CN101665777A - Bacillus cereus with heterotrophic nitrification-aerobic denitrification performance and N2O emission control method thereof - Google Patents
Bacillus cereus with heterotrophic nitrification-aerobic denitrification performance and N2O emission control method thereof Download PDFInfo
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Abstract
The invention discloses a bacillus cereus with heterotrophic nitrification-aerobic denitrification performance and an N2O emission control method thereof, wherein the accession number of the bacilluscereus is CGMCC NO.3047. The bacillus cereus can degrade nitrate under the aerobic condition and perform denitrification; and the bacillus cereus can also grow in the ammonia nitrogen wastewater, perform heterotrophic nitrification-aerobic denitrification by using ammonia nitrogen under the aerobic condition, obtain the gaseous product N2O with extremely low emission amount in the denitrificationprocess and achieve the purpose of N2O emission control from the biological point of view.
Description
Technical field
The present invention relates to the bacterial strain that a strain has heterotrophic nitrification and aerobic denitrification capability, specifically have the bacillus cereus CGMCC NO.3047 and the N thereof of heterotrophic nitrification-aerobic denitrification capability
2The biological control of O ease method.
Background technology
In the multiple denitrogenation method of having used at present, biological denitrificaion remains the main means of denitrogenation of waste water in handling, wherein to the nitrification bacteria Study on Denitrification, be to tradition nitrated-the denitrification theory abundant with break through.The heterotrophic nitrification effect is meant that heterotrophic microorganism is under aerobic condition, ammonia or organic nitrogen compound are oxidized to the process of azanol, nitrite and nitrate, the importance of heterotrophic nitrification effect receives publicity just day by day, especially the discovery that has the allotrophic nitrobacteria of aerobic denitrification characteristic is for sewage water denitrification has been introduced completely new concept.
The early stage patent of contriver " has the Dell Ford bacterium of aerobic denitrification capability and handles the method for waste water " ZL 200610140872.3, and this patent is a kind of relating to adopt Dell's Ford (Delftia tsuruhatensis) WXZ-9 CGMCC No.1797 bacterial strain to carry out the method that biological denitrificaion is handled waste water under aerobic condition.Patent of invention " has the Comamonas testosteroni of aerobic denitrification capability and handles the method for waste water " ZL200610140870.4, and this patent is a kind of relating to adopt Comamonas testosteroni (Comamonastestosteroni) WXZ-18 CGMCC No.1800 bacterial strain and be applied to the method that denitrogenation of waste water is handled.Patent of invention " has the pseudomonas putida of aerobic denitrification capability and handles the method for waste water " ZL200610140871.9, and this patent is a kind of relating to adopt pseudomonas putida (Pseudomonas putida) WXZ-4 CGMCC No.1798 bacterial strain and be applied to the method that denitrogenation of waste water is handled.Patent of invention " has the careless spirillum of aerobic denitrification capability and handles the method for waste water " ZL 200610140869.1, and this patent is a kind of relating to adopt careless spirillum (Herbaspirillum huttiense) WXZ-14 CGMCC No.1799 bacterial strain and be applied to the method that denitrogenation of waste water is handled.These related bacterial strains of these four patents have excellent aerobic denitrification capability, can remove nitrate and nitroso-group nitrogen in the waste water.
Bacterial strain with denitrification capability can carry out anti-nitration reaction, and the anti-nitration reaction process comprises 4 reduction steps (relating to five kinds of wherein back three kinds of materials of nitrogenous substances is gas), is shown below.4 reduction steps are carried out in the aerobic denitrification reaction exactly under aerobic conditions, finished by nitrate reductase Nar, nitrite reductase Nir, nitric oxide reduction enzyme Nor, Nitrous Oxide reductase enzyme Nos catalysis respectively.
Heterotrophic nitrification effect (heterotrophic nitrification) is the Biochemical processes that heterotrophic microorganism participates in the inorganic nitrogen oxidation, and its substrate is the inorganic states ammonia nitrogen.In recent years, have the investigator to propose more strict heterotrophic nitrification effect and be defined as (Papen, 1998): heterotrophic microorganism under aerobic condition with ammonia/ammonium or organic nitrogen is oxidized to azanol, the process of nitrite and nitrate, that is:
At present do not see that as yet genus bacillus has the report of heterotrophic nitrification-aerobic denitrification capability abroad, but the domestic preliminary study of being correlated with that had.Hao Guiyu has studied the heterotrophic nitrification effect of genus bacillus among the SBR, points out the SBR cycle continuously after the operation, and sludge concentration can be relatively stable, and genus bacillus can the aerobic and anaerobic environment of fine adaptation, becomes the dominant bacteria in the mud.In the domestication process, influent ammonium concentration is about 20mg/L, and maximum ammonia nitrogen removal frank is lower than 60%, but does not point out whether to have denitrification denitrogenation function actually, therefore can not judge whether to exist aerobic denitrification capability.The 1 strain allotrophic nitrobacteria Bacillus sp.LY that He Xia etc. separate from membrane bioreactor is through being accredited as genus bacillus.Ammonia nitrogen concentration be respectively 40,80 and 3 kinds of situations of 120mg/L under, after the 120h reaction, ammonia-N removal rate is respectively 100%, 85.7%, 73.7%.But the nitrification of this bacterial strain starts slow, just can reach denitrification effect preferably at 120h.
