CN102676431B - Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same - Google Patents
Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same Download PDFInfo
- Publication number
- CN102676431B CN102676431B CN2012101481669A CN201210148166A CN102676431B CN 102676431 B CN102676431 B CN 102676431B CN 2012101481669 A CN2012101481669 A CN 2012101481669A CN 201210148166 A CN201210148166 A CN 201210148166A CN 102676431 B CN102676431 B CN 102676431B
- Authority
- CN
- China
- Prior art keywords
- nitrogen
- umbrella grass
- cctcc
- water
- denitrifying bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses denitrifying bacteria and an aquatic plant-microbe combined repair method using the same. The denitrifying bacteria LR were collected in China Center for Type Culture Collection on May 3rd, 2011, and the collection number of the strains is CCTCC NO: M2011159. The plant-microbe combined repair method for a nitrogen eutrophic water body comprises the following steps of: planting cyperus alternifolius in the nitrogen eutrophic water body, and inoculating the denitrifying bacteria LR. The denitrifying bacteria LR and the cyperus alternifolius or cyperus alternifolius root exudates are combined for repairing the nitrogen eutrophic water body, and the strains LR has a promotion effect of repairing the nitrogen polluted water body through the cyperus alternifolius. The method for repairing the nitrogen eutrophic water body by combining the denitrifying bacteria LR and the cyperus alternifolius or the cyperus alternifolius root exudates is particularly suitable for treating the nitrogen eutrophic water body in nature.
Description
Technical field
The invention belongs to environment (water body) Pollution abatement technical field, relate to a kind of denitrifying bacterium and be used for water body plant-microorganism combined remediation method, be specifically related to a kind of denitrifying bacterium and be used for the plant-microorganism combined remediation method of water body nitrogen eutrophication.
Background technology
Along with socioeconomic development, stream pollution is the problem that all faces of each state in the world.The municipal pollution water body is administered the basic measures that are the protection city water resource, improve the ecological environment.But physics and chemistry restorative procedure commonly used is generally invested costliness, equipment is complicated and be not suitable for the repairing and treating that water body in large pollutes.Microorganism (denitrifying bacterium) recovery technique then is subjected to oxygen level in the water body, can utilizes the restrictions such as carbon source kind not to be used widely because of colony growth.So far, isolated denitrifying bacterium is most of from physical environment belongs to facultative anaerobe, under anaerobic or hypoxia with nitrate ion (NO
3 -) carry out denitrification as final electron acceptor(EA); Its denitrification is suppressed even stops under the oxygen concentration higher state.The respiration of root system of plant can reduce the oxygen level of root system zone water body or soil, promotes that denitrifying bacterium carries out denitrification.In addition, available carbon source kind is the important factor that affects denitrifying bacterium survival in the environment, and root system of plant can be secreted several kinds of carbon source (carbohydrate, amino acid, protein, organic acid), wherein contains the denitrifying bacterium necessary nutritive substance of surviving.Studies show that in a large number the denitrifying bacterium kind that is positioned at the plant rhizosphere district and quantity are apparently higher than other zones.Large quantity research has been carried out in plant or the microorganism repair carried out for water pollution at present, but less to the control research of water body nitrogen eutrophication by the plant-microorganism combined remediation technology.
Summary of the invention
The purpose of this invention is to provide a kind of denitrifying bacterium for water body nitrogen eutrophication control.
Another object of the present invention provides the application of this denitrifying bacterium.
Another purpose of the present invention provides the plant-microorganism combined remediation method that this denitrifying bacterium is used for water body nitrogen eutrophication.
Purpose of the present invention can be achieved through the following technical solutions:
One strain denitrifying bacterium LR, Classification And Nomenclature are pseudomonas LR(Pseudomonas sp.LR), be preserved in Chinese Typical Representative culture collection center on May 3rd, 2011, culture presevation number is CCTCC NO:M2011159.This pseudomonas LR obtains from the separation of Nanjing eutrophication rheophyte rhizosphere district soil.Main Biological is: G
-, thalline is shaft-like.Indole test, glucose fermentation, lactose fermentation and Starch Hydrolysis test are negative, and the V.P test is positive; The antibiotics resistance test shows that the LR bacterial strain have resistance to ammonia benzyl, erythromycin, paraxin, strepto-; Can utilize carbon source that Trisodium Citrate, sodium-acetate, maltose, sodium succinate and starch are arranged, can not utilize carbon source that lactose and sucrose are arranged.The Genbank accession number of its 16S rDNA is JQ652571.
