CN102864075A - Ammonia-oxidizing bacterium screening and testing reagent box, screening and testing method and bacterium sample separating method - Google Patents
Ammonia-oxidizing bacterium screening and testing reagent box, screening and testing method and bacterium sample separating method Download PDFInfo
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- CN102864075A CN102864075A CN2012103813669A CN201210381366A CN102864075A CN 102864075 A CN102864075 A CN 102864075A CN 2012103813669 A CN2012103813669 A CN 2012103813669A CN 201210381366 A CN201210381366 A CN 201210381366A CN 102864075 A CN102864075 A CN 102864075A
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Abstract
Disclosed are an ammonia-oxidizing bacterium screening and testing reagent box, a screening and testing method and a bacterium sample separating method. The ammonia-oxidizing bacterium screening and testing reagent box comprises a bacterium strain enrichment tube, ammonia-oxidizing testing tubes 1, 2 and 3, bacterium strain preservative liquid and color developers A and B. The screening and testing method includes inoculating bacterium strains to be detected to the ammonia-oxidizing testing tubes 1, 2 and 3 to culture after enriching the bacterium strains to be detected via the bacterium strain enrichment tube, and storing enrichment bacterium liquid by the bacterium strain preservative liquid simultaneously; adding the color developer A and B after culturing of the ammonia-oxidizing testing tubes and judging ammonia-oxidizing capability of the bacterium strains according to color development states. The bacterium sample separation method includes culturing bacterium samples, diluting, applying and separating. The ammonia-oxidizing bacterium screening and testing reagent box, the screening and testing method and the bacterium sample separating method has the advantages that effect of quickly screening and testing ammonia-oxidizing bacteria in high throughput is realized, one-stop operations of culturing, testing and preserving of the bacterium strains are realized.
Description
Technical field:
The invention belongs to a kind of bacteria screening authentication method and test kit, particularly a kind of ammonia oxidizing bacteria Screening and Identification test kit, Screening and Identification and bacterium sample separation method.
Background technology:
Ammonia nitrogen is one of important substance that causes water pollution.Contain a large amount of ammonia nitrogens in the municipal effluent at present, can cause serious environmental pollution if directly be discharged in the physical environment, cause the eutrophication in river, lake, water body self-purification ability is weakened.Biological denitrificaion is one of most economical effective means in the present denitrogenation of waste water, and the conversion of ammonia nitrogen depends on the effect of nitrated microorganism.Bio-denitrifying sewage comprises nitrated stage and denitrification stage.Wherein the nitrated stage comprises two stages: (1) is with NH
4+-N is oxidized to NO
2-The ammonia oxidation bacteria of-N (Ammonia oxidizing bacteria, AOB); (2) with NO
2--N is oxidized to NO
3-The NOB of-N (Nitrite oxidizing bacteria, NOB).Finish denitrogenation through the nitrate that nitrifying process forms by denitrification formation nitrogen effusion water body.In the nitrated stage, the ammonia oxidation process that is oxidized to nitrite nitrogen by ammonia nitrogen is the rate-limiting step of whole reaction, and the enrichment, the Fast Growth that therefore accelerate ammonia oxidation bacteria are present urgent problems.
Have at present a large amount of for the screening of ammonia oxidizing bacteria, the research of separation and Culture.Patent CN1884480A has described and has a kind ofly carried out enrichment with the extractum carnis substratum, and then picking list bacterium colony from the beef extract-peptone solid medium is inoculated into the ammonia oxidation substratum and obtains ammonia oxidizing bacteria.Patent CN 101434905A has described a kind of the cultivation by the method that progressively improves ammonia nitrogen loading can tolerate the active sludge that ammonia nitrogen in high density is loaded, again separation sieve menu bacterium from active sludge.Patent CN 102268386A has described and a kind of mud has been carried out being coated with silica gel plate after the enrichment, and picking list bacterium colony obtains ammonia oxidizing bacteria in that the flat board line that contains o-cresolsulfonphthalein is minute pure.The method of present various screening ammonia oxidizing bacterias has all obtained good result, but the Screening and Identification for ammonia oxidizing bacteria generally all adopts artificial medium to carry out indirect verification, less than verifying under Sewage Environment or result and Sewage Environment being carried out the match contrast.And adopt traditional verification method to have large, the inefficient problem of workload.
Summary of the invention:
The object of the invention is to overcome above-mentioned defective, a kind of ammonia oxidizing bacteria Screening and Identification test kit, process for screening and identifying and bacterium sample separation method are provided; The method is to have designed by experiment a series of artificial ammonia oxidations to identify that substratum carries out ammoxidation capability to microorganism and identifies, and through comparing match with the ammonia oxidation result of microorganism in municipal wastewater, medical sewage, final design has gone out 3 groups of artificial ammonia oxidations and has identified that substratum carries out the ammonia oxidation effect identification to microorganism, and its result can absolutely cover, simulate the ammonia oxidation effect of microorganism under Sewage Environment; This test kit is that the artificial evaluation substratum that will filter out is prepared into aseptic prefabricated pipe, and in conjunction with bacterial strain enrichment pipe, ammonia oxidation assessor, bacterial strain preservative fluid, developer a kind of can high-throughput, simple to operate, one-stop ammonia oxidizing bacteria Screening and Identification test kit fast.
