CN103865852B - One strain can the denitrifying bacteria of efficient denitrification and application thereof under low ratio of carbon to ammonium condition - Google Patents
One strain can the denitrifying bacteria of efficient denitrification and application thereof under low ratio of carbon to ammonium condition Download PDFInfo
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Abstract
The invention discloses a strain can the denitrifying bacteria of efficient denitrification and application thereof under low ratio of carbon to ammonium condition.Does is denitrifying bacteria provided by the present invention genus bacillus (Bacillus? sp.) DN-LWX4.It can be used for removing nitrate nitrogen, described genus bacillus (Bacillus? does is sp.) DN-LWX4 CGMCC at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center? No.8290.Genus bacillus (Bacillus of the present invention? sp.) DN-LWX4 can utilize the complete denitrifications of several kinds of carbon source such as glucose, ethanol, acetic acid, sodium acetate, initial NO
3 -when-N concentration is 110.13mg/L, NO
3 --N clearance all can reach 95%.The condition of carbon-nitrogen ratio 3:1 just can meet the demand of bacterium to carbon source, initial NO
3 -when-N concentration is 110.13mg/L, nitrate nitrogen clearance can reach 99.52%.
Description
Technical field
The present invention relates to a strain can the denitrifying bacteria of efficient denitrification and application thereof under low ratio of carbon to ammonium condition.
Background technology
Urban sewage reutilization alleviates shortage of water resources, water prevention environmental pollution, improves the effective way of quality of water environment.Municipal wastewater treatment plant secondary effluent through manipulation of regeneration can be used for the many aspects such as cities and towns are used mixedly, water source covering, view amusement, process water, agriculture, forestry, animal husbandry and fishery water.But due to nutritive substances such as the nitrogen that contains in municipal effluent secondary effluent, be the key factor causing body eutrophication and wawter bloom risk.Therefore, the nitrogen concentration controlled in reuse water is most important for surface water bodies such as urban sewage recycling feeder lakes.
For the improvement of nitrate pollution, biological denitrificaion is most economical effective denitrogenation mode.Biological denitrificaion is divided into two parts, nitrification and denitrification process, and nitrification is nitrate nitrogen by mineralized nitrogen; Denitrification the nitrate nitrogen in water body is converted into nitrite nitrogen and a series of intermediate gaseous product, finally becomes innocuous gas nitrogen and remove from water body, is the significant process of thorough denitrogenation from water body.Denitrification is realized by the vital movement of denitrifying bacteria, and therefore the discovery of efficient denitrifying bacteria is significant to biological denitrificaion.
Denitrifying bacteria is heterotrophic bacterium, and COD concentration is lower in municipal effluent secondary effluent, additional carbon is needed to meet denitrification, therefore, research types carbon sources, carbon-nitrogen ratio can find out effective carbon source more economically to the impact of denitrifying bacteria nitrogen removal characteristics, cost-saving.And municipal effluent secondary effluent carbon source kind is numerous and diverse, finds to utilize the denitrifying bacteria of several kinds of carbon source can improve the utilization ratio of carbon source, reduce the input of additional carbon, for engineer applied is cost-saving.
Summary of the invention
For above problem, the object of this invention is to provide a strain efficiency utilization several kinds of carbon source and realize thorough denitrifying bacterium, this bacterium also can realize complete denitrification under low ratio of carbon to ammonium condition, and denitrifying intermediate product nitrite nitrogen (NO
2 --N) accumulation volume few.
Denitrifying bacterium provided by the present invention is genus bacillus (Bacillussp.) DN-LWX4, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 29th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNo.8290.
The 16srDNA sequence of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is as sequence in sequence table 1, and the morphological specificity of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is as follows: bacterium colony circle, neat in edge, yellow transparent, surface smooth; Shaft-like, the long 30-40 μm of thalline, Gram-positive.The physiological and biochemical property of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is as shown in table 2.
Another object of the present invention is to provide a kind of denitrification microbial inoculum, and the activeconstituents of this denitrification microbial inoculum is described genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290.
