve concentration of nitrogen and phosphorus, to tame out the degradability of nitrogen phosphorus and the strong Black Liquor with Efficient Bacteria of tolerance;
(4) the multiple sieve of denitrifying phosphorus removing bacteria
Prepare each concentration gradient Nitrogen-and Phosphorus-containing plate culture medium, the bacterial strain of taming out is rule on plate culture medium separated, choose the bacterium colony growing on high concentration of nitrogen and phosphorus flat board, be nitrogen phosphorus efficiency degradation bacteria; This high efficient strain is seeded to slant medium and is stored in Biohazard Safety Equipment and store for future use.
As further scheme of the present invention, in step (2), the separation of described denitrifying phosphorus removing bacteria and purifying, specifically comprise the following steps:
1) first water sample is mixed, move into and fill in the triangular flask of sterilized water, vibration mixes, and dilution obtains series concentration diluting soln in proportion;
2) diluting soln is coated respectively on nitrogen phosphorus flat board to 25-35 ℃ of constant temperature culture 22-26 hour;
3) line is separated continuously to the flat board of higher concentration of nitrogen and phosphorus for the rare colony inoculation of choosing nitrogen phosphorus high concentration region in each flat board, until obtain the consistent bacterium colony of colony characteristics.
As further scheme of the present invention, described nitrogen phosphorus plate culture medium comprises: sodium succinate 4.72g/L, NaNO
20.05g/L, KH
2pO
40.09g/L, MgSO
47H
2o 1.00g/L, the pH value of nitrogen phosphorus plate culture medium is 7.2, substratum is 1 * 10
5pa sterilizing 20 min.
As further scheme of the present invention, described slant medium comprises: peptone 10g/L, and extractum carnis 5g/L, NaCl 5g/L, the pH value of slant medium is 7.0, substratum is 1 * 10
5pa sterilizing 20 min.
As further scheme of the present invention, the present invention can also adopt batch feeding technology to ferment when scale operation, to improve the efficiency of fermentation.
The present invention combines water ecological substance filtering device by denitrifying phosphorus removing bacteria and drops in actual sewage, investigates carrier surface forming biofilm property and the clearance to every pollutent of denitrification dephosphorization bacterial classification on water ecological substance filtering device.
Compare with conventional art, the present invention has the following advantages:
The denitrifying phosphorus removing bacteria that the present invention filters out integrates denitrogenation and dephosphorization, has solved the nonsynchronous contradiction of denitrification dephosphorization technique, in the water surrounding lower applicable to dissolved oxygen, C/N is out of proportion or carbon source is not enough.Scale operation at bacterial classification adopts fed-batch fermentation technology, is easy to automatic control, has reduced the probability of microbiological contamination.And it is strong that strain adapts to alkaline-resisting ability, can under highly basic (pH 9.5) condition, bring into play denitrification dephosphorization effect; Growth temperature range wide (10 ~ 60 ℃), optimum temperuture is between 25 ~ 35 ℃, and extreme temperature conditions (10 ℃ or 60 ℃) is little to its activity influence.In addition, the present invention can apply in various eutrophication waters, and recovers in 3d, can reduce efficiently the nitrogen and phosphorus content in water body.
Accompanying drawing explanation
Fig. 1 is that the present invention's extension is in use touched situation one;
Fig. 2 is that the present invention's extension is in use touched situation two;
Fig. 3 is that the present invention's extension is in use touched situation three;
Fig. 4 is that the present invention's extension is in use touched situation four.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
A screening method of cultivation for denitrification dephosphorization bacterial strain, comprises the following steps:
(1) sample collecting
The active sludge gathering from the aeration tank of dyeing waste water with aseptic bottle and waste water, the i.e. source of denitrification dephosphorization bacterial classification;
(2) separation of denitrifying phosphorus removing bacteria and purifying
Adopt dilution plate coating method and plate streak to carry out separation and purification, that is, first water sample is mixed, move into and fill in the triangular flask of sterilized water, vibration mixes, and dilution obtains series concentration diluting soln in proportion; Then diluting soln is coated respectively on nitrogen phosphorus flat board to 30 ℃ of constant temperature culture 24 hours; Line is separated continuously to the flat board of higher concentration of nitrogen and phosphorus for the rare colony inoculation of finally choosing nitrogen phosphorus high concentration region in each flat board, until obtain the consistent bacterium colony of colony characteristics, the bacterial classification that separation and purification is gone out is inoculated on slant medium and saves backup; Wherein, separation and purification vessel used and water are all processed through autoclaving;
(3) domestication of denitrifying phosphorus removing bacteria
Isolated bacterial classification is seeded to the waste water of pre-configured high concentration N-P, shaking culture, makes bacterial classification amount reproduction, eliminates the microorganism that is not suitable with nitrogen phosphorus environment simultaneously, progressively improve concentration of nitrogen and phosphorus, to tame out the degradability of nitrogen phosphorus and the strong Black Liquor with Efficient Bacteria of tolerance;
(4) the multiple sieve of denitrifying phosphorus removing bacteria
Prepare each concentration gradient Nitrogen-and Phosphorus-containing plate culture medium, the bacterial strain of taming out is rule on plate culture medium separated, choose the bacterium colony growing on high concentration of nitrogen and phosphorus flat board, be nitrogen phosphorus efficiency degradation bacteria; This high efficient strain is seeded to slant medium and is stored in Biohazard Safety Equipment and store for future use.
