CN102911997B - The method of biological phosphate-eliminating microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability - Google Patents
The method of biological phosphate-eliminating microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability Download PDFInfo
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- CN102911997B CN102911997B CN201210437874.4A CN201210437874A CN102911997B CN 102911997 B CN102911997 B CN 102911997B CN 201210437874 A CN201210437874 A CN 201210437874A CN 102911997 B CN102911997 B CN 102911997B
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Abstract
The method of biological phosphate-eliminating microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability, it relates to a kind of dephosphorization microorganism detection substratum and detects the method for dephosphorization microorganism dephosphorization ability.The invention solves existing detection substratum specific aim poor, substratum used is not suitable for biological phosphate-eliminating microorganism dephosphorization ability and detects, and dephosphorization microorganism phosphorus removing method problem improperly.Detection substratum is that anaerobism detects substratum, aerobic detection substratum; Method: one, prepare substratum; Two, the pure bacterium liquid of dephosphorization microorganism is got; Three, anaerobism detects substratum; Four, aerobic detection substratum; Six, anaerobism detects substratum; Seven, cyclical operation four to six 2 ~ 4 times, eight, dephosphorization ability detects; Nine, microscopy is carried out to pure bacterial strain.The inventive method is with strong points, and substratum is applicable to dephosphorization microbial growth and dephosphorization ability detects.
Description
Technical field
The present invention relates to a kind of method of dephosphorization microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability.
Background technology
The method of current detection biological phosphate-eliminating microorganism pure bacterial strain dephosphorization ability is cultivated at the shaking flask that the pure bacterial strain screened from Biological Phosphorus Removal System is placed in containing beef-protein medium, analyze the phosphate content in the bacterium liquid after cultivating again, thus obtain the dephosphorization ability of pure bacterial strain, but also there is following problem in the method: 1, this method specific aim is poor, it is enrichment and the reproductive process of pure bacterial strain, under this aerobic conditions, no matter which kind of microorganism can absorb carbon source in substrate and phosphoric acid salt carries out bacterial strain breeding, so the net result recorded untrue, 2, the nutrient broth medium Middle nutrition composition that adopts of this method is unreasonable, and the biological phosphate-eliminating microorganism after making to cultivate loses the dephosphorization ability under reactor operational conditions.
Summary of the invention
The method specific aim that the object of the invention is to solve existing detection biological phosphate-eliminating microorganism dephosphorization ability is poor, the irrational problem of medium nutrient content.And provide a kind of method of biological phosphate-eliminating microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability.
Biological phosphate-eliminating microorganism detection substratum of the present invention is two kinds of substratum that biological phosphate-eliminating Institute of Micro-biology carries out detecting successively, is respectively anaerobism detects substratum, aerobic detection substratum according to the order two kinds of substratum for detecting; Wherein often liter of anaerobism detects the (NH that substratum is NaAc by 0.3 ~ 0.5g, 270 ~ 290mg
4)
2sO
4, 27 ~ 29mg CaCl
22H
2the MgSO of O, 350 ~ 370mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 0.7mL, the VITAMIN liquid of 1.8 ~ 2.4mL and surplus, the pH value that anaerobism detects substratum is 6.9 ~ 7.4; Often liter of aerobic detection substratum is by the KH of 70 ~ 80mg
2pO
4, 125 ~ 135mg K
2hPO
4, 260 ~ 300mg (NH
4)
2sO
4, 26 ~ 30mg CaCl
22H
2the MgSO of O, 340 ~ 380mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 1.2mL, the VITAMIN liquid of 1.5 ~ 3mL and surplus, the pH value of aerobic detection substratum is 6.9 ~ 7.4.
Wherein, anaerobism detect component in substratum, liquid microelement in aerobic detection substratum and concentration as follows: FeCl
36H
2the concentration of O is 2.5 ~ 3.5g/L, H
3bO
3concentration be 0.25 ~ 0.35g/L, CuSO
45H
2the concentration of O is 0.05 ~ 0.07g/L, the concentration of KI is 0.32 ~ 0.4g/L, MnCl
24H
2the concentration of O is 0.2 ~ 0.28g/L, Na
2mO
42H
2the concentration of O is 0.1 ~ 0.14g/L, ZnSO
47H
2the concentration of O is 0.2 ~ 0.28g/L and CoCl
26H
2the concentration of O is 0.28 ~ 0.32g/L.