The objective of the invention is to be achieved through the following technical solutions: a kind of ammonia oxidizing bacteria Screening and Identification test kit is characterized in that: it is comprised of bacterial strain enrichment pipe, ammonia oxidation assessor 1-3, bacterial strain preservative fluid, developer A and developer B; Wherein:
The bacterial strain enrichment pipe:
In the bacterial strain enrichment pipe enrichment medium is housed, every liter of enrichment medium ingredient is as follows: ammonium sulfate 250-780mg, glucose 0.8-2.1g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 7.00-8.50 with yellow soda ash and the HCl of concentration 5%;
After enrichment medium preparation is finished and transferred pH, enrichment medium divided install in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds enrichment medium 0.8ml, after packing is complete, and 115 ℃ of sterilization 20-30min;
Ammonia oxidation assessor 1:
Ammonia oxidation is housed in the ammonia oxidation assessor 1 identifies substratum (1), every liter of ammonia oxidation identifies that substratum (1) ingredient is as follows: ammonium sulfate 250-780mg, glucose 0.1-1.2g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 7.00-8.50 with yellow soda ash and the HCl of concentration 5%; After ammonia oxidation identifies that substratum (1) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (1) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (1) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
Ammonia oxidation assessor 2:
Ammonia oxidation is housed in the ammonia oxidation assessor 2 identifies substratum (2), every liter of ammonia oxidation identifies that substratum (2) ingredient is as follows: ammonium sulfate 250-780mg, sodium-acetate 0.4-1.2g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (2) yellow soda ash and the HCl accent pH to 7.00-8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (2) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (2) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (2) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
Ammonia oxidation assessor 3:
Ammonia oxidation is housed in the ammonia oxidation assessor 3 identifies substratum (3), every liter of ammonia oxidation identifies that substratum (3) ingredient is as follows: ammonium sulfate 250-780mg, glucose 0-50 mg; Sodium-acetate 0-50 mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium carbonate 1-5g, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (3) yellow soda ash and the HCl accent pH to 7.00-8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (3) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (3) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (3) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
The bacterial strain preservative fluid:
Bacterial strain preservative fluid composition is as follows: glycerine 30-50ml, deionized water 50-70ml;
Developer A:
Developer A composition is as follows: Sulphanilic Acid 0.5-0.8g, 15% acetum 100ml;
Developer B:
Developer B composition is as follows: alpha-naphthylamine 0.1-0.3g, 15% acetum 100ml;
Wherein:
Every liter of micro-mother liquor A ingredient is as follows: ferrous sulfate 5g, EDETATE SODIUM 5g;
Wherein:
Every liter of micro-mother liquor B ingredient is as follows: EDETATE SODIUM 1.5g, ZnSO
4.7H
2O 430mg, CoCl
2.6H
2O 240mg, MnCl
2629mg, CuSO
4.5H
2O 250mg, Na
2MoO
4.2H
2O 220mg, NiCl
2.6H
2O 190 mg, H
3BO
314mg.
A kind of employing ammonia oxidizing bacteria Screening and Identification test kit carries out the ammonia oxidizing bacteria process for screening and identifying, it is characterized in that: undertaken by following step:
Step 1: the preparation of bacterium sample to be measured:
Bacterium sample to be measured is:
(a) through the sewage that separates, the bacterial strain in the mud;
(b) the not sewage of separation and purification, the flora in the mud;
Step 2:
The bacterium sample to be measured that step 1 is prepared is seeded in the bacterial strain enrichment pipe with transfering loop or pipettor, in the bacterial strain enrichment pipe enrichment medium is housed, bacterial strain enrichment pipe lid is tightened, culture condition is selected cultivation in static 12-16 hour under the 30-35 ℃ of temperature, or the bacterial strain enrichment pipe is kept flat into shaking table 30-35 ℃, 150-200rpm concussion cultivation in 12-16 hour;
Step 3:
The bacterial strain of step 2 enrichment is seeded to respectively in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3, and inoculum size is 10-60ul; The ammonia oxidation assessor is kept flat into shaking table, and 24-48h is cultivated in concussion under 30-35 ℃, 150-200rpm condition;
Step 4:
Inoculate adding 0.8ml bacterial strain preservative fluid in the residual bacterium liquid of complete backward bacterial strain enrichment pipe, the concussion mixing is put into-20 ℃ of Refrigerator stores;
Step 5:
The ammonia oxidation assessor of step 3 drips developer A 2-3 in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3 respectively and drips fully concussion after cultivating 24-48h, add respectively again developer B, 2-3 drips, and fully concussion is observed colour-change after leaving standstill 20min; Color pulverize redness or red-purple are that ammonia oxidation is positive, and with "+" expression, color is unchanged to be that ammonia oxidation is negative, with "-" expression;
Step 6: the result of step 5 is carried out record, and colour developing is the result compare by table 1, draws the bacterial strain results of property.
