CN103114062B - Denitrifying phosphate-accumulating organism with nitrogen and phosphorus removal functions and applications thereof - Google Patents

Denitrifying phosphate-accumulating organism with nitrogen and phosphorus removal functions and applications thereof Download PDF

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CN103114062B
CN103114062B CN201310032730.5A CN201310032730A CN103114062B CN 103114062 B CN103114062 B CN 103114062B CN 201310032730 A CN201310032730 A CN 201310032730A CN 103114062 B CN103114062 B CN 103114062B
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nitrogen
phosphorus
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denitrifying
bacterial strain
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CN103114062A (en
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裴海燕
孟盼盼
胡文容
邵媛媛
李政
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Shandong University
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Abstract

The invention relates to a denitrifying phosphate-accumulating organism with nitrogen and phosphorus removal functions and applications thereof. The strain is named as pseudomonas pseudoalcaligenes CL-1, belongs to pseudomonas pseudoalcaligenes, and is preserved in China Center for Type Culture Collection on June 13, 2012, and the preservation numbers is CCTCC NO: M2012225. The strain is gram-positive and belongs to brevibacterium, a bacterial colony is rounded, milk white, convex and glossy, and edges are smooth and transparent. The growth cycle of the organism is long, the nitrogen and phosphorus removal rates in the first 24h are respectively 56.93% and 43.56%, and the nitrogen and phosphorus removal rates in the whole growth cycle are respectively 75.6% and 57.5%. The strain has a complete denitrifying capacity, and can directly reduce inorganic nitrogen such as nitrate nitrogen and nitrite nitrogen and the like to harmless nitrogen gas discharged from water bodies. When the denitrifying phosphate-accumulating organism is used for treating nitrogen and phosphorus containing wastewater, the process is simple, the treatment effect is efficient and stable, and the operating cost is saved, meanwhile, no greenhouse gas such as NO and N2O and the like is produced, therefore, no air pollution is caused.

Description

One strain has Denitrifying Phosphate Accumulating Organisms and the application thereof of denitrogenation dephosphorizing dual capability
Technical field
The present invention relates to cultivation and application thereof that a strain has the amphimicrobian Denitrifying Phosphate Accumulating Organisms of complete denitrification enzyme system, belong to environmental microorganism field.
Background technology
Along with the fast development of China's economy, water environment pollution has become the subject matter that various circles of society pay close attention to.The principal pollutant of domestic water environment comprise the inorganic pollutants such as organism and total nitrogen, total phosphorus at present, what wherein water body is endangered to maximum is total nitrogen and total phosphorus, a large amount of nitrogen phosphorus enters the sharply deterioration that water body can cause water surrounding, its main harm is the eutrophication of water body, the ratio of China's eutrophication water has risen to 55% from 50% nearly ten years, and the water body ratio of oligotrophication reduces to 0.53% by 3.2%.Body eutrophication impels the amount reproduction of algae, and algae is after excessive multiplication, and death in a large number decays again, for the growth of heterotrophic microorganism provides abundant matrix, the amount reproduction of aerobic microbiological, has exhausted the dissolved oxygen in water body very soon, has a strong impact on the growth and breeding of fish; Algal bloom can also cause waterworks paralysis and tap water quality to worsen, and Taihu Lake wawter bloom is the most typical in recent years, and it has caused the shortage of tap water; Many algae can also produce toxin simultaneously, have had a strong impact on human health life; The eutrophication of water body will speed up the decline in lake in addition, promotes it to swamp, to develop.Therefore, how efficient, economic denitrogenation dephosphorizing has become the hot research direction in current water prevention and cure of pollution field.
Nitrogen and phosphorus pollution principal element in water surrounding is summed up as human social activity, and its main source comprises city domestic sewage, trade effluent and the residual waste water of agricultural irrigation etc.Nitrogen mainly contains two kinds of organonitrogen and inorganic nitrogens and has form in water body: organonitrogen has protein, many skins, amino acid and urea etc., and the nitrogen of this form is mainly derived from sanitary sewage, agricultural wastes (straw, excrement of animals etc.) and some trade effluent (as wool processing, process hides, printing and dyeing, food-processing etc.); Inorganic N forms is mainly ammonia nitrogen, nitric nitrogen, nitrite nitrogen (general designation nitrogen compound), and its main source is trade effluent, and the modality between inorganic nitrogen.The Main Morphology that phosphorus exists in water body is phosphoric acid salt (being called for short phosphorus or total phosphorus), according to its physical property, be divided into solvability and graininess two classes, by chemical property, can be divided into orthophosphoric acid salt, polymeric phosphate and organophosphate, its main source is sanitary sewage and agricultural effluent.