Described microbial inoculum also can comprise carrier.Described carrier can be solid carrier or liquid vehicle.Described solid carrier is mineral material, vegetable material or macromolecular compound; Described mineral material is at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; Described vegetable material is at least one in Semen Maydis powder, bean powder and starch; Described macromolecular compound is polyvinyl alcohol and/or polyglycol.Described liquid vehicle can be vegetables oil, mineral oil or water; Described organic solvent is decane and/or dodecane.In described microbial inoculum, described activeconstituents can with by cultivate viable cell, the fermented liquid of viable cell, the filtrate of cell culture or cell and filtrate the form of mixture exist.The formulation of described microbial inoculum can be multiple formulation, as liquor, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
As required, tensio-active agent (as polysorbas20, tween 80 etc.), tackiness agent, stablizer (as antioxidant), pH adjusting agent etc. can also be added in described microbial inoculum.
Denitrification biofilm containing described genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 or denitrification biofilm reactor also belong to protection scope of the present invention.
This denitrification biofilm specifically by hand-stuff or natural materials as carrier, described genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 adheres to membranoid substance that its surface of flocculation is formed.
Above-mentioned genus bacillus (Bacillussp.) DN-LWX4, above-mentioned denitrification microbial inoculum, above-mentioned denitrification biofilm or denitrification biofilm reactor are at removal nitrate nitrogen (NO
3 --N) and/or nitrite nitrogen (NO
2 --N) in application also belong to protection scope of the present invention.
Above-mentioned removal nitrate nitrogen (NO
3 --N) and/or nitrite nitrogen (NO
2 --N) can be the nitrate nitrogen (NO removed in liquid
3 --N) and/or nitrite nitrogen (NO
2 --N).Described liquid can be containing nitrate nitrogen (NO
3 --N) and/or nitrite nitrogen (NO
2 --N) any liquid, as containing nitrate nitrogen (NO
3 --N) and/or nitrite nitrogen (NO
2 --N) waste water.In one embodiment of the invention, described liquid is for using carbon source, KH
2pO
40.5g, MgSO
4.7H
2o0.2g, KNO
30.8g, micro-1mL and water are mixed with the liquid of 1 liter; The pH of described liquid is 7.0-7.2, and the nitrate nitrogen concentration of described liquid is 110.13mg/L, COD concentration is 1.87 ± 0.10g/L; Described carbon source is glucose, ethanol, acetic acid and/or sodium acetate.
Described genus bacillus (Bacillussp.) DN-LWX4 can cultivate in the substratum for cultivating genus bacillus.Its suitable culture conditions is: pH7.0, temperature 30 DEG C.
Genus bacillus of the present invention (Bacillussp.) DN-LWX4 is separated and obtains from anoxic denitrification biological filter, and the several kinds of carbon source such as glucose, ethanol, acetic acid, sodium acetate can be utilized to realize complete denitrification, initial NO
3 -when-N concentration is 110.1mg/L, NO
3 --N clearance all can reach 95%, and shaking flask Nitrite Nitrogen accumulated concentrations 72h can be down to 0.05mg/L(Fig. 4), accumulate less.Can be observed bubble in experimentation to produce, illustrate that denitrification is thorough.Under low ratio of carbon to ammonium condition, denitrification can carry out thoroughly, and the condition of carbon-nitrogen ratio 3:1 just can meet the demand of bacterium to carbon source, initial NO
3 -when-N concentration is 110.1mg/L, nitrate nitrogen clearance can reach 99.5%.Nitrogen element in this bacterial strain energy effective elimination water body, several kinds of carbon source can be utilized under low ratio of carbon to ammonium condition to realize denitrification, and treatment effect efficient stable, is widely used.
preservation explanation
Strain name: genus bacillus
Latin name: (Bacillussp.)
Strain number: DN-LWX4
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on September 29th, 2013
Register on the books numbering: CGMCCNo.8290 at preservation center
Accompanying drawing explanation
Fig. 1 is genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 nitrate removal ability under different carbon source.
Fig. 2 is the nitrite accumulation change of different carbon source substratum.
Fig. 3 is genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 nitrate removal ability under different carbon-nitrogen ratio.
Fig. 4 is the nitrite accumulation of different carbon-nitrogen ratio denitrification substratum.
Fig. 5 is genus bacillus (Bacillussp.) DN-LWX4 stereoscan photograph.
