CN101560470B - Culture medium for screening enhanced biological phosphorus removal functional bacteria - Google Patents
Culture medium for screening enhanced biological phosphorus removal functional bacteria Download PDFInfo
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- CN101560470B CN101560470B CN2009100720957A CN200910072095A CN101560470B CN 101560470 B CN101560470 B CN 101560470B CN 2009100720957 A CN2009100720957 A CN 2009100720957A CN 200910072095 A CN200910072095 A CN 200910072095A CN 101560470 B CN101560470 B CN 101560470B
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Abstract
The invention provides a culture medium for screening an enhanced biological phosphorus removal functional bacterium and a method for screening the enhanced biological phosphorus removal functional bacterium, and relates to a culture medium for screening a phosphorus removal functional bacterium and a method for screening the phosphorus removal functional bacterium. The culture medium and the method solve the problems that the prior method for screening the enhanced biological phosphorus removal functional bacterium has poor direction, the obtained enhanced biological phosphorus removal functional bacterium has small amount of strains, and the culture medium is not suitable for growth of the enhanced biological phosphorus removal functional bacterium. The culture medium for screening can be an anaerobic culture medium, an aerobic culture medium, a solid separation culture medium and a liquid separation culture medium. The method comprises the following steps: 1, preparing the culture mediums; 2, collecting mud; 3, culturing the anaerobic culture medium; 4, culturing the aerobic culture medium; 6, culturing the anaerobic culture medium; 7, circulating step 4 to step 6 for 2 to 4 times; 8, diluting in constant gradient; 9, culturing the solid separation culture medium; 10, culturing the liquid separation culture medium; 11, repeating step 8 to step 10 for 3 to 8 times; and 12, detecting. The method has strong direction, the functional bacterium obtained by screening has large amount, and the culture medium is suitable for growth of the functional bacterium.
Description
Technical field
The present invention relates to a kind of phosphorus removal functional bacteria screening substratum.
Background technology
At present the method for screening enhanced biological phosphorus removal functional bacteria be the mud that will from the enhanced biological phosphorus removal system, take out place contain beef-protein medium shake the bottle fermentation culture, again the bacterium liquid after the fermentation culture is diluted, coat solid tradition substratum then and screen the biological phosphorus removal functional bacteria that strengthened, but also there is following problem in this method: 1, this method specific aim is poor, so the nonreinforcement biological phosphorus removal functional bacteria is bred in a large number in culturing process, assorted bacterium grows, cause the ratio of enhanced biological phosphorus removal functional bacteria to descend, to separate the bacterial strain quantity of the biological phosphorus removal functional bacteria that strengthened few, only account for to separate about 15% of all strains examined number that obtains; 2, nutritive ingredient is unreasonable in the nutrient broth medium that this method adopted, and makes that the growth of enhanced biological phosphorus removal functional bacteria is restricted in the culturing process.
Summary of the invention
It is poor to the objective of the invention is for the method specific aim that solves existing screening enhanced biological phosphorus removal functional bacteria, and the bacterial strain quantity of the biological phosphorus removal functional bacteria that strengthened reaches the problem that used substratum is not suitable for the enhanced biological phosphorus removal functional bacteria growth less.Use substratum and provide a kind of enhanced biological phosphorus removal functional bacteria to screen.
Enhanced biological phosphorus removal functional bacteria of the present invention screening with substratum screen by enhanced biological phosphorus removal functional bacteria four kinds of substratum of usefulness successively, be respectively anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium according to four kinds of substratum of the order that is used to screen; The substratum of enhanced biological phosphorus removal functional bacteria screening usefulness is anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium; Wherein every liter of anaerobic culture medium is by the NaAC of 0.1～0.3g, (N-H of 130～150mg
4)
2SO
4, 13～15mg CaCl
22H
2The MgSO of O, 170～190mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.2～0.4mL, 0.8～1.2mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7～7.2; Every liter of aerobic culture medium is the KH by 130～150mg
2PO
4, 230～260mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, l2～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic culture medium is 7～7.2; Every liter of solid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The distilled water of the VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL, the agar of 14～16g and surplus is formed, and the pH value of solid separation culture medium is 7～7.2; Every liter of liquid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7～7.2.
