CN109929783A - The culture medium and cultural method of Rhodopseudomonas palustris - Google Patents
The culture medium and cultural method of Rhodopseudomonas palustris Download PDFInfo
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- CN109929783A CN109929783A CN201910322238.9A CN201910322238A CN109929783A CN 109929783 A CN109929783 A CN 109929783A CN 201910322238 A CN201910322238 A CN 201910322238A CN 109929783 A CN109929783 A CN 109929783A
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Abstract
The present invention relates to biological fields to provide a kind of culture medium for the low problem of the culture concentration of Rhodopseudomonas palustris, and the ingredient of culture medium includes: natrium malicum 2-2.7g, (NH4)2SO41.15-1.55g MgSO4·7H2O0.1-0.3g, CaCl2·H2O0.05-0.08g, KH2PO40.4-0.8g, K2HPO40.7-1g, ironic citrate 0.01-0.03g, EDTA0.01-0.03g, yeast extract 0.7-1.2g, trace element solution 0.8-1.3mL, vitamin solution 7-8mL, distilled water 1000mL.A kind of cultural method, comprising the following steps: A, culture medium preparation；B, prepared by bacterium solution；C, Spawn incubation.Using above-mentioned culture medium culture Rhodopseudomonas palustris, be conducive to improve the concentration for cultivating resulting Rhodopseudomonas palustris.
Description
Technical field
The present invention relates to biological pesticide technical fields, more specifically, it relates to a kind of culture of Rhodopseudomonas palustris
Base and cultural method.
Background technique
Rhodopseudomonas palustris belongs to one kind of photosynthetic bacteria, is rich in various bioactivators, and it is adaptable it is strong,
Be resistant to high concentration organic wastewater and stronger decomposition and inversion ability.There are many purposes of Rhodopseudomonas palustris, answered extensively
For fields such as agricultural, fishery, environmental protection, it can be used for purification of water quality, sewage treatment, feed grade bio-additive etc..
It since the application range of Rhodopseudomonas palustris is than wide, requires to use in many fields, it usually needs big
Amount breeding culture.But the culture medium of existing Rhodopseudomonas palustris and the resulting marsh of cultural method culture are red false single
The concentration of born of the same parents bacterium is relatively low, and expression activitiy is poor, it is difficult to adapt to production and need, therefore, still there is improved space.
Summary of the invention
In view of the deficienciess of the prior art, the first object of the present invention is to provide a kind of training of Rhodopseudomonas palustris
Base is supported, is had the advantages that higher, active preferable by the concentration of the resulting Rhodopseudomonas palustris of culture medium culture.
To achieve the above object, the present invention provides the following technical scheme that
A kind of culture medium of Rhodopseudomonas palustris, the ingredient of the culture medium include: natrium malicum 2-2.7g, (NH4)2SO41.15-1.55g MgSO4·7H2O0.1-0.3g, CaCl2·H2O0.05-0.08g, KH2PO40.4-0.8g,
K2HPO40.7-1g, ironic citrate 0.01-0.03g, EDTA0.01-0.03g, yeast extract 0.7-1.2g, trace element solution
0.8-1.3mL, vitamin solution 7-8mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.1-0.5g, (NH4)6Mo7O24·4H2O0.001-0.005g,
MnSO4·H2O0.001-0.004g, ZnSO40.001-0.003g, H3BO30.001-0.003g, EDTA0.03-0.07g,
CuSO4·5H2O0.001-0.004g, CaCl2·2H2O0.01-0.05g, distilled water 100mL；
The vitamin solution includes: niacin 0.1-0.4g, biotin 0.005-0.009g, niacinamide 0.1-0.4g, right
Aminobenzoic acid 0.1-0.3g, thiamine hydrochloride 0.2-0.7g, distilled water 1000mL.
The present invention is further arranged to: the ingredient of the culture medium includes: natrium malicum 2.4-2.6g, (NH4)2SO41.2-
1.35g MgSO4·7H2O0.1-0.2g, CaCl2·H2O0.06-0.07g, KH2PO40.5-0.7g, K2HPO40.8-0.9g, lemon
Lemon acid iron 0.01-0.02g, EDTA0.01-0.02g, yeast extract 0.9-1.1g, trace element solution 0.9-1.1mL, vitamin
Solution 7.5-7.7mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.2-0.3g, (NH4)6Mo7O24·4H2O0.001-0.003g,
MnSO4·H2O0.001-0.002g, ZnSO40.001-0.002g, H3BO30.001-0.002g, EDTA0.04-0.06g,
CuSO4·5H2O0.001-0.002g, CaCl2·2H2O0.01-0.03g, distilled water 100mL；
The vitamin solution includes: niacin 0.1-0.3g, biotin 0.007-0.008g, niacinamide 0.2-0.3g, right
Aminobenzoic acid 0.1-0.3g, thiamine hydrochloride 0.3-0.6g, distilled water 1000mL.
The present invention is further arranged to: the ingredient of the culture medium includes: natrium malicum 2.5g, (NH4)2SO41.25g
MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g, K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, ferment
Female medicinal extract 1.1g, trace element solution 1.05mL, vitamin solution 7.6mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.001g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g,
Distilled water 100mL；
The vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g,
Thiamine hydrochloride 0.5g, distilled water 1000mL.