In Fig. 1-Fig. 2, glucose represents glucose denitrification substratum, and ethanol represents ethanol denitrification substratum, and acetic acid represents acetic acid denitrification substratum, and sodium acetate represents sodium acetate denitrification substratum.
In Fig. 3-Fig. 4,1:3 represents 3:1 carbon-nitrogen ratio denitrification substratum, and 1:5 represents 5:1 carbon-nitrogen ratio denitrification substratum, and 1:7 represents 7:1 carbon-nitrogen ratio denitrification substratum, 1:10 represents 10:1 carbon-nitrogen ratio denitrification substratum, and 1:15 represents 15:1 carbon-nitrogen ratio denitrification substratum.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
COD in following embodiment adopts and connects China Tech skill COD rapid test method, and concrete grammar is as follows: INSTRUMENT MODEL: connect China Tech skill COD Quick testing instrument 5B-1 type; Reagent: connect China Tech skill COD water quality and monitor special consumptive material LH-DE-500 type; Determination step: get 2.5ml water sample to be measured in test tube, add 0.7mlD reagent, 4.8mlE reagent, mixing, COD Quick testing instrument 165 DEG C clears up 10min, cools 2min after taking-up, adds 2.5ml high purity water, abundant mixing, measures absorbancy after cooling under 610nm wavelength.COD concentration (mg/L)=Abs
610× 1505 × 3.
NO in following embodiment
3 --N concentration adopts peroxide potassium sulfate-ultraviolet spectrophotometry, surveys biological filter water outlet NO
3 -during-N concentration, direct taking-up water dilutes 25 times and measures, and measures substratum NO in microbial cultivation process
3 -during-N concentration, after centrifugal for bacteria suspension to be measured, getting supernatant liquor 0.22 μm of membrane filtration, doubly measuring filtering the liquid diluting 100 obtained.
NO in following embodiment
2 -n concentration: 1-(naphthyl)-quadrol spectrophotometry.
In the present invention, denitrogenation refers to Inorganic Nitrogen in Water Bodies NO
3 --N, NO
2 -the removal of-N.
NO in following embodiment
3 --N clearance=(NO before process
3 -nO after-N concentration-process
3 --N concentration) the front NO of/process
3 --N concentration.
In the present invention, carbon-nitrogen ratio is the ratio of COD concentration (mg/L) and nitrate nitrogen concentration (mg/L).
COD value before COD clearance in following embodiment=(before process the rear COD value of COD value-process)/process.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The isolation identification of embodiment 1, genus bacillus (Bacillussp.) DN-LWX4
One, the separation of genus bacillus (Bacillussp.) DN-LWX4
Denitrification filter pool (sampling the same day water quality in table 1) water outlet filtrate 10 is placed in and 40ml enrichment medium is housed (is mixed with the liquid of 1 liter with extractum carnis 5g, peptone 10g, saltpetre 1g and water, pH is 7.0-7.2) 100ml triangular flask in 30 DEG C of incubated overnight, allow microorganism dispersion enrichment and growth on filtrate, draw 1ml bacteria suspension in the centrifuge tube that 9ml sterilized water is housed, obtain extent of dilution 10
-1bacteria suspension, bacteria suspension sterilized water is diluted to 10 by Using such method
-15, obtain the bacteria suspension of a series of dilution gradient.Draw 0.1ml extent of dilution 10 respectively
-8--10
-15bacteria suspension (be mixed with the liquid of 1 liter with extractum carnis 5g, peptone 10g, saltpetre 1g, agar 18g and water to denitrifying bacteria isolation medium.PH is 7.0-7.2), even with glass stick coating, be inverted in 30 DEG C of cultivations, to growing obvious bacterium colony after sealed membrane sealing.