The present invention screens the method for enhanced biological phosphorus removal functional bacteria and carries out according to following steps: one, prepare anaerobic culture medium recited above, aerobic culture medium, solid separation culture medium and liquid separation culture medium; Two, collect sludge settling: the mud 60ml that gets aerobic end in the enhanced biological phosphorus removal reactor carries out centrifugal, abandons supernatant, stays precipitation, precipitation is placed the serum bottle of 100mL; Three, the anaerobic culture medium of 60ml step 1 preparation is fallen in the serum bottle of step 2 with serum bottle in sludge settling mix, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature then cultivation 23～25h; Four, support culture medium culturing well: the bacterium liquid after step 3 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Five, detest foster culture medium culturing: the bacterium liquid after step 4 is cultivated is centrifugal, remove supernatant, precipitation after centrifugal is put into the 100mL serum bottle of the anaerobic culture medium that contains 60ml step 1 preparation, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature cultivation 23～26h; Six, cyclical operation step 4 to five is 2～4 times, and active sludge is tamed, and obtains the bacterium liquid after anaerobic culture medium is cultivated; Seven, the bacterium liquid after step 6 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Eight, the bacterium liquid of getting after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; Nine, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; Ten, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature in the picking step 9 in the liquid separation culture medium of step 1 preparation; 11, repetitive operation step 8 to ten obtains pure bacterial strain 3～8 times; 12, the pure bacterial strain that step 9 is obtained carries out phosphorus removal property detection and microscopy, determines that the pure bacterial strain that screening obtains is an enhanced biological phosphorus removal functional bacteria.
In the culturing process of anaerobic culture medium, enhanced biological phosphorus removal functional bacteria decomposes intravital poly-phosphorus particle or the glycogen absorption of N aAC that releases energy and forms poly--beta-hydroxyl-alkanoates (PHAs); In the aerobic culture medium culturing process, enhanced biological phosphorus removal functional bacteria decompose in vivo PHAs absorb phosphoric acid salt or glycogen biosynthesis as energy derive; Nutritive ingredient in the substratum of the present invention is reasonable, compares with the beef-protein medium of existing screening usefulness, and the used substratum of the inventive method is fit to the growth of enhanced biological phosphorus removal.
In the screening method of the present invention in anaerobic culture medium static placement 22～26h, the obligate aerobic bacteria can be sieved, keep enhanced biological phosphorus removal functional bacteria, and then put into aerobic culture medium and cultivate, some of them just can not grown at the bacterial strain of anaerobic stages gathering PHA, and the enhanced biological phosphorus removal functional bacteria continued growth; And all cultivate the time about 1 day in anaerobic culture medium and in the aerobic culture medium, this ultimate limit state is not suitable for the growth of nonreinforcement biological phosphorus removal functional bacteria, has reduced assorted bacterium quantity; The present invention cultivates through constant gradient dilution, solid separation culture medium again and liquid separation culture medium is cultivated, further reduced the quantity of assorted bacterium, screening method of the present invention is with strong points, assorted bacterium quantity is few, to separate the enhanced biological phosphorus removal functional bacteria quantity that obtains many, account for and separate more than 70% of all strains examined number that obtains.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment enhanced biological phosphorus removal functional bacteria screening with substratum screen by enhanced biological phosphorus removal functional bacteria four kinds of substratum of usefulness successively, be respectively anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium according to four kinds of substratum of the order that is used to screen; Wherein every liter of anaerobic culture medium is by the NaAC of 0.1～0.3g, (NH of 130～150mg
4)
2SO
4, 13～15mg CaCl
22H
2The MgSO of O, 170～190mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.2～0.4mL, 0.8～1.2mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7～7.2; Every liter of aerobic culture medium is the KH by 130～150mg
2PO
4, 230～260mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, 12～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic culture medium is 7～7.2; Every liter of solid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The distilled water of the VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL, the agar of 14～16g and surplus is formed, and the pH value of solid separation culture medium is 7～7.2; Every liter of liquid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7～7.2.
Embodiment two: present embodiment and embodiment one are different is FeCl in the liquid microelement in anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium
3-6H
2The concentration of O is 1.5g/L, H
3BO
3Concentration be 0.15g/L, CuSO
45H
2The concentration of O is that the concentration of 0.03g/L, KI is 0.18g/L, MnCl
24H
2The concentration of O is 0.12g/L, Na
2MO
42H
2The concentration of O is 0.06g/L, ZnSO
47H
2The concentration of O is 0.12g/L and CoCl
26H
2The concentration of O is 0.15g/L.Other is identical with embodiment one.
Embodiment three: present embodiment is different with embodiment one or two is that VITAMIN liquid in anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium is the vitamins B of 50mg/L by concentration
1, concentration is the vitamins B of 50mg/L
2, concentration is the 100mg/L vitamins B
6, concentration is the vitamins B of 1g/L
12, concentration is that folic acid, the concentration of 20mg/L is the nicotinic acid of 50mg/L, concentration is that the vitamin H that the calcium pantothenate of 50mg/L, para-amino benzoic acid that concentration is 50mg/L and concentration are 20mg/L is formed.Other is identical with embodiment two.
