CN110484458A - It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage - Google Patents

It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage Download PDF

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CN110484458A
CN110484458A CN201810987440.9A CN201810987440A CN110484458A CN 110484458 A CN110484458 A CN 110484458A CN 201810987440 A CN201810987440 A CN 201810987440A CN 110484458 A CN110484458 A CN 110484458A
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acclimation
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胡智泉
吴丁山
刘冬啟
肖波
刘石明
包美玲
周志勤
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Huazhong University of Science and Technology
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Abstract

The invention belongs to sewage treatment and field of environment protection, handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage more particularly, to a kind of.The present invention has certain nitrogen by selection, the black and odorous water bed mud of phosphorus content is as strain source, cooperate suitable culture medium, successively it is enriched with by aerobic strain, aerobic primary dcreening operation, aerobic domestication culture obtains to low C/N the aerobic denitrification polyP bacteria than sewage while with good Nitrogen/Phosphorus Removal, because same strain has good denitrogenation and phosphor-removing effect simultaneously, to solve the contradiction of traditional denitrifying bacterium and dephosphorization bacterial contention carbon source, the present invention is simple to the acclimating method of aerobic denitrification polyP bacteria, operation is easy, acclimation period is short, screening efficiency is obviously improved.

Description

It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage
Technical field
The invention belongs to sewage treatment and field of environment protection, to handle low C/N better than sewage more particularly, to a kind of The acclimation and screening method of oxygen Denitrifying Phosphate Accumulating Organisms.
Background technique
As the eutrophication problem of water body is got worse, country puts into effect or standard supplemented with sewage discharge, especially It is that the discharge standards such as nitrogen, phosphorus are stringenter.The processing of traditional biological dephosphorization denitrogenation is substantially with biological nitration, denitrification and biology The combination of dephosphorization mechanism generally requires C/N >=15 to the more demanding of C/N, and in this type of process due to dephosphorization and denitrification It is two different Biochemical Mechanisms, there is fundamental contradictions, such as the competition of carbon source, the concentration of dissolved oxygen, sludge retention time. With C/N the and C/P ratio downward trend of municipal sewage, so that polyP bacteria and nitrobacteria, denitrifying bacterium fight for organic carbon The problem of source, is more significant, and urgent need is a kind of to adapt to Biological Nitrogen Removal Processe method of the low C/N than environment.
Currently, traditional biological denitrogenation mainly includes ammonification, nitrification and denitrification three phases.It is that amonifying bacteria is having first Under oxygen or anoxia condition, ammonia nitrogen is converted by organic nitrogen, nitrobacteria is under aerobic state, by NH4 +It is converted to NO2 -, reoxidize At NO3 -, last denitrifying bacteria is under anaerobic condition by NO2 -Or NO3 -It is reduced to nitrogen.Traditional dephosphorization process includes that anaerobism is released Phosphorus, anoxic or aerobic excess inhale two processes of phosphorus.PolyP bacteria under anaerobic, decomposes intracorporal polyphosphate and generates ATP, Enter intracellular synthesis PHB or PHA by the matrix that ATP absorbs acid-producing bacteria offer in the way of active transport.PolyP bacteria is aerobic Under the conditions of, disassembler intracorporal PHB or PHA and Exogenous ground substance generate proton motive force for external and are transported to synthesis ATP in vivo And nucleic acid, superfluous is aggregated into storage of cells object, the sludge that phosphorus is inhaled by excluding excess achievees the purpose that remove Phosphorus From Wastewater.
Traditional biological denitrification dephosphorization technique, such as A2O, SBR etc. relies on carbon source larger, and denitrifying bacterium and polyP bacteria are to carbon source There are competitive relations, and dephosphorization efficiency is low, and sludge yield is big.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, to handle low C/N better than sewage the present invention provides a kind of The acclimation and screening method of oxygen polyP bacteria, by separating, taming from black and odorous water bed mud, filter out a kind of aerobic phosphorous accumulating bacterium, The strain has good dephosphorization denitrification effect, thus solves nitrobacteria, denitrifying bacterium in traditional denitrification dephosphorization technique and removes Phosphorus bacterium fights for the contradictory relationship of carbon source, and solves traditional denitrogenation dephosphorizing and rely on greatly carbon source, and dephosphorization efficiency is low, sludge produces Measure big technical problem.