Component in VITAMIN liquid and concentration as follows: vitamins B
1concentration be 80 ~ 120mg/L, vitamins B
2concentration is 80 ~ 120mg/L, vitamins B
6concentration be 180 ~ 220mg/L, vitamins B
12concentration be 1.8 ~ 2.2g/L, the concentration of folic acid is 36 ~ 44mg/L, the concentration of nicotinic acid is 80 ~ 120mg/L, the concentration that the concentration of calcium pantothenate is 80 ~ 120mg/L, the concentration of para-amino benzoic acid is 80 ~ 120mg/L and vitamin H is 36 ~ 44mg/L.
The method that the present invention detects biological phosphate-eliminating microorganism dephosphorization ability is carried out according to following steps: one, prepare anaerobism recited above and detect substratum, aerobic detection substratum; Two, the aerobic detection substratum that bacterium liquid that 1mL is separated the pure bacterial strain obtained is placed in the preparation of 50mL step one is got, cultivate 1 ~ 1.5 day in 16 ~ 20 DEG C of shaking tables, pour in the centrifuge tube of 50mL by cultivating the bacterium liquid obtained again, under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, abandon supernatant and stay precipitation; Three, the anaerobism adding the preparation of 30ml step one again in 50mL centrifuge tube detects substratum mixing, is filled with nitrogen 10 minutes in 50mL centrifuge tube, seals the shaking table being placed in 17 ~ 21 DEG C and cultivates 1.5 ~ 2.5 hours; Four, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Five, in the centrifuge tube of step 4 50mL, the aerobic detection substratum mixing of 30ml step one preparation is added again, aeration 1.5 ~ 2.5 hours at ambient temperature; Six, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Seven, cyclical operation step 3 to six carries out the domestication of pure bacterial strain for 10 ~ 15 times; Eight, the anaerobism adding step one preparation in the 50mL centrifuge tube containing bacterial strain after domestication detects substratum 30ml and mixes, and is filled with nitrogen 10 minutes in the centrifuge tube of 50mL, seals the shaking table being placed in 17 ~ 21 DEG C and cultivates 1.5 ~ 2.5 hours; Nine, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Ten, in the centrifuge tube of step 8 50mL, aerobic detection substratum is added, cultivate 1.5 ~ 2.5 hours, with Ammonium Molybdate Spectrophotometric Method for Determination, the phosphorus concentration cultivated in the bacterium liquid obtained is the phosphorus concentration in b, aerobic detection substratum is a, the dephosphorizing rate of pure bacterial strain is (1-b/a) × 100%, namely determines the phosphorus removal property of biological phosphate-eliminating microorganism; Wherein the volume ratio of aerobic detection substratum and centrifuge tube is 0.5:1.11, microscopy is carried out to the pure bacterial strain that step 9 obtains, determine that detecting the pure bacterial strain obtained is biological phosphate-eliminating microorganism.