Embodiment four: the method for present embodiment screening enhanced biological phosphorus removal functional bacteria is carried out according to following steps: one, preparation as a concrete described anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium implemented; Two, collect sludge settling: the mud 60ml that gets aerobic end in the enhanced biological phosphorus removal reactor carries out centrifugal, abandons supernatant, stays precipitation, precipitation is placed the serum bottle of 100mL; Three, the anaerobic culture medium of 60ml step 1 preparation is fallen in the serum bottle of step 2 with serum bottle in sludge settling mix, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature then cultivation 23～25h; Four, support culture medium culturing well: the bacterium liquid after step 3 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Five, detest foster culture medium culturing: the bacterium liquid after step 4 is cultivated is centrifugal, remove supernatant, precipitation after centrifugal is put into the 100mL serum bottle of the anaerobic culture medium that contains 60ml step 1 preparation, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature cultivation 23～26h; Six, cyclical operation step 4 to five is 2～4 times, and active sludge is tamed, and obtains the bacterium liquid after anaerobic culture medium is cultivated; Seven, the bacterium liquid after step 6 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Eight, the bacterium liquid of getting after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; Nine, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; Ten, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature in the picking step 9 in the liquid separation culture medium of step 1 preparation; 11, repetitive operation step 8 to ten obtains pure bacterial strain 3～8 times; 12, the pure bacterial strain that step 9 is obtained carries out phosphorus removal property detection and microscopy, determines that the pure bacterial strain that screening obtains is an enhanced biological phosphorus removal functional bacteria.
Every liter of anaerobic culture medium is by the NaAC of 0.1～0.3g, (NH of 130～150mg in the present embodiment step 1
4)
2SO
4, 13～15mg CaCl
22H
2The MgSO of O, 170～190mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.2～0.4mL, 0.8～1.2mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7～7.2; Every liter of aerobic culture medium is the KH by 130～150mg
2PO
4, 230～260mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, 12～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic culture medium is 7～7.2; Every liter of solid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The distilled water of the VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL, the agar of 14～16g and surplus is formed, and the pH value of solid separation culture medium is 7～7.2; Every liter of liquid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7～7.2.
FeCl in the liquid microelement in present embodiment anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium
3-6H
2The concentration of O is 1.5g/L, H
3BO
3Concentration be 0.15g/L, CuSO
45H
2The concentration of O is that the concentration of 0.03g/L, KI is 0.18g/L, MnCl
24H
2The concentration of O is 0.12g/L, Na
2MO
42H
2The concentration of O is 0.06g/L, ZnSO
47H
2The concentration of O is 0.12g/L and CoCl
26H
2The concentration of O is 0.15g/L.
VITAMIN liquid in present embodiment anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium is the vitamins B of 50mg/L by concentration
1, concentration is the vitamins B of 50mg/L
2, concentration is the 100mg/L vitamins B
6, concentration is the vitamins B of 1g/L
12, concentration is that folic acid, the concentration of 20mg/L is the nicotinic acid of 50mg/L, concentration is that the vitamin H that the calcium pantothenate of 50mg/L, para-amino benzoic acid that concentration is 50mg/L and concentration are 20mg/L is formed.
In the present embodiment step 4 precipitation after centrifugal is put into the bottle of the aerobic culture medium that contains the step 1 preparation, sealed with ventilative filter membrane then.
Embodiment five: present embodiment and embodiment four are different is that mud in the step 2 is under 4000～5000 rev/mins the condition at rotating speed, centrifugal 15～20 minutes.Other step and parameter are identical with embodiment four.
Embodiment six: what present embodiment was different with embodiment four or five is that centrifugal rotational speed is 4000～5000 rev/mins in step 4, step 5 and the step 7, and centrifugation time is 15～20 minutes.Other step and parameter are identical with embodiment four.
Embodiment seven: present embodiment and embodiment six are different is that the dilution gradient multiple of the medium gradient dilution of step 9 is 10
1～10
9Doubly.Other step and parameter are identical with embodiment six.