To achieve the above object, according to one aspect of the present invention, provide that a kind of to handle low C/N aerobic more anti-than sewage The acclimation and screening method for nitrifying polyP bacteria, includes the following steps:
(1) Enrichment of bacteria: black and odorous water bed mud being inoculated into enriched medium and is cultivated, and is made thin in the bed mud Bacterium is enriched with, and enriched medium suspension is obtained；
(2) aerobic denitrification polyP bacteria primary dcreening operation: by the enriched medium inoculation of suspension liquid in logarithmic phase to solid It is cultivated on culture medium, until the solid culture primary surface grows bacterium colony；
(3) the different single colonie difference of form the domestication of aerobic denitrification polyP bacteria: is selected from the solid medium It is inoculated into denitrification culture medium and is cultivated, it is induced to carry out aerobic denitrification denitrogenation dephosphorizing, obtain domestication culture medium and suspend Liquid.
(4) identification of aerobic denitrification polyP bacteria: by the domestication culture medium inoculation of suspension liquid to the anti-nitre of bromthymol blue Change and cultivated on solid medium, so that culture medium is become blue bacterium colony is aerobic denitrification polyP bacteria；
(5) secondary screening and preservation of aerobic denitrification polyP bacteria: by the colony inoculation of the blue region to rich Nitrogen-and Phosphorus-containing It identifies in culture medium, the bacterium colony in logarithmic growth phase and with best Nitrogen/Phosphorus Removal is taken to save backup.
Preferably, the acclimation and screening method, further comprises the steps of:
(6) it takes and is cultivated in the identification culture medium inoculation of suspension liquid to solid medium of logarithmic phase, until the solid Media surface grows bacterium colony；
(7) step (3) is repeated to step (5), is saved backup until obtaining the stable bacterium colony of Nitrogen/Phosphorus Removal.
Preferably, a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage, including walk as follows It is rapid:
(1) Enrichment of bacteria: the black and odorous water bed mud belonged to containing Pseudomonas stutzeri is inoculated into enriched medium and is carried out Culture, is enriched with the bacterium in the bed mud, obtains enriched medium suspension.
(2) aerobic denitrification polyP bacteria primary dcreening operation: by the enriched medium inoculation of suspension liquid in logarithmic phase to solid It is cultivated on culture medium, until the solid culture primary surface grows bacterium colony；
(3) the different single colonie difference of form the domestication of aerobic denitrification polyP bacteria: is selected from the solid medium It is inoculated into denitrification culture medium and is cultivated, it is induced to carry out aerobic denitrification denitrogenation dephosphorizing, obtain domestication culture medium and suspend Liquid.
(4) identification of aerobic denitrification polyP bacteria: by the domestication culture medium inoculation of suspension liquid to the anti-nitre of bromthymol blue Change and cultivated on solid medium, so that culture medium is become blue bacterium colony is aerobic denitrification polyP bacteria；
(5) secondary screening and preservation of aerobic denitrification polyP bacteria: by the colony inoculation of the blue region to rich Nitrogen-and Phosphorus-containing It identifies in culture medium, the bacterium colony in logarithmic growth phase and with best Nitrogen/Phosphorus Removal is taken to save backup；It is described to have most The bacterium colony of good Nitrogen/Phosphorus Removal is strain Pseudomonas stutzeri ATCC17588, and full genome sequencing accession number is CP002881。
Preferably, the black and odorous water bed mud 156.3-162.4mg/kg containing total phosphorus, overlying water total phosphorus are 1.4- 1.89mg/L PO4 3-- P is 1.13-1.5mg/L, NH4 +- N is 9.22-22.3mg/L, NO3 -- N is 2.6-7.4mg/L, NO2 --N For 0.01-0.22mg/L.
Preferably, step (1) the enrichment culture based formulas is 5-8g/L peptone, 5-8g/L yeast extract leachate and 5- 10g/LNaCI。
Preferably, OD600 >=0.8 when step (2) described logarithmic phase.
Preferably, step (2) condition of culture are as follows: cultivated 48-72 hours in 25-30 DEG C of constant incubator.
Preferably, step (2) is diluted the suspension, and taking extension rate respectively is 105、106、107Again described Suspension 0.1mL is inoculated into the solid medium.
Preferably, step (2) the solid culture based formulas is 5-8g/L CH3COONa、0.0659-0.1g/L KH2PO4、0.722-1g/L KNO3、0.10-0.20g/L MgSO4·7H2O, 20g/L agar and 1-3mL/L microelement.