In the culturing process of anaerobic culture medium, the polyphosphate particle in biological phosphate-eliminating microbial decomposition body or glycogen release energy absorb NaAc form poly-beta-hydroxyl-alkanoates (PHAs); In aerobic culture medium culturing process, biological phosphate-eliminating microbial decomposition in vivo PHAs absorb phosphoric acid salt or glycogen biosynthesis as energy derive; Nutritive ingredient in substratum of the present invention is reasonable, compared with the beef-protein medium of existing detection, the inventive method fully takes into account biological phosphate-eliminating microorganism growing environment in the reactor, there is dephosphorization ability in domesticated organism dephosphorization microorganism, and detection substratum used is applicable to biological phosphate-eliminating microbial growth and domestication.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the method for testing performance of pure bacterial strain carries out according to following steps: one, prepare anaerobism as claimed in claim 1 and detect substratum, aerobic detection substratum; Two, the aerobic detection substratum that bacterium liquid that 1mL is separated the pure bacterial strain obtained is placed in the preparation of 50mL step one is got, cultivate 1 ~ 1.5 day in 16 ~ 20 DEG C of shaking tables, pour in the centrifuge tube of 50mL by cultivating the bacterium liquid obtained again, under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, abandon supernatant and stay precipitation; Three, the anaerobism adding the preparation of 30ml step one again in 50mL centrifuge tube detects substratum mixing, is filled with nitrogen 10 minutes in 50mL centrifuge tube, seals the shaking table being placed in 17 ~ 21 DEG C and cultivates 1.5 ~ 2.5 hours; Four, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Five, in the centrifuge tube of step 4 50mL, the aerobic detection substratum mixing of 30ml step one preparation is added again, aeration 1.5 ~ 2.5 hours at ambient temperature; Six, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Seven, cyclical operation step 3 to six carries out the domestication of pure bacterial strain for 10 ~ 15 times; Eight, the anaerobism adding step one preparation in the 50mL centrifuge tube containing bacterial strain after domestication detects substratum 30ml and mixes, and is filled with nitrogen 10 minutes in the centrifuge tube of 50mL, seals the shaking table being placed in 17 ~ 21 DEG C and cultivates 1.5 ~ 2.5 hours; Nine, centrifuge tube to be placed under the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, to abandon supernatant and stay precipitation; Ten, in the centrifuge tube of step 8 50mL, aerobic detection substratum is added, cultivate 1.5 ~ 2.5 hours, with Ammonium Molybdate Spectrophotometric Method for Determination, the phosphorus concentration cultivated in the bacterium liquid obtained is the phosphorus concentration in b, aerobic detection substratum is a, the dephosphorizing rate of pure bacterial strain is (1-b/a) × 100%, namely determines the phosphorus removal property of biological phosphate-eliminating microorganism; Wherein the volume ratio of aerobic detection substratum and centrifuge tube is 0.5:1; 11, microscopy is carried out to the pure bacterial strain that step 9 obtains, determine that detecting the pure bacterial strain obtained is biological phosphate-eliminating microorganism.
In present embodiment step one, often liter of anaerobism detects the (NH that substratum is NaAc by 0.3 ~ 0.5g, 270 ~ 290mg
4)
2sO
4, 27 ~ 29mg CaCl
22H
2the MgSO of O, 350 ~ 370mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 0.7mL, the VITAMIN liquid of 1.8 ~ 2.4mL and surplus, the pH value that anaerobism detects substratum is 6.9 ~ 7.4; Often liter of aerobic detection substratum is by the KH of 70 ~ 80mg
2pO
4, 125 ~ 135mg K
2hPO
4, 260 ~ 300mg (NH
4)
2sO
4, 26 ~ 30mg CaCl
22H
2the MgSO of O, 340 ~ 380mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 1.2mL, the VITAMIN liquid of 1.5 ~ 3mL and surplus, the pH value of aerobic detection substratum is 6.9 ~ 7.4.
FeCl during wherein anaerobism detects in substratum and aerobic detection substratum liquid microelement
36H
2the concentration of O is 2.5 ~ 3.5g/L, H
3bO
3concentration be 0.25 ~ 0.35g/L, CuSO
45H
2the concentration of O is 0.05 ~ 0.07g/L, the concentration of KI is 0.32 ~ 0.4g/L, MnCl
24H
2the concentration of O is 0.2 ~ 0.28g/L, Na
2mO
42H
2the concentration of O is 0.1 ~ 0.14g/L, ZnSO
47H
2the concentration of O is 0.2 ~ 0.28g/L and CoCl
26H
2the concentration of O is 0.28 ~ 0.32g/L.The VITAMIN liquid that anaerobism detects in substratum and aerobic detection substratum is the vitamins B of 80 ~ 120mg/L by concentration
1, concentration is the vitamins B of 80 ~ 120mg/L
2, concentration is 180 ~ 220mg/L vitamins B
6, concentration is the vitamins B of 1.8 ~ 2.2g/L
12, concentration is the folic acid of 36 ~ 44mg/L, concentration is the nicotinic acid of 80 ~ 120mg/L, the para-amino benzoic acid that concentration is the calcium pantothenate of 80 ~ 120mg/L, concentration is 80 ~ 120mg/L and concentration are the vitamin H composition of 36 ~ 44mg/L.