Because main task is the nitrogen of removing in the waste water in this area, so in general people only are retained in denitrification to the focus of above-mentioned existing aerobic denitrification bacterial strain and can reach the purpose of removing nitrogen in the waste water to gaseous product and get final product.Because the toxicity of gas NO is very big, bacterial strain preserves from for self, just finds in the research in early days, and reductase enzyme Nir and Nor are basic to be occurred in pairs, and therefore general denitrogenation product is N
2O or N
2But along with the arrival of Global warming crisis, people pay close attention to greenhouse gases, CO more and more
2Be our known greenhouse gases, but N
2The monomer Greenhouse effect of O compare CO
2Be eager to excel 310 times.In addition N in the atmosphere
2O can also be decomposed into the bigger gas NO of toxicity through uviolizing; Can also react with ozone molecule, damage the ozone layer.For this reason, those skilled in the art are devoted to study the product N that how to reduce denitrification process always
2O, wherein the method for biology control promptly selects the bacterial strain of better effects if denitrification reaction can be proceeded to N fully
2, such bacterial strain should have four kinds of whole enzymes of above-mentioned reaction formula, has not yet to see relevant N
2The biological control ease of O bacterial strain report.
Existing other denitrogenation products N
2The technology control ease that has that the O control is escaped is reported, as passing through to regulate the process conditions of denitrification reaction, as temperature, pH or manufacturing radiation atmosphere etc.These method principles are to reach by the activity that improves Nitrous Oxide reductase enzyme Nos in the denitrogenation flora to realize control N substantially
2The purpose of O discharging.But technology control ease method can not solve the problem that some bacterial strain lacks Nitrous Oxide reductase enzyme Nos at all, promptly can not fundamentally solve N
2The problem that can O be reduced, so the process conditions change often improves N
2The ratio that O produces is very limited, the general back N that improves
2O accounts for and remove 10～30% of nitrogen amount from water body.Our scheme is to seek a kind of bacterial strain, has four kinds of whole enzymes of above-mentioned reaction formula, and Nor reductase enzyme site needs very near Nos reductase enzyme site the feasible N that produces
2Can be N by Nos reductase enzyme catalytic reduction rapidly in the time of O
2Because if can not be N by Nos reductase enzyme catalytic reduction rapidly
2, gas N
2O is in case produce and might evaporate in the air rapidly.
In sum, screen and both generate N
2The O amount is few, can guarantee the efficient bacterial strain of certain nitric efficiency again, is purpose of the present invention.
Summary of the invention
The object of the present invention is to provide a strain both also had been the bacterial strain that nitrogenous source is grown with the ammonia nitrogen with nitrate; It is inoculated in the conventional aerobic sludge, can realize the direct denitrogenation of nitrogen-containing wastewater, can solve that denitrogenation need utilize different nitrification and denitrification floras in traditional wastewater treatment, can solve because of different flora biological nature differences, need the more important thing is N in this bacterial strain denitrogenation product in anaerobic environment, nitrated problem as denitrification in the aerobic environment staging treating
2The O generating capacity only below 5/10000ths, can be realized N effectively
2The biological control ease of O.
Invention thinking of the present invention is that screening has N
2The efficient heterotrophic nitrification-aerobic denitrification bacterial strain of the spontaneous growth advantage of the biological control ease of O characteristic, and to the bacterial strain discriminating of checking order.With the bacterial strain that screening obtains, be inoculated in the waste water that contains nitrate, it can carry out the aerobic denitrification denitrogenation effectively; With this inoculation in the ammonia nitrogen waste water of different concns, and by differential responses condition test (dissolved oxygen, carbon-nitrogen ratio, pH etc.), also can carry out aerobic denitrogenation effectively, confirm that this bacterial strain is a strain heterotrophic nitrification-aerobic denitrification bacterium, and adaptability is more widely arranged.
Technical scheme of the present invention is: a strain has the bacillus cereus (Bacillus cereus) of heterotrophic nitrification-aerobic denitrification capability, on April 30th, 2009, be preserved in Beijing that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; address: Datun Road, Chaoyang District, Beijing City, its preserving number are CGMCCNO.3047.