Preferably, step (3) the denitrification culture medium prescription is 5-8g/L CH3COONa、0.0659-0.1g/L KH2PO4、0.722-1g/LKNO3、0.10-0.20g/L MgSO4.7H2O, 1-3ml/L microelement.
Preferably, step (3) condition of culture is to cultivate 72h in 30 DEG C of water-bath constant temperature oscillators.
Preferably, step (4) the bromthymol blue denitrification solid culture based formulas is 5-8g/L CH3COONa、 0.0659-0.1g/LKH2PO4、0.722-1g/LKNO3、0.10-0.20g/L MgSO4·7H2O, 1-3ml microelement/L, 20g/L agar and 10g/L bromthymol blue.
Preferably, step (4) condition of culture is that 48-72h is cultivated in constant incubator under the conditions of 25-30 DEG C.
Preferably, step (5) is described identifies that culture medium prescription is 5-8g/L CH3COONa、0.722-2.888g/L KNO3、 0.0659-0.1g/L KH2PO4、0.20g/L MgSO4·7H2O and 1-3mL/L microelement；It or is 5-8g/L CH3COONa、0.493-1.972/L NaNO2、0.0659-0.1g/LKH2PO4、0.10-0.20g/LMgSO4·7H2O and 1-3mL Microelement/L.
Preferably, the trace element formula are as follows: contain 10-15g EDTA, 0.10-0.20g in every liter of microelement ZnSO4、1.0-2.0g MnCl2·4H2O、0.10-0.5g FeSO4·7H2O、0.10-0.5g CuSO4·5H2O、0.10- 0.50g CoCl2·6H2O、0.10-0.20g Na2MoO4·2H2O and 0.10-0.20g CaCl2。
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show Beneficial effect:
The present invention, which passes through, selects the black and odorous water bed mud with certain the content of nitrogen and phosphorous as strain source, and cooperation is suitable Culture medium successively obtains by the negative enrichment of aerobic strain, aerobic primary dcreening operation, aerobic domestication culture while having good denitrogenation dephosphorizing The aerobic denitrification polyP bacteria of effect, same strain have good denitrogenation and phosphor-removing effect simultaneously, solve traditional anti- The contradiction of nitrifier and dephosphorization bacterial contention carbon source, the present invention is simple to the acclimating method of aerobic denitrification polyP bacteria, operation It is easy, acclimation period is short, and screening efficiency is obviously improved.
The aerobic denitrification polyP bacteria that the present invention tames out is that it can be directly with NO3 -Or NO2 -It is efficiently gone for electron acceptor Except the nitrogen phosphorus in low C/N ratio (C/N≤4) sewage, achieve the effect that synchronous denitrification dephosphorizing, to NO3 -- N and NO2 -The removal rate of-N Reach 100%, to PO4 3-Removal rate be more than 84%.Compared to traditional denitrification dephosphorization technique, denitrogenation dephosphorizing is greatly improved Efficiency and the tolerance of microorganism have biggish economic benefit and environmental benefit.
The present invention selects to contain multiple-microorganism and especially contains the black and odorous water bed mud of Pseudomonas stutzeri category as bacterium Kind source, cooperates suitable culture medium, is successively obtained simultaneously by the enrichment of aerobic strain, aerobic primary dcreening operation, aerobic domestication culture Aerobic denitrification polyP bacteria with good Nitrogen/Phosphorus Removal, the bacterium are Pseudomonas stutzeri ATCC17588, It is with good Nitrogen/Phosphorus Removal.
Detailed description of the invention
Fig. 1 is aerobic denitrification polyP bacteria domesticating device schematic diagram.
Fig. 2 is adaptation situation of the aerobic denitrification polyP bacteria to different C/N ratios.
Fig. 3 is aerobic denitrification polyP bacteria to NO3 -- N and PO4 3-The removal situation of-P.
Fig. 4 is aerobic denitrification polyP bacteria to NO2 -- N and PO4 3-The removal situation of-P.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below Not constituting a conflict with each other can be combined with each other.
Screening acclimation method of the low C/N than the aerobic denitrification polyP bacteria of sewage is handled the present invention provides a kind of, including Following steps:
(1) Enrichment of bacteria: the black and odorous water bed mud belonged to containing Pseudomonas stutzeri is inoculated into enriched medium and is carried out Culture, obtains enriched medium suspension；Purpose is to be enriched with the bacterium in bed mud, is convenient for subsequent screening.