Table 1: ammonia oxidation qualification result analytical table
A kind of employing ammonia oxidizing bacteria Screening and Identification test kit carries out the ammonia oxidizing bacteria process for screening and identifying, and it is characterized in that: the separation method of said bacterium sample to be measured is undertaken by following step:
Step 1:
The collection of ammonia oxidizing bacteria bacterium sample is cultivated: comprise the sample collection cultivation of autotrophy ammonia oxidizing bacteria bacterium and the collection of heterotrophic ammonia-oxidizing Bacteria sample cultivation;
(1), the collection of autotrophy ammonia oxidizing bacteria bacterium sample is cultivated: with sewage, the flora sample in the mud of separation and purification do not mix with equal-volume autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution, putting into the shaking table concussion cultivates, temperature 20-30 ℃, rotating speed 150-200rpm, concussion was cultivated 5-7 days;
(2), the collection of heterotrophic ammonia-oxidizing Bacteria sample is cultivated: with sewage, the flora sample in the mud of separation and purification do not mix with equal-volume heterotrophic ammonia-oxidizing Bacteria sample nutrient solution, putting into the shaking table concussion cultivates, temperature 20-30 ℃, rotating speed 150-200rpm, concussion was cultivated 1-3 days;
Step 2:
Cultivating bacterium liquid dilution spread separates:
Will be through step 1(1) the bacterium liquid cultivated of autotrophy ammonia oxidizing bacteria bacterium sample collection carry out gradient dilution to 10
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the autotrophy ammonia oxidizing bacteria solid separation culture medium and separate, 20-30 ℃ of cultivation 5-10 days;
Will be through step 1(2) the bacterium liquid cultivated of heterotrophic ammonia-oxidizing Bacteria sample collection carry out gradient dilution to 10
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the heterotrophic ammonia-oxidizing bacterial solids isolation medium and separate, 20-30 ℃ of cultivation 2-3 days.
Said autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution is:
Every liter of autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution ingredient is ammonium sulfate 400-1200mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium carbonate 1-10g, sodium bicarbonate 50-200mg, sal epsom 100-200mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, pH 7.00-8.50.
Said heterotrophic ammonia-oxidizing Bacteria sample nutrient solution is:
Every liter of heterotrophic ammonia-oxidizing Bacteria sample nutrient solution ingredient is ammonium sulfate 400-1200mg, glucose 0.5-3.0g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 100-200mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, pH 7.00-8.50.
Said autotrophy ammonia oxidizing bacteria solid separation culture medium is:
Every liter of autotrophy ammonia oxidizing bacteria solid separation culture medium ingredient is ammonium sulfate 400-1200mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 200mg, trace element mother liquor A 1ml, trace element mother liquor B 1ml, agar 1.2%-2.0%, pH 7.00-8.50.
Said heterotrophic ammonia-oxidizing bacterial solids isolation medium is:
Every liter of heterotrophic ammonia-oxidizing bacterial solids isolation medium ingredient is ammonium sulfate 400-1200mg, glucose 0.5-3.0g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 200mg, micro-mother liquor A 1ml, micro-mother liquor B 1ml, agar 1.2%-2.0%, pH 7.00-8.50.
Advantage and beneficial effect that ammonia oxidizing bacteria Screening and Identification test kit disclosed by the invention compared with prior art has are:
Reach specifically the effect that ammonia oxidizing bacteria is quick, high flux screening is identified, realize spawn culture, evaluation, the one-stop operation of preservation; Especially the screening of autotrophy and peculiar ammonia oxidizing bacteria in sewage, active sludge, aquaculture water, natural water, the soil there are good specific aim and effect.
Embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used for explanation the present invention and is not used in and limits the scope of the invention.Need to prove: should guarantee that operating environment and relevant appliance sterilize before the operation.The said raw material of the present invention is the commercially available prod if no special instructions.
Embodiment 1:
The autotrophy ammonia oxidizing bacteria separates, Screening and Identification
Screening autotrophy ammonia oxidizing bacteria from active sludge, sample picks up from the active sludge in Haihe River Hospital, Tianjin City aeration tank, and gathers municipal wastewater as test kit effect comparison sample.
Step 1: the collection of autotrophy ammonia oxidizing bacteria bacterium sample is cultivated: the sample active sludge mixed with equal-volume autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution, puts into the shaking table concussion and cultivate, and 27 ℃ of temperature, rotating speed 180rpm, concussion was cultivated 7 days; Every liter of autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution ingredient is ammonium sulfate 578mg, potassium primary phosphate 730mg, Sodium phosphate dibasic 1.10g, calcium carbonate 5g, sodium bicarbonate 60mg, sal epsom 200mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, pH 8.00;
Step 2: cultivate bacterium liquid dilution spread and separate: will carry out gradient dilution to 10 through the bacterium liquid that step 1 is cultivated from anaerobic ammonium oxidation
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the autotrophy ammonia oxidizing bacteria solid separation culture medium and separate, 25 ℃ of cultivations 7 days; Every liter of autotrophy ammonia oxidizing bacteria solid separation culture medium ingredient is ammonium sulfate 578mg, potassium primary phosphate 730mg, Sodium phosphate dibasic 1.10g, calcium chloride 50mg, sodium bicarbonate 60mg, sal epsom 200mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, agar 2.0%, pH 8.00;