An above-mentioned strain has the bacillus cereus CGMCC NO.3047 of heterotrophic nitrification-aerobic denitrification capability, and this bacterial strain 16SrRNA has the nucleotide sequence shown in SEQID No.1, and sequence length is 1404bp.
A kind of N
2The biology control ease method of O, it is that above-mentioned bacillus cereus CGMCC NO.3047 is inoculated in the ammonia nitrogen waste water.Need constant temperature to keep 30 ℃ before the inoculation, shaking speed 120～160r/min carries out the bacterial strain activation, activates after 12～16 hours, and the bacterial strain in vegetative period of taking the logarithm is that 5% inoculum size is inoculated in the ammonia nitrogen waste water with percent by volume.Reactivation process can not influence the bacterial classification performance, can only make that final effect is better.
Above-mentioned N
2The biology control ease method of O, the condition that described bacillus cereus CGMCC NO.3047 is inoculated in the ammonia nitrogen waste water is: be sole carbon source with the trisodium citrate, ammonium sulfate is only nitrogen source, temperature=20～40 ℃, COD/N=15～30, pH=5.0～9.0, rotating speed=90～180r/min, NH
4 +-N starting point concentration is about 80～120mg/L.
Bacterial strain CGMCC NO.3047 of the present invention has following feature: (1) colony characteristics: cultivated on the BTB flat board 2～3 days, bacterium colony is circular, and glossiness is fine, yellow, and the center does not have projection.(2) cell morphological characteristic: be suitable for about pH neutrality, normal temperature is cultivated down, and cell is shaft-like, Gram-negative.(3) the 16SrRNA gene sequence characteristic of bacillus cereus (Bacillus cereus) CGMCC NO.3047: its 16SrRNA has the nucleotide sequence shown in the sequence table, sequence length is that sequence length is 1404bp, and the accession number in Genbank is EF440610.
With reference to " the content of the outstanding Bacteria Identification handbook of uncle (the 8th edition), according to its morphological specificity and physiological and biochemical property, and, identify that this bacterial strain (CGMCCNo.) is a bacillus cereus (Bacillus cereus) according to its retrieval of 16S rDNA gene order in Genbank.
This bacterial strain CGMCC NO.3047 is inoculated in the nitrate simulated wastewater, is 100r/min at rotating speed, and corresponding dissolved oxygen concentration is 4.2～5.0mg/L; Nitrate concentration is about 10～130mg/L; Carbon-nitrogen ratio 10～25, pH value are under 6.5～7.5 the condition, this bacterium is carried out denitrification capability measure.The result shows: NO after 4 days
3 --N reduction ratio after 78.0%, 4 day total nitrogen (TN) clearance be 76.8%, this bacterial strain can effectively carry out denitrification, the grown cell of this bacterial strain, cell suspending liquid all can be nitrogenous source growth with the nitrate.
This bacterial strain CGMCC N0.3047 is inoculated in the ammonia nitrogen simulated wastewater, tolerance degree to dissolved oxygen, temperature is strong, at rotating speed is under 90～180r/min (corresponding dissolved oxygen concentration is 25%～98% saturated dissolved oxygen) condition, temperature is 20～40 ℃, and carbon-nitrogen ratio is 15～30, and pH is 5.0～9.0, react under the condition of initial ammonia nitrogen concentration 20～120mg/L, after finding 24h, ammonia-N removal rate reaches 96.3%, and nitrogen removal rate reaches 93.81%.Do not have the accumulation of nitrate, nitroso-group nitrogen in the process, this bacterial strain can carry out the heterotrophic nitrification denitrogenation effectively.
This bacterial strain CGMCC NO.3047 is a strain heterotrophic nitrification-aerobic denitrification bacterial strain.Specifically, the present invention relates to a strain bacillus cereus (Bacillus cereus) CGMCC NO.3047, the grown cell of this bacterial strain, cell suspending liquid all can be the nitrogenous source growth with the ammonia nitrogen, also can be the nitrogenous source growth with the nitrate, it can realize synchronous nitration and denitrification under aerobic condition, make nitrogen compound reduction in the waste water, reach the purpose of denitrogenation, the while is obnoxious flavour product N in denitrification process after testing
2The effusion of O is few, only accounts for and remove 0.047% of nitrogen from water body.
Advantage of the present invention and beneficial effect are:
1, this bacterial strain CGMCC NO.3047 is inoculated in the waste water that contains the different concns ammonia nitrogen, selects different condition such as dissolved oxygen, COD/N, pH etc. to experimentize in the different experiments scope, and calculate nitrated ability.Experimental result shows, this bacterial strain is at reaction rotating speed 90～180rpm, being equivalent to dissolved oxygen concentration is 25%～98% of saturated dissolved oxygen, COD/N=15～30, ammonia nitrogen waste water concentration are at 20～120mg/L, in the scope of experiment of pH=5.0～9.0, its ammonia nitrogen maximum removal rate can reach 51.91mg/ (Ld), behind the 24h, ammonia nitrogen removal frank can reach 96.3%, and nitrogen removal rate reaches 93.81%.
