CN102899263B - Facultative anaerobic denitrifying bacteria having complete denitrification enzyme systems and use thereof - Google Patents
Facultative anaerobic denitrifying bacteria having complete denitrification enzyme systems and use thereof Download PDFInfo
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- CN102899263B CN102899263B CN201210201671.5A CN201210201671A CN102899263B CN 102899263 B CN102899263 B CN 102899263B CN 201210201671 A CN201210201671 A CN 201210201671A CN 102899263 B CN102899263 B CN 102899263B
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Abstract
The invention discloses facultative anaerobic denitrifying bacteria having complete denitrification enzyme systems and a use thereof. The facultative anaerobic denitrifying bacteria are named as Pseudomonas sp. XP-2, belong to pseudomonas and have a preservation number of CCTCC M 2011431. The optimal denitrification temperature of the facultative anaerobic denitrifying bacteria is in a range of 10 to 30 DEG C. In a pH value of 6-9, the facultative anaerobic denitrifying bacteria have a high-efficiency denitrification activity. When a ratio of C/N (mol/mol) is more than 3, a denitrification rate of the facultative anaerobic denitrifying bacteria is more than 90%. In an oxygen micro-dissolution environment, the facultative anaerobic denitrifying bacteria have the highest denitrification activity. Denitrification end product gas is nitrogen and thus it is proved that the facultative anaerobic denitrifying bacteria have a complete denitrification capability, and can directly reduce inorganic nitrogen substances such as nitrates and nitrites in water into harmless nitrogen, wherein the harmless nitrogen is discharged from the water. The facultative anaerobic denitrifying bacteria have a wide carbon source spectrum and can utilize a plurality of carbon sources. A performance test of the facultative anaerobic denitrifying bacteria proves that a nitrogen-containing waste water treatment technology adopting the facultative anaerobic denitrifying bacteria has simple processes and good and stable treatment effects, saves an operation cost, does not produce greenhouse gas such as No and N2O, and does not cause air pollution.
Description
Technical field
The present invention relates to a strain and there is amphimicrobian denitrifying bacterium of complete denitrification enzyme system and uses thereof, belong to field of environment microorganism.
Background technology
Along with the development of industrial and agricultural production and the raising of people's living standard, the quantity discharged of China's nitrogen-containing pollutant increases sharply, and large small size pond becomes in " collecting post " of these pollutents, and Water quality sharply worsens.At present, all parts of the country have established large quantities of biological wastewater treatment engineering, have effectively contained the impetus that water pollution is continuous worsening.But the discharge water nitrogen content after sewage second-level process is still higher, become a serious environomental pollution source.After secondary biochemical treatment, in water outlet, ammonia nitrogen and nitric nitrogen are the main existence forms of nitrogen.Nitrate pollution can cause serious harm to the productive life of water surrounding and people, is mainly manifested in: (1) accelerates the eutrophication of water body; (2) difficulty of Water purification is increased; (3) dissolved oxygen in water body is consumed; (4) to the toxic effect of hydrobiont.Therefore, the improvement to nitrogen in sewage must be strengthened.
For the improvement of nitrate pollution, biological denitrificaion is most economical effective Treatment process.Bio-denitrification technology is made up of nitration processes and denitrification process usually.Nitrogen, to remove ammonia nitrogen, although can be nitrate mineralized nitrogen, can not thoroughly remove by nitration processes from water body; Denitrification process then fundamentally can eliminate the pollution of water body nitrogen to environment.Denitrification is denitrifying bacterium with nitric nitrogen or nitrite nitrogen for terminal electron acceptor, by electron transmission under the effect of denitrification enzyme system, nitric nitrogen or nitrite nitrogen is progressively reduced to the process of nitrogen.From NO
3 -revert to N
2denitrification reaction usually by following four steps
Composition: NO
3 -→ NO
2 -→ NO → N
2o → N
2
General when anoxic, the first-selected nitrate of amphimicrobian denitrifying bacteria carries out its respiration, by NO
3 --N is reduced to N
2, biochemical reaction formula is:
NO
3 -+6H
++5e
-＝1/2N
2(g)+3H
2O
Denitrifying bacterium is the main body that denitrification occurs, and verifies kind and the characteristic of denitrifying bacterium, gets their nutritional condition and envrionment conditions clear, will contribute to research, the development and application of wastewater biological denitrificaion technique.