By adopting the above technical scheme, culture medium is cooperatively formed by using the above component, be conducive to as the red false unit cell in marsh
Bacterium provides nutriment, so that Rhodopseudomonas palustris is preferably grown, cultivates the resulting red vacation in marsh to be conducive to improve
The concentration and activity of monad, meanwhile, so that the ability for cultivating the reduction water body COD of resulting Rhodopseudomonas palustris increases
By force；By the ratio cooperation of each component in control culture medium, be conducive between each component preferably coordinated and red for marsh
The growth of pseudomonad provides nutriment, is conducive to Rhodopseudomonas palustris and preferably grows, so that culture is resulting
The concentration and activity of Rhodopseudomonas palustris improve, meanwhile, be conducive to the reduction water body COD's for improving Rhodopseudomonas palustris
Ability.
The present invention is further arranged to: the ingredient of the culture medium further include: pork liver extracting solution 2-2.5mL.
By adopting the above technical scheme, rich in pork liver extracting solution by being added pork liver extracting solution in the medium
Protein is conducive to provide nutriment for the growth of Rhodopseudomonas palustris, so that Rhodopseudomonas palustris is preferably grown,
To be conducive to improve the concentration and activity of cultivating resulting Rhodopseudomonas palustris.
The present invention is further arranged to: the ingredient of the culture medium further include: ennel oil 1-1.5mL.
By adopting the above technical scheme, by the way that ennel oil is added in the medium, ennel oil has broad spectrum activity to fungi
Antibacterial action be conducive to improve and cultivate resulting Rhodopseudomonas palustris so that culture medium is not easy by fungal contamination
Purity be not easy so that cultivating resulting Rhodopseudomonas palustris by other fungal contaminations.
In view of the deficienciess of the prior art, the second object of the present invention is to provide a kind of training of Rhodopseudomonas palustris
The method of supporting, has the advantages that the concentration for cultivating resulting Rhodopseudomonas palustris is higher, active preferable.
To achieve the above object, the present invention provides the following technical scheme that
A kind of cultural method of the Rhodopseudomonas palustris using above-mentioned culture medium, comprising the following steps:
A, preparation of culture medium: each component of culture medium is dissolved in distilled water one by one, adjust pH value to 6.8-7.2, through high pressure
After sterilizing and cooling down, culture medium is moved into sterilized closed container, immediately closed container, is placed in shady place and stores for future use；
B, bacterium solution prepare: take culture presevation number be M209077 Rhodopseudomonas palustris 10-20g be added 100-200mL without
In bacterium water, 30min is sufficiently vibrated after standing 20min, forms bacterium solution；
C, Spawn incubation: B is prepared into resulting bacterium solution addition A and is prepared in resulting culture medium, then culture medium is placed in light
It is cultivated 10-15 days under conditions of according to intensity be 500-600lx, temperature is 20-30 DEG C, can cultivate to obtain the red false unit cell in marsh
Bacterium；
Step A and step B is operated before step C, and the operation order of step A and step B does not limit.
By adopting the above technical scheme, by using above-mentioned culture medium culture Rhodopseudomonas palustris, meanwhile, it is trained by control
Feeding intensity of illumination, temperature and incubation time, is conducive to Rhodopseudomonas palustris and preferably grows, so that culture gained
Rhodopseudomonas palustris concentration and activity it is higher, meanwhile, also advantageously improve the resulting Rhodopseudomonas palustris of culture
Reduction water body COD ability；It can be prepared by culture medium by directly mixing each component of culture medium, by the way that bacterium solution is direct
Addition can cultivate Rhodopseudomonas palustris into culture medium, simple and quick, conveniently.
The present invention is further arranged to: in the step A, controlling autoclaved temperature is 113 DEG C -122 DEG C, and control is high
The time of pressure sterilizing is 10-15min.
By adopting the above technical scheme, it is 113 DEG C -122 DEG C by controlling autoclaved temperature, and controls autoclaved
Time is 10-15min, other miscellaneous bacterias be conducive in culture medium are removed more complete, so that being not easy to deposit in culture medium
In other strains, to be conducive to improve the purity for cultivating resulting Rhodopseudomonas palustris.
The present invention is further arranged to: in the step B, sterile water uses the sterile water with bead.
By adopting the above technical scheme, the bacterium solution of Rhodopseudomonas palustris is prepared by using the sterile water with bead, benefit
With collision of the bead in oscillatory process, be conducive to Rhodopseudomonas palustris being dispersed into single bacterium colony, so that the red vacation in marsh
The dispersion of monad is more uniform, so that Rhodopseudomonas palustris is not easy to compete with one another for growing during the cultivation process, has
Grow more preferably conducive to Rhodopseudomonas palustris, so that cultivating the concentration of resulting Rhodopseudomonas palustris and activity mentions
It is high.
The present invention is further arranged to: in the step C, the intensity of illumination control of culture is 550-560lx, temperature control
It is 28 DEG C -30 DEG C, incubation time is 10-12 days.