The different single bacterium colony of picking form is to incubated overnight in enrichment medium, and repeatedly purifying on flat board, till basis of microscopic observation display is without miscellaneous bacteria, obtains the bacterial strain of purifying.The experiment of nitrate reduction aerogenesis is carried out to the bacterial strain after purifying, pick out denitrification thorough, aerogenesis is many, and the fast inoculation of aerogenesis saves backup to slant medium (be mixed with the liquid of 1 liter with extractum carnis 5g, peptone 10g, saltpetre 1g, agar 18g and water, pH is 7.0-7.2).The denitrification strain screened being numbered to DN-LWX4 is thorough, and aerogenesis is many, and the fast bacterial strain of aerogenesis carries out the experiment of following nitrate reduction aerogenesis: in Boiling tube, add 25mL denitrification substratum (often liter of denitrification substratum: CH
3cOONa2g; KH
2pO
40.4g; ; MgSO
4.7H
2o0.6g; CaCl
2.2H
2o0.07g; KNO
31g; Tris damping fluid 12mL; Trace element 1mL(solvent is high purity water, solute and concentration as follows: H
3bO
32.86g/L; ZnSO
4.7H
2o0.22g/L; CuSO
4.5H
2o0.08g/L; MnSO
4.4H
2o2.03g/L; Na
2moO
4.2H
2o1.26g/L; PH7.0); 1L is settled to high purity water; PH controls at 7.0-7.2) sterilizing 20 minutes under 115 DEG C of conditions, inoculate 1mL bacterium liquid in each test tube, add the fluid-tight of 1mL paraffin, silica gel plug sealing is placed on 30 DEG C of constant incubators and cultivates, and measures NO after 72h
3-, NO
2-concentration (centrifugal rear 0.22 μm of membrane filtration), NO
3--N clearance 87.2%, has bubble to produce in test tube.
Table 1 samples Inlet and outlet water water quality on the same day
Two, the qualification of genus bacillus (Bacillussp.) DN-LWX4
1,16srDNA sequence homology analysis
(1) DNA extraction
Bacterial strain DN-LWX4, after 30 DEG C of enrichment culture 24h, gets 2mL bacteria suspension in centrifuge tube, and under room temperature, the centrifugal 5min of 12000r/min, abandons supernatant liquor.In the bacterial sediment obtained, add 567 μ LTEbuffer, piping and druming makes it Eddy diffusion repeatedly, adds 30 μ L10%SDS and 10 μ L Proteinase Ks afterwards, after mixing, and 37 DEG C of incubation 1h.Then add 100 μ L5moL/LNaCl solution, fully mix, then add 80 μ LCTAB/NaCl solution, incubation 10min at mixing latter 65 DEG C.Add in centrifuge tube and all solution isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1) added before, mix the centrifugal 10min of rear 12000r/min.In transfer centrifuge tube in the new 2mL centrifuge tube of supernatant liquor to, add the Virahol of 0.6-0.8 times of volume, mix gently to DNA and precipitate, the centrifugal 5min of 12000r/min afterwards, and abandon supernatant liquor.By the precipitation 1mL70% washing with alcohol obtained, the centrifugal 10min of 12000r/min, obtains DNA precipitation after abandoning supernatant liquor.After DNA is precipitated seasoning by the super clean bench of cleaning, add 95 μ LTEbuffer, put into-20 DEG C of Refrigerator stores.
(2) pcr amplification
Get 1 μ L bacterial strain DN-LWX4DNA extracting solution as template, adopt bacterial universal primers (upstream primer 27F:5 '-AGAGTTTGATCMTGGCTCAG-3 ' [M=C, A]; Downstream primer 1492R:5 '-CGGYTACCTTGTTACGACTT-3 ' [Y=T, C]) carry out pcr amplification.Pcr amplification reaction system is 25 μ L(2*PCRmixbuffer12.5 μ L, each 0.2 μm of ol/L of upstream and downstream primer, and DNA profiling 1 μ L, ddH2O mend to 25 μ l).PCR primer 1.5% agarose gel electrophoresis detects output and specificity.Then cloning and sequencing is carried out.
(3) 16SrDNA sequential analysis:
The 16srDNA sequence of bacterial strain DN-LWX4 refers to sequence 1 in sequence table.Bacterial strain DN-LWX4 and BacilluskorlensisstrainZLC-26(type strain) sequence similarity 97.8%; Determine that it is bacillus (Bacillussp.).
2, Morphological Identification
Logarithmic phase will be in, and bacterium colony size is stablized, above-mentioned steps one be separated and the bacterial strain DN-LWX4 that obtains of purifying carry out single bacterium colony state observation, mainly comprise the size of bacterium colony, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), colony edge state (whether neat, irregular, radial etc.).
For the bacterial strain DN-LWX4 being in logarithmic phase, after smear staining, adopt the form of observation by light microscope thalline.