Embodiment eight: present embodiment and embodiment four, five or seven are different is that the method for testing performance of pure bacterial strain in the step 12 carries out according to following steps: described anaerobic culture medium of step 1 and liquid separation culture medium in A, the preparation embodiment four; B, get the bacterium liquid that 1mL embodiment four separates the pure bacterial strain that obtains and place the liquid separation culture medium of 40mL steps A preparation, in 16～20 ℃ of shaking tables, cultivated 1～1.5 day, the bacterium liquid that again cultivation is obtained is poured in the centrifuge tube of 50mL, under 4000～5000 rev/mins condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; C, the anaerobism that 50mL adding 30ml steps A is prepared in centrifuge tube again detect the substratum mixing, charge into nitrogen 5 minutes in the 50mL centrifuge tube, and sealing places 16～20 ℃ shaking table to cultivate 1.5～2.5h; D, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; E, the aerobic detection substratum mixing that adding 30ml steps A is prepared in the centrifuge tube of step D 50mL again, aeration is 1.5～2.5 hours at ambient temperature; F, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; G, cyclical operation step C to F10～15 times carry out the domestication of bacterial classification; H, the anaerobism that the adding steps A is prepared in the 50mL centrifuge tube that contains domestication back bacterial strain detect substratum 30ml and mix, and charge into nitrogen 5 minutes in the centrifuge tube of 50mL, and sealing places 16～20 ℃ shaking table to cultivate 1.5～2.5 hours; I, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; J, in the centrifuge tube of step H50mL, add aerobic detection substratum, cultivated 1.5～2.5 hours, the phosphorus concentration of cultivating phosphorus concentration in the bacterium liquid obtain with the ammonium molybdate spectrophotometry and being in b, the aerobic detection substratum is a, the dephosphorizing rate of pure bacterial strain is 1-b/a, has promptly determined the phosphorus removal property of biological phosphorus removal functional bacteria; The volume ratio of wherein aerobic detection substratum and centrifuge tube is 0.5: 1; Every liter of aerobic detection substratum is the KH by 35～40mg among step e and the step I
2PO
4, 62～68mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, 12～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic detection substratum is 7～7.2.Other step and parameter are identical with embodiment four, five or seven.
Every liter of anaerobic culture medium is by the NaAC of 0.1～0.3g, (NH of 130～150mg in the present embodiment step 1
4)
2SO
4, 13～15mg CaCl
22H
2The MgSO of O, 170～190mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.2～0.4mL, 0.8～1.2mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7～7.2; Every liter of liquid separation culture medium is by the NaAC of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7～7.2.
FeCl in the liquid microelement in anaerobic culture medium and the liquid separation culture medium wherein
3-6H
2The concentration of O is 1.5g/L, H
3BO
3Concentration be 0.15g/L, CuSO
45H
2The concentration of O is that the concentration of 0.03g/L, KI is 0.18g/L, MnCl
24H
2The concentration of O is 0.12g/L, Na
2MO
42H
2The concentration of O is 0.06g/L, ZnSO
47H
2The concentration of O is 0.12g/L and CoCl
26H
2The concentration of O is 0.15g/L; VITAMIN liquid in anaerobic culture medium and the liquid separation culture medium is the vitamins B of 50mg/L by concentration
1, concentration is the vitamins B of 50mg/L
2, concentration is the 100mg/L vitamins B
6, concentration is the vitamins B of 1g/L
12, concentration is that folic acid, the concentration of 20mg/L is the nicotinic acid of 50mg/L, concentration is that the vitamin H that the calcium pantothenate of 50mg/L, para-amino benzoic acid that concentration is 50mg/L and concentration are 20mg/L is formed.
In the 50mL centrifuge tube, add the extreme two-phase substratum of the aerobic detection of 30ml among the present embodiment step J, aeration is 1.5～2 hours at ambient temperature, sampling 0.6ml when cultivating 20 minutes, 40 minutes, 60 minutes, 90 minutes and 120 minutes respectively, place the 1.5ml centrifuge tube, 4000～5000 rev/mins after centrifugal 5 minutes, get the 0.5ml supernatant liquor respectively in another 1.5ml centrifuge tube, with the phosphorus concentration in the ammonium molybdate spectrophotometry bacterium liquid, it is b that record detects the highest phosphorus concentration that obtains.
Embodiment nine: what present embodiment and embodiment eight were different is in the step 12 pure bacterial strain to be carried out microscopy, observe to determine that by microscopy it is tetramer form that embodiment four is separated the form that obtains pure bacterial strain, promptly determine pure bacterial strain and be the fusca xylanase in the enhanced biological phosphorus removal functional bacteria.Other steps and parameter are identical with embodiment eight.