The denitrification of bacterium completes under the katalysis of various reductase enzyme, and the active height of each reaction enzymes system is subject to the impact of the conditions such as temperature, pH value, dissolved oxygen, C/N ratio.Theoretically, denitrification requires that C/N is 4.6 (mass ratioes), but also can consume dissolved organic carbon due to the growth and breeding of cell, under practical situation, requires C/N>4.6.Therefore, in most cases, dissolved organic carbon becomes the limiting factor of denitrification in water surrounding.PH value is also affect the important environmental factor of denitrification rate and denitrification end product one.Concerning the growth of denitrifying bacterium, optimal ph is 6.5 ~ 7.5.Within the scope of this pH, denitrification rate is maximum, and when pH value is not at optimum range, denitrification rate reduces.Temperature of reaction in biological wastewater treatment, to microbial growth, breed in close relations, temperature dominates the solubleness etc. of enzymatic reaction kinetics, microorganism growth speed and compound, thus play keying action to the Degradation and Transformation of pollutent, the existence of dissolved oxygen (DO) has a significant impact denitrification process.If the dissolved oxygen of reactor is too much, will to the dissimilation generation restraining effect of denitrifying bacteria.Therefore the factors such as temperature, pH, dissolved oxygen, C/N ratio play efficient denitrification activity impact on denitrification bacterial strain is investigated, and then obtain efficient denitrification bacterial strain, realize the economic denitrogenation of waste water high-efficiency, to solving, day by day serious water surrounding nitrate pollution problem is significant.
It is the enzyme of nitrite that many bacteriums have nitrate reduction, can realize the first step and transform, but will complete thorough denitrogenation, then require that bacterium must have complete denitrification enzyme system.Verify denitrifying bacterium and whether there is the prerequisite that complete efficient denitrification enzyme system is again the denitrogenation of employing biological denitrification technique.Research finds that bacterium of the present invention has complete denitrification enzyme system, efficiently can remove the nitrogen in water body, be that nitrogen excretes, realize waste water high-efficiency denitrogenation by nitrate-nitrogen reduction, makes contributions for solving the day by day serious pollution problem of nitrogenous effluent to environment.
Summary of the invention
For above problem, the object of this invention is to provide the efficient amphimicrobian denitrifying bacterium that a strain has complete denitrification enzyme system.This bacterium has denitrification enzyme system completely, thoroughly can remove the nitrogen in water body, not produce NO, N
2the obnoxious flavoures such as O.
Another object of the present invention is to provide a kind of culture condition, enables this bacterium show stronger heterotrophic denitrification active.
Another object of the present invention is to provide a kind of culture condition, makes this bacterium show denitrification activity completely.
The present invention can be achieved through the following technical solutions:
Bacterial classification in the present invention is a facultative anaerobic denitrifying bacterium, this Strain Designation is Pseudomonas stutzeri XP-2, belong to Pseudomonas stutzeri, be preserved in China typical culture collection center on November 27th, 2011, its deposit number is: CCTCCM2011431.Its mycology is characterized as: cell is rod-short, Gram-negative bacteria, and bacterium colony is circular, light yellow, concave, glossy, neat in edge.This bacterial strain can utilize several kinds of carbon source, wherein with sodium acetate for the suitableeest carbon source.Optimal temperature carries out denitrogenation under the micro-oxygen conditions of 10 ~ 30 DEG C, and best C/N (mol/mol) is 3, and the pH value range be applicable to is 6.0 ~ 9.0.Denitrogenation with this understanding, without nitrite accumulation.
This bacterial strain can nitrogen element in effective elimination water body, and be applicable to the process of higher concentration nitrite or nitrate waste water, this bacterial strain has adaptability widely.Its best denitrogenation condition is: temperature 10 ~ 30 DEG C, pH6.0 ~ 9.0, and best C/N is 3, and dissolved oxygen is slightly soluble oxygen condition (< 0.5mg/L).