The ultimate principle of bio-denitrifying sewage is exactly organonitrogen to be converted on the basis of ammonia-state nitrogen, first utilizes aerobic section through nitrification, by the synergy of nitrobacteria and nitrite bacteria, by NH 3be converted into with under anoxia condition by denitrification just (through anti-nitrosification) and (through denitrification) is reduced to nitrogen, overflows the water surface and is discharged into atmosphere, participates in the circulation of nature nitrogen.In water, nitrogenous substances reduces in a large number, reduces the potentially danger of water outlet, reaches the object of denitrogenation from waste water.Waste water is in biological treatment, under anaerobic, the growth of polyP bacteria is suppressed, for self growth just discharges the poly-phosphate in its cell, produce simultaneously and utilize the simple required energy of solvability organic substrate in waste water, claim that this process is the release of phosphorus.Enter after aerobic environment, vigor is fully recovered, and when making full use of matrix, absorbs the orthophosphoric acid salt of a large amount of solubilised state from waste water, thereby completes the process of poly-phosphorus.The microorganism that these are absorbed to a large amount of phosphorus is removed from waste water, can reach the object of dephosphorization.
The competition to organic carbon source due to polyP bacteria and denitrifying bacterium, at present a lot of denitrification dephosphorization techniques can not reach the best effect of denitrogenation and dephosphorization simultaneously, for this reason, many software engineering researchers invent various novel materials, new technology, new installation, derive many new biological denitrification phosphorous removal techniques to realize maximum denitrogenation and dephosphorization simultaneously, yet can not fundamentally solve this difficult problem all the time.Therefore, the best effect of denitrogenation and dephosphorization be realized simultaneously, thinking must be changed, not only just from aspect researchs such as research and development technique and adjust operation parameters, also must be from mechanism and the usefulness of microbiological angle research and inquirement biological carbon and phosphorous removal.
At present existing investigator has found to have the microorganism species of denitrogenation simultaneously and Removal, be referred to as Denitrifying Phosphate Accumulating Organisms (DPB), determined that this is that a class can be under anaerobic, poly-phosphorus in decomposer, and absorb easily biodegradable organics and be stored in thalline with poly-β-hydroxybutyric acid (PHB) form, under anoxia condition, PHB in decomposer, and take nitrate nitrogen and take the photograph the microorganism species of phosphorus as electron acceptor(EA), utilize the biological nature of DPB, successfully solved the problem of denitrifier and polyP bacteria competition carbon source in traditional biological denitrification dephosphorization technique.Yet this type of bacterium and characteristic research thereof are all in the junior stage, do not have systematic authentication method, conveniently do not cultivate acclimation yet.Verify kind and the characteristic of denitrification phosphorus-collecting bacterium, define their nutritional condition and envrionment conditions, will contribute to research, the development and application of wastewater biological denitrificaion dephosphorization process.
The denitrification phosphorus-collecting effect of bacterium is to complete under the katalysis of various reductase enzymes, and the active height of each reaction enzymes system is subject to the impact of the conditions such as temperature, pH value.Temperature of reaction in biological wastewater treatment, to microbial growth, breed in close relationsly, temperature is being arranged the solubleness of enzymatic reaction kinetics, microorganism growth speed and compound etc., thereby the Degradation and Transformation of pollutent is played to keying action.The factors such as investigation temperature, pH compare are brought into play the impact of efficient denitrification activity on denitrification bacterial strain, and then obtain denitrifying polyphosphate-accumulating organisms, realize the denitrogenation dephosphorizing of waste water high-efficiency economy, significant to solving day by day serious water surrounding nitrogen and phosphorus pollution problem.