Embodiment ten: present embodiment and embodiment four are different is that the method for screening enhanced biological phosphorus removal functional bacteria is carried out according to following steps: one, preparation is as embodiment one described anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium; Two, collect sludge settling: the mud 60ml that gets aerobic end in the enhanced biological phosphorus removal reactor carries out centrifugal, abandons supernatant, stays precipitation, precipitation is placed the serum bottle of 100mL; Three, anaerobic culture medium is cultivated: with the anaerobic culture medium of 60ml step 1 preparation fall in the serum bottle in step 2 with serum bottle in sludge settling mix, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature then cultivation 23～25h; Four, support culture medium culturing well: the bacterium liquid after step 3 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Five, detest foster culture medium culturing: the bacterium liquid after step 4 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 100mL triangular flask of the anaerobic culture medium that contains the preparation of 60ml step 1, static at ambient temperature cultivation 23～26h; Six, support culture medium culturing well: the bacterium liquid after step 5 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Seven, detest foster culture medium culturing: the bacterium liquid after step 6 is cultivated is centrifugal, remove supernatant, precipitation after centrifugal is put into the 100mL serum bottle of the anaerobic culture medium that contains 60ml step 1 preparation, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature cultivation 23～26h; Eight, support culture medium culturing well: the bacterium liquid after step 7 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; Nine, the bacterium liquid after step 8 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 100mL triangular flask of the anaerobic culture medium that contains the preparation of 60ml step 1, static at ambient temperature cultivation 23～26h; Ten, the bacterium liquid after step 9 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 23～26h at ambient temperature; 11, the bacterium liquid of getting after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; 12, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; 13, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature and is obtained pure bacterial strain in the liquid separation culture medium of step 1 preparation in the picking step 9; 14, the bacterium liquid of getting again after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; 15, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; 16, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature and is obtained pure bacterial strain in the liquid separation culture medium of step 1 preparation in the picking step 9; 17, the bacterium liquid of getting again after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; 18, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; 19, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature and is obtained pure bacterial strain in the liquid separation culture medium of step 1 preparation in the picking step 9; 20, the bacterium liquid of getting again after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; 21, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 20～24h at ambient temperature dilution; 22, the single colony inoculation on the solid separation culture medium is cultivated 20～24h at ambient temperature and is obtained pure bacterial strain in the liquid separation culture medium of step 1 preparation in the picking step 9; 23, the pure bacterial strain that step 10 six, 19 and 22 is obtained carries out phosphorus removal property detection and microscopy, determines that the pure bacterial strain that screening obtains is an enhanced biological phosphorus removal functional bacteria.Other steps and parameter are identical with embodiment four.
Embodiment 11: the method for present embodiment screening enhanced biological phosphorus removal functional bacteria is carried out according to following steps: one, preparation is as embodiment one described anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium; Two, collect sludge settling: the mud 60ml that gets aerobic end in the enhanced biological phosphorus removal reactor carries out centrifugal, abandons supernatant, stays precipitation, precipitation is placed the serum bottle of 100mL; Three, anaerobic culture medium is cultivated: the anaerobic culture medium of 60ml step 1 preparation is fallen in the serum bottle of step 2, mix with sludge settling in the serum bottle, charged into nitrogen 5 minutes in serum bottle, static at ambient temperature then cultivation 25h; Four, support culture medium culturing well: the bacterium liquid after step 3 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 26h at ambient temperature; Five, detest foster culture medium culturing: the bacterium liquid after step 4 is cultivated is centrifugal, remove supernatant, precipitation after centrifugal is put into the 100mL serum bottle of the anaerobic culture medium that contains 60ml step 1 preparation, in serum bottle, charged into nitrogen 5 minutes, static at ambient temperature cultivation 23～26h; Six, cyclical operation step 4 to five is 3 times, and active sludge is tamed, and obtains the bacterium liquid after anaerobic culture medium is cultivated; Seven, the bacterium liquid after step 6 is cultivated is centrifugal, removes supernatant, and the precipitation after centrifugal is put into the 150mL triangular flask of the aerobic culture medium of the step 1 preparation that contains 75ml, and shaking table is cultivated 25h at ambient temperature; Eight, the bacterium liquid of getting after 1mL step 7 shaking table is cultivated carries out the constant gradient dilution; Nine, be inoculated in the solid separation culture medium of step 1 preparation with the bacterium liquid of spread plate, cultivate 23h at ambient temperature dilution; Ten, the single colony inoculation on the solid separation culture medium is cultivated 22h at ambient temperature in the picking step 9 in the liquid separation culture medium of step 1 preparation; 11, repetitive operation step 8 to ten obtains pure bacterial strain 5 times; 12, the pure bacterial strain that step 9 is obtained carries out phosphorus removal property detection and microscopy, determines that the pure bacterial strain that screening obtains is an enhanced biological phosphorus removal functional bacteria.