During embody rule, using method is:
(1) with LB substratum activated strains before using: by the Pseudomonas stutzeri XP-2 transfering loop scraping 1 ring lawn that slant medium is preserved, inoculate in the 250mL Erlenmeyer flask that 100mL LB substratum is housed, 12h is cultivated in 30 DEG C of shaking table concussions, can obtain bacterium liquid.
(2) the bacterium liquid after activation is inoculated in pending nitrogenous effluent, dosage is be subject to processing wastewater volume 10%, pH value range is between 7.0 ~ 9.0, and room temperature quiescent culture, timing sampling detects nitre nitrogen in water sample and removes the accumulation of situation and nitrite.
(3) detection method
CODcr: potassium dichromate process;
TN: peroxide potassium sulfate-ultraviolet spectrophotometry;
NO
3 --N: ultraviolet spectrophotometry;
NO
2 --N:N-(1-naphthyl)-quadrol light-intensity method.
OD value: adopt UV-2450 ultraviolet spectrophotometer (Shimadzu), measure the OD value of nutrient solution under wavelength 600nm.
Bacterial strain of the present invention is from Nansi Lake Cattail Plant rhizosphere soil, screen a kind of denitrifying bacterium with efficient denitrification activity obtained, and can remove nitrite nitrogen in water body and nitrate nitrogen by denitrification.This bacterial strain has denitrifying capacity completely, nitric nitrogen progressively can be reduced to nitrogen and discharge water body, be applicable to the process of higher concentration nitrite or nitrate waste water, this bacterial strain has carbon source spectrum widely, can several kinds of carbon source be utilized, there is suitability widely.Use this bacterial strain process waster water process simple, denitrogenation is thorough, effect stability, saves running cost.
Denitrogenation in the present invention refers to the removal of Inorganic Nitrogen in Water Bodies, and inorganic nitrogen refers to NH
4 +, NO
3 --N and NO
2 --N.
C/N in the present invention is than referring to the ratio of carbon with the amount of substance of nitrogen element.
Slightly soluble oxygen condition in the present invention refer to waste water leave standstill and under the condition of ingress of air, dissolved oxygen concentration is at below 0.5mg/L.
Bacterial strain of the present invention has the following advantages:
(1) bacterial strain of the present invention has denitrification enzyme system completely, can be nitrogen by nitric nitrogen direct-reduction, realizes thorough denitrogenation and not polluted air.
(2) bacterial strain of the present invention has very high denitrification activity, can be used for process higher concentration nitrite or nitrate waste water.
(3) bacterial strain of the present invention has carbon source spectrum widely, can utilize several kinds of carbon source, have good adaptability.
Accompanying drawing explanation
Strain Designation of the present invention is Pseudomonas stutzeri XP-2, belongs to Pseudomonas stutzeri, and be preserved in China typical culture collection center on November 27th, 2011, its deposit number is: CCTCCM2011431.
The stereoscan photograph of Fig. 1: pseudomonas XP-2 thalline.
Fig. 2: the growth curve of pseudomonas XP-2 and nitrogen removal rate.
Embodiment
The isolation identification of embodiment 1 bacterial strain of the present invention:
(1) substratum:
A, strains separation, purifying, storage medium (/L):
CH
3cOONa, 2g; Peptone, 15g; Yeast extract paste, 3g; Glucose, 1g; NaCl, 6g; Agar, 12g; KNO
3, 1.5g; PH controls 7.0 ~ 7.2.
B, bacterial strain screening, denitrogenation substratum (DM:Denitrifying Medium) (/L):
CH
3cOONa, 2g; KH
2pO
4, 0.4g; MgSO
47H
2o, 0.6g; CaCl
22H
2o, 0.07g; KNO
3, 1g; Tris damping fluid 12mL; Trace element 2mL; PH controls 7.0 ~ 7.2.
C, LB substratum (/L):
Peptone, 10g; Yeast extract paste, 5g; Sodium-chlor, 10g; PH controls 7.5.
