CN105925508A - Aerobic denitrifying pseudomonas and application thereof - Google Patents
Aerobic denitrifying pseudomonas and application thereof Download PDFInfo
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Abstract
The invention discloses Pseudomonas (sp.) HC-1. The (sp.) HC-1is preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 11924, and the preservation date of December 24, 2015. The Pseudomonas (sp.) HC-1 has high aerobic denitrifying property, and is applied to sewage biological nitrogen removal process, the total nitrogen removal rate reaches 88.2 percent, and the characteristics have great significance in the field of water treatment.
Description
Technical field
The invention belongs to bioengineering, field of environment engineering technology, relate to a kind of aerobic denitrifying bacteria and application thereof, simultaneously
The denitrification efficiency of this bacterial strain is provided.
Background technology
Currently, along with national economy develop rapidly with urbanization process deepen continuously, urban life and the row of industrial wastewater
Putting total amount also increasing year by year, agricultural chemicals, chemical fertilizer and synthetic detergent etc. are widely used so that the nutrient concentrations in water body
Constantly raising, wherein nitrogen is the one of the main reasons causing body eutrophication.Conventional biochemical processing process can be effectively
Reduce BOD and SS in sewage, but when the nutrients such as simultaneous N, P in sewage when, sewage can only be removed
The N element of middle 30-40%, a large amount of nitric wastewaters will be directly discharged into environment water.
Traditional biological denitrificaion includes aerobic nitrification and two processes of anoxic denitrification: first under aerobic condition, nitrite
Being nitrite by ammonium oxidation, then cultured water is further oxidized to nitrate nitrogen by Nitromonas；The most under anoxic conditions,
Nitrogen as nitrate or nitrite is reduced into the GN 2 or N by denitrifying bacterium2O.Aerobic denitrification has the advantage that (1) exists
Denitrification is carried out so that synchronous nitration and denitrification (SND) is possibly realized under aerobic conditions；(2) product of nitration reaction can be direct
Become the substrate of anti-nitration reaction, it is to avoid the accumulation of incubation nitrite and the nitrate suppression to nitration reaction, add
Speed nitrification and denitrification process；Meanwhile, denitrification can compensate the basicity that nitration reaction consumes, and maintains reaction system pH
Value stabilization, reduces operation easier and operating cost；(3) major part aerobic denitrifying bacteria adaptability is relatively strong, fast growth,
Yield is high and requires relatively low to dissolved oxygen concentration, and denitrification speed is fast and thorough, is suitable for administering large area polluted by nitrogen waters.
Therefore, aerobic denitrification technology enjoys researcher to pay close attention to as a kind of brand-new denitrogenation technology.
But, up to the present, aerobic denitrifying bacteria be concentrated mainly on Pseudomonas Pseudomonas, Bacillus Pseudomonas and
Aeromonas Pseudomonas, bacterial classification is the most single, and research focuses mostly in terms of degrading genes and mechanism of degradation, actual answers effect
The best.
Summary of the invention
The first object of the present invention is the pseudomonad providing a kind of aerobic denitrification effective.
The second object of the present invention is the application providing this pseudomonad in water processes.
The third object of the present invention is the application providing this pseudomonad in aerobic denitrification water processes.
In order to solve above-mentioned technical problem, the technical solution adopted in the present invention is: a kind of pseudomonad, its Classification And Nomenclature is
Pseudomonad (Pseudomonas sp.) HC-1, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution address 3, Institute of Microorganism, Academia Sinica, postal
Political affairs are encoded to 100101, and its deposit number is CGMCC No.11924, and preservation date is on December 24th, 2015.
The Physiology and biochemistry of pseudomonad (Pseudomonas sp.) HC-1 identifies reference " the outstanding Bacteria Identification handbook of uncle " the 8th edition
Operation, physiologically active feature is as follows: pseudomonad (Pseudomonas sp.) HC-1 is non-fermented Gram-negative bacteria, specially
Property aerobic bacteria, growth temperature range 25-42 DEG C, optimum growth temperature is 25-30 DEG C, particularly this bacterium do not grow at 4 DEG C and
Can grow at 42 DEG C；Thalline a length of 1.5-3.0 μm, width is 0.5-0.8 μm, and Dan becomes short chain in pairs or once in a while,
Thalline has 1-3 root flagellum, without gemma；This bacterial strain need not organic growth factor；Nutrition variation: the growth of single bacterial strain
Available 76-82 kind or more different organic compound.