Every liter of anaerobic culture medium is by the NaAC of the 0.2g, (NH of 140mg in the present embodiment step 1
4)
2SO
4, 14mg CaCl
22H
2The MgSO of O, 180mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.3mL, 1mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7.2; Every liter of aerobic culture medium is the KH by 140mg
2PO
4, 250mg K
2HPO
4, 150mg (NH
4)
2SO
4, 14mg CaCl
22H
2The MgSO of O, 180mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.4mL, 1mL and the distilled water of surplus are formed, and the pH value of aerobic culture medium is 7.2; Every liter of solid separation culture medium is by the NaAC of 0.2g, the KH of 35mg
2PO
4, 64mg K
2HPO
4, 1.3g (NH
4)
2SO
4, 140mg CaCl
22H
2The MgSO of O, 1.8g
47H
2The distilled water of the VITAMIN liquid of the liquid microelement of O, 3mL, 10mL, the agar of 15g and surplus is formed, and the pH value of solid separation culture medium is 7.2; Every liter of liquid separation culture medium is by the NaAC of 0.2g, the KH of 35mg
2PO
4, 65mg K
2HPO
4, 1.3g (NH
4)
2SO
4, 150mg CaCl
22H
2The MgSO of O, 1.8g
47H
2The VITAMIN liquid of the liquid microelement of O, 3mL, 10mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7.2.
FeCl in the liquid microelement in present embodiment anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium
3-6H
2The concentration of O is 1.5g/L, H
3BO
3Concentration be 0.15g/L, CuSO
45H
2The concentration of O is that the concentration of 0.03g/L, KI is 0.18g/L, MnCl
24H
2The concentration of O is 0.12g/L, Na
2MO
42H
2The concentration of O is 0.06g/L, ZnSO
47H
2The concentration of O is 0.12g/L and CoCl
26H
2The concentration of O is 0.15g/L.
VITAMIN liquid in present embodiment anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium is the vitamins B of 50mg/L by concentration
1, concentration is the vitamins B of 50mg/L
2, concentration is the 100mg/L vitamins B
6, concentration is the vitamins B of 1g/L
12, concentration is that folic acid, the concentration of 20mg/L is the nicotinic acid of 50mg/L, concentration is that the vitamin H that the calcium pantothenate of 50mg/L, para-amino benzoic acid that concentration is 50mg/L and concentration are 20mg/L is formed.
The method for testing performance of pure bacterial strain carries out according to following steps in the present embodiment step 12: described anaerobic culture medium of step 1 and liquid separation culture medium in A, the preparation embodiment four; B, get the bacterium liquid that 1mL embodiment four separates the pure bacterial strain that obtains and place the liquid separation culture medium of 40mL steps A preparation, in 16～20 ℃ of shaking tables, cultivated 1～1.5 day, the bacterium liquid that again cultivation is obtained is poured in the centrifuge tube of 50mL, under 4000～5000 rev/mins condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; C, the anaerobism that 50mL adding 30ml steps A is prepared in centrifuge tube again detect the substratum mixing, charge into nitrogen 5 minutes in the 50mL centrifuge tube, and sealing places 16～20 ℃ shaking table to cultivate 1.5～2.5h; D, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; E, the aerobic detection substratum mixing that adding 30ml steps A is prepared in the centrifuge tube of step D 50mL again, aeration is 1.5～2.5 hours at ambient temperature; F, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; G, cyclical operation step C to F10～15 times carry out the domestication of bacterial classification; H, the anaerobism that the adding steps A is prepared in the 50mL centrifuge tube that contains domestication back bacterial strain detect substratum 30ml and mix, and charge into nitrogen 5 minutes in the centrifuge tube of 50mL, and sealing places 16～20 ℃ shaking table to cultivate 1.5～2.5 hours; I, centrifuge tube placed under 4000～5000 rev/mins the condition centrifugal 15～20 minutes, abandon supernatant and stay precipitation; J, in the centrifuge tube of step H50mL, add aerobic detection substratum, cultivated 1.5～2.5 hours, the phosphorus concentration of cultivating phosphorus concentration in the bacterium liquid obtain with the ammonium molybdate spectrophotometry and being in b, the aerobic detection substratum is a, the dephosphorizing rate of pure bacterial strain is 1-b/a, has promptly determined the phosphorus removal property of biological phosphorus removal functional bacteria; The volume ratio of wherein aerobic detection substratum and centrifuge tube is 0.5: 1; Every liter of aerobic detection substratum is the KH by 35～40mg among step e and the step I
2PO
4, 62～68mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, 12～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic detection substratum is 7～7.2.
In the present embodiment step 12 pure bacterial strain is carried out microscopy, observe to determine that by microscopy it is tetramer form that present embodiment is separated the form that obtains pure bacterial strain, promptly determine pure bacterial strain and be the fusca xylanase in the enhanced biological phosphorus removal functional bacteria.
The screening process of present embodiment is all carried out in Bechtop, and observes the variation of acetate in the anaerobic culture medium in the step 3, phosphatic variation in the aerobic culture medium in the step 4; Wherein remain on about 25mg in anaerobism cultivation stage acetate absorbed dose, all remain on about 2.0mg in aerobic cultivation stage phosphate absorption amount, eliminated the NOT-function bacterium in the mud in present embodiment step 3, four the culturing process, and enhanced biological phosphorus removal functional bacteria continued growth in the mud; The assorted bacterium quantity of present embodiment is few, and the used substratum of present embodiment is fit to the growth of enhanced biological phosphorus removal functional bacteria.