The present invention selects black and odorous water bed mud as strain source, in order to guarantee that domestication obtains aerobic denitrification polyP bacteria Nitrogen/Phosphorus Removal, the black and odorous water bed mud that the present invention selects preferably contain total phosphorus 156.3-162.4mg/kg, overlying water total phosphorus For 1.4-1.89mg/L, PO4 3-- P is 1.13-1.5mg/L, NH4 +- N is 9.22-22.3mg/L, NO3 -- N is 2.6-7.4mg/L, NO2 -- N is 0.01-0.22mg/L.
Enrichment culture based formulas is preferably 5-8g/L peptone, 5-10g/L yeast extract leachate and 5-10g/L NaCI.
(2) described enriched medium suspension (i.e. suspension of logarithmic phase will aerobic denitrification polyP bacteria primary dcreening operation: be in Middle bacterium is in logarithmic growth phase, corresponding OD600 >=0.8) it is inoculated on solid medium in 25-30 DEG C of constant incubator Culture 48-72 hours, until the solid culture primary surface grows bacterium colony；Preferably mode is diluted suspension, respectively Taking extension rate is 105、106、107The suspension 0.1mL again, which is inoculated into the solid medium, carries out above-mentioned culture. Solid culture based formulas is preferably 5-8g/L CH3COONa、0.0659-0.10g/L KH2PO4、0.722-2.888g/L KNO3、 0.10-0.20g/LMgSO4·7H2O, 20g/L agar and 1-3mL/L microelement.Colonial morphology clearly culture medium is selected, it is right Its different bacterium colony carries out label of taking pictures, and is successively named as A bacterium, B bacterium, C bacterium, D bacterium etc..
(3) the different single colonie (picking of form the domestication of aerobic denitrification polyP bacteria: is selected from the solid medium Single A bacterium, B bacterium, C bacterium, D bacterium etc.) it is inoculated into denitrification culture medium respectively and cultivates 72h in 30 DEG C of water-bath constant temperature oscillators, It induces it to carry out aerobic denitrification denitrogenation dephosphorizing, obtains domestication culture medium suspension.The denitrification culture medium prescription is preferably 5-8g/L CH3COONa、0.0659-0.1g/L KH2PO4、0.722-1g/LKNO3、0.10-0.20g/L MgSO4.7H2O、1- 3ml/L microelement.
(4) identification of aerobic denitrification polyP bacteria: by the domestication culture medium inoculation of suspension liquid to bromthymol blue (BTB) 48-72h is cultivated in constant incubator under the conditions of 25-30 DEG C on denitrification solid medium, until bacterium colony is grown, in indigo plant The bacterium colony in color region is aerobic denitrification polyP bacteria；BTB denitrification solid culture based formulas is 5-8gCH3COONa/L、 0.0659-0.10g KH2PO4/L、0.722-2.888gKNO3/L、0.10-0.20gMgSO4·7H2O/L, 1-3ml microelement/ L, 20g/L agar and 10g/LBTB.
(5) secondary screening and preservation of aerobic denitrification polyP bacteria: by the colony inoculation of the blue region to rich Nitrogen-and Phosphorus-containing It identifies in culture medium, such as 6 hours at regular intervals detections OD600, PO4 3--P、NO3 --N、NO2 -The content of-N, detection are de- Nitrogen phosphor-removing effect；The bacterium colony in logarithmic growth phase and with best Nitrogen/Phosphorus Removal is taken to save backup.Identification culture medium is matched Side is 5-8g/L CH3COONa、0.722-2.888g/L KNO3、0.0659-0.10g/L KH2PO4、0.10-0.20g/L MgSO4·7H2O and 1-3mL/L microelement；It or is 5-8g/L CH3COONa、0.493-1.972g/LNaNO2、0.0659- 0.10g/LKH2PO4、0.10-0.20g/LMgSO4·7H2O and 1-3mL/L microelement.By the detection of OD600, bacterium is understood The growth curve fallen.Its denitrogenation of the bacterium that the present invention tames and phosphor-removing effect have positive correlation, multiple in blue When being in logarithmic phase or logarithmic phase latter stage after the bacterium colony in region is identified, wherein the bacterium colony with highest nitrogen removal efficiency, There is highest phosphorus removal efficiency simultaneously.Taking the bacterium with highest denitrification percent or dephosphorizing rate is best Nitrogen/Phosphorus Removal Bacterium colony.