Result shows, above-mentioned steps one is separated and bacterium colony circle of the bacterial strain DN-LWX4 that obtains of purifying, neat in edge, yellow transparent, surperficial smooth; Shaft-like, the long 30-40 μm of thalline, Gram-positive (Fig. 5).
3, analysis of physio biochemical characteristics
VITEK2 Gram-negative bacteria identification card (BCL) and Mei Liai VITEK2 automatic bacterial assessing instrument is used to measure the physiological and biochemical property of bacterial strain DN-LWX14.The physiological and biochemical property measurement result of bacterial strain DN-LWX4 and bacterial strain BacilluskorlensisstrainZLC-26 is as shown in table 2.
The physiological and biochemical property of table 2 bacterial strain DN-LWX4 and BacilluskorlensisstrainZLC-26
Note :+represent positive;-represent negative; (+) represents the weak positive; (-) represents weak feminine gender.The physiological and biochemical property of BacilluskorlensisstrainZLC-26 is the result of following bibliographical information: LeiZhang, etal.Bacilluskorlensissp.nov., amoderatelyhalotolerantbacteriumisolatedfromasandsoilsam pleinChina.InternationalJournalofSystematicandEvolutiona ryMicrobiology (2009), 59,1787 – 1792.
According to above-mentioned 16srDNA sequence homology analysis, morphological specificity and physiological and biochemical property result, bacterial strain DN-LWX4 step one separation and purification obtained is accredited as genus bacillus, called after Bacillussp..This genus bacillus (Bacillussp.) DN-LWX4 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 09 29th, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNo.8290.
The preparation of embodiment 2, denitrification microbial inoculum
By slant medium, (often liter of slant medium is mixed with the liquid of 1 liter; PH is 7.0-7.2; Sterilizing 20 minutes under 115 DEG C of conditions) transfering loop picking one ring of upper genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 sterilizing of preserving is seeded to and 40ml enrichment medium is housed (often liter of enrichment medium is mixed with the liquid of 1 liter, and pH is 7.0-7.2; Sterilizing 20 minutes under 115 DEG C of conditions) 100ml Erlenmeyer flask in, 12h are cultivated in the concussion of 30 DEG C of shaking tables, and the bacterium liquid of acquisition is denitrification microbial inoculum, and the content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 in this denitrification microbial inoculum is 1 × 10
15cfu/ml.
Embodiment 3, types carbon sources are on the denitrifying impact of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290
Preparation NO
3 --N concentration is 110.13mg/L, and COD concentration is four kinds of carbon source denitrification substratum (carbon-nitrogen ratio is 17:1) of 1.87 ± 0.10g/L, glucose denitrification substratum respectively, sodium acetate denitrification substratum, acetic acid denitrification substratum and ethanol denitrification substratum.Often liter of these four kinds of carbon source denitrification substratum are all with carbon source, KH
2pO
40.5g, MgSO
4.7H
2o0.2g, KNO
30.8g, micro-1mL(solvent are high purity water, solute and concentration as follows: H
3bO
32.86g/L; ZnSO
4.7H
2o0.22g/L; CuSO
4.5H
2o0.08g/L; MnSO
4.4H
2o2.03g/L; Na
2moO
4.2H
2o1.26g/L; PH7.0) and water be mixed with the liquid of 1 liter; PH is 7.0-7.2.Carbon source in glucose denitrification substratum is glucose, and the carbon source in sodium acetate denitrification substratum is sodium acetate, and the carbon source in acetic acid denitrification substratum is acetic acid, and the carbon source in ethanol denitrification substratum is ethanol.In these four kinds of carbon source denitrification substratum, the addition of carbon source will meet and makes the COD concentration of these four kinds of substratum be 1.87 ± 0.10g/L.