2, being inoculated in the waste water that contains ammonia nitrogen at this bacterial strain CGMCC NO.3047, is being sole carbon source with the trisodium citrate, and ammonium sulfate is only nitrogen source, temperature=20～40 ℃, COD/N=15～30, pH=5.0～9.0, rotating speed=90～180r/min, NH
4 +-N starting point concentration is about 20～120mg/L, after aeroseal is cultivated 96h, and NH
4 +-N clearance is 90.52%, and nitrogen removal rate is 91.54%.The N that produces
2O gas is 0.00945mg, accounts for 0.047% of the nitrogen that removes from water body; After 96h is cultivated in the pure oxygen sealing, NH
4 +-N clearance is 91.45%, and nitrogen removal rate is 92.49%.The N that produces
2O is 0.00425mg, accounts for 0.024% of the nitrogen that removes from water body.
3, the bacillus cereus CGMCC NO.3047 that relates among the present invention cultivate initial nitrate concentration 10～130mg/L, and this bacterial strain has heterotrophic nitrification-aerobic denitrification capability and N concurrently when the nutrient solution dissolved oxygen concentration is 2～7mg/L
2The biology control ease ability of O.
4, adopt aeroseal and pure oxygen to cultivate two conditions, confirm that these two conditions are to N
2The generation influence of O is very little, N
2The generation of O all accounts for the less than 0.05% that removes total nitrogen from water body, shows that this bacterial strain has very strong Nitrous Oxide reducing power, can reach N really
2The control of O ease purpose shows that simultaneously aeroseal is provided by dissolved oxygen control that the oxygen that provides just can the satisfy strain culturing condition of escaping.
5, bacillus cereus provided by the invention (Bacillus cereus) CGMCC NO.3047 can grow in nitrate wastewater, and degraded utilizes nitrate under aerobic condition, carries out the denitrification denitrogenation effect; Also can in ammonia nitrogen waste water, grow, under aerobic condition, utilize ammonia nitrogen to carry out the heterotrophic nitrification-aerobic denitrification denitrogenation, and gaseous product N in the denitrification process
2The O escaped quantity is few; Also can in the nutritional medium that with beef peptone, nutrient agar medium etc. is carbon source, nitrogenous source, grow.
Description of drawings
Fig. 1 is for being mensuration N in the experiment
2The used special diplopore of O is cultivated and is shaken a bottle synoptic diagram;
Fig. 2 measures the N that produces in the wastewater biological denitrificaion process for the GC-ECD method
2O gas typical case color atlas.
Embodiment
The screening of embodiment 1 bacillus cereus CGMCC NO.3047
This bacterial strain comes from this laboratory, with nitrate is substrate, the active sludge among the SBR aeration reactor A (total nitrogen TN clearance 60.0～83.0%, COD clearance 85.0～90.0%) under the domestication of high-solubility oxygen condition, strain number is WXZ-8, and deposit number is CGMCC NO.3047.
Utilize dibromothymolsulfonphthalein contained in the BTB substratum to meet alkali and become blue characteristics (as producing neutralization indicator), adopt gradient dilution and coating method that the mixed solution among this laboratory reaction device A is evenly coated the BTB substratum, after the 30 ℃ of aerobic cultivation of constant temperature a couple of days, the aerobic denitrifying bacteria of blue haloing and colonial morphology single bacterium colony inequality as primary dcreening operation appears in substratum around the picking, and the purifying of bacterial strain is carried out in line on flat board.The pure bacterial strain that the line screening obtains is drawn the inclined-plane and is preserved in test tube.Then bacterial strain is carried out denitrification capability test (weighing with the nitrate clearance), sift out pure aerobic denitrifying bacteria again.
The aerobic denitrification bacterial strain that filters out is inoculated in the heterotrophic nitrification substratum, measure the nitrated performance (nitrated performance is weighed with the clearance of ammonia nitrogen removal frank and total nitrogen) of bacterial strain, choose ammonia nitrogen removal frank higher bacterial strain WXZ-8, so far think to filter out the heterotrophic nitrification-aerobic denitrification bacterial strain.