(6) it takes and is cultivated in the identification culture medium inoculation of suspension liquid to solid medium of logarithmic phase, until the solid Media surface grows bacterium colony；
(7) step (3) is repeated to step (5), until PO4 3--P、NO3 --N、NO2 -- N removal effect is obvious and removes Rate keeps stablizing i.e. domestication work and completes.
Wherein trace element formula is that every liter of microelement contains: 10-15gEDTA, 0.10-0.20gZnSO4、1.0- 2.0gMnCl2·4H2O、0.10-0.5g FeSO4·7H2O、0.10-0.5gCuSO4·5H2O、0.10-0.50g CoCl2· 6H2O、0.10-0.20gNa2MoO4·2H2O and 0.10-0.20gCaCl2。
The aerobic denitrifying bacteria that the present invention is separated from black and odorous water bed mud is identified as Pseudomonas Stutzeri ATCC17588, it is CP002881 that accession number, which is sequenced, in full genome.
The single colonie tamed out according to the screening of above-mentioned acclimating method is inoculated into expand in culture medium and is cultivated, then will Expansion culture medium inoculation of suspension liquid in logarithmic growth phase is handled into the sewage containing nitrogen phosphorus, tests Nitrogen/Phosphorus Removal.
Acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage is handled the present invention provides a kind of, is used Acclimation and screening method of the invention can get the bacterium colony with good Nitrogen/Phosphorus Removal.This method passes through to black and odorous water bottom Microorganism in mud is manually enriched with, select single bacterium colony carried out in rich in phosphorus and culture using nitrate nitrogen as only nitrogen source it is tame and docile Change, forces microorganism that can only breed using nitrate nitrogen as nitrogen source for its own, it is made gradually to adapt to the denitrification dephosphorization item of setting Part, directive breeding have the microorganism of higher degradation capability to nitrogen phosphorus.Due to only having nitrate nitrogen and phosphate radical etc. necessary in culture medium Nutriment can be utilized, and enable the bacterium with NO3 -- N or NO2 -- N is electron acceptor, has dephosphorization while denitrogenation Effect, reach the dual-purpose effect of a bacterium, overcome conventional denitrification bacterium and polyP bacteria and nutrient (such as carbon source) is striven Take by force and the problem of nitrite accumulation plays inhibiting effect to dephosphorization.The process of taming does not need anaerobism, aerobic alternate run simultaneously, It is easy to operate and lower to sewage-treatment plant requirement, it does not need to be aerated, it is only necessary to which great amount of cost is saved in appropriate stirring.
The aerobic denitrification polyP bacteria that the present invention tames out, while there is denitrogenation and phosphor-removing effect, carbon source contention is not present Problem carries out denitrogenation dephosphorizing processing mainly for low C/N ratio (C/N≤4) sewage in city, can save carbon source and the energy, substantially Degree improves denitrogenation dephosphorizing efficiency, has biggish application value.
As a preferred embodiment, the present invention is to the Heisui River Sediments belonged to containing Pseudomonas stutzeri according to the method Carry out acclimation and screening processing, actually separation, domestication and the sieve to Pseudomonas stutzeri ATCC17588 bacterial strain The method of choosing.Bacterium Nitrogen/Phosphorus Removal after domestication is preferable.The following are embodiments:
Embodiment 1
(1) strain choose: the HuaZhong Science University east school district river Hu Xi be typical black and odorous water, with bottom sampler take river bed with One liter of sludge, saves under the conditions of -4 DEG C at lower 10cm.Bed mud 156.3-162.4mg/kg containing total phosphorus, overlying water total phosphorus are 1.4-1.89mg/L, PO4 3-- P is 1.13-1.5mg/L, NH4 +- N is 9.22-22.3mg/L, NO3 -- N is 2.6-7.4mg/L, NO2 -- N is 0.01-0.22mg/L.
(2) it the preparation and inoculation of enriched medium: takes 100mL enrichment culture to be based in 250mL conical flask, and is inoculated with 1mL Bed mud small size shaken cultivation 72h in 30 DEG C of water-bath constant temperature oscillators, device are shown in Fig. 1, and 1 indicates 250mL conical flask, and 2 indicate water Bathe constant temperature oscillator.The formula of enriched medium is 5g peptone/L, 5g yeast extract leachate/L, 10gNaCl/L.