(2) separation of pseudomonas XP-2 bacterial strain, purifying:
Take 10g Cattail Plant rhizosphere soil sample and be placed in 250mL triangular flask, add 100mL sterilized water and several granulated glass spherees, shaking table vibration 15min makes soil sample be uniformly dispersed.After soil sample dispersion, after leaving standstill 5min, draw 1mL soil supension to 9mL diluent (sterilized water), obtain 10
-2extent of dilution suspension, is diluted to 10 by 10 times of dilution methods successively
-7, obtained each extent of dilution soil supension thus.
Draw each extent of dilution suspension 0.1mL respectively (to have placed on denitrifying bacterium separation solid medium and spent the night, without varied bacteria growing), with glass slicker by even for soil supension coating.Afterwards flat board is inverted, puts into 30 DEG C of constant incubators, be cultured to and grow obvious bacterium colony.
Picking single strain, pseudomonas XP-2 flat board repeatedly rule in purifying the present invention, till basis of microscopic observation display is without miscellaneous bacteria, purifying is complete now can to think bacterial strain.Isolated bacterial strain is good at the cultured on solid medium containing nitrate, has potential Denitrification Characteristics, is seeded to slant medium and saves backup.
(3) the nitrate reduction aerogenesis test of pseudomonas XP-2
For understanding denitrifying capacity and the characteristic of bacterial strain of the present invention, the test of nitrate reduction aerogenesis is carried out to bacterial strain of the present invention.
Inoculation: the denitrification liquid nutrient medium adding 10mL sterilizing in small test tube (specification: 15mm × 100mm), bacterium colony 1 ring after purifying with transfering loop picking in small test tube, stirring and evenly mixing.Add the sealing of the whiteruss after the sterilizing of 1mL afterwards.Using the test tube of non-inoculating strain as blank.After making, all test tubes are together inserted 30 DEG C of constant incubator quiescent culture.After cultivating for some time, observe and find there is bubble formation between paraffin and substratum, prove that bacterial strain of the present invention has denitrifying capacity.
(4) colony morphology characteristic of pseudomonas XP-2:
Culture medium A was cultivated after 2 days, grows circular colonies, light yellow, concave, glossy, neat in edge.
(5) morphological features of pseudomonas XP-2:
Scanning electron microscopic observation shows that thalline is rod-short, 550nm ~ 640nm × 1100nm ~ 2000nm.
(6) pcr amplification of 16S rDNA and order-checking:
Laboratory apparatus: compact centrifuge (Eppendorf, rotating speed >12000r/min); Electrophoresis apparatus (Liuyi Instruments Plant, Beijing); PCR thermal cycling amplification instrument (Eppendorf); Gel imaging instrument (Bio-Rad company of the U.S.).
Experimental technique: direct picking thalline from the fresh inclined-plane of strain X P-2, is added in the centrifuge tube containing 100 μ L distilled waters, after vortex mixing, thermo-cracking bacteria suspension take genomic dna as template amplification 16S rDNA, and amplimer is a pair universal primer.
Forward primer is 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ';
Reverse primer is 1492R:5 '-GGTTACCTTGTTA CGACTT-3 '.
PCR reaction is carried out in 50 μ L systems.Consisting of of reaction system: template DNA (50ng/ μ L) 2 μ L; DNTP mixture 4 μ L; Taq archaeal dna polymerase 0.25 μ L; Forward primer 2 μ L; Reverse primer 2 μ L; Distilled water 34.75 μ L.
Pcr amplification condition: 95 DEG C of sex change 5min; 95 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min30s, circulate 30 times; 72 DEG C extend 10min, 4 DEG C of preservations.The 16S rDNA amplified production of sepharose with 1% to bacterial strain does electrophoresis detection, cuts adhesive tape after checking, reclaims test kit (Shanghai Sheng Gong biotechnology company limited) purified pcr product with DNA gel.Pcr amplification product after recovery entrusts High Technology Center of academy of agricultural sciences of Shandong Province to check order.
(7) 16S rDNA sequential analysis and Phylogenetic Analysis:
The 16S rDNA length obtaining XP-2 bacterial strain after order-checking is the sequence of 1430bp, be submitted to Genbank and other bacterial strains are compared, find that the evolutionary distance of strain X P-2 and Pseudomonas sp. is the most close, determine that it belongs to Rhodopseudomonas, called after Pseudomonas stutzeri XP-2.