When by adopting the above technical scheme, by the intensity of illumination, temperature and culture of control culture Rhodopseudomonas palustris
Between, be conducive to Rhodopseudomonas palustris and preferably grow, so that the speed of growth of Rhodopseudomonas palustris is accelerated, to be conducive to
Improve the concentration and activity for cultivating resulting Rhodopseudomonas palustris.
The present invention is further arranged to: in the step C, culture medium being placed under anoxia condition and is cultivated.
By adopting the above technical scheme, by cultivating Rhodopseudomonas palustris under anoxic conditions, it is red false single to be conducive to marsh
Born of the same parents bacterium preferably grows, and is conducive to the speed of growth for accelerating Rhodopseudomonas palustris, so that cultivating the red false unit cell in resulting marsh
The activity and concentration of bacterium improve.
In conclusion the invention has the following advantages:
1. cooperatively forming culture medium by using different component, be conducive to provide nutriment for Rhodopseudomonas palustris,
Be conducive to improve the concentration and activity for cultivating resulting Rhodopseudomonas palustris, meanwhile, so that cultivating the red vacation in resulting marsh
The ability enhancing of the reduction water body COD of monad；
2. being conducive between each component preferably coordinated by the ratio cooperation of each component in control culture medium and being
The growth of Rhodopseudomonas palustris provides nutriment, so that cultivating the concentration of resulting Rhodopseudomonas palustris and activity mentions
Height, meanwhile, be conducive to the ability for the reduction water body COD for improving Rhodopseudomonas palustris；
3. by using above-mentioned culture medium culture Rhodopseudomonas palustris, meanwhile, pass through the intensity of illumination of control culture, temperature
Degree and incubation time so that cultivate resulting Rhodopseudomonas palustris concentration and activity it is higher, meanwhile, also help and mention
Height cultivates the ability of the reduction water body COD of resulting Rhodopseudomonas palustris；
4. can be prepared by culture medium by directly mixing each component of culture medium, by the way that bacterium solution to be directly added to cultivate
Rhodopseudomonas palustris can be cultivated in base, it is simple and quick, conveniently.
Specific embodiment
With reference to embodiments, invention is further described in detail.
Embodiment 1
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2g, (NH4)2SO41.15g MgSO4·7H2O0.3g, CaCl2·H2O0.05g, KH2PO40.8g,
K2HPO41g, ironic citrate 0.025g, EDTA0.03g, yeast extract 0.7g, trace element solution 1.3mL, vitamin solution
7mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.1g, (NH4)6Mo7O24·4H2O0.004g, MnSO4·
H2O0.004g, ZnSO40.003g, H3BO30.0025g, EDTA0.07g, CuSO4·5H2O0.003g, CaCl2·2H2O0.05g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.35g, biotin 0.009g, niacinamide 0.1g, p-aminobenzoic acid 0.3g, salt
Allithiamine element 0.2g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 6.8, it is uniform by being blended
Culture medium through 110 DEG C temperature high pressure sterilization 17min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In 100mL sterile water, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 20 DEG C of illumination constant temperature incubation rooms of 500lx intensity of illumination and are cultivated 13 days,
After the completion of culture, test tube is taken out, and counts the red points occurred in test tube, is averaged.Wherein, each red points show
Culture obtains single Rhodopseudomonas palustris, since the dilution of the dilution for culture is 1:109, and take out 1mL dilution
Liquid is placed in culture medium and cultivates, then n red point represents cultivate the viable bacteria concentration of obtained Rhodopseudomonas palustris as n hundred million/
ml。
Embodiment 2
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.7g, (NH4)2SO41.55g MgSO4·7H2O0.25g, CaCl2·H2O0.08g, KH2PO40.4g,
K2HPO40.7g, ironic citrate 0.03g, EDTA0.025g, yeast extract 1.2g, trace element solution 0.8mL, vitamin solution
8mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.5g, (NH4)6Mo7O24·4H2O0.005g, MnSO4·
H2O0.003g, ZnSO40.0025g, H3BO30.003g, EDTA0.03g, CuSO4·5H2O0.004g, CaCl2·2H2O0.04g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.4g, biotin 0.005g, niacinamide 0.4g, p-aminobenzoic acid 0.1g, hydrochloric acid
Thiamine 0.7g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.2, it is uniform by being blended
Culture medium through 113 DEG C temperature high pressure sterilization 15min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 28 DEG C of illumination constant temperature incubation rooms of 550lx intensity of illumination and are cultivated 10 days,
Controlling culture environment simultaneously is anoxia condition, after the completion of culture, takes out test tube, and counts the red points occurred in test tube, is taken
Average value.Wherein, each red points show that culture obtains single Rhodopseudomonas palustris, due to the dilution for culture
Dilution is 1:109, and taking-up 1mL dilution is placed in culture medium and cultivates, then it is red to represent the marsh that culture obtains for n red point
The viable bacteria concentration of pseudomonad is hundred million/ml of n.