The nitrogen phosphorus plate culture medium configuration of wherein, selecting is as follows:
Sodium succinate 4.72g/L, NaNO
20.05g/L, KH
2pO
40.09g/L, MgSO
47H
2o 1.00g/L, the pH value of nitrogen phosphorus plate culture medium is 7.2, substratum is 1 * 10
5pa sterilizing 20 min.
Selected slant medium configuration is as follows:
Peptone 10g/L, extractum carnis 5g/L, NaCl 5g/L, the pH value of slant medium is 7.0, substratum is 1 * 10
5pa sterilizing 20 min.
Embodiment 2
The use of the denitrifying phosphorus removing bacteria associating water ecological substance filtering device that the present invention's screening is cultivated:
As Figure 1-4, in certain river black smelly section carried out the biofilm of 3 months and to the black smelly section of test of carrying out treatment effect of river.River course experimental project segment length 500m, equal wide 8m, dark 1.5m, water quality is bad V class water, during test, evenly throws in 500m in river course
2water ecological substance filtering device, throw in 10 of denitrification dephosphorization microbial inoculums, after 8 days, complete the biofilm of microorganism, after 3 months, test section water quality obviously promotes, test section repair before and after water quality monitoring (WQM) in Table 1.
table 1 test section is repaired front and back water monitoring data
Consequently show, the present invention is to the COD in sewage
cr, BOD
5, ammonia nitrogen, TP and TN clearance be respectively 70%, 78%, 84%, 74% and 82%.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and in the situation that not deviating from spirit of the present invention or essential characteristic, can realize the present invention with other specific form.Therefore, no matter from which point, all should regard embodiment as exemplary, and be nonrestrictive, scope of the present invention is limited by claims rather than above-mentioned explanation, is therefore intended to include in the present invention dropping on the implication that is equal to important document of claim and all changes in scope.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should make specification sheets as a whole, and the technical scheme in each embodiment also can, through appropriately combined, form other embodiments that it will be appreciated by those skilled in the art that.
Claims (10)
1. a denitrification dephosphorization bacterial strain, is characterized in that, selected bacterial strain is amphimicrobe, under anaerobic condition and aerobic condition, all can use.
2. a kind of denitrification dephosphorization bacterial strain according to claim 1, is characterized in that, selected bacterial strain is 6.5 ~ 9.5 at pH, and temperature is to grow under 10 ~ 60 ℃ of conditions.
3. a kind of denitrification dephosphorization bacterial strain according to claim 1, is characterized in that, selected bacterial strain is 6.7 ~ 7.3 at pH, and temperature is to grow under 25 ~ 35 ℃ of conditions.
4. a kind of denitrification dephosphorization bacterial strain according to claim 1, is characterized in that, selected bacterial strain can be recovered in 3d.
5. a screening method of cultivation for the denitrification dephosphorization bacterial strain as described in claim 1-4 any one, is characterized in that, comprises the following steps:
(1) sample collecting
The active sludge gathering from the aeration tank of dyeing waste water with aseptic bottle and waste water, the i.e. source of denitrification dephosphorization bacterial classification;
(2) separation of denitrifying phosphorus removing bacteria and purifying
Adopt dilution plate coating method and plate streak to carry out separation and purification, the bacterial classification that separation and purification is gone out is inoculated on slant medium and saves backup; Wherein, separation and purification vessel used and water are all processed through autoclaving;
(3) domestication of denitrifying phosphorus removing bacteria
Isolated bacterial classification is seeded to the waste water of pre-configured high concentration N-P, shaking culture, makes bacterial classification amount reproduction, eliminates the microorganism that is not suitable with nitrogen phosphorus environment simultaneously, progressively improve concentration of nitrogen and phosphorus, to tame out the degradability of nitrogen phosphorus and the strong Black Liquor with Efficient Bacteria of tolerance;
(4) the multiple sieve of denitrifying phosphorus removing bacteria
Prepare each concentration gradient Nitrogen-and Phosphorus-containing plate culture medium, the bacterial strain of taming out is rule on plate culture medium separated, choose the bacterium colony growing on high concentration of nitrogen and phosphorus flat board, be nitrogen phosphorus efficiency degradation bacteria; This high efficient strain is seeded to slant medium and is stored in Biohazard Safety Equipment and store for future use.