The cultivation of embodiment 2 bacterial strain of the present invention
(1) substratum used
A, bacterial strain storage medium (/L): peptone, 5g; Yeast extract paste, 3g; Glucose, 1g; NaCl, 6g; Agar, 12g; KNO
3, 1.5g; PH controls 7.0 ~ 7.2.
B, bacterial strain denitrification substratum (DM:Denitrifying Medium) (/L): CH
3cOONa, 2g; KH
2pO
4, 0.4g; MgSO
47H
2o, 0.6g; CaCl
22H
2o, 0.07g; KNO
3, 1g; Tris damping fluid 12mL; Trace element 2mL; PH controls 7.0 ~ 7.2.
Before above-mentioned substratum uses, 121 DEG C, sterilizing 20 minutes.
(2) culture condition
By the pseudomonas XP-2 transfering loop scraping 1 ring lawn preserved on storage medium inclined-plane, inoculate in the 250mL Erlenmeyer flask that the sterilized LB substratum of 100mL is housed, 30 DEG C of constant-temperature shaking culture 12h, can obtain seed liquor.During experiment, seed liquor is inoculated in denitrification substratum or waste water by the inoculum size by 10%.
The best denitrogenation condition of embodiment 3 bacterial strain of the present invention
Sterilized for 100mL denitrification substratum is loaded in 250mL triangular flask, according to inoculum size access kind of the daughter bacteria liquid of 10%, quiescent culture.Within the scope of 10 ~ 30 DEG C, pseudomonas XP-2 can remove the total nitrogen of more than 80%, and 25 DEG C time, nitrogen removal rate is the highest, reaches 95%.The total nitrogen of about 140mg/L, between 6 ~ 9, can be dropped to below 10mg/L, and not have nitrite accumulation by the pH value that bacterial strain of the present invention adapts to.The suitableeest C/N (mol/mol) of bacterial strain of the present invention is than being 3, and when C/N >=3, the clearance of total nitrogen can reach more than 90%.The micro dissolution oxygen environment of quiescent culture is best suited for the performance of bacterial strain denitrification activity of the present invention.
Embodiment 4 bacterial strain of the present invention has denitrification enzyme system completely
In the triangular flask of 1L, fill the denitrification substratum being vaccinated with 10% seed liquor, sealing bottleneck, left at room temperature is cultivated.The gas discharged in experimentation is collected, then by chromatography of gases mass spectrometry analyser (GC-MS) analytical gas composition with collection and confinement of gases bag.It is 28 that result shows this molecular weight gas, and the highest with the similarity of nitrogen, infers that this gas is N
2, without other gas.Proving that bacterial strain of the present invention has denitrification enzyme system completely, can be N by nitric nitrogen direct-reduction
2, denitrification activity completely can be played, there is no NO, N
2the generation of the poisonous and harmful intermediate gas such as O.Therefore, this bacterial strain is suitable for engineer applied.
Claims (3)
1. a strain has the amphimicrobian denitrifying bacterium of complete denitrification enzyme system, it is characterized in that: this Strain Designation is Pseudomonas stutzeri XP-2, belong to denitrifying bacterium, there is denitrifying capacity completely, be preserved in China typical culture collection center on November 27th, 2011, its deposit number is: CCTCCM2011431.
2. the amphimicrobian denitrifying bacterium with complete denitrification enzyme system according to claim 1, its Denitrification Characteristics is: have denitrification enzyme system completely, can be nitrogen by nitric nitrogen direct-reduction, generate without other obnoxious flavoures.
3. the amphimicrobian denitrifying bacterium with complete denitrification enzyme system according to claim 2, is characterized in that: denitrogenation condition is: temperature is 10 ~ 30 DEG C, pH value 6 ~ 9, C/N >=3, and dissolved oxygen is slightly soluble oxygen condition, and dissolved oxygen concentration is less than 0.5mg/L.
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| CN113502247B (en) * | 2021-07-12 | 2023-08-08 | 徐韡卿 | Composite functional microbial agent for total nitrogen removal of high-salt wastewater and application thereof |
| CN114774320B (en) * | 2022-04-27 | 2023-12-05 | 武汉轻工大学 | Denitrifying microorganism and application thereof |
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