Embodiment 3
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.4g, (NH4)2SO41.3g, MgSO4·7H2O0.1g, CaCl2·H2O0.07g, KH2PO40.7g,
K2HPO40.8g, ironic citrate 0.01g, EDTA0.02g, yeast extract 0.9g, trace element solution 1mL, vitamin solution
7.7mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.003g, MnSO4·
H2O0.001g, ZnSO40.0015g, H3BO30.001g, EDTA0.06g, CuSO4·5H2O0.001g, CaCl2·2H2O0.03g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.1g, biotin 0.0075g, niacinamide 0.2g, p-aminobenzoic acid 0.3g, salt
Allithiamine element 0.3g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.2, it is uniform by being blended
Culture medium through 125 DEG C temperature high pressure sterilization 7min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In 100mL sterile water, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 30 DEG C of illumination constant temperature incubation rooms of 600lx intensity of illumination and are cultivated 15 days,
After the completion of culture, test tube is taken out, and counts the red points occurred in test tube, is averaged.Wherein, each red points show
Culture obtains single Rhodopseudomonas palustris, since the dilution of the dilution for culture is 1:109, and take out 1mL dilution
Liquid is placed in culture medium and cultivates, then n red point represents cultivate the viable bacteria concentration of obtained Rhodopseudomonas palustris as n hundred million/
ml。
Embodiment 4
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.5g, (NH4)2SO41.2g, MgSO4·7H2O0.15g, CaCl2·H2O0.065g, KH2PO40.5g,
K2HPO40.85g, ironic citrate 0.02g, EDTA0.015g, yeast extract 1.1g, trace element solution 0.9mL, vitamin solution
7.5mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.25g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.002g, ZnSO40.002g, H3BO30.0015g, EDTA0.04g, CuSO4·5H2O0.002g, CaCl2·2H2O0.01g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.4g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 6.8, it is uniform by being blended
Culture medium through 113 DEG C temperature high pressure sterilization 15min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 28 DEG C of illumination constant temperature incubation rooms of 550lx intensity of illumination and are cultivated 10 days,
Controlling culture environment simultaneously is anoxia condition, after the completion of culture, takes out test tube, and counts the red points occurred in test tube, is taken
Average value.Wherein, each red points show that culture obtains single Rhodopseudomonas palustris, due to the dilution for culture
Dilution is 1:109, and taking-up 1mL dilution is placed in culture medium and cultivates, then it is red to represent the marsh that culture obtains for n red point
The viable bacteria concentration of pseudomonad is hundred million/ml of n.
Embodiment 5
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.6g, (NH4)2SO41.35g MgSO4·7H2O0.2g, CaCl2·H2O0.06g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.015g, EDTA0.01g, yeast extract 1g, trace element solution 1.1mL, vitamin solution
7.6mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.3g, (NH4)6Mo7O24·4H2O0.003g, MnSO4·
H2O0.0015g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.0015g, CaCl2·
2H2O0.02g, distilled water 100mL；
Vitamin solution includes: niacin 0.3g, biotin 0.007g, niacinamide 0.25g, p-aminobenzoic acid 0.1g, salt
Allithiamine element 0.5g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.2, it is uniform by being blended
Culture medium through 122 DEG C temperature high pressure sterilization 10min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 30 DEG C of illumination constant temperature incubation rooms of 560lx intensity of illumination and are cultivated 12 days,
Controlling culture environment simultaneously is anoxia condition, after the completion of culture, takes out test tube, and counts the red points occurred in test tube, is taken
Average value.Wherein, each red points show that culture obtains single Rhodopseudomonas palustris, due to the dilution for culture
Dilution is 1:109, and taking-up 1mL dilution is placed in culture medium and cultivates, then it is red to represent the marsh that culture obtains for n red point
The viable bacteria concentration of pseudomonad is hundred million/ml of n.
Embodiment 6
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, yeast extract 1.1g, trace element solution 1.05mL, vitamin solution
7.6mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.001g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.5g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.0, it is uniform by being blended
Culture medium through 125 DEG C temperature high pressure sterilization 7min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In 100mL sterile water, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 25 DEG C of illumination constant temperature incubation rooms of 540lx intensity of illumination and are cultivated 14 days,
After the completion of culture, test tube is taken out, and counts the red points occurred in test tube, is averaged.Wherein, each red points show
Culture obtains single Rhodopseudomonas palustris, since the dilution of the dilution for culture is 1:109, and take out 1mL dilution
Liquid is placed in culture medium and cultivates, then n red point represents cultivate the viable bacteria concentration of obtained Rhodopseudomonas palustris as n hundred million/
ml。
Embodiment 7
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, yeast extract 1.1g, trace element solution 1.05mL, vitamin solution
7.6mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.001g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.5g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.0, it is uniform by being blended
Culture medium through 118 DEG C temperature high pressure sterilization 13min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 29 DEG C of illumination constant temperature incubation rooms of 555lx intensity of illumination and are cultivated 11 days,
Controlling culture environment simultaneously is anoxia condition, after the completion of culture, takes out test tube, and counts the red points occurred in test tube, is taken
Average value.Wherein, each red points show that culture obtains single Rhodopseudomonas palustris, due to the dilution for culture
Dilution is 1:109, and taking-up 1mL dilution is placed in culture medium and cultivates, then it is red to represent the marsh that culture obtains for n red point
The viable bacteria concentration of pseudomonad is hundred million/ml of n.