Embodiment 2: the measuring method of bacillus cereus CGMCC NO.3047 aerobic denitrification capability
In order to prevent to bring assorted bacterium in the simulated wastewater into, nitrate simulated wastewater I and nitrate simulated wastewater II be deactivation 20min in high-temperature sterilization pot 0.1MP all.Get nitrate simulated wastewater I after the 50mL sterilization in the 100mL Erlenmeyer flask, to insert nitrate simulated wastewater I with bacillus cereus (Bacillus cereus) the CGMCC NO.3047 that whiteruss is sealed up for safekeeping, place gas bath constant temperature shaking table, constant temperature keeps 30 ℃, and shaking speed 120～160r/min carries out the bacterial strain activation.Activate after 12～16 hours, the inoculum size with 5% is inoculated into carries out denitrification, nitrogen removal performance mensuration in the 250mL Erlenmeyer flask that 200mL nitrate simulated wastewater II is housed.Every 24 hours, filter wastewater sample 5mL in the extraction Erlenmeyer flask with the pin type strainer that 0.2 μ m blend fiber film is housed, measure NO
2 --N and NO
3 --N concentration.By calculating NO
3 --N reduction ratio and total nitrogen (ammonia nitrogen, nitrate and nitrite concentration sum in the waste water) clearance is analyzed the NO of bacterial strain
3 --N reducing property and nitrogen removal performance.
Nitrate simulated wastewater I (/L): sodium succinate: 8.5g, KNO
3: 1.0g, KH
2PO
4: 1.0g, FeCl
26H
2O:0.5g, CaCl
27H
2O:0.2g, MgSO
47H
2O:1.0g.
Nitrate simulated wastewater II (/L): sodium succinate: 2.4g, KNO
3: 0.722g, KH
2PO
4: 1.0g, MgSO
47H
2O:1.0g.
Embodiment 3: the aerobic denitrification of bacillus cereus CGMCC NO.3047 is measured
Waste water aerobic denitrification condition is: dissolved oxygen concentration, realize that by control different rotating speeds rpm (r/min) rotating speed is 100r/min, and corresponding dissolved oxygen concentration is 4.2～5.0mg/L; Nitrate concentration is 126mg/L; Carbon-nitrogen ratio 10～25, pH value are under 6.5～7.5 the condition, this bacterium CGMCC NO.3047 is carried out denitrification capability measure.
Carry out denitrification capability according to embodiment 2 methods and measure test-results: NO after 4 days
3 --N reduction ratio after 78.0%, 4 day TN (total nitrogen is nitrate and nitroso-group nitrogen sum) clearance be 76.8%.
Embodiment 4: the measuring method of bacillus cereus CGMCC NO.3047 heterotrophic nitrification-aerobic denitrification capability
In order to prevent to bring assorted bacterium in the simulated wastewater into, ammonia nitrogen simulated wastewater I and ammonia nitrogen simulated wastewater II be deactivation 20min in high-temperature sterilization pot 0.1MPa all.To insert ammonia nitrogen simulated wastewater I with bacillus cereus (Bacilluscereus) the CGMCC NO.3047 that whiteruss is sealed up for safekeeping, and place gas bath constant temperature shaking table, constant temperature keeps 30 ℃, and shaking speed 120～160r/min carries out the bacterial strain activation.Activate after 12～16 hours, the inoculum size with 5% is inoculated into carries out nitrated, nitrogen removal performance mensuration in the 250mL Erlenmeyer flask that 200ml ammonia nitrogen simulated wastewater II is housed.Every 24 hours, filter wastewater sample 5mL in the extraction Erlenmeyer flask with the pin type strainer that 0.2 μ m blend fiber film is housed, measure NH
4 +-N, NO
2 --N and NO
3 --N concentration.Analyze the nitrated performance and the nitrogen removal performance of bacterial strain by the clearance that calculates ammonia nitrogen removal frank and total nitrogen (ammonia nitrogen in the waste water, nitrate and nitrite concentration sum).
Ammonia nitrogen simulated wastewater I (/L): (NH
4)
2SO
4: 0.47g, KH
2PO
4: 1g, FeCl
26H
2O:1.25g, CaCl
27H
2O:0.2g, MgSO
47H
2O:1g, trisodium citrate (C
6H
5Na
3O
72H
2O): 5.1g, the carbon source consumption is regulated according to orthogonal experiment design COD/N ratio, and regulates initial pH value simultaneously.
Ammonia nitrogen simulated wastewater II (/L): (NH
4)
2SO
4Be nitrogenous source, trisodium citrate (C
6H
5Na
3O
72H
2O) be carbon source, (annotate: the nitrogenous source consumption requires to regulate according to the difference of initial ammonia nitrogen concentration in the experiment, and the carbon source consumption is regulated according to experimental design COD/N ratio), Vickers salts solution 50ml; Be dissolved in water, replenish distilled water to 1L, and regulate initial pH value simultaneously.Wherein the Vickers salts solution (/L): K
2HPO
4: 5.0g; FeSO
47H
2O:0.05g; NaCl:2.5g; MgSO
47H
2O:2.5g; MnSO
44H
2O:0.05g adds water and is settled to 1L after the dissolving.