Described preserving number is the application of denitrifying bacterium LR in the Pollution abatement of nitrogen eutrophication water of CCTCC NO:M2011159.
Described preserving number is denitrifying bacterium LR associating umbrella grass (Cyperus alternifoliusL.) application in the Pollution abatement of nitrogen eutrophication water of CCTCC NO:M2011159.
A kind of the nitrogen eutrophication water is carried out the method that plant-microorganism is united reparation, plantation umbrella grass in the water body of nitrogen eutrophication, inoculating described preserving number is the denitrifying bacterium LR of CCTCC NO:M2011159; Wherein every premium on currency body plantation umbrella grass 7～15 strains, the preserving number that every premium on currency body inoculum density is 106cFu/ml is the denitrifying bacterium LR bacterium liquid 10ml of CCTCC NO:M2011159.
A kind of method of administering nitrogen eutrophic water body pollution, adding the umbrella grass roots in the water body of nitrogen eutrophication is secretory product, and the inoculation preserving number is CCTCC NO:M2011159 denitrifying bacterium LR, and it is secretory product that every premium on currency body adds 50ml umbrella grass roots, and every premium on currency body inoculum density is 10
6The preserving number of cFu/ml is the denitrifying bacterium LR bacterium liquid 10ml of CCTCC NO:M2011159, and umbrella grass roots of interpolation in per two days is secretory product after the inoculation, and each every premium on currency body adds 40～60ml, and preferred each every premium on currency body adds 50ml;
Wherein, the umbrella grass roots is after deionized water drip washing, is 75-80% in relative humidity, and temperature is 25 ℃, and intensity of illumination is that the interior deionized water that uses of the climatic chamber of 3000Lx～5000Lx soaks root system of plant 4～7h, and is concentrated into 1/10/ volume with Rotary Evaporators; Described umbrella grass roots system is 1g: 10～30ml with the mass volume ratio of the deionized water that is used for root immersion, preferred 1g: 20ml.
The standard of N element eutrophication liquid of the present invention is announced in 2004 by WHO (WHO), its standard is 11.3mg-N/l(Horing, H.and Chapman, D.:Nitrates and nitrites in drinking water:World Health Organization Drinking Water Series., IWA Publishing, London (2004) .); Being nitrogen content is called N element eutrophication liquid more than or equal to the liquid of 11.3mg-N/l, and nitrogen content is called N element eutrophication water more than or equal to the water body of 11.3mg-N/l.
Beneficial effect:
The present invention obtains filtering out a strain from the separation of Nanjing eutrophication rheophyte rhizosphere district soil and has efficient denitrifying bacterium---pseudomonas LR, this bacterial strain is that the final degradation rate of nitrate reaches more than 90% in the substratum of carbon source to Citrate trianion, and certain denitrification ability is also arranged with this bacterial strain enlarged culturing, for the treatment of high NO under higher nitrogen level
2 -Or high NO
3 -Waste water, with significant.
On this basis, the present invention is that secretory product is united for repairing the nitrogen eutrophication water with pseudomonas LR and umbrella grass or umbrella grass roots, and bacterial strain LR repairs the nitrate pollution water body to the umbrella grass and has promoter action.Pseudomonas LR provided by the invention and umbrella grass or umbrella grass roots are that the method that secretory product is united for reparation nitrogen eutrophication water is specially adapted to natural nitrogen eutrophied water treatment.The method so that in the sewage denitrifying bacterium survival rate significantly improve, increased substantially the denitrification rate of repair system, saved the cost that external source is added carbon source.
Description of drawings
Fig. 1 bacterial strain LR aerogenesis is measured photo.
The bacterium colony photo of Fig. 2 bacterial strain LR on the BTB flat board.
The removal situation of Fig. 3 bacterial strain LR nitric nitrogen.
The different carbon sources of Fig. 4 are on the impact of bacterial strain denitrification denitrogenation.
Fig. 5 different nitrogen sources is to bacterial strain LR affects on the growth.
Fig. 6 C/N is on the denitrification denitrogenation (A) of bacterial strain LR and the impact of growth (B).
The denitrification rate of Fig. 7 element eutrophication liquid different treatment group take the Hoagland nutritive medium as N.
The denitrification rate of Fig. 8 element eutrophication liquid different treatment group take extraneous sewage as N.
Element eutrophication liquid external source interpolation umbrella grass roots is the experiment of secretory product denitrification to Fig. 9 take extraneous sewage as N.