In 50mL centrifuge tube, the aerobic detection substratum of 30ml is added in present embodiment step 7, aeration 1.5 ~ 2 hours at ambient temperature, 0.6ml is sampled to when 20 minutes, 40 minutes, 60 minutes, 90 minutes and 120 minutes respectively in cultivation, be placed in 1.5ml centrifuge tube, 5000 ~ 6000 revs/min after centrifugal 10 minutes, get 0.5ml supernatant liquor respectively in another 1.5ml centrifuge tube, with the phosphorus concentration in Ammonium Molybdate Spectrophotometric Method for Determination bacterium liquid, it is b that record detects the highest phosphorus concentration obtained.
Embodiment two: present embodiment and embodiment one carry out microscopy unlike in step 11 to pure bacterial strain, observed by microscopy and determine that the form that embodiment four separation obtains pure bacterial strain is tetramer form, namely determine that pure bacterial strain is the fusca xylanase in biological phosphate-eliminating microorganism.Other steps and parameter identical with embodiment one.
The testing process of present embodiment is all carried out in Bechtop, and the anaerobism of observing in step 3 detects the change of acetic acid in substratum, phosphatic change in the aerobic detection substratum in step 4; Wherein remain on about 30mg in Anaerobic culturel stage acetic acid absorbed dose, all about 3.8mg is remained in aerobic cultivation stage phosphate absorption amount, present embodiment step 3, four culturing process in biological phosphate-eliminating microorganism tamed, at Anaerobic culturel stage release phosphoric acid salt, and phosphoric acid salt can be absorbed at aerobic cultivation stage; Present embodiment with strong points, present embodiment substratum used is applicable to the domestication of biological phosphate-eliminating microorganism and the detection of dephosphorization ability.
Present embodiment carries out Performance Detection and microscopy to 32 strain biological phosphate-eliminating microorganisms, and detected result is as shown in table 1, wherein dephosphorizing rate lower than 20% be non-dephosphorization microorganism.
Table 1
| Kind | Bacterial strain | Dephosphorizing rate (%) |
| Acinetobacter sp. | P38 | 8.9 |
| Acinetobacter sp. | P19 | 23 |
| Acinetobacter sp. | P4 | 100 |
| Acinetobacter sp. | P10 | 90.2 |
| Acinetobacter sp. | P43 | 28.5 |
| Acinetobacter sp. | P48 | 33.6 |
| Acinetobacter sp. | P23 | 41 |
| Acinetobacter sp. | P22 | 37.5 |
| Acinetobacter sp. | P13 | 98.4 |
| Acinetobacter sp. | P42 | 32 |
| Acinetobacter sp. | P47 | 42.8 |
| Acinetobacter sp. | P21 | 34.5 |
| Acinetobacter sp. | P33 | 65 |
| Acinetobacter sp. | P46 | 21.1 |
| Delftia sp. | P1 | 66 |
| Delftia sp. | P3 | 100 |
| Delftia sp. | P45 | 33 |
| Delftia sp. | P24 | 36.2 |
| Delftia sp. | P40 | 6.5 |
| Delftia sp. | P11 | 52.5 |
| Delftia sp. | P39 | 27.7 |
| Delftia sp. | P30 | 11.5 |
| Pseudomonas sp. | P2 | 97.3 |
| Pseudomonas sp. | P34 | 13.5 |
| Pseudomonas sp. | P12 | 72 |
| Pseudomonas sp. | P22 | 95.8 |
| Pseudomonas sp. | P7 | 92.5 |
| Pseudomonas sp. | P16 | 96 |
| Aeromonas sp. | P35 | 14.6 |
| Brevibacillus sp. | P26 | 84 |
| Stenotrophomonas sp. | P8 | 96.4 |
| Paracoccus sp. | P20 | 82.5 |
As can be seen from Table 1, the biological phosphate-eliminating microorganism dephosphorization mechanism of present embodiment domestication is machine-processed consistent with its dephosphorization in the reactor, and dephosphorizing rate can reach 100%, and phosphor-removing effect is good.