Present embodiment is divided into from having obtained 33 strain bacterium, this 33 strain bacterium is checked order respectively, sequencing result shows, it is the bacterial strain of Dell Ford Pseudomonas (Delftia sp) that 8 strain bacterium are arranged, have 6 strain bacterium be germ/Pseudomonas aeruginosa belong to (Pseudomonas sp) _ bacterial strain, it is the bacterial strain of acinetobacter (Acinetobacter sp) that 15 strain bacterium are arranged, it is the bacterial strain of oligotrophy zygosaccharomyces (Stenotrophomonas sp) that 1 strain bacterium is arranged, it is the bacterial strain of paracoccus (Paracoccus sp) that 1 strain bacterium is arranged, it is the bacterial strain of Aeromonas (Aeromonas sp) that 1 strain bacterium is arranged, and it is that bacillus brevis belongs to the bacterial strain of (Brevibacillus sp) that 1 strain bacterium is arranged; Present embodiment is separated to be had 14 strain bacterium to have tangible dephosphorization ability in the bacterial strain obtained to be polyP bacteria, to account for 42.4% of total sieve bacterium number; Observe to determine separate the bacterial strain that obtains having in the pure bacterial strain tetramer form by microscopy 11 strain bacterium are arranged, it is fusca xylanase that 11 strain bacterium are promptly arranged, account for 33.3% of total sieve bacterium number, also on the books in the article of a piece " development of enhanced biological phosphorus removal: microcosmic and macroscopic view " by name on Oehmen A and " Water Research ", delivering about fusca xylanase.Present embodiment is separated the enhanced biological phosphorus removal functional bacteria obtain and is accounted for and separate 75.7% of all strains examined that obtains, and the quantity of enhanced biological phosphorus removal functional bacteria is many; And adopt existing beef-protein medium screening enhanced biological phosphorus removal functional bacteria method because specific aim is poor, used beef-protein medium substratum is not suitable for enhanced biological phosphorus removal functional bacteria, assorted bacterium is many, the quantity of the change biological phosphorus removal functional bacteria that this method separation obtains is few, only is about 15%.
The 33 strain bacterium that present embodiment is obtained carry out Performance Detection, and detected result is as shown in table 1, and wherein dephosphorizing rate is lower than 20% the non-phosphorus removal functional bacteria that is.
Table 1
| Kind | Bacterial strain | Dephosphorizing rate (%) |
| Acinetobacter sp. | J1-1 | 31.5 |
| Acinetobacter sp. | J1-2 | 40.5 |
| Acinetobacter sp. | J2 | 20 |
| Delftia sp. | J3-1 | 32.5 |
| Acinetobacter sp. | J3-2 | 27.5 |
| Acinetobacter sp. | J3-3 | 31.5 |
| Delftia sp. | J3-4 | 7.5 |
| Delftia sp. | J3-5 | 16 |
| Acinetobacter sp. | J4 | 9.5 |
| Aeromonas sp. | J5 | 15 |
| Pseudomonas sp. | J6-1 | 12.5 |
| Acinetobacter sp. | J6-2 | 9 |
| Acinetobacter sp. | J6-3 | 64 |
| Delftia sp. | J7-1 | 12.5 |
[0037]
| Brevibacillus sp. | J7-2 | 82 |
| Delftia sp. | J7-3 | 35 |
| Acinetobacter sp. | J7-4 | 40.5 |
| Acinetobacter sp. | H1 | 37 |
| Acinetobacter sp. | H2-1 | 33.5 |
| Acinetobacter sp. | H2-2 | 22.5 |
| Acinetobacter sp. | H3-1 | 97.2 |
| Pseudomonas sp. | H3-2 | 70 |
[0037]
| Brevibacillus sp. | J7-2 | 82 |
| Delftia sp. | H3-3 | 50.5 |
| Acinetobacter sp. | H4-1 | 89 |
| Pseudomonas sp. | H4-2 | 96 |
| Stenotrophomonas sp. | H5-1 | 97.4 |
| Pseudomonas sp. | H5-2 | 91.6 |
| Pseudomonas sp. | H5-3 | 97 |
| Paracoccus sp. | H6 | 81.5 |
| Acinetobacter sp. | H7 | 100 |
| Delftia sp. | C1-1 | 100 |
| Pseudomonas sp. | C1-2 | 98.6 |
| Delftia sp. | C4 | 67 |
As can be seen from Table 1, the dephosphorizing rate of the enhanced biological phosphorus removal functional bacteria that the present embodiment screening obtains can reach 100%, and phosphor-removing effect is good.