Biomaterial preservation information
Denitrifying bacterium LR, Classification And Nomenclature is pseudomonas LR(Pseudomonas sp.LR) be preserved in Chinese Typical Representative culture collection center, be called for short CCTCC, the preservation address is Wuhan, China Wuhan University, preservation date is on May 3rd, 2011, and deposit number is CCTCC NO:M2011159.
Embodiment
1.1 strain separating and screening
Test waterplant root system, sewage work's bed mud and Lianyun Harbour water paddy soil that used pedotheque picks up from limit, some river courses, Nanjing, limit, lake.Choose eugonic plant, shake off top layer bulk soil after, remainder is as plant rhizosphere soil.
Take by weighing the root system of plant pedotheque 10g of collection, put into the sterilized water that 90mL contains granulated glass sphere, vibration 20min leaves standstill 20-30s, makes bacteria suspension.Inoculum size with 10% is received this bacteria suspension in the DM substratum, 28 ℃ of cultivations.Get the 1mL bacteria suspension under the aseptic condition and put into Sheng 9mL sterilized water Glass tubing, mixing makes 10 more successively
-2, 10
-3, 10
-4, 10
-5, 10
-6Various concentration dilution liquid.
Get the diluent of the above-mentioned different concns of 0.1mL and coat respectively on the BTB differential medium, each concentration arranges respectively three repetitions.Cultivate 1-3d for 28 ℃, single bacterium colony of the different shape of blue haloing is arranged around the picking, carry out purifying through repeatedly ruling.
The bacterial strain that picking is separated, the purifying of on flat board, repeatedly ruling, until microscopically observe to show without till the miscellaneous bacteria, purifying is complete can to think bacterial strain this moment.Isolated bacterial strain is containing well-grown on the solid medium of nitrate, has potential Denitrification Characteristics, is bacterial strain to be sieved, and all is seeded to slant medium and saves backup.
Carry out multiple sieve with accessing respectively the Giltay substratum behind the bacterial strain of purifying, cultivate 2-14d for 28 ℃.The bacterial strain that select that aerogenesis is fast, bubble is many further detects the content of nitrite nitrogen and total nitrogen in the substratum.Preserve filtering out the strong bacterial strain inclined-plane of denitrification activity.
The cultivation of using in above-mentioned separation, the screening process and prescription are:
Denitrification basis (DM) substratum (Li Yan etc., 2008): KNO
32.0g, K
2HPO
41.0g, MgSO
47H
2O 0.2g, Trisodium Citrate 5g, distilled water 1000mL, pH value 7.2.
Bromothymol blue (BTB) differential medium (TAKAYA etc., 2003): L-aspartoyl amino acid 1.0g, KNO
31.0g, KH
2PO
41.0g, FeCl
36H
2O 0.05g, CaCl
22H
2O 0.2g, MgSO
47H
2O 1.0g, agar 20g, 1% bromothymol blue 1mL, distilled water 1000mL, pH value 7.0-7.3.
Giltay substratum (soil microorganisms research association, 1983):
A solution: KNO
32.0g, L-aspartoyl amino acid 1.0g, 1% bromothymol blue 5mL, distilled water 500mL;
B solution: Trisodium Citrate 8.5g, KH
2PO
41.0g, FeCl
36H
2O 0.05g, CaCl
22H
2O 0.2g, MgSO
47H
2O1.0g, distilled water 500mL;
Mix A, B two solution, adjust pH 7.0-7.2.
Sieve again by concentration and separation, BTB differential medium primary dcreening operation and Gailty substratum, finally from from Nanjing eutrophication rheophyte rhizosphere district pedotheque, isolating the strongest aerogenesis denitrifying bacterium of a strain denitrification activity, bacterial strain called after LR, and LR delivered the center preservation of Chinese Typical Representative culture collection, culture presevation number is CCTCC NO:M2011159.Aerogenesis photo such as Fig. 1.
1.2 denitrifying bacterium Determination of Physiological And Biochemical Indices
The evaluation of bacterial strain is with reference to " carry out common bacteria system identification handbook (eastern elegant pearl etc., 2001) and " Bergey ' s Manual of Determinative Bacteriology " (Buchanan and Gibbons, 1986).
Bacterial strain LR is gram negative bacillus, and it is circular to form white on the BTB solid medium flat board, and flat opaque, the edge shows slightly projection, dry lacklustre bacterium colony.Bacterium colony photo such as Fig. 2.