Step 3: bacterial strain ammoxidation capability Screening and Identification:
A. choose the flat board of colony number about 1-300 that separates through the dull and stereotyped coating of step 2, single bacterium colony on the flat board is carried out mark, be total to 237 single bacterium colonies of mark, with transfering loop with in the bacterial strain enrichment pipe of single colony inoculation in the test kit, in the bacterial strain enrichment pipe enrichment medium is housed, bacterial strain enrichment pipe lid is tightened, kept flat into 30 ℃ of shaking tables, 170rpm concussion and cultivated in 12-16 hour; Wherein:
Every liter of enrichment medium ingredient is as follows: ammonium sulfate 580mg, glucose 2.0g, potassium primary phosphate 680mg, Sodium phosphate dibasic 1.0g, sal epsom 150mg, calcium chloride 30mg, sodium bicarbonate 80mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, above substratum is transferred pH to 7.50 with yellow soda ash and the HCl of concentration 5%; After enrichment medium preparation is finished and transferred pH, enrichment medium divided install in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds enrichment medium 0.8ml, after packing is complete, and 115 ℃ of sterilization 20-30min;
B. the bacterium liquid with step a enrichment is seeded to respectively in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3, and inoculum size is 20ul, ammonia oxidation is housed respectively in the ammonia oxidation assessor 1,2,3 identifies substratum 1,2,3; The ammonia oxidation assessor is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition; Wherein:
Ammonia oxidation is housed in the ammonia oxidation assessor 1 identifies substratum (1), every liter of ammonia oxidation identifies that substratum (1) ingredient is as follows: ammonium sulfate 780mg, glucose 0.1g, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, sal epsom 100mg, calcium chloride 5mg, sodium bicarbonate 20mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 7.00 with yellow soda ash and the HCl of concentration 5%; After ammonia oxidation identifies that substratum (1) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (1) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (1) 0.8ml, the complete rear 115 ℃ of sterilization 25min of packing;
Ammonia oxidation is housed in the ammonia oxidation assessor 2 identifies substratum (2), every liter of ammonia oxidation identifies that substratum (2) ingredient is as follows: ammonium sulfate 780mg, sodium-acetate 0.4g, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, sal epsom 100mg, calcium chloride 5mg, sodium bicarbonate 20mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (2) yellow soda ash and the HCl accent pH to 7.00 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (2) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (2) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (2) 0.8ml, the complete rear 115 ℃ of sterilization 25min of packing;
Ammonia oxidation is housed in the ammonia oxidation assessor 3 identifies substratum (3), every liter of ammonia oxidation identifies that substratum (3) ingredient is as follows: ammonium sulfate 780mg, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, sal epsom 100mg, calcium carbonate 2g, sodium bicarbonate 20mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, substratum (3) yellow soda ash and the HCl accent pH to 7.00 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (3) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (3) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (3) 0.8ml, the complete rear 115 ℃ of sterilization 25min of packing;
C. with municipal wastewater as test kit effect comparison sample: municipal wastewater COD is 152 mg/L, and ammonia nitrogen is 25.3 mg/L, minute is filled in the 2ml cryopreservation tube minute loading amount 0.8ml/g pipe, 115 ℃ of sterilization 25min; The bacterium liquid of step a enrichment is seeded in the municipal wastewater, and inoculum size is 20ul, and cryopreservation tube is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition;
D. inoculate complete after, remaining bacterium liquid adds 0.8ml bacterial strain preservative fluid in the step a bacterial strain enrichment pipe, the concussion mixing is put into-20 ℃ of Refrigerator stores;
E. ammonia oxidation assessor and sewage contrast: respectively to the ammonia oxidation assessor 1,2 after step b and c cultivate 48h, 3 and the municipal wastewater pipe in drip developer A 2-3 and drip, fully concussion, add respectively developer B 2-3 again and drip, fully concussion is observed colour-change after leaving standstill 20min; Color pulverize redness or red-purple are that ammonia oxidation is positive, and with "+" expression, color is unchanged to be that ammonia oxidation is negative, with "-" expression;
F. interpretation of result: through color reaction, have 38 strains in the single bacterium colony of 237 strains and produce positive reaction, positive findings sees Table 2:
Table 2: Haihe River hospital autotrophy ammonia oxidizing bacteria positive findings table
By contrasting with sewage, the bacterial strain that screens is the ammonia oxidation positive in sewage bacterial strain all is positive in the ammonia oxidation assessor; The 38 strain ammonia oxidizing bacterias that filter out by the ammonia oxidation assessor have 29 strains that ammoxidation is arranged under Sewage Environment, and positive ratio is greater than 75%.
Embodiment 2:
The autotrophy ammonia oxidizing bacteria separates, Screening and Identification
Screening autotrophy ammonia oxidizing bacteria from active sludge, the active sludge sample picks up from Jizhuangzi Waste Water Treatment Plant, Tianjin aeration tank.