Claims (3)
1. detect the method for biological phosphate-eliminating microorganism dephosphorization ability, the method detecting biological phosphate-eliminating microorganism dephosphorization ability is carried out according to following steps:
(1) preparation is respectively aerobic detection substratum for the order two kinds of substratum detected, anaerobism detects substratum;
(2) get the aerobic detection substratum that the bacterium liquid being separated the pure bacterial strain obtained is placed in (1) preparation, cultivate, then pour in centrifuge tube by cultivating the bacterium liquid obtained, centrifugal, abandon supernatant and stay precipitation;
(3) in centrifuge tube, add the anaerobism detection substratum mixing that step one is prepared again, in centrifuge tube, be filled with nitrogen, sealing is placed in shaking table and cultivates;
(4) by centrifugal for centrifuge tube 10 ~ 15 minutes, abandon supernatant and stay precipitation; Under centrifuge tube being placed in the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, abandon supernatant and stay precipitation;
(5) in the centrifuge tube of step (4), the aerobic detection substratum mixing of preparation in (1) is added again, aeration at ambient temperature;
(6) by centrifugal for centrifuge tube 10 ~ 15 minutes, abandon supernatant and stay precipitation; Under centrifuge tube being placed in the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, abandon supernatant and stay precipitation;
(7) cyclical operation step (3)-(6) carries out the domestication of pure bacterial strain for 10 ~ 15 times;
(8) anaerobism adding preparation in (1) in the centrifuge tube containing bacterial strain after domestication detects substratum and mixes, and in centrifuge tube, be filled with nitrogen, sealing is placed in shaking table and cultivates; The anaerobism adding step one preparation in the 50mL centrifuge tube containing bacterial strain after domestication detects substratum 30ml and mixes, and is filled with nitrogen 10 minutes in the centrifuge tube of 50mL, seals the shaking table being placed in 17 ~ 21 DEG C and cultivates 1.5 ~ 2.5 hours;
(9) by centrifugal for centrifuge tube 10 ~ 15 minutes, abandon supernatant and stay precipitation; Under centrifuge tube being placed in the condition of 5000 ~ 6000 revs/min centrifugal 10 ~ 15 minutes, abandon supernatant and stay precipitation;
(10) in the centrifuge tube of step (8), aerobic detection substratum is added, cultivate, with Ammonium Molybdate Spectrophotometric Method for Determination, the phosphorus concentration cultivated in the bacterium liquid obtained is the phosphorus concentration in b, aerobic detection substratum is a, the dephosphorizing rate of pure bacterial strain is (1-b/a) × 100%, namely determines the phosphorus removal property of biological phosphate-eliminating microorganism; Wherein the volume ratio of aerobic detection substratum and centrifuge tube is 0.5:1;
(11) microscopy is carried out to the pure bacterial strain that step (9) obtains, determine that detecting the pure bacterial strain obtained is the micro-life of biological phosphate-eliminating; Wherein often liter of anaerobism detects the (NH that substratum is NaAc by 0.3 ~ 0.5g, 270 ~ 290mg
4)
2sO
4, 27 ~ 29mg CaCl
22H
2the MgSO of O, 350 ~ 370mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 0.7mL, the VITAMIN liquid of 1.8 ~ 2.4mL and surplus, the pH value that anaerobism detects substratum is 6.9 ~ 7.4; Often liter of aerobic detection substratum is by the KH of 70 ~ 80mg
2pO
4, 125 ~ 135mg K
2hPO
4, 260 ~ 300mg (NH
4)
2sO
4, 26 ~ 30mg CaCl
22H
2the MgSO of O, 340 ~ 380mg
47H
2the distilled water composition of the liquid microelement of O, 0.5 ~ 1.2mL, the VITAMIN liquid of 1.5 ~ 3mL and surplus, the pH value of aerobic detection substratum is 6.9 ~ 7.4.
2. the method for detection biological phosphate-eliminating microorganism dephosphorization ability according to claim 1, method, it is characterized in that same, in step (5), the aerobic detection substratum mixing that 30ml step (1) is prepared is added, aeration 1.5 ~ 2.5 hours at ambient temperature in the centrifuge tube of step (4) 50mL.
3. the method for detection biological phosphate-eliminating microorganism dephosphorization ability according to claim 1, it is characterized in that carrying out microscopy to the pure bacterial strain in step (11), determining to be separated by microscopy observation the form obtaining pure bacterial strain is tetramer form, namely determines that pure bacterial strain is the fusca xylanase in biological phosphate-eliminating microorganism.
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