Under embodiment 5～10 different culture condition, the heterotrophic nitrification-aerobic denitrification capability of bacillus cereus CGMCC NO.3047 is measured
Carry out the heterotrophic nitrification performance measurement according to embodiment 4 methods, 6 test-results of bacillus cereus (Bacillus cereus) CGMCC NO.3047 see Table 1.
Nitrated denitrification test condition of the waste water of table 1 embodiment 5～10 and nitric efficiency
| Numbering | ??COD ??/N | Temperature/℃ | The pH value | Rotating speed/rpm | Ammonia nitrogen starting point concentration (mg/L) | Ammonia nitrogen removal frank/% after 4 days | TN clearance % after 4 days |
| Embodiment 5 | ??25 | ??20 | ??9.0 | ??180 | ??98 | ??86.48 | ??87.50 |
| Embodiment 6 | ??15 | ??30 | ??7.0 | ??180 | ??100 | ??59.43 | ??62.79 |
| Embodiment 7 | ??20 | ??30 | ??9.0 | ??90 | ??106 | ??69.86 | ??72.52 |
| Embodiment 8 | ??25 | ??30 | ??5.0 | ??130 | ??100 | ??67.64 | ??70.97 |
| Embodiment 9 | ??20 | ??35 | ??5.0 | ??180 | ??114 | ??80.68 | ??81.55 |
| Embodiment 10 | ??20 | ??30 | ??7.0 | ??180 | ??36 | ??98.71 | ??99.23 |
As can be seen from Table 1, to the ammonia nitrogen removal ability influence factor of bacillus cereus (Bacillus cereus) bacterial strain COD/N most importantly, be dissolved oxygen and pH secondly, the Temperature Influence minimum.
Embodiment 11: under preferred culture condition, the heterotrophic nitrification-aerobic denitrification capability of bacillus cereus CGMCC NO.3047 is measured
Adopt preferably nitrated, the preferred culture condition of denitrification: temperature is that 30 ℃, COD/N are 25, initial pH value is 9.0, rotating speed is 180r/min.Under this condition, bacillus cereus (Bacillus cereus) CGMCC NO.3047 ammonia nitrogen removal frank when 24h reaches maximum, is 96.30%.The TN clearance reaches 93.81% during 24h.This bacterial strain logarithmic phase occurs in initial 24 hours, and it is maximum that the thalli growth amount reached at the 24th hour, OD
600Value is 1.094.The removal of TN and NH
4 +The removal of-N has identical trend, reaches 95.21% at the 16th hour TN clearance, and the NO of minute quantity is only arranged in whole nitrifying process
3 --N, NO
2 -The accumulation of-N, bacterial strain in nitrifying process with NH
4 +-N is oxidized to NO
3 --N, NO
2 --N, and be removed by the aerobic denitrification effect.
Embodiment 12: under the aeroseal flask culture condition, be carbon source with the trisodium citrate, ammonium sulfate is only nitrogen source, to N in the bacillus cereus CGMCC NO.3047 heterotrophic nitrification process
2The biology control ease technology of O
In order to prevent to bring assorted bacterium in the simulated wastewater into, ammonia nitrogen simulated wastewater I and ammonia nitrogen simulated wastewater II be deactivation 20min in high-temperature sterilization pot 0.1MPa all.To insert ammonia nitrogen simulated wastewater I with bacillus cereus (Bacilluscereus) the CGMCC NO.3047 that whiteruss is sealed up for safekeeping, and place gas bath constant temperature shaking table, constant temperature keeps 30 ℃, and shaking speed 120～160r/min carries out the bacterial strain activation.Activate after 12～16 hours, the inoculum size with 5% is inoculated into the 1L that 200ml ammonia nitrogen simulated wastewater II is housed and has in the triangular flask of sealing rubber plug and carry out nitrated ability, denitrogenation product N
2The O eudiometry.At 20～40 ℃,, pH=7.0～9.0, rotating speed is 90～180r/min, COD/N is under 15～30 the condition, with cultivating assay determination under the gas bath constant temperature shaking table.Under the similarity condition, blank wastewater sample (not meeting the ammonia nitrogen simulated wastewater II of bacterial strain) is reference test as a comparison.
Every 24 hours, slowly at the uniform velocity extract escaping gas in the Erlenmeyer flask with 10ml locking-type sampler, direct analysis detects N
2O.With extracting liquid sample, measure NH simultaneously
4 +-N, NO
2 --N and NO
3 --N concentration.By calculating ammonia nitrogen removal frank, nitrogen removal rate and N
2The gas yield of O is analyzed the nitrated performance and the nitrogen removal performance of bacterial strain.