Embodiment 8
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, pork liver extracting solution 2mL, ennel oil 1.5mL, yeast extract 1.1g are micro-
Secondary element solution 1.05mL, vitamin solution 7.6mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.001g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.5g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.0, it is uniform by being blended
Culture medium through 125 DEG C temperature high pressure sterilization 7min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In 100mL sterile water, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 25 DEG C of illumination constant temperature incubation rooms of 540lx intensity of illumination and are cultivated 14 days,
After the completion of culture, test tube is taken out, and counts the red points occurred in test tube, is averaged.Wherein, each red points show
Culture obtains single Rhodopseudomonas palustris, since the dilution of the dilution for culture is 1:109, and take out 1mL dilution
Liquid is placed in culture medium and cultivates, then n red point represents cultivate the viable bacteria concentration of obtained Rhodopseudomonas palustris as n hundred million/
ml。
Embodiment 9
A kind of culture medium of Rhodopseudomonas palustris, including following component:
Natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, yeast extract 1.1g, trace element solution 1.05mL, vitamin solution
7.6mL, distilled water 1000mL；
Wherein, trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·
H2O0.001g, ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g,
Distilled water 100mL；
Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.5g, distilled water 1000mL.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.0, it is uniform by being blended
Culture medium through 118 DEG C temperature high pressure sterilization 13min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
Above 5 vaccinated test tubes are placed in 29 DEG C of illumination constant temperature incubation rooms of 555lx intensity of illumination and are cultivated 11 days,
Controlling culture environment simultaneously is anoxia condition, after the completion of culture, takes out test tube, and counts the red points occurred in test tube, is taken
Average value.Wherein, each red points show that culture obtains single Rhodopseudomonas palustris, due to the dilution for culture
Dilution is 1:109, and taking-up 1mL dilution is placed in culture medium and cultivates, then it is red to represent the marsh that culture obtains for n red point
The viable bacteria concentration of pseudomonad is hundred million/ml of n.
Comparative example 1
Use notification number in the Chinese patent application of CN105543142B " a kind of efficiently to reduce the marsh water body COD red
Culture medium culture Rhodopseudomonas palustris in the culture medium of pseudomonad ".
A kind of culture medium of Rhodopseudomonas palustris, including following component:
NH4SO41.5g, MgCl20.5g, NaHCO35.3g, K2HPO40.8g, NaCl2.3g, yeast extract 1.1g, micro member
Plain growth-promoting object mother liquor 43mL, water 1000mL；
Wherein, microelement growth-promoting object mother liquor includes: Na2EDTA503mg, FeSO4·7H2O203mg, ZnSO4·
7H2O13mg, MnSO4·4H2O2.3mg, H3BO353mg, CoCl2·2H2O23mg,NiCl2·6H2O7.3mg, CuCl2·
6H2O0.8mg, NaMoO4·2H2O4.3mg, CaCl2The above ingredient is dissolved in 1000mL water by 203mg one by one, is adjusted pH and is
4.7, filtration sterilization.
The cultural method of Rhodopseudomonas palustris, comprising the following steps:
A, prepared by culture medium, specific as follows:
The mentioned component of culture medium is dissolved in distilled water one by one by quality, and adjusting pH value is 7.3, it is uniform by being blended
Culture medium through 118 DEG C temperature high pressure sterilization 13min and after cooling down, 9mL culture medium is drawn with 10mL aseptic straw sterile working,
It is moved into a series of sterilized 10mL test tubes with plastic spiral turncap respectively, and tightens plastic cap immediately, be stored in shady place,
It is spare.
B, prepared by bacterium solution, specific as follows:
Use sterile metal spoon accurately weigh 10g culture presevation number for the Rhodopseudomonas palustris of M209077 be added to
In sterile water of the 100mL with bead, 30min is sufficiently vibrated after standing 20min, forms bacterium solution.
C, Spawn incubation, specific as follows:
5mL bacterium solution is drawn using aseptic straw to be added into 45mL sterile water, is mixed to form the dilution that dilution is 1:10
Liquid, then be taken out the dilution that 1mL dilution is 1:10 and be added in 9mL sterile water, being mixed to form dilution is the dilute of 1:100
Liquid is released, is repeated above operation, respectively obtaining dilution is 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109It is dilute
Liquid is released, and each dilution must replace sterile tube.
1mL aseptic straw is used to draw 1mL dilution as 1:109Dilution, be added A in prepare fill 9mL without
In the test tube with plastic spiral turncap of bacterium culture medium, repetition connects 5 test tubes, tightens plastic cap after inoculation immediately and shakes up.