Three repetitions are established in experiment, repeat to get 8 500mL Erlenmeyer flasks at every turn, are divided into four groups, often organize 2 500mL Erlenmeyer flasks, are handled as follows respectively simultaneously:
Glucose is the process of sole carbon source denitrification: in each 500mL Erlenmeyer flask, to add under 450mL glucose denitrification substratum 115 DEG C of conditions sterilizing 20 minutes, and (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
Sodium acetate is the process of sole carbon source denitrification: in each 500mL Erlenmeyer flask, to add under 450mL sodium acetate denitrification substratum 115 DEG C of conditions sterilizing 20 minutes, and (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
Acetic acid is the process of sole carbon source denitrification: in each 500mL Erlenmeyer flask, to add under 450mL acetic acid denitrification substratum 115 DEG C of conditions sterilizing 20 minutes, and (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
Ethanol is the process of sole carbon source denitrification: in each 500mL Erlenmeyer flask, to add under 450mL ethanol denitrification substratum 115 DEG C of conditions sterilizing 20 minutes, and (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
Result shows at initial medium NO
3 --N concentration is 110.13mg/L, COD concentration is under the condition of 1.87 ± 0.10g/L, and genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is to glucose denitrification substratum, ethanol denitrification substratum, acetic acid denitrification substratum and the sodium acetate denitrification substratum NO of 60 hours
3 --N clearance is respectively 96.6%, 99.9%, 98.2%, 98.4%(Fig. 1), 72h nitrite nitrogen accumulated concentrations is respectively 5.20mg/L, 0.07mg/L, 0.05mg/L and 2.12mg/L(Fig. 2).
Embodiment 4, carbon-nitrogen ratio are on the denitrifying impact of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290
Preparation NO
3 --N concentration is 110.13mg/L, five kinds of carbon-nitrogen ratio denitrification substratum that carbon-nitrogen ratio is different, 3:1 carbon-nitrogen ratio denitrification substratum respectively, 5:1 carbon-nitrogen ratio denitrification substratum, 7:1 carbon-nitrogen ratio denitrification substratum, 10:1 carbon-nitrogen ratio denitrification substratum and 15:1 carbon-nitrogen ratio denitrification substratum.Often liter of these five kinds of carbon-nitrogen ratio denitrification substratum are all with sodium acetate, KH
2pO
40.5g, MgSO
4.7H
2o0.2g, KNO
30.8g, micro-1mL(solvent are high purity water, solute and concentration as follows: H
3bO
32.86g/L; ZnSO
4.7H
2o0.22g/L; CuSO
4.5H
2o0.08g/L; MnSO
4.4H
2o2.03g/L; Na
2moO
4.2H
2o1.26g/L; PH7.0) and water be mixed with the liquid of 1 liter; PH is 7.0-7.2.Wherein, in 3:1 carbon-nitrogen ratio denitrification substratum, the addition of sodium acetate will meet the carbon-nitrogen ratio making 3:1 carbon-nitrogen ratio denitrification substratum is 3:1, in 5:1 carbon-nitrogen ratio denitrification substratum, the addition of sodium acetate will meet the carbon-nitrogen ratio making 5:1 carbon-nitrogen ratio denitrification substratum is 5:1, in 7:1 carbon-nitrogen ratio denitrification substratum, the addition of sodium acetate will meet the carbon-nitrogen ratio making 7:1 carbon-nitrogen ratio denitrification substratum is 7:1, in 10:1 carbon-nitrogen ratio denitrification substratum, the addition of sodium acetate will meet the carbon-nitrogen ratio making 10:1 carbon-nitrogen ratio denitrification substratum is 10:1, in 15:1 carbon-nitrogen ratio denitrification substratum, the addition of sodium acetate will meet the carbon-nitrogen ratio making 15:1 carbon-nitrogen ratio denitrification substratum is 15:1.