Bacterial strain LR belongs to facultative anaerobe, and under oxygen free condition, denitrifying bacteria as final electron acceptor(EA), is finished the nitrate metabolism with nitric nitrogen, Gram-negative bacteria, and VP is positive, and litmus milk is peptonized.Bacterial strain LR can utilize the sucrose fermentation.Concrete outcome sees Table 1.
The physiological and biochemical property of table 1 bacterial strain LR
Annotate: the result is positive for "+" table; The result is negative for "-" table
1.3 denitrifying bacterium 16S rRNA gene sequencing is identified
(1) extracts bacteria total DNA
(2) pcr amplification
The preparation of PCR reaction system is as shown in table 2.
The preparation of table 2 PCR reaction system
The PCR working procedure is: 95 ℃ of denaturation 5min, and 94 ℃ are extended 30sec, 52 ℃ of renaturation 30sec, 72 ℃ are extended 1.5min, 30 circulations, 72 ℃ are extended 10min; 10 ℃ of insulation 5min.
The primer that is used for the PCR reaction of 16S rDNA is a pair of universal primer (Zhai Qian etc., 2007):
Forward primer is 27F:5'-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID NO.1);
Reverse primer is 1492R:5 '-GGTTACCTTGTTACGACTT-3 ' (SEQ ID NO.2).
4 ℃ of preservations of PCR product.
(3) mensuration of 16Sr rna gene sequence and similarity analysis
Through extraction and the pcr amplification to bacterial strain LR DNA, obtained certain-length and be the dna fragmentation about 1.5kb, the PCR product send the company of specialty to reclaim purifying and sequencing, the 16S rRNA gene that sequencing result shows bacterial strain LR is 1436 bases altogether, by blast program and GenBank(http: //www.ncbi.nlm.nih.gov) and RDP(http: //rdp.cme.msu.edu) in existing Related Bacteria 16S rRNA gene order carry out the similarity comparison.According to sequencing result, utilize the homologous sequence that finds in the related softwares such as clustalxl.83, Mega4 and GenBank and the RDP database to compare, find that bacterial strain LR and pseudomonas homology are the highest, morphological specificity, Analysis of The Physiological And Biochemical Properties and 16S rRNA dna homolog sequence according to this bacterial strain compare, and LR is accredited as pseudomonas (Pseudomona sp.).
The Denitrification Characteristics research of embodiment 2 pseudomonas LR
2.1 denitrification ability is measured
Getting 28 ℃ of bacteria suspension 8mL that cultivate 36h is inoculated in the sterilized DM nutrient solution of 500mL (triangle is bottled), mix, be sub-packed in again in the sterilizing test tubes, each 10mL, 28 ℃ of cultivations are taken a sample during respectively at 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, 72h, 4 ℃ of Refrigerator stores are put in taking-up, 3 of each taking-ups are treated all to take, and measure the nitric nitrogen clearance.
When bacterial classification inoculation behind the fresh DM substratum, bacterium is growth and breeding immediately not, and will be through adjusting after a while and adapting to, and with synthetic plurality of enzymes, and improve enzyme system in the body and other composition of cell.Short lag phase explanation is stronger to the adaptive faculty of environment.Different bacteriums has different growth characteristics, changes by measuring nitric nitrogen, can reflect the denitrification ability of different times bacterium.
The LR denitrification ability is seen Fig. 3.As seen from the figure, the whole growth cycle of bacterial strain LR is about 3 days, and logarithmic phase is longer, just is in the steady growth phase about 54 hours.Its denitrification mainly occurs in the logarithmic phase (12-54h) of its growth, and the time length is longer.
2.2 Carbon and nitrogen sources test
Carbon source test: do not add respectively the 0.5%(massfraction in adding the denitrification substratum of carbon source) sodium acetate, Trisodium Citrate, sodium succinate, sucrose, lactose, starch, maltose is as unique carbon source, other components unchanged.With each substratum for preparing, get 10mL and divide to install in the test tube and sterilize, afterwards with 10% the bacterium amount that connects access logarithmic phase pseudomonas LR activation thalline.28 ℃ of constant temperature leave standstill cultivation, observe, record the aerogenesis phenomenon, and sampling detects TN behind the 2d, analyzes different carbon sources to the impact of the denitrification ability of bacterial strain.
As seen from Figure 4, three bacterial strains all have comparatively widely carbon source spectrum, can utilize carbon source kind various, and when cultivating 2d, bacterial strain LR is take Trisodium Citrate as carbon source the time, and the nitrogen clearance is more than 70% two days later.