N
2O adopts the GC-ECD method to measure Varian 3800 gas chromatographs; 10mci63Ni electron capture detector (ECD); The stainless steel column of 3m * 3mm is filled solid Porapak Q post, 80/100 order.The comprehensive chromatographic condition of selecting for use: detector temperature (T
D) (350 ℃), separator column temperature (T
C) (50 ℃), flow rate of carrier gas (f
C) (20mL/min), have higher sensitivity and tolerance range with this understanding, circulation ratio better (relative standard deviation C.V=1.05%, n=23).As shown in Figure 2, according to above-mentioned test condition as can be seen, O
2And N
2The O resolution is bigger, illustrates that gaseous constituent is clear, and following result is measured precisely.
Experimental result shows: under the aeroseal culture condition, bacillus cereus (Bacillus cereus) CGMCC NO.3047 bacterial strain is being sole carbon source with the trisodium citrate, and ammonium sulfate is only nitrogen source, temperature=30 ℃, COD/N=25, pH=8.0, rotating speed=160r/min, NH
4 +-N starting point concentration is 100mg/L, under the experiment condition, and NH behind the 96h
4 +-N clearance is 90.52%, nitrogen removal rate=91.54%, the N of generation
2O is 0.00945mg, accounts for 0.047% of the nitrogen that removes from water body.
Embodiment 13: under the pure oxygen air-tight bottle culture condition, be carbon source with the trisodium citrate, ammonium sulfate is only nitrogen source, to the biology control ease technology of N2O in the bacillus cereus CGMCC NO.3047 heterotrophic nitrification process
Different with embodiment 11, in this test 200ml ammonia nitrogen simulated wastewater II inserted in the triangular flask that 1L has the double aperture seal rubber plug and carry out nitrated ability, denitrogenation product N
2The O eudiometry.As shown in Figure 1, for being mensuration N in the experiment
2The used special diplopore of O is cultivated and is shaken a bottle synoptic diagram; Wherein this cultivation is shaken bottle and is comprised diplopore rubber plug 1, gas sampling mouth 2, valve 3, liquid sampling mouth 4, air outlet 5 (the rubber hose the other end that has a clip in succession Glass tubing) and inlet mouth 6 (the rubber hose the other end that has a clip in succession Glass tubing).
In the cultivation, high purity oxygen gas with 99.999% is injected in the culturing bottle with identical flow velocity, the oxygenation time is 60 seconds (notes: by the experimental verification in early stage, 40 seconds oxygenation time can guarantee to contain in the 1L bottle oxygen more than 95%), with the in succession rubber hose of Glass tubing of clamp, carry out shaking table then and cultivate at once.Every 24 hours, slowly at the uniform velocity extract escaping gas in the Erlenmeyer flask with 10ml locking-type sampler, direct analysis detects N
2O.With extracting liquid sample, measure NH simultaneously
4 +-N, NO
2 --N and NO
3 --N concentration.By calculating ammonia nitrogen removal frank, nitrogen removal rate and N
2The gas yield of O is analyzed the nitrated performance and the nitrogen removal performance of bacterial strain.
Experimental result shows: bacillus cereus (Bacillus cereus) bacterial strain is being sole carbon source with the trisodium citrate, and ammonium sulfate is only nitrogen source, temperature=30 ℃, COD/N=25, pH=8.0, rotating speed=160r/min, NH
4 +-N starting point concentration is under the 100mg/L experiment condition, NH behind the 96h
4 +-N clearance is 91.45%, nitrogen removal rate=92.49%, the N of generation
2O is 0.00425mg, accounts for 0.024% of the nitrogen that removes from water body.
Embodiment 14: the pcr amplification and the sequencing of the 16SrRNA gene of bacillus cereus CGMCC NO.3047
Bacillus cereus WXZ-8 (Bacillus cereus) is inoculated in the 100 μ L distilled waters, and boiling water bath boils 5min, carries out colony PCR amplification.The primer that is used for 16SrRNAPCR reaction is a pair of universal primer, and promptly forward primer is that f27 (5 '-AGAGTTGATCCTGGCTAG-3 ') and reverse primer are r1492 (5 '-GGTTACCTTCGACTT-3 ').Primer is synthetic by Beijing China big-and-middle living development in science and technology company limited.PCR reaction system (50 μ L): 10 * Buffer, 5 μ L, dNTPs4 μ L, each 1 μ L of primer f27 and r1492, distilled water 39 μ L add dna profiling 0.5 μ L, Taq enzyme 0.5 μ L behind the centrifugal mixing.The PCR response procedures comprises: (1) 94 ℃ of pre-sex change 5min; (2) 94 ℃ of sex change 1min; (3) 50 ℃ of annealing 1min; (4) 72 ℃ are extended 2min; Carry out 25 circulations; At last once more 72 ℃ extend 10min. agarose gel electrophoresis (1 * TAE electrophoretic buffers, 1% gel) analyzes PCR result, transfer to Beijing China big-and-middle living development in science and technology company limited and carry out the 16SrRNA order-checking, 16SrRNA gene order length is 1404bp, and the accession number in Genbank is EF440610.