Step 1 among the method that the collection of ammonia oxidizing bacteria bacterium sample is cultivated, separated and the embodiment 1, step 2 are identical, and embodiment 2 selects 364 strains list bacterium colony altogether; Wherein:
Every liter of autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution ingredient is ammonium sulfate 1200mg, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, calcium carbonate 1g, sodium bicarbonate 50mg, sal epsom 100mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, pH 7.00;
Every liter of autotrophy ammonia oxidizing bacteria solid separation culture medium ingredient is ammonium sulfate 1200mg, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, calcium chloride 40mg, sodium bicarbonate 50mg, sal epsom 200mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, agar 1.2%, pH 7.00;
The ammonia oxidizing bacteria Screening and Identification is identical with step 3 among the embodiment 1; Wherein:
Every liter of enrichment medium ingredient is as follows: ammonium sulfate 780mg, glucose 0.8g, potassium primary phosphate 480mg, Sodium phosphate dibasic 0.7g, sal epsom 100mg, calcium chloride 5mg, sodium bicarbonate 20mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, above substratum is transferred pH to 8.00 with yellow soda ash and the HCl of concentration 5%; After enrichment medium preparation is finished and transferred pH, enrichment medium divided install in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds enrichment medium 0.8ml, after packing is complete, and 115 ℃ of sterilization 25min;
Every liter of ammonia oxidation identifies that substratum (1) ingredient is as follows: ammonium sulfate 500mg, glucose 0.6g, potassium primary phosphate 600mg, Sodium phosphate dibasic 1.0g, sal epsom 150mg, calcium chloride 35mg, sodium bicarbonate 70mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, above substratum is transferred pH to 8.00 with yellow soda ash and the HCl of concentration 5%; After ammonia oxidation identifies that substratum (1) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (1) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (1) 0.8ml, the complete rear 115 ℃ of sterilization 30min of packing;
Every liter of ammonia oxidation identifies that substratum (2) ingredient is as follows: ammonium sulfate 500mg, sodium-acetate 0.6g, potassium primary phosphate 600mg, Sodium phosphate dibasic 1.0g, sal epsom 150mg, calcium chloride 35mg, sodium bicarbonate 70mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, substratum (2) yellow soda ash and the HCl accent pH to 8.00 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (2) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (2) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (2) 0.8ml, the complete rear 115 ℃ of sterilization 30min of packing;
Every liter of ammonia oxidation identifies that substratum (3) ingredient is as follows: ammonium sulfate 500mg, glucose 25 mg, sodium-acetate 25mg, potassium primary phosphate 600mg, Sodium phosphate dibasic 1.0g, sal epsom 150mg, calcium carbonate 3g, sodium bicarbonate 70mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (3) yellow soda ash and the HCl accent pH to 8.00 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (3) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (3) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (3) 0.8ml, the complete rear 115 ℃ of sterilization 30min of packing;
Through color reaction, have 43 strains to present the ammonia oxidation positive, positive findings such as table 3 in the single bacterium colony of 364 strains:
Table 3: discipline village's Sewage Plant autotrophy ammonia oxidizing bacteria positive findings table
By contrasting with sewage, the bacterial strain that screens is the ammonia oxidation positive in sewage bacterial strain all is positive in the ammonia oxidation assessor; The 43 strain ammonia oxidizing bacterias that filter out by the ammonia oxidation assessor have 36 strains that ammoxidation is arranged under Sewage Environment, and positive ratio is greater than 80%.
Embodiment 3:
The heterotrophic ammonia-oxidizing bacterium separates, Screening and Identification
Screening heterotrophic ammonia-oxidizing bacterium from active sludge, sample picks up from the active sludge in Haihe River Hospital, Tianjin City aeration tank, and gathers municipal wastewater as test kit effect comparison sample.
Step 1: the collection of heterotrophic ammonia-oxidizing Bacteria sample is cultivated: the sample active sludge mixed with equal-volume heterotrophic ammonia-oxidizing Bacteria sample nutrient solution, puts into the shaking table concussion and cultivate, and 27 ℃ of temperature, rotating speed 150rpm, concussion was cultivated 1 day; Every liter of heterotrophic ammonia-oxidizing Bacteria sample nutrient solution ingredient is ammonium sulfate 498mg, glucose 2.1g, potassium primary phosphate 730mg, Sodium phosphate dibasic 1.10g, calcium chloride 80mg, sodium bicarbonate 60mg, sal epsom 200mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, pH 8.00;
Step 2: cultivate bacterium liquid dilution spread and separate: will carry out gradient dilution to 10 through the bacterium liquid that step 1 heterotrophic ammonia-oxidizing is cultivated
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the heterotrophic ammonia-oxidizing bacterial solids isolation medium and separate, 27 ℃ of cultivations 2 days; Every liter of heterotrophic ammonia-oxidizing bacterial solids isolation medium ingredient is ammonium sulfate 498mg, glucose 2.1 g, potassium primary phosphate 730mg, Sodium phosphate dibasic 1.10g, calcium chloride 80mg, sodium bicarbonate 60mg, sal epsom 200mg, micro-mother liquor A 1ml, micro-mother liquor B 1ml, agar 1.5%, pH 8.00;
Step 3: bacterial strain ammoxidation capability Screening and Identification:
A. choose the flat board of colony number about 1-300 that separates through the dull and stereotyped coating of step 2, single bacterium colony on the flat board is carried out mark, be total to 308 single bacterium colonies of mark, with transfering loop with in the bacterial strain enrichment pipe of single colony inoculation in the test kit, in the bacterial strain enrichment pipe enrichment medium is housed, bacterial strain enrichment pipe lid is tightened, kept flat into 30 ℃ of shaking tables, 170rpm concussion and cultivated in 12-16 hour; Wherein:
Every liter of enrichment medium ingredient is as follows: ammonium sulfate 250mg, glucose 2.1g, potassium primary phosphate 890mg, Sodium phosphate dibasic 1.5g, sal epsom 200mg, calcium chloride 50mg, sodium bicarbonate 120mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, above substratum is transferred pH to 8.50 with yellow soda ash and the HCl of concentration 5%; After enrichment medium preparation is finished and transferred pH, enrichment medium divided install in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds enrichment medium 0.8ml, after packing is complete, and 115 ℃ of sterilization 30min;
B. the bacterium liquid with step a enrichment is seeded to respectively in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3, and inoculum size is 40ul, ammonia oxidation is housed respectively in the ammonia oxidation assessor 1,2,3 identifies substratum 1,2,3; The ammonia oxidation assessor is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition; Wherein:
Ammonia oxidation is housed in the ammonia oxidation assessor 1 identifies substratum (1), every liter of ammonia oxidation identifies that substratum (1) ingredient is as follows: ammonium sulfate 250mg, glucose 0.4g, potassium primary phosphate 890mg, Sodium phosphate dibasic 1.5g, sal epsom 200mg, calcium chloride 50mg, sodium bicarbonate 120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 8.50 with yellow soda ash and the HCl of concentration 5%; After ammonia oxidation identifies that substratum (1) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (1) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (1) 0.8ml, the complete rear 115 ℃ of sterilization 20min of packing;
Ammonia oxidation is housed in the ammonia oxidation assessor 2 identifies substratum (2), every liter of ammonia oxidation identifies that substratum (2) ingredient is as follows: ammonium sulfate 250mg, sodium-acetate 1.2g, potassium primary phosphate 890mg, Sodium phosphate dibasic 1.5g, sal epsom 200mg, calcium chloride 50mg, sodium bicarbonate 120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (2) yellow soda ash and the HCl accent pH to 8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (2) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (2) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (2) 0.8ml, the complete rear 115 ℃ of sterilization 20min of packing;
Ammonia oxidation is housed in the ammonia oxidation assessor 3 identifies substratum (3), every liter of ammonia oxidation identifies that substratum (3) ingredient is as follows: ammonium sulfate 250mg, glucose 50 mg; Sodium-acetate 50mg, potassium primary phosphate 890mg, Sodium phosphate dibasic 1.5g, sal epsom 200mg, calcium carbonate 5g, sodium bicarbonate 120mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, substratum (3) yellow soda ash and the HCl accent pH to 8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (3) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (3) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (3) 0.8ml, the complete rear 115 ℃ of sterilization 20min of packing;
C. with municipal wastewater as test kit effect comparison sample: municipal wastewater COD is 184mg/L, and ammonia nitrogen is 31mg/L, minute is filled in the 2ml cryopreservation tube minute loading amount 0.8ml/ pipe, 115 ℃ of sterilization 25min; The bacterium liquid of step a enrichment is seeded in the municipal wastewater, and inoculum size is 60ul; Cryopreservation tube is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition;
D. inoculate complete after, remaining bacterium liquid adds 0.8ml bacterial strain preservative fluid in the step a bacterial strain enrichment pipe, the concussion mixing is put into-20 ℃ of Refrigerator stores;
E. ammonia oxidation assessor and sewage contrast: respectively to the ammonia oxidation assessor 1,2 after step b and c cultivate 48h, 3 and sewage in drip developer A 2-3 and drip, fully concussion, add respectively developer B 2-3 again and drip, fully concussion is observed colour-change after leaving standstill 20min; Color pulverize redness or red-purple are that ammonia oxidation is positive, and with "+" expression, color is unchanged to be that ammonia oxidation is negative, with "-" expression;
F. interpretation of result: through color reaction, have 21 strains in the single bacterium colony of 308 strains and produce positive reaction, positive findings such as table 4:
Table 4: Haihe River hospital heterotrophic ammonia-oxidizing positive for bacteria is table as a result
By contrasting with sewage, the bacterial strain that screens is the ammonia oxidation positive in sewage bacterial strain all is positive in the ammonia oxidation assessor; The 21 strain ammonia oxidizing bacterias that filter out by the ammonia oxidation assessor have 13 strains that ammoxidation is arranged under Sewage Environment, and positive ratio is greater than 60%.
Embodiment 4:
Heterotrophic ammonia-oxidizing bacterium separation screening
Screening heterotrophic ammonia-oxidizing bacterium from active sludge, the active sludge sample picks up from Jizhuangzi Waste Water Treatment Plant, Tianjin aeration tank.
Step 1 among the method that the collection of ammonia oxidizing bacteria bacterium sample is cultivated, separated and step and the embodiment 3, step 2 are identical, and embodiment 4 selects 387 strains list bacterium colony altogether;
Ammonia oxidizing bacteria ability Screening and Identification is identical with step 3 among the embodiment 3;
Through color reaction, have 25 strains to present the ammonia oxidation positive, positive findings such as table 5 in the single bacterium colony of 387 strains:
Table 5: discipline village's Sewage Plant heterotrophic ammonia-oxidizing positive for bacteria is table as a result
By contrasting with sewage, the bacterial strain that screens is the ammonia oxidation positive in sewage bacterial strain all is positive in the ammonia oxidation assessor; The 25 strain ammonia oxidizing bacterias that filter out by the ammonia oxidation assessor have 17 strains that ammoxidation is arranged under Sewage Environment, and positive ratio is greater than 65%.
Embodiment 5:
Do not separate flora sample ammonia oxidation effect identification
Take 23 of water samples from different landscape river course, Tianjin, river course for sewage discharge, the complete rear incubation chamber cryopreservation of in time putting into of sample collecting; Sample is delivered to rapidly the experiment place carry out 4 ℃ of preservations, and carry out the ammonia oxidation Performance Testing; Gather simultaneously municipal wastewater as test kit effect comparison sample;
A. the water sample fetched is drawn the 40ul sample with pipettor and be inoculated in the bacterial strain enrichment pipe in the test kit, in the bacterial strain enrichment pipe enrichment medium is housed, bacterial strain enrichment pipe lid is tightened, keep flat into 30 ℃ of shaking tables, 170rpm concussion and cultivated in 12 hours;
B. the bacterium liquid with step a enrichment is seeded to respectively in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3, and inoculum size is 40ul, ammonia oxidation is housed respectively in the ammonia oxidation assessor 1,2,3 identifies substratum 1,2,3; The ammonia oxidation assessor is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition;
C. with municipal wastewater as test kit effect comparison sample: municipal wastewater COD is 167mg/L, and ammonia nitrogen is 28.6 mg/L, minute is filled in the 2ml cryopreservation tube minute loading amount 0.8ml/ pipe, 115 ℃ of sterilization 25min; The bacterium liquid of step a enrichment is seeded in the municipal wastewater, and inoculum size is 30ul; Cryopreservation tube is kept flat into shaking table, and 48h is cultivated in concussion under 30 ℃, 170rpm condition;
D. inoculate complete after, remaining bacterium liquid adds 0.8ml bacterial strain preservative fluid in the step a bacterial strain enrichment pipe, the concussion mixing is put into-20 ℃ of Refrigerator stores;
E. ammonia oxidation assessor and sewage contrast: behind step b and c cultivation 48h, respectively to ammonia oxidation assessor 1,2,3 and sewage in drip developer A 2-3 and drip, fully concussion adds respectively developer B 2-3 again and drips, fully concussion is observed colour-change after leaving standstill 20min; Color pulverize redness or red-purple are that ammonia oxidation is positive, and with "+" expression, color is unchanged to be that ammonia oxidation is negative, with "-" expression.