Nitrogenous source test: take Trisodium Citrate as carbon source, in adding the denitrification basic medium of nitrogenous source, do not add respectively the 0.1%(massfraction) saltpetre, Sodium Nitrite, ethanamide, ammonium chloride, urea as sole carbon source, the substratum rear inoculation logarithmic phase pseudomonas LR activation thalline that goes out can be got rid of and connects the impact that liquid is brought nitrogenous source into.28 ℃ of constant temperature leave standstill cultivates 2d, by measuring OD
600Changing conditions, the upgrowth situation of indicator under the different nitrogen sources condition.
As can be seen from Figure 5, for the nitrogenous source of 5 kinds of different shapes that adopt in the test, bacterial strain LR has all shown certain adaptability, and the biomass that wherein utilizes saltpetre to reach is maximum.There are some researches show, because the toxic action of nitrite, some bacterial strain can not utilize nitrite growth (Zhang Guangya etc., 2005), the pseudomonas LR that the present invention filters out can utilize the nitrite growth, the diversity that nitrogenous source utilizes, illustrate that it can adapt to the water quality of different nitrogen pollution type, applies thereby be conducive to it.
2.3 carbon-nitrogen ratio is on the impact of denitrogenation
With KNO
3Be only nitrogen source, Trisodium Citrate is carbon source and the energy, investigates different C/N(mol ratios) on the impact of denitrifying bacterium growth and denitrification ability.In the 250mL Erlenmeyer flask, add substratum after the 200mL sterilization, the inoculation liquid of 1% inoculation preculture ld by volume, add rubber stopper seal after (the airtight cultivation of deep layer) put into shaking culture case (140r min
-1) 28 ℃ of constant temperature culture.Initial pH value is adjusted to 7.0, and keeping TN content is lgL
-1, by regulating the add-on of Trisodium Citrate, make C/N be respectively l, 3,5,7,10 and 15.Timing sampling detects OD
600, TN, analyze C/N to the impact of denitrification process.
Fig. 6 (A) and (B) reacted respectively bacterial strain LR under different C/N conditions the nitrogen purification efficiency and the changing conditions of biomass.Along with the increase of C/N, the biomass of bacterial strain LR and nitrogen removal rate all present the trend of rising.When C/N was 7, the biomass of bacterial strain LR was maximum, and it is also the highest that nitrogen is purified percentage.As C/N〉10 the time, biomass and remove percentage and all begin to descend.
The umbrella grass roots is the collection method of secretory product: take by weighing 10g left and right sides umbrella grass, wash several times with the root system of deionized water with the umbrella grass, then blot the moisture on surface with filter paper, plant is put into 200mL distilled water, it is (25 ± l) ℃ in temperature, intensity of illumination is 4000Lx, and relative humidity is when collecting about 6h in the phytotron of 75-80%, behind the taking-up plant, immediately with the root exudates solution filter, adopt Rotary Evaporators to be concentrated into 20ml, save backup under-20 ℃, or directly save backup.
Choose growing way identical, at Hoagland nutritive medium (Ca (NO
3)
2, 945mg/l; KNO
3, 607mg/l; (NH
4)
3PO
4, 115mg/l; MgSO
4, 493mg/l; 2.5ml/l ferrous solution: FeSO
47H
2O, 5.56g/l; Na
2EDTA, 7.46g/l; 5ml/l trace element: Sodium Tetraborate, 6.2mg/l; MnSO
4H
2O, 22.3mg/l; ZnSO
4, 8.6mg/l; Na
2MoO
42H
2O, 0.25mg/l; CuSO
45H
2O, 0.025mg/l; CoCl
26H
2O, 0.025mg/l) 10 days umbrella of inner preculture grass (Cypcrus alternifoliusL.), be transferred in the fresh Hoagland nutritive medium of 400ml, external source is added Trisodium Citrate as carbon source, and transferring to carbon-nitrogen ratio is 7.Choose the thalline LR that grows to logarithmic phase, be resuspended in the deionized water with behind the distilled water centrifuge washing three times, cell concentration transfers to 10
6CFu/ml 1/100 is seeded in the Hoagland nutritive medium by volume.Test arranges four treatment group: bacterium LR, and umbrella grass (not adding LR bacterium liquid), LR+ umbrella grass roots is secretory product, LR+ umbrella grass.Umbrella grass treatment group is added the identical umbrella grass of three strain growing ways with every group of LR+ umbrella grass treatment group, and LR+ umbrella grass roots is that the umbrella grass roots of secretory product treatment group interpolation Hoagland nutritive medium 10% volume (being 40ml) is secretory product; Each treatment group arranges five repetitions.After processing, used residual nitrogen content in the alkaline alkaline potassium per-sulfate digestion Water by Ultraviolet Spectrophotometry body on the the 1st, 2,3,7 day.And got 1ml solution at the 7th day from each treatment group and add the liquid D M substratum that contains 50mg/l ammonia benzyl and 50mg/l erythromycin 28 ℃ to and cultivated 2 days.Drawing the 0.5ml nutrient solution is applied on the BTB flat board that contains 50mg/l paraxin.The blue colonies that grows on the flat board is chosen and is shaken bacterium and carry genomic dna, carries out 16s rDNA sequence amplification and send the order-checking of order-checking company.LR+ umbrella grass treatment group denitrification rate was significantly higher than LR treatment group (Fig. 7) after the result showed 7 days, reached 96%.All treatment group (do not add LR umbrella grass treatment group except) can both be separated to bacterial strain LR(table 3 after 7 days).