By above 5 vaccinated test tubes be placed in 40W incandescent lamp, 30 DEG C, culture with equidistant 18cm illumination cultivation 11 days,
After the completion of culture, test tube is taken out, and counts the red points occurred in test tube, is averaged.Wherein, each red points show
Culture obtains single Rhodopseudomonas palustris, since the dilution of the dilution for culture is 1:109, and take out 1mL dilution
Liquid is placed in culture medium and cultivates, then n red point represents cultivate the viable bacteria concentration of obtained Rhodopseudomonas palustris as n hundred million/
ml。
Experiment 1
A certain amount of sewage is taken, is detected according to GB11914-1989 " the measurement dichromate titration of water chemical oxygen demand " dirty
COD in waterCrThe identical sewage of COD content is respectively charged into the glass flask of 1000mL a series of, each by content (mg/L)
750mL sewage is added in flask, then is separately added into above embodiments and the resulting marsh of comparative example culture in each flask
Red pseudomonas about 4,000,000,000, after 15min, again according to GB11914-1989 " the measurement dichromate titration of water chemical oxygen demand "
Detect the COD in sewageCrContent (mg/L), and according to the [(COD of sewage before Rhodopseudomonas palustris is addedCrContent-addition natural pond
COD of sewage after damp red pseudomonasCrContent)/be added Rhodopseudomonas palustris before COD of sewageCrContent] * 100% calculating
Obtain CODCrRemoval rate (%).
The above detection data is shown in Table 1.
Table 1
It can be obtained according to the data comparison of embodiment 1-9 in table 1 and comparative example 1, embodiment 1-9 is all made of culture of the invention
Base component and cultural method culture Rhodopseudomonas palustris, comparative example 1 use existing nutrient media components and cultural method culture
Rhodopseudomonas palustris, and the concentration that embodiment 1-9 cultivates resulting Rhodopseudomonas palustris is above comparative example 1, implements
The COD of example 1-9CrRemoval rate is above comparative example 1, illustrates to train by using nutrient media components of the invention and cultural method
Rhodopseudomonas palustris is supported, is conducive to preferably provide nutriment for the growth of Rhodopseudomonas palustris, to be conducive to mention
Height cultivates the concentration and activity of resulting Rhodopseudomonas palustris, so that cultivating the quantity of resulting Rhodopseudomonas palustris more
It is more, meanwhile, also advantageously improve the reduction water body COD of Rhodopseudomonas palustrisCrAbility so that the COD of sewageCrRemoval rate
It improves.
According to embodiment 1 in table 1 and embodiment 2, embodiment 3 and embodiment 4 and 5, embodiment 6 and embodiment 7, embodiment
8 can obtain with the data comparison of embodiment 9, implement 1 with the compositional range of each component of the culture medium of embodiment 2 in following model
In enclosing: natrium malicum 2-2.7g, (NH4)2SO41.15-1.55g MgSO4·7H2O0.1-0.3g, CaCl2·H2O0.05-
0.08g, KH2PO40.4-0.8g, K2HPO40.7-1g, ironic citrate 0.01-0.03g, EDTA0.01-0.03g, yeast extract
0.7-1.2g, trace element solution 0.8-1.3mL, vitamin solution 7-8mL, distilled water 1000mL, wherein trace element solution
It include: ironic citrate 0.1-0.5g, (NH4)6Mo7O24·4H2O0.001-0.005g, MnSO4·H2O0.001-0.004g,
ZnSO40.001-0.003g, H3BO30.001-0.003g, EDTA0.03-0.07g, CuSO4·5H2O0.001-0.004g,
CaCl2·2H2O0.01-0.05g, distilled water 100mL, vitamin solution include: niacin 0.1-0.4g, biotin 0.005-
0.009g, niacinamide 0.1-0.4g, p-aminobenzoic acid 0.1-0.3g, thiamine hydrochloride 0.2-0.7g, distilled water 1000mL are real
The compositional range of each component of the culture medium of a 3-5 is applied in following range: natrium malicum 2.4-2.6g, (NH4)2SO41.2-
1.35g MgSO4·7H2O0.1-0.2g, CaCl2·H2O0.06-0.07g, KH2PO40.5-0.7g, K2HPO40.8-0.9g, lemon
Lemon acid iron 0.01-0.02g, EDTA0.01-0.02g, yeast extract 0.9-1.1g, trace element solution 0.9-1.1mL, vitamin
Solution 7.5-7.7mL, distilled water 1000mL, wherein trace element solution includes: ironic citrate 0.2-0.3g, (NH4)6Mo7O24·4H2O0.001-0.003g, MnSO4·H2O0.001-0.002g, ZnSO40.001-0.002g, H3BO30.001-
0.002g, EDTA0.04-0.06g, CuSO4·5H2O0.001-0.002g, CaCl2·2H2O0.01-0.03g, distilled water
100mL, vitamin solution include: niacin 0.1-0.3g, biotin 0.007-0.008g, niacinamide 0.2-0.3g, p-aminophenyl
Formic acid 0.1-0.3g, thiamine hydrochloride 0.3-0.6g, distilled water 1000mL, and the group of each component of the culture medium of embodiment 1-2
Divide range not in the range of embodiment 3-5；Embodiment 6 is identical as the component of the culture medium of embodiment 7 and constituent content；It is real
It is identical as the component of the culture medium of embodiment 9 and constituent content to apply example 8；