Claims (3)
1. enhanced biological phosphorus removal functional bacteria screens and uses substratum, it is characterized in that described screening substratum screen by enhanced biological phosphorus removal functional bacteria four kinds of substratum of usefulness successively, be respectively anaerobic culture medium, aerobic culture medium, solid separation culture medium and liquid separation culture medium according to four kinds of substratum of the order that is used to screen; Wherein every liter of anaerobic culture medium is by the NaAc of 0.1～0.3g, (NH of 130～150mg
4)
2SO
4, 13～15mg CaCl
22H
2The MgSO of O, 170～190mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.2～0.4mL, 0.8～1.2mL and the distilled water of surplus are formed, and the pH value of anaerobic culture medium is 7～7.2; Every liter of aerobic culture medium is the KH by 130～150mg
2PO
4, 230～260mg K
2HPO
4, 120～160mg (NH
4)
2SO
4, 12～16mg CaCl
22H
2The MgSO of O, 160～200mg
47H
2The VITAMIN liquid of the liquid microelement of O, 0.1～0.6mL, 0.5～1.5mL and the distilled water of surplus are formed, and the pH value of aerobic culture medium is 7～7.2; Every liter of solid separation culture medium is by the NaAc of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The distilled water of the VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL, the agar of 14～16g and surplus is formed, and the pH value of solid separation culture medium is 7～7.2; Every liter of liquid separation culture medium is by the NaAc of 0.1～0.3g, the KH of 32～40mg
2PO
4, 62～68mg K
2HPO
4, 1.2～1.4g (NH
4)
2SO
4, 120～160mg CaCl
22H
2The MgSO of O, 1.6～2g
47H
2The VITAMIN liquid of the liquid microelement of O, 1～6mL, 5～15mL and the distilled water of surplus are formed, and the pH value of liquid separation culture medium is 7～7.2.
2. substratum is used in enhanced biological phosphorus removal functional bacteria screening according to claim 1, it is characterized in that FeCl in the liquid microelement in anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium
36H
2The concentration of O is 1.5g/L, H
3BO
3Concentration be 0.15g/L, CuSO
45H
2The concentration of O is that the concentration of 0.03g/L, KI is 0.18g/L, MnCl
24H
2The concentration of O is 0.12g/L, Na
2MO
42H
2The concentration of O is 0.06g/L, ZnSO
47H
2The concentration of O is 0.12g/L and CoCl
26H
2The concentration of O is 0.15g/L.
3. substratum is used in enhanced biological phosphorus removal functional bacteria screening according to claim 1 and 2, it is characterized in that vitamins B in the VITAMIN liquid in anaerobic culture medium, aerobic culture medium, solid separation culture medium and the liquid separation culture medium
1Concentration be 50mg/L, vitamins B
2Concentration is 50mg/L, vitamins B
6Concentration be 100mg/L, vitamins B
12Concentration be that the concentration of 1g/L, folic acid is that the concentration of 20mg/L, nicotinic acid is 50mg/L, the concentration of calcium pantothenate is that the concentration of 50mg/L, para-amino benzoic acid is that the concentration of 50mg/L and vitamin H is 20mg/L.
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| CN102911997B (en) * | 2012-11-06 | 2015-08-12 | 沈阳建筑大学 | The method of biological phosphate-eliminating microorganism detection substratum and detection biological phosphate-eliminating microorganism dephosphorization ability |
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| CN105200115A (en) * | 2015-11-06 | 2015-12-30 | 沈阳建筑大学 | Culture medium for bacterium screening of biological dephosphorization function bacterium and bacterium screening method for reinforced dephosphorization |
| CN108359617B (en) * | 2017-12-29 | 2021-03-02 | 浙江双良商达环保有限公司 | Acinetobacter CL05 and application thereof in village and town sewage dephosphorization treatment |
| CN109055224A (en) * | 2018-09-17 | 2018-12-21 | 中车环境科技有限公司 | A kind of fast enriching cultural method of efficient anaerobic dephosphorization bacterial |
| CN111394251B (en) * | 2020-04-03 | 2022-03-15 | 苏州科技大学 | Method for enriching and separating efficient phosphorus-accumulating bacteria from active sludge of biofilm reactor |
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Application publication date: 20091021 Assignee: Jiangsu Dingze Environmental Engineering Co., Ltd. Assignor: Harbin Institute of Technology Contract record no.: 2013320000466 Denomination of invention: Culture medium for screening enhanced biological phosphorus removal functional bacteria and method for screening enhanced biological phosphorus removal functional bacteria Granted publication date: 20101013 License type: Exclusive License Record date: 20130527 |
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