The enzyme that it is nitrite that many bacteriums have nitrate reduction, can realize the first step and transform, but will complete thorough denitrogenation, requires bacterium must have complete denitrification enzyme system.Verify denitrification phosphorus-collecting bacterium whether have complete again efficiently denitrification enzyme system be the prerequisite that adopts biological denitrification technique denitrogenation dephosphorizing.Research finds that bacterium of the present invention has complete denitrification enzyme system, can efficiently remove the nitrogen in water body, by nitrate-nitrogen reduction, be that nitrogen is discharged water body, realize the high-efficient denitrification and dephosphorization of waste water, for solving day by day serious Nitrogen-and Phosphorus-containing waste water, the pollution problem of environment is made contributions.
Summary of the invention
For above problem, the object of this invention is to provide a strain and there is the efficient amphimicrobian denitrification phosphorus-collecting bacterium that complete denitrification enzyme is.This bacterium has denitrification enzyme system completely, when can thoroughly remove the nitrogen phosphorus in water body, does not produce NO, N 2the obnoxious flavoures such as O.
Another object of the present invention is to provide application and the application method of this Denitrifying Phosphate Accumulating Organisms, makes this bacterium show denitrification dephosphorization activity completely.
The present invention is achieved through the following technical solutions:
One strain has the Denitrifying Phosphate Accumulating Organisms of denitrogenation dephosphorizing dual capability, this bacterial strain called after pseudomonas pseudoalcaligenes CL-1, Pseudomonas pseudoalcaligenes CL-1, belong to pseudomonas pseudoalcaligenes, on June 13rd, 2012, be preserved in Chinese Typical Representative culture collection center, its deposit number is CCTCC NO:M2012225, preservation address: Wuhan, China Wuhan University.Its mycology is characterized as: cell is rod-short, gram-positive microorganism, and bacterium colony circle, oyster white, convexity, glossy, the smooth of the edge is transparent.Its best denitrogenation condition is: 25~30 ℃ of temperature, pH7.0~8.0.Denitrogenation with this understanding, without nitrite accumulation.
The application of the Denitrifying Phosphate Accumulating Organisms with denitrogenation dephosphorizing dual capability in wastewater treatment.During concrete application, method is:
(1) before use, with LB substratum, activate bacterial strain: by transfering loop scraping 1 ring lawn for the pseudomonas pseudoalcaligenes CL-1 preserving on slant medium, inoculate in the 250mL Erlenmeyer flask that 100mL LB substratum is housed, 12h is cultivated in 30 ℃ of shaking table concussions, can obtain bacterium liquid (OD 600=1.0 ± 0.01);
(2) the bacterium liquid after activation is inoculated in pending Nitrogen-and Phosphorus-containing waste water, dosage is to be subject to processing 10% of wastewater volume, and pH value scope is between 7.0~9.0, and the standing cultivation of room temperature is more than 192h.
Front 24h every 8h sampling once, detects the accumulation of nitre nitrogen in water sample, total phosphorus removal situation and nitrite afterwards every 24h timing sampling.
Detection method
TP: molybdate-ultraviolet spectrophotometry;
NO 3 --N: ultraviolet spectrophotometry;
Bacterial strain of the present invention is to utilize two mud method SBR to cultivate domestication to oxidation ditch active sludge, a kind of denitrification phosphorus-collecting bacterium with high-efficient denitrification and dephosphorization activity that screening obtains, can remove nitrite nitrogen and the nitrate nitrogen in water body by denitrification, in decomposer, gather phosphorus simultaneously, and absorb easily biodegradable organics and be stored in thalline with PHB form, under anoxia condition, PHB in decomposer, and take nitrate nitrogen and take the photograph phosphorus as electron acceptor(EA).This bacterial strain has denitrifying capacity completely, nitric nitrogen progressively can be reduced to nitrogen and discharge water body, is applicable to the processing of higher concentration nitrite or nitrate waste water.Use this bacterial strain to process waster water process simple, denitrogenation is thorough, and effect stability is saved running cost.
Denitrogenation in the present invention refers to the removal of Inorganic Nitrogen in Water Bodies, and inorganic nitrogen refers to NO 3 --N and NO 2 --N.
Bacterial strain of the present invention has the following advantages:
(1) bacterial strain of the present invention has completely denitrification enzyme system, can be nitrogen by nitric nitrogen direct-reduction, realizes thorough denitrogenation and polluted air not.
(2) bacterial strain of the present invention has very high denitrification activity, can be used for processing the processing that is applicable to higher concentration nitrite or nitrate waste water.