Different time sections LR survival condition in table 3 Hoagland nutritive medium and the sewage
" ND " expression does not detect in the upper table, and "+" expression can be separated to bacterial strain LR, and expression "-" is not separated to bacterial strain LR.
Embodiment 4. take extraneous sewage as N element eutrophication liquid implement plant-microorganism and unite reparation
Sewage is taken from Nanjing one body eutrophication river course, and the sewage nitrogen content is 16.3mg-N/l, and measuring method is identical with detection method among the embodiment 3 all to be with alkaline alkaline potassium per-sulfate digestion determined by ultraviolet spectrophotometry (with reference to GB GB11894-89).The selection of umbrella grass, thalline, the umbrella grass roots is the collection method of secretory product, the treatment group setting, nitrogen content is measured with embodiment 3 in the water body, and test water body volume is 400ml.Carried out thalline in 1,2,3,7 day after processing and separate, separating step is with embodiment 4.The result shows that LR+ umbrella grass treatment group nitrogen-removing rate is significantly higher than other three groups, wherein adding separately thalline LR treatment group denitrification rate is 7%, and several time period no significant differences, LR+ umbrella grass roots is that the denitrification rate of secretory product treatment group second day is 45%, denitrification rate two days later and second day no significant difference (Fig. 8).The thalline separating resulting shows: LR+ umbrella grass beginning treatment group can be separated to bacterial strain LR eventually, four time periods all separate less than bacterial strain LR after adding bacterial strain LR separately, LR+ umbrella grass roots is that after processing the 1st, 2 day of secretory product treatment group can be separated to thalline LR, can't be separated to thalline LR(table 3 at the 3rd day and the 7th day).Infer tentatively that accordingly the umbrella grass is root exudates to the promoter action of LR.
Element eutrophication liquid external source interpolation umbrella grass roots is the experiment of secretory product denitrification to embodiment 5. take extraneous sewage as N
Adopt used sewage among the embodiment 4, the water body volume is 400ml.The selection of umbrella grass, thalline, the umbrella grass roots is the collection method of secretory product, and nitrogen content is measured with embodiment 4 in the water body, three treatment group is set: thalline LR treatment group, thalline LR+ umbrella grass roots is the secretory product treatment group, and thalline LR+ umbrella grass roots is secretory product (20ml/2 days) treatment group.Wherein thalline LR treatment group is only added thalline LR in sewage: choose the thalline LR that grows to logarithmic phase, be resuspended in the deionized water with behind the distilled water centrifuge washing three times, cell concentration transfers to 10
6CFu/ml 1/100 is seeded in the 400ml sewage by volume; Thalline LR+ umbrella grass roots be the secretory product treatment group when processing except add with the thalline LR of thalline LR treatment group with concentration, also add 20ml umbrella grass roots border secretory product; Thalline LR+ umbrella grass roots is that secretory product (20ml/2 days) treatment group is except interpolation and the thalline LR of thalline LR treatment group with concentration, after adding first 20ml umbrella grass secretory product, added a umbrella grass roots border secretory product in per two days, front two groups replace with the equal volume deionized water.Experimental result shows: thalline LR+ umbrella grass roots is that the nitrogen clearance of secretory product (20ml/2 days) treatment group after 7 days is the highest, thalline LR treatment group denitrification rate only is 7%, thalline LR+ umbrella grass roots is the secretory product treatment group at the 1st, 2 day denitrification rate and thalline LR+ umbrella grass roots is secretory product (20ml/2 days) treatment group there was no significant difference, but compared there was no significant difference with the 7th day denitrification rate with second day in its 3rd day, and showed that the denitrification of thalline stopped (Fig. 9) two days later.From the thalline separating resulting: LR+ umbrella grass roots is that after processing 1,2,3,7 day of secretory product (20ml/2 days) treatment group is all separable to thalline LR, treatment group LR all fails to isolate LR at four time point places, treatment group LR+ umbrella grass roots is that LR can be isolated in after processing the 1st, 2 day of secretory product from water body, but fails to isolate thalline LR(table 4 at the 3rd day and the 7th day).