Gram's staining shows as feminine gender: bacterial strain carries out Gram's staining, oxidizing ferment, catalase, glucose oxidative fermentation,
Producing indoles, V.P. measures, and M.R. measures, gelatin liquefaction, Starch Hydrolysis, and citrate utilizes, and nitrate reduction etc. is main
Physiological and biochemical index is tested, and result is as shown in table 1.
The physiological and biochemical index of table 1 pseudomonad (Pseudomonas sp.) HC-1
Described pseudomonad (Pseudomonas sp.) HC-1 is the application in water processes.
Described pseudomonad (Pseudomonas sp.) HC-1 is the application in nitrogenous effluent treatment.
Concrete application process, comprises the following steps: by the pseudomonad (Pseudomonas sp.) described in claim 1
HC-1 colony inoculation, in culture medium, cultivates 12-48h at 25 DEG C-42 DEG C, and the zymotic fluid obtained is as application microbial inoculum.
Preferably, described aerobic denitrification culture medium prescription is: sodium succinate 4.7g/L, disodium hydrogen phosphate 7.9g/L, phosphorus
Acid dihydride potassium 1.5g/L, ammonium chloride 0.3g/L, magnesium sulfate 0.1g/L, potassium nitrate 1.5g/L, trace element 1ml, remaining
Amount is water, pH=7.2～7.5；Described trace element formula is: EDTA 50g/L, ferrous sulfate 5.0g/L, zinc sulfate 2.2
G/L, ammonium molybdate 1.1g/L, calcium chloride 5.5g/L, copper sulphate 1.57g/L, manganese chloride 5.06g/L, cobalt chloride 1.61g/L,
Surplus is water.
Beneficial effect:
Pseudomonad provided by the present invention (Pseudomonas sp.) HC-1 has higher aerobic denitrification capability, is used for
Bio-denitrifying sewage technique nitrogen removal rate is up to 88.2%, and these features are significant in water treatment field.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of pseudomonad (Pseudomonas sp.) HC-1；
Fig. 2 is pseudomonad (Pseudomonas sp.) HC-1 removal usefulness figure to total nitrogen.
Detailed description of the invention
The application of pseudomonad of the present invention (Pseudomonas sp.) HC-1 is elaborated below in conjunction with experimental example.
The aerobic denitrification culture medium prescription that the present invention uses is: sodium succinate 4.7g/L, disodium hydrogen phosphate 7.9g/L, phosphorus
Acid dihydride potassium 1.5g/L, ammonium chloride 0.3g/L, magnesium sulfate 0.1g/L, potassium nitrate 1.5g/L, trace element 1ml, remaining
Amount is water, pH=7.2～7.5；Described trace element formula is: EDTA 50g/L, ferrous sulfate 5.0g/L, zinc sulfate 2.2
G/L, ammonium molybdate 1.1g/L, calcium chloride 5.5g/L, copper sulphate 1.57g/L, manganese chloride 5.06g/L, cobalt chloride 1.61g/L,
Surplus is water.
Described nitrate reduction culture medium prescription is: beef extract 3g/L, peptone 5g/L, KNO31g/L、pH 7.4；Warp
121 DEG C of high pressure steam sterilization 20min.
Embodiment 1
Pseudomonad, its Classification And Nomenclature is pseudomonad (Pseudomonas sp.) HC-1, is deposited in Chinese microorganism strain
Preservation administration committee common micro-organisms center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution address 3, in
Institute of microbiology of the academy of sciences of state, postcode is 100101, and its deposit number is CGMCC No.11924, preservation
Date is on December 24th, 2015.
Described pseudomonad (Pseudomonas sp.) HC-1 obtains in the following manner:
A. the activated sludge at anoxic section end of sbr reactor device stable operation two weeks is taken as separation mud；
B. dilution mixture flat band method is used to separate: the bacterial strain separating oneself is placed in denitrification under aerobic condition and cultivates
Carrying out denitrification test in base, auxiliary carries out nitrate reduction aerogenesis test and Babes-Ernst bodies dyeing (methylene blue staining method) simultaneously
Inspection.It is positive and aerogenesis for nitrate reductase, and the bacterium that can reduce TN concentration under aerobic condition is the present invention
The aerobic denitrifying bacteria obtained.