(3) it aerobic denitrification polyP bacteria primary dcreening operation: takes 1mL to be in the enriched medium suspension of logarithmic phase, uses deionized water It is diluted to 10,10 respectively2、103、104、105、106、107Times, respectively take the 10 of 0.1mL4、105、106、107Times dilution is inoculated into Solid medium, culture to culture medium grows clear bacterium colony under the conditions of 30 DEG C.Solid culture based formulas are as follows: 5g/L CH3COONa、0.0659g/L KH2PO4、0.722g/L KNO3、0.20g/L MgSO4·7H2O, the micro member of 20g/L agar, 2ml (every liter of microelement includes 15gEDTA, 0.2gZnSO to element4、1.5gMnCl2·4H2O、0.5g FeSO4·7H2O、 0.5gCuSO4·5H2O、0.3g CoCl2·6H2O、0.2gNa2MoO4·2H2O、0.1gCaCl2).Select colonial morphology clearly Two kinds of culture medium, label of taking pictures is carried out to its bacterium colony, is respectively designated as A bacterium and B bacterium.
(4) the domestication culture of aerobic denitrification polyP bacteria: taking 100mL denitrification culture to be based in 250mL conical flask, respectively The single A bacterium of picking and B bacterium are inoculated into denitrification culture medium and are tamed, and but small oscillations are trained in 30 DEG C of water-bath constant temperature oscillators Support 72h.Denitrification culture medium prescription is 5g/L CH3COONa、0.0659g/L KH2PO4、0.722g/L KNO3、0.20g/L MgSO4·7H2O, 2ml microelement.
(5) identification of aerobic denitrification polyP bacteria: 0.1mlA bacterium and the 0.1ml B bacterium denitrification culture of logarithmic phase are taken respectively Base inoculation of suspension liquid bromthymol blue solid medium (abbreviation BTB culture medium), culture to culture medium is grown under the conditions of 30 DEG C Clear bacterium colony.BTB culture medium purpose is to examine the presence of aerobic denitrifying bacteria herein, and BTB is as acid-base indicator, alkaline environment In will become blue, when denitrifying bacteria generate NH3When, culture medium locally will form alkaline environment, therefore culture medium is made to become blue Bacterium colony be aerobic denitrifying bacteria.BTB culture medium prescription is every liter and contains 5gCH3COONa、0.0659gKH2PO4、 0.722gKNO3、0.20gMgSO4·7H2O, 2ml microelement, 20g/L agar, 10g/L BTB.BTB culture medium needs 121 DEG C Sterilize 20min.
(6) secondary screening and preservation of aerobic denitrification polyP bacteria: training of the colony inoculation of picking blue region to rich Nitrogen-and Phosphorus-containing It supports in base, detects OD every 6h600、PO4 3--P、NO3 --N、NO2 -- N, is in logarithmic growth stationary phase for 24 hours, and detection takes off at this time Nitrogen phosphor-removing effect selects the optimal strain of Nitrogen/Phosphorus Removal to be saved with freeze-drying.The method acclimation and screening comes out thin Bacterium is aerobic denitrification polyP bacteria.It is identified, it is Pseudomonas stutzeri ATCC17588, full genome sequencing is stepped on Record number is CP002881.
2, with following verification experimental verification beneficial effects of the present invention
(1) it tests one: the adaptability to low C/N ratio simulation sewage being examined to carry out as follows:
One, the expansion culture of aerobic denitrification polyP bacteria: the single aerobic denitrification polyP bacteria bacterium of picking in an aseptic environment It falls to be inoculated into and expand in culture medium, cultivated in water-bath constant temperature oscillator under the conditions of 30 DEG C, detect OD600 value every 6h.Expand Big culture medium prescription is 10g peptone/L, 5g yeast extract leachate/L, 10gNaCl/L, with 1moL/LNaOH and 1moL/LHCl Adjust pH to 7.0-7.2.
Two, configuration C/N ratio be respectively 2,4,6,8,10 simulation sewage: weigh respectively 2.34g, 4.79g, 7.04g, 9.36g、11.7gCH3COONa and 0.0659gKH2PO4、2.888gKNO3、0.20gMgSO4·7H2O, 2ml microelement is dissolved in In 1L deionized water, take the configured sewage of 100mL in 150mL conical flask respectively.
Three, 1mL is taken to be in the expansion culture medium inoculation of suspension liquid of logarithmic phase (OD600 >=0.8) to different C/N ratios respectively In conical flask, is cultivated in water-bath constant temperature oscillator under the conditions of 30 DEG C, monitor PO afterwards for 24 hours4 3--P、NO3 -N content.