Different time sections LR survival condition in table 4 sewage
"+" expression can be separated to bacterial strain LR in the upper table, and expression "-" is not separated to bacterial strain LR.
Related " bacterial strain LR " refers to that all deposit number is the pseudomonas LR of CCTCC NO:M2011159 among the present invention.
Claims (6)
1. a strain denitrifying bacterium LR, Classification And Nomenclature is pseudomonas LR(
PseudomonasSp.LR), be preserved in Chinese Typical Representative culture collection center on May 3rd, 2011, culture presevation number is CCTCC NO:M2011159.
2. preserving number claimed in claim 1 is the application of denitrifying bacterium LR in the Pollution abatement of nitrogen eutrophication water of CCTCC NO:M2011159.
Preserving number claimed in claim 1 be CCTCC NO:M2011159 denitrifying bacterium LR associating umbrella grass (
Cyperus alternifoliusL.) application in the Pollution abatement of nitrogen eutrophication water.
4. one kind is carried out the method that plant-microorganism is united reparation to the nitrogen eutrophication water, it is characterized in that plantation umbrella grass in the water body of nitrogen eutrophication, and inoculating described preserving number is the denitrifying bacterium LR of CCTCC NO:M2011159; Wherein every premium on currency body plantation umbrella grass 7 ~ 15 strains, every premium on currency body inoculum density is 10
6The preserving number of cFu/ml is the denitrifying bacterium LR bacterium liquid 10ml of CCTCC NO:M2011159.
5. method of administering nitrogen eutrophic water body pollution, it is characterized in that adding the umbrella grass roots in the water body of nitrogen eutrophication is secretory product, and the inoculation preserving number is CCTCC NO:M2011159 denitrifying bacterium LR, it is secretory product that every premium on currency body adds 50ml umbrella grass roots, and every premium on currency body inoculum density is 10
6The preserving number of cFu/ml is the denitrifying bacterium LR bacterium liquid 10ml of CCTCC NO:M2011159, and umbrella grass roots of interpolation in per two days is secretory product after the inoculation, and each every premium on currency body adds 40 ~ 60ml;
Wherein, described umbrella grass roots is that secretory product is collected preparation by the following method: the umbrella grass roots is after deionized water drip washing, be 75%-80% in relative humidity, temperature is 25 ℃, intensity of illumination is that the interior deionized water that uses of the climatic chamber of 3000Lx ~ 5000Lx soaks root system of plant 4 ~ 7h, and is concentrated into 1/10 volume with Rotary Evaporators; Described umbrella grass roots system is 1g:10 ~ 30ml with the mass volume ratio of the deionized water that is used for root immersion.