Concrete separating step is: take 10mL activated sludge to equipped with 90mL sterilized water from sbr reactor device anoxic section end
In triangular flask, add sterile glass beads；Being put in shaking table by triangular flask and fully vibrate, making bacterium is that unicellular is scattered in
In water.Then, the sludge bacteria suspension in triangular flask take in the test tube that lmL suspension accesses equipped with 9mL sterilized water,
To 10-1The bacteria suspension of gradient, continues this step, obtains 10 successively-2、10-3、10-4……10-7The bacteria suspension of gradient, respectively takes
LmL water sample puts into the culture dish kind equipped with the aerobic denitrification culture medium containing agar；Culture dish is poured into when culture medium coagulates but soon
In, mixing, after after culture medium solidifying, inversion culture dish is cultivated 2～3 days in 30 DEG C of constant incubators, choose respectively not
Same form, bacterium colony colonies typical clearly, the mark also single bacterium colony of picking carries out three ride separation on plating medium,
Repeat 3～4 times to the consistent single bacterium colony of colony characteristics, the single bacterium colony of last picking, be transferred on ready test tube slant
With standby；Above-mentioned all operations is the most aseptically carried out.
C. the bacterial strain obtained screening carries out Molecular Identification, sequentially includes the following steps:
Extract bacterial strain DNA, through PCR expand after, then utilize glue reclaim kit (reclaim purified pcr product,
Carrying out afterwards cloning, converting, screening positive clone daughter colony, check order after expanding and cultivating, sequencing result position records length
Degree is the sequence of 1450bp, and as shown in sequence table Seq ID No:1, its sequence is committed to GenBank, to determine bacterial strain
Race relation, be ultimately determined to pseudomonas (Pseudomonas sp.).By combining morphological features, growth
Condition, Physiology and biochemistry qualification result its be pseudomonad (Pseudomonas sp.) HC-1.
Concretely comprising the following steps of described PCR amplification procedure:
1) PCR system sets up (25 μ L):
2) PCR program setting:
95 DEG C of 3min of denaturation
95 DEG C of denaturations 5min, 94 DEG C of sex change lmin, 58 DEG C of renaturation 30s, 72 DEG C extend 3min, totally 30 circulations,
Last 72 DEG C extend 10min；
3) primer sequence:
Primer 15 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '
Primer 25 '-AGCGGATAACAATTTCACACAGGA-3 '.
D. the bacterial strain obtained screening carries out Physiology and biochemistry qualification, sequentially includes the following steps:
The Physiology and biochemistry of bacterial strain identifies that qualification result is: pseudomonad with reference to " the outstanding Bacteria Identification handbook of uncle " the 8th edition operation
(Pseudomonas sp.) HC-1 is non-fermented Gram-negative bacteria, obligate aerobes, growth temperature range 25-42 DEG C,
Optimum growth temperature is 25-30 DEG C, and particularly this bacterium does not grows at 4 DEG C and can grow at 42 DEG C；Thalline is a length of
1.5-3.0 μm, width is 0.5-0.8 μm, Dan, becomes short chain, thalline to have 1-3 root flagellum, without gemma in pairs or once in a while；
This bacterial strain need not organic growth factor；Nutrition variation: the growth of single bacterial strain may utilize 76-82 kind or more different
Organic compound.
Gram's staining shows as feminine gender: bacterial strain carries out Gram's staining, oxidizing ferment, catalase, glucose oxidative fermentation,
Producing indoles, V.P. measures, and M.R. measures, gelatin liquefaction, Starch Hydrolysis, and citrate utilizes, and nitrate reduction etc. is main
Physiological and biochemical index is tested, and result is as shown in table 1.
The physiological and biochemical index of table 1 pseudomonad (Pseudomonas sp.) HC-1
E. denitrification test
Pseudomonad (Pseudomonas sp.) HC-1 separating oneself enters under aerobic condition in denitrification culture medium
Row denitrification test, auxiliary carries out nitrate reduction aerogenesis test and Babes-Ernst bodies dyeing (methylene blue staining method) inspection simultaneously；
Be positive and aerogenesis for nitrate reductase, and the bacterium of TN concentration can be reduced under aerobic condition, it was demonstrated that its be aerobic instead
Nitrifier.
Concrete test is as follows: by pseudomonad (Pseudomonas sp.), HC-1 activates according to a conventional method, enrichment culture.
After enrichment culture completes, pipette 10mL suspension to 30 DEG C of 160rpm/min in 100mL aerobic denitrification culture medium
Under the conditions of cultivate 24h；After cultivation completes, 8000rpm is centrifuged 10min, takes supernatant and measures ammonia nitrogen, nitrate nitrogen, nitrite nitrogen
And the content of TN, the results are shown in Table 2.
Table 2 assay result
Nitrate reduction aerogenesis is tested: takes pseudomonad (Pseudomonas sp.) HC-1 and trains on solid agar slants
Bacterial classification after Yanging, is seeded in the nitrate reduction culture medium of band Du Shi tubule, and each bacterial strain makees 2 parallel tests, simultaneously
Additionally stay 2 pipes not inoculate to compare.