Embodiment 1, embodiment 3, embodiment 6, step A in embodiment 8 autoclave temperature at 113 DEG C -122 DEG C
Range outside, sterilization time is all made of sterile water and forms bacterium solution also outside the range of 10-15min in step B, in step C
Culture intensity of illumination controls in the range of 500-600lx and not in the range of 550-560lx, and cultivation temperature controls
In the range of 20 DEG C -30 DEG C and not in the range of 28 DEG C -30 DEG C, incubation time is controlled at 13-15 days；Embodiment 2 is implemented
Example 4 and 5, embodiment 7, step A in embodiment 9 autoclave temperature control in the range of 113 DEG C -122 DEG C, when sterilizing
Between also control in the range of 10-15min, bacterium solution is formed using the sterile water with bead in step B, the culture in step C
Intensity of illumination controls in the range of 550-560lx, and cultivation temperature controls in the range of 28 DEG C -30 DEG C, incubation time control
At 10-12 days, and culture environment control was anaerobic condition, and the concentration and COD of the Rhodopseudomonas palustris of embodiment 2CrIt goes
Except rate is above embodiment 1, the concentration and COD of the Rhodopseudomonas palustris of embodiment 4 and embodiment 5CrRemoval rate is high
In embodiment 3, the concentration and COD of the Rhodopseudomonas palustris of embodiment 7CrRemoval rate is above embodiment 6, embodiment
The concentration and COD of 9 Rhodopseudomonas palustrisCrRemoval rate is above embodiment 8, illustrates the high pressure by rate-determining steps A
In the range of 113 DEG C -122 DEG C, control sterilization time also in the range of 10-15min, is conducive in culture medium sterilising temp
Miscellaneous bacteria be removed more complete so that Rhodopseudomonas palustris is not readily susceptible to the shadow of other miscellaneous bacterias during the cultivation process
It rings, is conducive to improve the purity for cultivating resulting Rhodopseudomonas palustris, so that Rhodopseudomonas palustris is preferably grown, in turn
Be conducive to improve the concentration for cultivating resulting Rhodopseudomonas palustris；By using the sterile water shape with bead in stepb
At bacterium solution, using bead colliding with each other in oscillatory process, is conducive to Rhodopseudomonas palustris and better disperses come, have
It is preferably grown conducive to Rhodopseudomonas palustris, to be conducive to improve the concentration for cultivating resulting Rhodopseudomonas palustris；It is logical
The culture intensity of illumination in rate-determining steps C is crossed in the range of 550-560lx, controls range of the cultivation temperature at 28 DEG C -30 DEG C
It is interior, incubation time is controlled in the range of 10-12 days, and controlling culture environment is anaerobic condition, be conducive to the red false unit cell in marsh
Bacterium preferably grows, so that the concentration for cultivating resulting Rhodopseudomonas palustris improves.
According to embodiment 1, embodiment 3, embodiment 6, the data comparison of embodiment 8 and embodiment 2,
Embodiment 4-5, embodiment 7, embodiment 9 data comparison can obtain, embodiment 1, embodiment 3, embodiment 6, implement
The cultural method of example 8 is essentially identical, embodiment 2, embodiment 4-5, embodiment 7, the basic phase of cultural method of embodiment 9
Together, implement the compositional range of each component of the culture medium of 1 and embodiment 2 in following range: natrium malicum 2-2.7g,
(NH4)2SO41.15-1.55g MgSO4·7H2O0.1-0.3g, CaCl2·H2O0.05-0.08g, KH2PO40.4-0.8g,
K2HPO40.7-1g, ironic citrate 0.01-0.03g, EDTA0.01-0.03g, yeast extract 0.7-1.2g, trace element solution
0.8-1.3mL, vitamin solution 7-8mL, distilled water 1000mL, wherein trace element solution includes: ironic citrate 0.1-
0.5g, (NH4)6Mo7O24·4H2O0.001-0.005g, MnSO4·H2O0.001-0.004g, ZnSO40.001-0.003g,
H3BO30.001-0.003g, EDTA0.03-0.07g, CuSO4·5H2O0.001-0.004g, CaCl2·2H2O0.01-0.05g,
Distilled water 100mL, vitamin solution include: niacin 0.1-0.4g, biotin 0.005-0.009g, niacinamide 0.1-0.4g, right
Aminobenzoic acid 0.1-0.3g, thiamine hydrochloride 0.2-0.7g, distilled water 1000mL, each component of the culture medium of embodiment 3-5
Compositional range in following range: natrium malicum 2.4-2.6g, (NH4)2SO41.2-1.35g, MgSO4·7H2O0.1-
0.2g, CaCl2·H2O0.06-0.07g, KH2PO40.5-0.7g, K2HPO40.8-0.9g, ironic citrate 0.01-0.02g,
EDTA0.01-0.02g, yeast extract 0.9-1.1g, trace element solution 0.9-1.1mL, vitamin solution 7.5-7.7mL steam
Distilled water 1000mL, wherein trace element solution includes: ironic citrate 0.2-0.3g, (NH4)6Mo7O24·4H2O0.001-
0.003g, MnSO4·H2O0.001-0.002g, ZnSO40.001-0.002g, H3BO30.001-0.002g, EDTA0.04-
0.06g, CuSO4·5H2O0.001-0.002g, CaCl2·2H2O0.01-0.03g, distilled water 100mL, vitamin solution packet