(3) bacterial strain of the present invention has very high dephosphorization activity, can be used for processing the processing that is applicable to higher concentration phosphorus-containing wastewater.Accompanying drawing explanation
Fig. 1: the stereoscan photograph of pseudomonas pseudoalcaligenes CL-1 thalline.
Fig. 2: the growth curve of pseudomonas pseudoalcaligenes CL-1.
Fig. 3: the denitrogenation dephosphorizing curve of pseudomonas pseudoalcaligenes CL-1.
Fig. 4: the denitrogenation dephosphorizing efficiency of pseudomonas pseudoalcaligenes CL-1 and temperature variation relation.
Fig. 5: the denitrogenation dephosphorizing curve of pseudomonas pseudoalcaligenes CL-1 and pH variation relation.
Fig. 6: the aerogenesis collection of illustrative plates of pseudomonas pseudoalcaligenes CL-1.
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 is controlled at 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 is controlled at 7.0~7.2.
C, LB substratum (/L):
Peptone, 10g; Yeast extract paste, 5g; Sodium-chlor, 10g; PH is controlled at 7.5.
(2) separation of pseudomonas CL-1 bacterial strain, purifying:
Adopt method of dilution butteron on plate and plate streak to carry out separation and purification.First with aseptic transfer pipet, get the mud mixed liquid 10mL of SBR anoxic while finishing, be placed in the aseptic triangular flask that several granulated glass spherees are housed, add sterilized water 90mL, make 10 -1the bacteria suspension of concentration stirs 20min and breaks up zoogloea on magnetic stirring apparatus.
With liquid-transfering gun, draw respectively 0.5ml sample in the test tube of 4.5mL sterilized water is housed, mix, the rest may be inferred, and finally drawing extent of dilution is 10 -4diluent, with the amount of every dull and stereotyped 0.2mL, be coated with four obligate substratum, with aseptic triangular glass scraper, in media surface, be evenly coated with.Afterwards flat board is inverted, is put into 30 ℃ of constant incubators, be cultured to and grow obvious bacterium colony.
Picking single strain, the purifying of repeatedly ruling on flat board, until micro-Microscopic observation shows without miscellaneous bacteria, purifying is complete now can to think bacterial strain.Isolated bacterial strain is containing well-grown on the solid medium of nitrate, has potential Denitrification Characteristics, and that after Babes-Ernst bodies dyeing, in thalline, have atrament is the pseudomonas pseudoalcaligenes CL-1 in the present invention, is seeded to slant medium and saves backup.
(3) the nitrate reduction aerogenesis of pseudomonas pseudoalcaligenes CL-1 test
For understanding denitrifying capacity and the characteristic of bacterial strain of the present invention, bacterial strain of the present invention is carried out to the test of nitrate reduction aerogenesis.
Be inoculated in small test tube (specification: add the denitrification liquid nutrient medium of 10mL sterilizing 15mm * 100mm), the bacterium colony 1 after purifying with transfering loop picking encircles to small test tube, stirring and evenly mixing.Add afterwards the whiteruss sealing after the sterilizing of 1mL.Using the test tube of inoculating strain not as blank.After making, all test tubes are together inserted to 30 ℃ of standing cultivations of constant incubator.Cultivate after 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 pseudoalcaligenes CL-1:
In culture medium A, cultivate after 2 days, grow circular bacterium colony, oyster white, convexity, glossy, the smooth of the edge is transparent.
(5) morphological features of pseudomonas pseudoalcaligenes CL-1:
Scanning electron microscopic observation shows that thalline is rod-short, 182nm~260nm * 1145nm~1458nm.
(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 (U.S. Bio-Rad company).
Experimental technique: direct picking thalline from the fresh inclined-plane of bacterial strain CL-1, be added to containing in the centrifuge tube of 100 μ L distilled waters, after vortex mixes, thermo-cracking bacteria suspension, take genomic dna as template amplification 16S rDNA, and amplimer is a pair of 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 ℃ of sex change 5min; 95 ℃ of 30s, 55 ℃ of 45s, 72 ℃ of 1min30s, circulate 30 times; 72 ℃ are extended 10min, 4 ℃ of preservations.Sepharose with 1% is done electrophoresis detection to the 16S rDNA amplified production of bacterial strain, cuts adhesive tape after checking, with DNA gel, reclaims test kit (Shanghai Sheng Gong biotechnology company limited) purified pcr product.Pcr amplification product after recovery entrusts High Technology Center, academy of agricultural sciences, Shandong Province to check order.