6. the screening method of cultivation of a kind of denitrification dephosphorization bacterial strain according to claim 5, is characterized in that, in step (2), the separation of described denitrifying phosphorus removing bacteria and purifying, specifically comprise the following steps:
1) first water sample is mixed, move into and fill in the triangular flask of sterilized water, vibration mixes, and dilution obtains series concentration diluting soln in proportion;
2) diluting soln is coated respectively on nitrogen phosphorus flat board to 25-35 ℃ of constant temperature culture 22-26 hour;
3) line is separated continuously to the flat board of higher concentration of nitrogen and phosphorus for the rare colony inoculation of choosing nitrogen phosphorus high concentration region in each flat board, until obtain the consistent bacterium colony of colony characteristics.
7. the screening method of cultivation of a kind of denitrification dephosphorization bacterial strain according to claim 5, is characterized in that, described nitrogen phosphorus plate culture medium comprises: sodium succinate 4.72g/L, NaNO
20.05g/L, KH
2pO
40.09g/L, MgSO
47H
2o 1.00g/L, the pH value of nitrogen phosphorus plate culture medium is 7.2, substratum is 1 * 10
5pa sterilizing 20 min.
8. the screening method of cultivation of a kind of denitrification dephosphorization bacterial strain according to claim 5, is characterized in that, described slant medium comprises: peptone 10g/L, and extractum carnis 5g/L, NaCl 5g/L, the pH value of slant medium is 7.0, substratum is 1 * 10
5pa sterilizing 20 min.
9. the screening method of cultivation of a kind of denitrification dephosphorization bacterial strain according to claim 5, is characterized in that, can also adopt batch feeding technology to ferment when scale operation.
10. an application for the denitrification dephosphorization bacterial strain as described in claim 1-9 any one, is characterized in that, the application by denitrification dephosphorization bacterial strain associating water ecological substance filtering device in eutrophication water.
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| CN106623406A (en) * | 2016-12-23 | 2017-05-10 | 浙江大学 | Waste water and acid soil combined remediation method |
| CN107628684A (en) * | 2017-10-31 | 2018-01-26 | 沈阳建筑大学 | A kind of apparatus and method of improvement MBBR PROCESS FOR TREATMENT low carbon-nitrogen ratio sewages |
| CN108587949A (en) * | 2018-04-13 | 2018-09-28 | 太原科技大学 | One plant of medium temperature aerobic denitrification denitrification and dephosphorization bacterial and its separation method and application |
| CN110982732A (en) * | 2019-11-13 | 2020-04-10 | 重庆理工大学 | Salt-tolerant high-ammonia-nitrogen-resistant heterotrophic nitrification-aerobic denitrification composite microbial agent and preparation and application thereof |
| CN114940957A (en) * | 2022-05-11 | 2022-08-26 | 江苏科技大学 | Paracoccus pantotrophus with facultative denitrification synchronous nitrogen and phosphorus removal performance |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106623406A (en) * | 2016-12-23 | 2017-05-10 | 浙江大学 | Waste water and acid soil combined remediation method |
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| CN107628684A (en) * | 2017-10-31 | 2018-01-26 | 沈阳建筑大学 | A kind of apparatus and method of improvement MBBR PROCESS FOR TREATMENT low carbon-nitrogen ratio sewages |
| CN108587949A (en) * | 2018-04-13 | 2018-09-28 | 太原科技大学 | One plant of medium temperature aerobic denitrification denitrification and dephosphorization bacterial and its separation method and application |
| CN108587949B (en) * | 2018-04-13 | 2020-06-16 | 太原科技大学 | Medium-temperature aerobic denitrification phosphorus removal bacterium and separation method and application thereof |
| CN110982732A (en) * | 2019-11-13 | 2020-04-10 | 重庆理工大学 | Salt-tolerant high-ammonia-nitrogen-resistant heterotrophic nitrification-aerobic denitrification composite microbial agent and preparation and application thereof |
| CN114940957A (en) * | 2022-05-11 | 2022-08-26 | 江苏科技大学 | Paracoccus pantotrophus with facultative denitrification synchronous nitrogen and phosphorus removal performance |
| CN114940957B (en) * | 2022-05-11 | 2024-01-30 | 江苏科技大学 | Paracoccus ubitus with facultative denitrification synchronous denitrification and dephosphorization performances |
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Application publication date: 20140806 |