When treatment effect is shown in Fig. 2: C/N ratio >=4, to NO3 -The removal rate of-N is maintained at 90%, to PO4 3-The removal rate of-P reaches To 80%, under conditions of C/N ratio=4, to NO3 --N、PO4 3-- P removal rate with higher can adapt to the water of low C/N ratio Matter requirement.
(2) test two: Nitrogen/Phosphorus Removal test procedure is as follows:
One, configuration is with NO3 -- N or NO2 -- N is the simulation sewage of only nitrogen source: weighing 2.888gKNO3(or 1.972gNaNO2) and 5gCH3COONa、0.0659gKH2PO4、0.20gMgSO4·7H2O, 2ml microelement is dissolved in 1L deionization In water, take the configured solution of 100mL in 150mL conical flask respectively.
Two, 1mL is taken to be in the expansion culture medium inoculation of suspension liquid of logarithmic phase (OD600 >=0.8) to above-mentioned conical flask respectively In, it is cultivated in water-bath constant temperature oscillator under the conditions of 30 DEG C, monitors PO every 12h4 3--P、NO3 -- N or NO2 -N content.
Fig. 3 is aerobic denitrification polyP bacteria to NO3 -- N and PO4 3-The removal situation of-P: NO when for 24 hours3 -- N removal rate reaches To 100%, removal efficiency is up to 16.7mg/h.L and keeps stable thereafter.The PO in 36h4 3-Removal rate reach highest 83.74%, removal efficiency 0.525mg/h.L are discharged with the decline of microorganism without apparent phosphorus.
Fig. 4 is aerobic denitrification polyP bacteria to NO2 -- N and PO4 3-The removal situation of-P: the NO in 48h2 -The removal rate of-N Reach 100%, removal efficiency is up to 8.355mg/h.L, keeps stablizing thereafter.It is gradually increased in 0-72h dephosphorization amount, in 72h Removal rate reaches best 81.64%.
Comparative example 1
Using the river Hu Xi supernatant as strain source, by process identical with present case and method, the strain screened Denitrification effect is preferable, reaches 80% or so, but to the removal effect of phosphorus only 30% or so, this may be because bed mud is compared with supernatant Phosphorus rich in has carried out natural abundance to aerobic denitrification polyP bacteria.
Comparative example 2
Using certain urban wastewater treatment firm clarifier sludge as strain source, acclimating, discovery sieve are carried out using this method Select obtained strain Nitrogen/Phosphorus Removal bad, it may be possible to because bacterium is in aerobic state always in this method, and sewage Treatment plant's sludge will achieve the purpose that denitrogenation dephosphorizing by anaerobic and aerobic (or anoxic) alternate run, therefore in sewage treatment plant It is difficult to screen the high-efficient bacterial strain of denitrogenation dephosphorizing.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include Within protection scope of the present invention.

Claims (10)

1. a kind of handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage, which is characterized in that including as follows Step:
(1) Enrichment of bacteria: black and odorous water bed mud being inoculated into enriched medium and is cultivated, and obtains the bacterium in the bed mud To enrichment, enriched medium suspension is obtained；
(2) aerobic denitrification polyP bacteria primary dcreening operation: by the enriched medium inoculation of suspension liquid in logarithmic phase to solid culture It is cultivated on base, until the solid culture primary surface grows bacterium colony；
(3) domestication of aerobic denitrification polyP bacteria: the different single colonie of form is selected from the solid medium and is inoculated with respectively It is cultivated into denitrification culture medium, it is induced to carry out aerobic denitrification denitrogenation dephosphorizing, obtain domestication culture medium suspension；
(4) identification of aerobic denitrification polyP bacteria: the domestication culture medium inoculation of suspension liquid is consolidated to bromthymol blue denitrification It is cultivated on body culture medium, so that culture medium is become blue bacterium colony is aerobic denitrification polyP bacteria；
(5) secondary screening and preservation of aerobic denitrification polyP bacteria: by the colony inoculation of the blue region to the identification of rich Nitrogen-and Phosphorus-containing In culture medium, the bacterium colony in logarithmic growth phase and with best Nitrogen/Phosphorus Removal is taken to save backup.
2. acclimation and screening method as described in claim 1, which is characterized in that further comprise the steps of:
(6) it takes and is cultivated in the identification culture medium inoculation of suspension liquid to solid medium of logarithmic phase, until the solid culture Primary surface grows bacterium colony；
(7) step (3) is repeated to step (5), is saved backup until obtaining the stable bacterium colony of Nitrogen/Phosphorus Removal.