F. interpretation of result: through color reaction, 23 water sample detection results such as table 6:
Table 6: river course water sample ammonia oxidizing bacteria detected result table
Identify to have 16 samples to present the ammonia oxidation positive through the ammonia oxidation identification kit, wherein have 8 water samples to have the ammonia oxidation effect in sewage, sewage ammonia oxidation positive rate is 50%, and the test kit detected result of these 8 samples all is positive, without undetected.
Claims (7)
1. ammonia oxidizing bacteria Screening and Identification test kit, it is characterized in that: it is comprised of bacterial strain enrichment pipe, ammonia oxidation assessor 1-3, bacterial strain preservative fluid, developer A and developer B; Wherein:
The bacterial strain enrichment pipe:
In the bacterial strain enrichment pipe enrichment medium is housed, every liter of enrichment medium ingredient is as follows: ammonium sulfate 250-780mg, glucose 0.8-2.1g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 7.00-8.50 with yellow soda ash and the HCl of concentration 5%;
After enrichment medium preparation is finished and transferred pH, enrichment medium divided install in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds enrichment medium 0.8ml, after packing is complete, and 115 ℃ of sterilization 20-30min;
Ammonia oxidation assessor 1:
Ammonia oxidation is housed in the ammonia oxidation assessor 1 identifies substratum (1), every liter of ammonia oxidation identifies that substratum (1) ingredient is as follows: ammonium sulfate 250-780mg, glucose 0.1-1.2g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, above substratum is transferred pH to 7.00-8.50 with yellow soda ash and the HCl of concentration 5%; After ammonia oxidation identifies that substratum (1) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (1) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (1) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
Ammonia oxidation assessor 2:
Ammonia oxidation is housed in the ammonia oxidation assessor 2 identifies substratum (2), every liter of ammonia oxidation identifies that substratum (2) ingredient is as follows: ammonium sulfate 250-780mg, sodium-acetate 0.4-1.2g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium chloride 5-50mg, sodium bicarbonate 20-120mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, substratum (2) yellow soda ash and the HCl accent pH to 7.00-8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (2) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (2) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (2) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
Ammonia oxidation assessor 3:
Ammonia oxidation is housed in the ammonia oxidation assessor 3 identifies substratum (3), every liter of ammonia oxidation identifies that substratum (3) ingredient is as follows: ammonium sulfate 250-780mg, glucose 0-50 mg, sodium-acetate 0-50 mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, sal epsom 100-200mg, calcium carbonate 1-5g, sodium bicarbonate 20-120mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, substratum (3) yellow soda ash and the HCl accent pH to 7.00-8.50 of concentration 5% are identified in above ammonia oxidation; After ammonia oxidation identifies that substratum (3) preparation is finished and transferred pH, ammonia oxidation is identified that substratum (3) minute installs in the 1.8ml screw socket cryopreservation tube, every cryopreservation tube adds ammonia oxidation and identifies substratum (3) 0.8ml, the complete rear 115 ℃ of sterilization 20-30min of packing;
The bacterial strain preservative fluid:
Bacterial strain preservative fluid composition is as follows: glycerine 30-50ml, deionized water 50-70ml;
Developer A:
Developer A composition is as follows: Sulphanilic Acid 0.5-0.8g, 15% acetum 100ml;
Developer B:
Developer B composition is as follows: alpha-naphthylamine 0.1-0.3g, 15% acetum 100ml;
Wherein:
Every liter of micro-mother liquor A ingredient is as follows: ferrous sulfate 5g, EDETATE SODIUM 5g;
Wherein:
Every liter of micro-mother liquor B ingredient is as follows: EDETATE SODIUM 1.5g, ZnSO
4.7H
2O 430mg, CoCl
2.6H
2O 240mg, MnCl
2629mg, CuSO
4.5H
2O 250mg, Na
2MoO
4.2H
2O 220mg, NiCl
2.6H
2O 190 mg, H
3BO
314mg.