(7) 16S rDNA sequential analysis and Phylogenetic Analysis:
The sequence that the 16S rDNA length that obtains CL-1 bacterial strain after order-checking is 1430bp, be submitted to Genbank and other bacterial strains are compared, the evolutionary distance of finding bacterial strain CL-1 and Pseudomonas sp. is the most approaching, determines that it belongs to Rhodopseudomonas, called after Pseudomonas sp.CL-1.
The cultivation of embodiment 2 bacterial strains of the present invention
(1) substratum using
A, bacterial strain storage medium (/L): peptone, 5g; Yeast extract paste, 3g; Glucose, 1g; NaCl, 6g; Agar, 12g; KNO 3, 1.5g; PH is controlled at 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 is controlled at 7.0~7.2.
Before above-mentioned substratum is used, 121 ℃, sterilizing 20 minutes.
(2) culture condition
By transfering loop scraping 1 ring lawn for the pseudomonas pseudoalcaligenes CL-1 preserving on storage medium inclined-plane, inoculate in the 250mL Erlenmeyer flask that the sterilized LB substratum of 100mL is housed, 30 ℃ of constant-temperature shaking culture 12h, can obtain seed liquor.During experiment, the inoculum size by 10% is inoculated in seed liquor in denitrification substratum or waste water.
The best denitrogenation dephosphorizing condition of embodiment 3 bacterial strains of the present invention
The sterilized denitrification substratum of 100mL is packed in 250mL triangular flask into inoculum size access kind of the daughter bacteria liquid according to 10%, standing cultivation.Its best denitrification temperature is between 25-30 ℃, and optimum temperuture is 25 ℃, and optimum pH is 7~8.The growth cycle of this bacterium is long, and 48h enters logarithmic phase later, and 96h enters stationary phase later, and 96~120h, substantially in stationary phase, enters decline phase after 120h.Before this bacterial strain, 24h removal efficiency of nitrogen and phosphorus is respectively 56.93%, 43.56%, and whole experimentation removal efficiency of nitrogen and phosphorus is respectively 75.6% and 57.5%.
Embodiment 4 bacterial strain of the present invention has denitrification enzyme system completely
In the triangular flask of 1L, fill the denitrification substratum of having inoculated 10% seed liquor, sealing bottleneck, standing cultivation under room temperature.With collection and confinement of gases bag, collect the gas discharging in experimentation, then by chromatography of gases mass spectrometry analyser (GC-MS) analytical gas composition.Result shows that this molecular weight gas is 28, 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, can bring into play denitrification activity completely, there is no NO, N 2the generation of the poisonous and harmful intermediate product gases such as O.Therefore, this bacterial strain is more suitable for engineering application.

Claims (3)

1. a strain has the Denitrifying Phosphate Accumulating Organisms of denitrogenation dephosphorizing dual capability, it is characterized in that: this bacterial strain called after pseudomonas pseudoalcaligenes CL-1, Pseudomonas pseudoalcaligenes CL-1, belong to pseudomonas pseudoalcaligenes, on June 13rd, 2012, be preserved in Chinese Typical Representative culture collection center, its deposit number is CCTCC NO:M2012225.
2. the application of the Denitrifying Phosphate Accumulating Organisms with denitrogenation dephosphorizing dual capability claimed in claim 1 in wastewater treatment.
3. the application method of the Denitrifying Phosphate Accumulating Organisms with denitrogenation dephosphorizing dual capability claimed in claim 2 in wastewater treatment, is characterized in that, comprises that step is as follows:
(1) before use, with LB substratum, activate bacterial strain: by transfering loop scraping 1 ring lawn for the above-mentioned pseudomonas CL-1 preserving on slant medium, inoculate in the 250mL Erlenmeyer flask that 100mL LB substratum is housed, 12h is cultivated in 30 ℃ of shaking table concussions, can obtain bacterium liquid, OD 600=1.0 ± 0.01;
(2) the bacterium liquid after activation is inoculated in pending Nitrogen-and Phosphorus-containing waste water, dosage is to be subject to processing 10% of wastewater volume, and pH value scope is between 7.0~9.0, more than the standing 192h of room temperature.
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