30 DEG C are incubated, respectively 1 day, 3 days, testing result after 5 days: check in Du Shi tubule whether aerogenesis, as
Bubble is had to show have nitrogen to produce；Adding a small amount of nutrient solution in colorimetric porcelain dish, it is (right that instillation 1-2 drips Griess reagent A liquid
Aminobenzenesulfonic acid 0.5g；About 10% spirit of vinegar 150ml) and B liquid (α-aniline 0.1g；About 10% spirit of vinegar 150ml；
H2O 20ml)；In control tube, same addition A liquid and each 1-2 of B liquid drip.If solution becomes red, orange or brown etc.,
Indicate that nitrite exists, positive for nitrate reduction；As redfree occurs that then can add 1-2 drips diphenylamines reagent (0.5g
Diphenylamines is dissolved in the dense H of 100ml2SO4, and add 20ml distilled water diluting, and it is stored in brown bottle), if do not reacted in blueness
The most still for positive reaction, if reacting in blueness, it is negative reaction.
Embodiment 2 pseudomonad (Pseudomonas sp.) HC-1 denitrification efficiency measures
Pseudomonad of the present invention (Pseudomonas sp.) HC-1 can be used for saprobia take off with gas chromatography as carbon source
Nitrogen.Pseudomonad (Pseudomonas sp.) HC-1 biological denitrificaion ability is detected, pseudomonad of the present invention
(Pseudomonas sp.) HC-1 bacterial strain carries out following functional verification:
1) pseudomonad (Pseudomonas sp.) HC-1 growth curve measures.
According to the standard method measuring growth curve of bacteria, the growth curve of pseudomonad (Pseudomonas sp.) HC-1 is entered
Row measures, and takes nutrient solution every 2h, uses photoelectric turbidimetry, measures the OD of bacterium solution at 660nm wavelength660(Optical
Density), then through 0.22pm filtering with microporous membrane, detect filtrate TN, the index such as pH value.Obtain pseudomonad
(Pseudomonas sp.) HC-1 growth curve is as shown in Figure 1.
From figure 1 it appears that the growth curve ratio of pseudomonad (Pseudomonas sp.) HC-1 is more typically, incubation period
Shorter, for about 2h, this activity being possibly due to pseudomonad (Pseudomonas sp.) HC-1 is relatively strong, and inoculation
Condition of culture front and back is similar, and after access, within a short period of time is i.e. suitable for new environment.The exponential phase of bacterial strain is about
Stationary phase and decline phase is initially entered after 4-10h, 12h.
2) pseudomonad (Pseudomonas sp.) HC-1 denitrification efficiency measures
Pseudomonad (Pseudomonas sp.) HC-1 is conventionally activated, enrichment culture.Enrichment culture completes
After pipette 10ml suspension to containing 100ml without agar aerobic denitrification culture medium 250ml conical flask in,
24h is cultivated under the conditions of 30 DEG C of 160rpm/min.Taking nutrient solution every 2h, 8000rpm is centrifuged 10min, takes supernatant and surveys
Determining the content of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and TN, result is as shown in Figure 2.
Figure it is seen that bacterial strain HC-1 has good aerobic denitrification effect.
In general conventional denitrification theory is thought, owing to dissolved oxygen can compete electron acceptor, dissolved oxygen (DO) with nitrate nitrogen
Existence denitrification process can be played inhibitory action, thus suppress denitrifying and carry out.But, aerobic denitrification theory
Proposing to have changed traditional concept, its theory thinks that therefore bacterial strain may utilize nitre owing to there is pericentral siphon nitrate reductase in thalline
State nitrogen and oxygen carry out co-respiration simultaneously as electron acceptor.In sum, pseudomonad (Pseudomonas sp.) HC-1
For efficient aerobic denitrifying bacteria.
Present invention is not limited only to the content of the respective embodiments described above, the combination of one of them or several detailed description of the invention with
Sample can also realize the purpose of invention.
Embodiment 3
Picking pseudomonad (Pseudomonas sp.) HC-1 bacterium colony, colony inoculation is in aerobic denitrification culture medium, at 25 DEG C
Lower cultivation 24h, the zymotic fluid obtained is as application microbial inoculum.This application microbial inoculum is applied to nitrogenous effluent treatment, and 24h TN goes
Except rate: 87.2%.