2. one kind is adopted ammonia oxidizing bacteria Screening and Identification test kit claimed in claim 1 to carry out the ammonia oxidizing bacteria process for screening and identifying, it is characterized in that: undertaken by following step:
Step 1: the preparation of bacterium sample to be measured:
Bacterium sample to be measured is:
(a) through the sewage that separates, the bacterial strain in the mud;
(b) the not sewage of separation and purification, the flora in the mud;
Step 2:
The bacterium sample to be measured that step 1 is prepared is seeded in the bacterial strain enrichment pipe with transfering loop or pipettor, in the bacterial strain enrichment pipe enrichment medium is housed, bacterial strain enrichment pipe lid is tightened, culture condition is selected cultivation in static 12-16 hour under the 30-35 ℃ of temperature, or the bacterial strain enrichment pipe is kept flat into shaking table 30-35 ℃, 150-200rpm concussion cultivation in 12-16 hour;
Step 3:
The bacterial strain of step 2 enrichment is seeded to respectively in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3, and inoculum size is 10-60ul; The ammonia oxidation assessor is kept flat into shaking table, and 24-48h is cultivated in concussion under 30-35 ℃, 150-200rpm condition;
Step 4:
Inoculate adding 0.8ml bacterial strain preservative fluid in the residual bacterium liquid of complete backward bacterial strain enrichment pipe, the concussion mixing is put into-20 ℃ of Refrigerator stores;
Step 5:
The ammonia oxidation assessor of step 3 drips developer A 2-3 in ammonia oxidation assessor 1, ammonia oxidation assessor 2, the ammonia oxidation assessor 3 respectively and drips fully concussion after cultivating 24-48h, add respectively again developer B, 2-3 drips, and fully concussion is observed colour-change after leaving standstill 20min; Color pulverize redness or red-purple are that ammonia oxidation is positive, and with "+" expression, color is unchanged to be that ammonia oxidation is negative, with "-" expression;
Step 6: the result of step 5 is carried out record, and colour developing is the result compare by table 1, draws the bacterial strain results of property.
Table 1: ammonia oxidation qualification result analytical table
3. one kind is adopted the described ammonia oxidizing bacteria Screening and Identification of claim 2 test kit to carry out the ammonia oxidizing bacteria process for screening and identifying, and it is characterized in that: the separation method of said bacterium sample to be measured is undertaken by following step:
Step 1:
The collection of ammonia oxidizing bacteria bacterium sample is cultivated: comprise the sample collection cultivation of autotrophy ammonia oxidizing bacteria bacterium and the collection of heterotrophic ammonia-oxidizing Bacteria sample cultivation;
(1), the collection of autotrophy ammonia oxidizing bacteria bacterium sample is cultivated: with sewage, the flora sample in the mud of separation and purification do not mix with equal-volume autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution, putting into the shaking table concussion cultivates, temperature 20-30 ℃, rotating speed 150-200rpm, concussion was cultivated 5-7 days;
(2), the collection of heterotrophic ammonia-oxidizing Bacteria sample is cultivated: with sewage, the flora sample in the mud of separation and purification do not mix with equal-volume heterotrophic ammonia-oxidizing Bacteria sample nutrient solution, putting into the shaking table concussion cultivates, temperature 20-30 ℃, rotating speed 150-200rpm, concussion was cultivated 1-3 days;
Step 2:
Cultivating bacterium liquid dilution spread separates:
Will be through step 1(1) the bacterium liquid cultivated of autotrophy ammonia oxidizing bacteria bacterium sample collection carry out gradient dilution to 10
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the autotrophy ammonia oxidizing bacteria solid separation culture medium and separate, 20-30 ℃ of cultivation 5-10 days;
Will be through step 1(2) the bacterium liquid cultivated of heterotrophic ammonia-oxidizing Bacteria sample collection carry out gradient dilution to 10
-4, 10
-5, 10
-6, 10
-7Doubly, respectively get 80ul and be inoculated into and carry out flat board coating on the heterotrophic ammonia-oxidizing bacterial solids isolation medium and separate, 20-30 ℃ of cultivation 2-3 days.
4. bacterium sample separation method as claimed in claim 3, it is characterized in that: said autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution is:
Every liter of autotrophy ammonia oxidizing bacteria bacterium sample nutrient solution ingredient is ammonium sulfate 400-1200mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium carbonate 1-10g, sodium bicarbonate 50-200mg, sal epsom 100-200mg, micro-mother liquor A 1ml, trace element mother liquor B 1ml, pH 7.00-8.50.
5. bacterium sample separation method as claimed in claim 3, it is characterized in that: said heterotrophic ammonia-oxidizing Bacteria sample nutrient solution is:
Every liter of heterotrophic ammonia-oxidizing Bacteria sample nutrient solution ingredient is ammonium sulfate 400-1200mg, glucose 0.5-3.0g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 100-200mg, trace element mother liquor A 1ml, micro-mother liquor B 1ml, pH 7.00-8.50.
6. bacterium sample separation method as claimed in claim 3, it is characterized in that: said autotrophy ammonia oxidizing bacteria solid separation culture medium is:
Every liter of autotrophy ammonia oxidizing bacteria solid separation culture medium ingredient is ammonium sulfate 400-1200mg, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 200mg, trace element mother liquor A 1ml, trace element mother liquor B 1ml, agar 1.2%-2.0%, pH 7.00-8.50.
7. bacterium sample separation method as claimed in claim 3, it is characterized in that: said heterotrophic ammonia-oxidizing bacterial solids isolation medium is:
Every liter of heterotrophic ammonia-oxidizing bacterial solids isolation medium ingredient is ammonium sulfate 400-1200mg, glucose 0.5-3.0g, potassium primary phosphate 480-890mg, Sodium phosphate dibasic 0.7-1.5g, calcium chloride 40-120mg, sodium bicarbonate 50-200mg, sal epsom 200mg, micro-mother liquor A 1ml, micro-mother liquor B 1ml, agar 1.2%-2.0%, pH 7.00-8.50.
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