6. the method for improvement nitrogen eutrophic water body pollution according to claim 5 is characterized in that described umbrella grass roots system and the mass volume ratio of the deionized water that is used for root immersion are 1g:20ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101481669A CN102676431B (en) | 2012-05-14 | 2012-05-14 | Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101481669A CN102676431B (en) | 2012-05-14 | 2012-05-14 | Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102676431A CN102676431A (en) | 2012-09-19 |
| CN102676431B true CN102676431B (en) | 2013-05-29 |
Family
ID=46808987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101481669A Expired - Fee Related CN102676431B (en) | 2012-05-14 | 2012-05-14 | Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102676431B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525730B (en) * | 2013-10-14 | 2015-12-02 | 青岛蔚蓝生物集团有限公司 | A kind of otitis pseudomonas strains and application thereof |
| CN103588306B (en) * | 2013-10-29 | 2015-02-11 | 中国科学院南京土壤研究所 | Application of fatty acid amide compound |
| CN104556349B (en) * | 2014-12-11 | 2016-11-30 | 浙江海洋学院 | A kind of external source plant nutrient strengthening raw water biological pretreatment technique |
| CN107827240B (en) * | 2017-04-21 | 2020-12-11 | 湖南大学 | In-situ remediation method for jointly treating poor water body by using pseudomonas loaded by roots of lemna minor |
| CN107285482B (en) * | 2017-08-05 | 2020-09-25 | 贵州天保生态股份有限公司 | Environment-friendly enzyme for purifying eutrophic water and preparation method thereof |
-
2012
- 2012-05-14 CN CN2012101481669A patent/CN102676431B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| 反硝化细菌的筛选及其亚硝酸盐降解特性研究;罗秀针等;《福建农业学报》;20101231;第25卷(第4期);513-516 * |
| 好氧反硝化菌Pseudomonas aeruginosaNO62 筛选分离与性质鉴定;蔡亚君等;《长江大学学报(自然科学版) 》;20110131;第8卷(第1期);240-243 * |
| 王怡.水生植物对城市生活污水的净化能力研究.《中国优秀博硕士学位论文全文数据库(硕士)工程科技I辑》.2005,(第08期),B027-136. * |
| 罗秀针等.反硝化细菌的筛选及其亚硝酸盐降解特性研究.《福建农业学报》.2010,第25卷(第4期),513-516. |
| 蔡亚君等.好氧反硝化菌Pseudomonas aeruginosaNO62 筛选分离与性质鉴定.《长江大学学报(自然科学版) 》.2011,第8卷(第1期),240-243. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102676431A (en) | 2012-09-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105255782B (en) | There is fiber bacterium and the purposes of reducing power to Cr VI | |
| CN111423996B (en) | Complex microbial inoculant, preparation method thereof and application thereof in soil remediation field | |
| CN105170628B (en) | Method for repairing lead-polluted soil by combining plants and microorganisms | |
| CN110819556B (en) | Rhizobium and microbial inoculum and application thereof | |
| CN107384840B (en) | Drought-resistant growth-promoting compound microbial inoculum and application thereof | |
| CN102676431B (en) | Denitrifying bacteria and aquatic plant-microbe combined rehabilitation method using same | |
| CN104673715A (en) | Enteric bacilli with fixing effect on cadmium capable of promoting plant growth and application of enteric bacilli | |
| CN102851237B (en) | Ensiferadhaerens and application thereof | |
| CN104263682B (en) | Plant-growth-promoting endophytic bacterium having polycyclic aromatic hydrocarbons degrading function and application thereof | |
| CN105199979A (en) | Bacillus thuringiensis YLX-4 and application thereof | |
| CN116948882B (en) | Achromobacter madepuralensis and application thereof | |
| CN106399176A (en) | Paenibacillus and its application in water body purification | |
| CN109576177B (en) | Chinese micro-rod strain SM8 and application thereof in salt tolerance and growth promotion | |
| CN111117909A (en) | Strain capable of resisting multiple heavy metals and promoting plant growth and application thereof | |
| CN112760262B (en) | Erythromycin degradation composite microbial inoculum and preparation method and application thereof | |
| CN110257293A (en) | P. amylolyticus KY15, microbial inoculum, application and the product using it | |
| CN112280694B (en) | Plant endophytic fungus phomopsis D2G7 and application thereof | |
| CN113801817A (en) | Phosphate solubilizing bacteria 3-1 and application thereof in dissolving phosphate | |
| CN108841748A (en) | Sinorhizobium nitrogen-fixing bacteria strain H6 and its application | |
| CN118480465A (en) | Bacillus pumilus for saline-alkali soil crops and application thereof | |
| CN104845898B (en) | Providence (Providencia sp.) 2D of one high-efficiency degradation dibutyl phthalate | |
| CN106591173A (en) | Bacillus flexus HL-37 capable of activating soil heavy metal cadmium, and applications thereof | |
| CN113897320B (en) | Halophyte with salt-tolerant growth-promoting function and application thereof | |
| CN112011487B (en) | Aroma-like bacterial strain C40 capable of degrading phenol and having growth promoting function and application thereof | |
| CN110157637A (en) | Enterobacteria Z1 and klebsiella Z2 composite bacteria agent removal high nitrogen pollutant effluents and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 211225 Lishui County, Nanjing City, white horse town national agricultural science and Technology Park, Nanjing Agricultural University, Applicant after: Nanjing Agricultural University Address before: Weigang Xuanwu District of Nanjing Jiangsu province 210095 No. 1 Applicant before: Nanjing Agricultural University |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130529 Termination date: 20170514 |