Sequence table
＜110〉Beijing Technology and Business University
＜120〉have the bacillus cereus and the N thereof of heterotrophic nitrification-aerobic denitrification capability
2The biology control ease method of O
<130>
<160>1
<170>PatentIn?version?3.5
<210>1
<211>1406
<212>DNA
＜213〉artificial sequence
<400>1
catgcagtcg?aacggtaaca?ggtcttcgga?cgctgacgag?tggcgaacgg?gtgagtaata?60
catcggaacg?tgcccagtcg?tgggggataa?ctactcgaaa?gagtagctaa?taccgcatac?120
gatctgagga?tgaaagcggg?ggaccttcgg?gcctcgcgcg?attggagcgg?ccgatggcag?180
attaggtagt?tggtgggata?aaagcttacc?aagccgacga?tctgtagctg?gtctgagagg?240
acgaccagcc?acactgggac?tgagacacgg?cccagactcc?tacgggaggc?agcagtgggg?300
aattttggac?aatgggcgaa?agcctgatcc?agcaatgccg?cgtgcaggat?gaaggccttc?360
gggttgtaaa?ctgcttttgt?acggaacgaa?aaagctcctt?ctaatacagg?gggcccatga?420
cggtaccgta?agaataagca?ccggctaact?acgtgccagc?agccgcggta?atacgtaggg?480
tgcgagcgtt?aatcggaatt?actgggcgta?aagcgtgcgc?aggcggttat?gtaagacaga?540
tgtgaaatcc?ccgggctcaa?cctgggaact?gcatttgtga?ctgcatggct?agagtacggt?600
agagggggat?ggaattccgc?gtgtagcagt?gaaatgcgta?gatatgcgga?ggaacaccga?660
tggcgaaggc?aatcccctgg?acctgtactg?acgctcatgc?acgaaagcgt?ggggagcaaa?720
caggattaga?taccctggta?gtccacgccc?taaacgatgt?caactggttg?ttgggaatta?780
gttttctcag?taacgaagct?aacgcgtgaa?gttgaccgcc?tggggagtac?ggccgcaagg?840
ttgaaactca?aaggaattga?cggggacccg?cacaagcggt?ggatgatgtg?gtttaattcg?900
atgcaacgcg?aaaaacctta?cccacctttg?acatggcagg?aagtttccag?agatggattc?960
gtgctcgaaa?gagaacctgc?acacaggtgc?tgcatggctg?tcgtcagctc?gtgtcgtgag??1020
atgttgggtt?aagtcccgca?acgagcgcaa?cccttgtcat?tagttgctac?attcagttga??1080
gcactctaat?gagactgccg?gtgacaaacc?ggaggaaggt?ggggatgacg?tcaagtcctc??1140
atggccctta?taggtggggc?tacacacgtc?atacaatggc?tggtacagag?ggttgccaac??1200
ccgcgagggg?gagctaatcc?cataaaacca?gtcgtagtcc?ggatcgcagt?ctgcaactcg??1260
actgcgtgaa?gtcggaatcg?ctagtaatcg?cggatcagca?tgccgcggtg?aatacgttcc??1320
cgggtcttgt?acacaccgcc?cgtcacacca?tgggagcggg?tctcgccaga?agtaggtagc??1380
ctaaccgcaa?ggagggcgct?accacg???????????????????????????????????????1406
Claims (5)
1, a strain has the bacillus cereus (Bacillus cereus) of heterotrophic nitrification-aerobic denitrification capability, it is characterized in that described bacillus cereus deposit number is CGMCC NO.3047.
2, the bacillus cereus CGMCC NO.3047 with heterotrophic nitrification-aerobic denitrification capability according to claim 1 is characterized in that this bacterial strain 16SrRNA has the nucleotide sequence shown in SEQ ID No.1, and sequence length is 1404bp.
3, a kind of denitrogenation product N
2The biological control of O ease method is characterized in that, claim 1 or 2 described bacillus cereus CGMCC NO.3047 are inoculated in the ammonia nitrogen waste water.
4, N according to claim 3
2The biological control of O ease method is characterized in that need constant temperature to keep 20～35 ℃ before the inoculation, shaking speed 120～160r/min carries out the bacterial strain activation, activates after 12～16 hours, and the bacterial strain in the vegetative period of taking the logarithm, the inoculum size with 5% is inoculated in the ammonia nitrogen waste water.
5, N according to claim 4
2The biological control of O ease method, it is characterized in that, the condition that described bacillus cereus CGMCC NO.3047 is inoculated in ammonia nitrogen waste water is: be sole carbon source with the trisodium citrate, ammonium sulfate is only nitrogen source, temperature=20～40 ℃, COD/N=15～30, pH=5.0～9.0, rotating speed=90～180r/min, NH4
+-N starting point concentration is 80～120mg/L.
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