3. acclimation and screening method as described in claim 1, which is characterized in that black and odorous water bed mud 156.3- containing total phosphorus 162.4mg/kg, overlying water total phosphorus are 1.4-1.89mg/L, PO4 3-- P is 1.13-1.5mg/L, NH4 +- N is 9.22- 22.3mg/L NO3 -- N is 2.6-7.4mg/L, NO2 -- N is 0.01-0.22mg/L.
4. acclimation and screening method as described in claim 1, which is characterized in that step (1) the enrichment culture based formulas is 5- 8g/L peptone, 5-8g/L yeast extract leachate and 5-10g/LNaCI.
5. acclimation and screening method as described in claim 1, which is characterized in that step (2) described condition of culture are as follows: in 25-30 DEG C constant incubator in cultivate 48-72 hours.
6. acclimation and screening method as described in claim 1, which is characterized in that step (2) the solid culture based formulas is 5- 8g/L CH3COONa、0.0659-0.1g/L KH2PO4、0.722-1g/L KNO3、0.10-0.20g/LMgSO4·7H2O、20g/ L agar and 1-3mL/L microelement；The trace element formula are as follows: in every liter of microelement containing 10-15g EDTA, 0.10-0.20g ZnSO4、1.0-2.0g MnCl2·4H2O、0.10-0.5g FeSO4·7H2O、0.10-0.5g CuSO4· 5H2O、0.10-0.50g CoCl2·6H2O、0.10-0.20g Na2MoO4·2H2O and 0.10-0.20g CaCl2。
7. acclimation and screening method as described in claim 1, which is characterized in that step (3) the denitrification culture medium prescription is 5-8g/L CH3COONa、0.0659-0.1g/LKH2PO4、0.722-1g/L KNO3、0.10-0.20g/L MgSO4.7H2O、1- 3ml/L microelement；The trace element formula are as follows: contain 10-15g EDTA, 0.10-0.20g in every liter of microelement ZnSO4、1.0-2.0g MnCl2·4H2O、0.10-0.5g FeSO4·7H2O、0.10-0.5g CuSO4·5H2O、0.10- 0.50g CoCl2·6H2O、0.10-0.20gNa2MoO4·2H2O and 0.10-0.20g CaCl2。
8. acclimation and screening method as described in claim 1, which is characterized in that step (3) condition of culture is in 30 DEG C of water 72h is cultivated in bath constant temperature oscillator.
9. acclimation and screening method as described in claim 1, which is characterized in that step (4) the bromthymol blue denitrification is solid Body culture medium prescription is 5-8g/L CH3COONa、0.0659-0.1g/L KH2PO4、0.722-1g/L KNO3、0.10-0.20g/ L MgSO4·7H2O, 1-3ml microelement/L, 20g/L agar and 10g/L bromthymol blue；The trace element formula are as follows: every It rises and contains 10-15g EDTA, 0.10-0.20g ZnSO in the microelement4、1.0-2.0g MnCl2·4H2O、0.10-0.5g FeSO4·7H2O、0.10-0.5g CuSO4·5H2O、0.10-0.50g CoCl2·6H2O、0.10-0.20g Na2MoO4· 2H2O and 0.10-0.20g CaCl2；Step (4) condition of culture is to cultivate in constant incubator under the conditions of 25-30 DEG C 48-72h。
10. acclimation and screening method as described in claim 1, which is characterized in that step (5) is described to identify that culture medium prescription is 5- 8g/L CH3COONa、0.722-2.888g/LKNO3、0.0659-0.1g/L KH2PO4、0.20g/L MgSO4·7H2O and 1- 3mL/L microelement；It or is 5-8g/L CH3COONa、0.493-1.972/L NaNO2、0.0659-0.1g/L KH2PO4、 0.10-0.20g/L MgSO4·7H2O and 1-3mL microelement/L；The trace element formula are as follows: in every liter of microelement Contain 10-15g EDTA, 0.10-0.20g ZnSO4、1.0-2.0g MnCl2·4H2O、0.10-0.5g FeSO4·7H2O、 0.10-0.5g CuSO4·5H2O、0.10-0.50g CoCl2·6H2O、0.10-0.20g Na2MoO4·2H2O and 0.10- 0.20g CaCl2。
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