Embodiment 4
Picking pseudomonad (Pseudomonas sp.) HC-1 bacterium colony, colony inoculation is in aerobic denitrification culture medium, at 42 DEG C
Lower cultivation 48h, the zymotic fluid obtained is as application microbial inoculum.This application microbial inoculum is applied to nitrogenous effluent treatment, and 24h TN goes
Except rate: 88.5%.
Embodiment 5
Picking pseudomonad (Pseudomonas sp.) HC-1 bacterium colony, colony inoculation is in aerobic denitrification culture medium, at 30 DEG C
Lower cultivation 12h, the zymotic fluid obtained is as application microbial inoculum.This application microbial inoculum is applied to nitrogenous effluent treatment, and 24h TN goes
Except rate: 88.0%.
Claims (5)
1. pseudomonad (Pseudomonas sp.) HC-1, is deposited in China Committee for Culture Collection of Microorganisms the most micro-
Bio-Centers, its deposit number is CGMCC No.11924, and preservation date is on December 24th, 2015.
2. the application in water processes of the pseudomonad described in claim 1.
3. the application in nitrogenous effluent treatment of the pseudomonad described in claim 1.
Apply the most as claimed in claim 3, it is characterised in that: comprise the following steps: by the false list described in claim 1
Born of the same parents bacterium (Pseudomonas sp.) HC-1 colony inoculation, in aerobic denitrification culture medium, cultivates 12-48h at 25 DEG C-42 DEG C,
The zymotic fluid obtained is as application microbial inoculum.
Apply the most as claimed in claim 3, it is characterised in that: described aerobic denitrification culture medium prescription is: sodium succinate
4.7g/L, disodium hydrogen phosphate 7.9g/L, potassium dihydrogen phosphate 1.5g/L, ammonium chloride 0.3g/L, magnesium sulfate 0.1g/L, nitric acid
Potassium 1.5g/L, trace element 1ml, surplus is water, pH=7.2～7.5；Described trace element formula is: EDTA 50g/L,
Ferrous sulfate 5.0g/L, zinc sulfate 2.2g/L, ammonium molybdate 1.1g/L, calcium chloride 5.5g/L, copper sulphate 1.57g/L, chlorine
Changing manganese 5.06g/L, cobalt chloride 1.61g/L, surplus is water.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951658A (en) * | 2020-01-15 | 2020-04-03 | 广州市微生物研究所 | Pseudomonas and application thereof |
| CN112625942A (en) * | 2020-12-01 | 2021-04-09 | 华南理工大学 | Aerobic denitrifying bacterium and application thereof |
| CN113801824A (en) * | 2021-10-14 | 2021-12-17 | 中交和美环境生态建设有限公司 | Pseudomonas Y1 with efficient heterotrophic nitrification and aerobic denitrification function and embedded pellet and application thereof |
| CN114107136A (en) * | 2021-12-06 | 2022-03-01 | 华北电力大学 | Pseudomonas stutzeri with aerobic denitrification and greenhouse gas emission reduction functions |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039225A (en) * | 2015-08-25 | 2015-11-11 | 哈尔滨工业大学 | Aerobic denitrifying bacterium strain and application thereof |
| CN105420165A (en) * | 2015-12-31 | 2016-03-23 | 云南大学 | Aerobic denitrifying bacteria and applications therefor |
-
2016
- 2016-06-07 CN CN201610396898.8A patent/CN105925508A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039225A (en) * | 2015-08-25 | 2015-11-11 | 哈尔滨工业大学 | Aerobic denitrifying bacterium strain and application thereof |
| CN105420165A (en) * | 2015-12-31 | 2016-03-23 | 云南大学 | Aerobic denitrifying bacteria and applications therefor |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951658A (en) * | 2020-01-15 | 2020-04-03 | 广州市微生物研究所 | Pseudomonas and application thereof |
| CN112625942A (en) * | 2020-12-01 | 2021-04-09 | 华南理工大学 | Aerobic denitrifying bacterium and application thereof |
| CN113801824A (en) * | 2021-10-14 | 2021-12-17 | 中交和美环境生态建设有限公司 | Pseudomonas Y1 with efficient heterotrophic nitrification and aerobic denitrification function and embedded pellet and application thereof |
| CN114107136A (en) * | 2021-12-06 | 2022-03-01 | 华北电力大学 | Pseudomonas stutzeri with aerobic denitrification and greenhouse gas emission reduction functions |
| CN114107136B (en) * | 2021-12-06 | 2023-10-13 | 华北电力大学 | Pseudomonas stutzeri with aerobic denitrification and greenhouse gas emission reduction functions |
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