Three repetitions are established in experiment, repeat to get 10 500mL Erlenmeyer flasks at every turn, are divided into five groups, often organize 2 500mL Erlenmeyer flasks, are handled as follows respectively simultaneously:
3:1 carbon-nitrogen ratio denitrification substratum denitrification process: to add under 3:1 carbon-nitrogen ratio denitrification substratum 115 DEG C of conditions of 450mL sterilizing 20 minutes in each 500mL Erlenmeyer flask, (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
5:1 carbon-nitrogen ratio denitrification substratum denitrification process: to add under 5:1 carbon-nitrogen ratio denitrification substratum 115 DEG C of conditions of 450mL sterilizing 20 minutes in each 500mL Erlenmeyer flask, (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
7:1 carbon-nitrogen ratio denitrification substratum denitrification process: to add under 7:1 carbon-nitrogen ratio denitrification substratum 115 DEG C of conditions of 450mL sterilizing 20 minutes in each 500mL Erlenmeyer flask, (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.。
10:1 carbon-nitrogen ratio denitrification substratum denitrification process: to add under 10:1 carbon-nitrogen ratio denitrification substratum 115 DEG C of conditions of 450mL sterilizing 20 minutes in each 500mL Erlenmeyer flask, (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
15:1 carbon-nitrogen ratio denitrification substratum denitrification process: to add under 15:1 carbon-nitrogen ratio denitrification substratum 115 DEG C of conditions of 450mL sterilizing 20 minutes in each 500mL Erlenmeyer flask, (content of genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 is 1 × 10 to inoculate the denitrification microbial inoculum of 6mL embodiment 2 in each Erlenmeyer flask
15cfu/ml), add preservative film after sealed membrane sealing and completely cut off outside air, be placed in 30 DEG C of constant incubators and cultivate, from inoculation (the inoculation moment is decided to be 0 hour), measure the NO of bacteria suspension every 4h
3 --N concentration, NO
2--N concentration (bacteria suspension centrifugal after get supernatant liquor 0.22 μm of membrane filtration after measure NO
3 --N concentration and NO
2--N concentration), the COD(bacteria suspension of bacteria suspension is not centrifugal, do not filter), determine bacterial strain denitrification ability.
Initial medium NO in experiment
3 --N concentration is 110.13mg/L, arranges the carbon-nitrogen ratio that 3:1,5:1,7:1,10:1,15:1 five is different.Under result shows different carbon-nitrogen ratio, final NO3-N-clearance is close, 3:1 carbon-nitrogen ratio denitrification substratum, 5:1 carbon-nitrogen ratio denitrification substratum, 60 hours NO3-N-clearances of 7:1 carbon-nitrogen ratio denitrification substratum, 10:1 carbon-nitrogen ratio denitrification substratum and 15:1 carbon-nitrogen ratio denitrification substratum be respectively 99.52%, 99.52%, 99.52%, 98.61% and 98.42%, 72h nitrite nitrogen accumulated concentrations be respectively 0.70mg/L, 0.11mg/L, 0.18mg/L, 0.27mg/L, 2.12mg/L(Fig. 4); Complete denitrification is got final product when genus bacillus (Bacillussp.) DN-LWX4CGMCCNo.8290 carbon-nitrogen ratio 3:1 is described.
Claims (10)
1. genus bacillus (Bacillussp.) DN-LWX4 is removing the application in nitrate nitrogen, and described genus bacillus (Bacillussp.) DN-LWX4 is CGMCCNo.8290 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the application of genus bacillus (Bacillussp.) DN-LWX4 in biological denitrificaion, described genus bacillus (Bacillussp.) DN-LWX4 is CGMCCNo.8290 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
3. genus bacillus (Bacillussp.) DN-LWX4 is preparing the application in denitrification microbial inoculum, and described genus bacillus (Bacillussp.) DN-LWX4 is CGMCCNo.8290 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
4. genus bacillus (Bacillussp.) DN-LWX4 is preparing the application in denitrification biofilm or denitrification biofilm reactor, and described genus bacillus (Bacillussp.) DN-LWX4 is CGMCCNo.8290 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
5. genus bacillus (Bacillussp.) DN-LWX4, it is CGMCCNo.8290 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
6. a denitrification microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is genus bacillus according to claim 5 (Bacillussp.) DN-LWX4.
7. the denitrification biofilm containing genus bacillus according to claim 5 (Bacillussp.) DN-LWX4 or denitrification biofilm reactor.
8. denitrification microbial inoculum according to claim 6, denitrification biofilm according to claim 7 or denitrification biofilm reactor are removing the application in nitrate nitrogen.
9. cultivating the method for genus bacillus according to claim 5 (Bacillussp.) DN-LWX4, comprising described genus bacillus according to claim 5 (Bacillussp.) DN-LWX4 for cultivating the step of cultivating in the substratum of genus bacillus.
10. the preparation method of denitrification microbial inoculum according to claim 6, denitrification biofilm according to claim 7 or denitrification biofilm reactor, comprises and genus bacillus according to claim 5 (Bacillussp.) DN-LWX4 is obtained denitrification microbial inoculum, denitrification biofilm or denitrification biofilm reactor as activeconstituents.
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