It includes: niacin 0.1-0.3g, biotin 0.007-0.008g, niacinamide 0.2-0.3g, p-aminobenzoic acid 0.1-0.3g, hydrochloric acid sulphur
Amine element 0.3-0.6g, distilled water 1000mL, and the compositional range of each component of the culture medium of embodiment 1-2 is not in embodiment 3-5
In the range of；Embodiment 6 and the component and constituent content of the culture medium of embodiment 7 are equal are as follows: natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g, K2HPO40.9g, ironic citrate 0.01g,
EDTA0.01g, yeast extract 1.1g, trace element solution 1.05mL, vitamin solution 7.6mL, distilled water 1000mL；Wherein,
Trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·H2O0.001g,
ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g, distilled water
100mL；Vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid sulphur
Amine element 0.5g, distilled water 1000mL；The culture of the component ratio embodiment 6 and embodiment 7 of the culture medium of embodiment 8 and embodiment 9
Base component increases pork liver extracting solution and ennel oil, and embodiment 8 cultivate the concentration of resulting Rhodopseudomonas palustris with
And CODCrRemoval rate is above embodiment 6 and is higher than embodiment 1 higher than embodiment 3, and it is red false single that embodiment 9 cultivates resulting marsh
The concentration and COD of born of the same parents bacteriumCrRemoval rate is above embodiment 7 and is higher than embodiment 2 higher than embodiment 4-5, illustrates to pass through control
The ratio of culture medium each component cooperates, and is conducive to each component preferably coordinated and provides for the growth of Rhodopseudomonas palustris
Nutriment is preferably grown to be conducive to Rhodopseudomonas palustris, so that cultivating the dense of resulting Rhodopseudomonas palustris
Du Genggao, activity are more preferable, meanwhile, so that cultivating the removal water body COD of resulting Rhodopseudomonas palustrisCrAbility it is stronger.Separately
Outside, the COD of embodiment 8 and embodiment 6CrRemoval rate is compared, and increase rate is little, the COD of embodiment 9 and embodiment 7CrRemoval
Rate is compared, and increase rate is also little, is illustrated by the way that pork liver extracting solution and ennel oil, major function are added in the medium
It is to improve the concentration for cultivating resulting Rhodopseudomonas palustris.
The embodiment of present embodiment is presently preferred embodiments of the present invention, not limits protection of the invention according to this
Range, therefore: the equivalence changes that all structures under this invention, shape, principle are done, should all be covered by protection scope of the present invention it
It is interior.
Claims (10)
1. a kind of culture medium of Rhodopseudomonas palustris, it is characterized in that: the ingredient of the culture medium includes: natrium malicum 2-
2.7g, (NH4)2SO41.15-1.55g MgSO4·7H2O0.1-0.3g, CaCl2·H2O0.05-0.08g, KH2PO40.4-
0.8g, K2HPO40.7-1g, ironic citrate 0.01-0.03g, EDTA0.01-0.03g, yeast extract 0.7-1.2g, microelement
Solution 0.8-1.3mL, vitamin solution 7-8mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.1-0.5g, (NH4)6Mo7O24·4H2O0.001-0.005g, MnSO4·
H2O0.001-0.004g, ZnSO40.001-0.003g, H3BO30.001-0.003g, EDTA0.03-0.07g, CuSO4·
5H2O0.001-0.004g, CaCl2·2H2O0.01-0.05g, distilled water 100mL；
The vitamin solution includes: niacin 0.1-0.4g, biotin 0.005-0.009g, niacinamide 0.1-0.4g, to amino
Benzoic acid 0.1-0.3g, thiamine hydrochloride 0.2-0.7g, distilled water 1000mL.
2. the culture medium of Rhodopseudomonas palustris according to claim 1, it is characterized in that: the culture medium at subpackage
It includes: natrium malicum 2.4-2.6g, (NH4)2SO41.2-1.35g, MgSO4·7H2O0.1-0.2g, CaCl2·H2O0.06-
0.07g, KH2PO40.5-0.7g, K2HPO40.8-0.9g, ironic citrate 0.01-0.02g, EDTA0.01-0.02g, yeast extract
0.9-1.1g, trace element solution 0.9-1.1mL, vitamin solution 7.5-7.7mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.2-0.3g, (NH4)6Mo7O24·4H2O0.001-0.003g, MnSO4·
H2O0.001-0.002g, ZnSO40.001-0.002g, H3BO30.001-0.002g, EDTA0.04-0.06g, CuSO4·
5H2O0.001-0.002g, CaCl2·2H2O0.01-0.03g, distilled water 100mL；
The vitamin solution includes: niacin 0.1-0.3g, biotin 0.007-0.008g, niacinamide 0.2-0.3g, to amino
Benzoic acid 0.1-0.3g, thiamine hydrochloride 0.3-0.6g, distilled water 1000mL.
3. the culture medium of Rhodopseudomonas palustris according to claim 2, it is characterized in that: the culture medium at subpackage
It includes: natrium malicum 2.5g, (NH4)2SO41.25g MgSO4·7H2O0.2g, CaCl2·H2O0.07g, KH2PO40.6g,
K2HPO40.9g, ironic citrate 0.01g, EDTA0.01g, yeast extract 1.1g, trace element solution 1.05mL, vitamin solution
7.6mL, distilled water 1000mL；
The trace element solution includes: ironic citrate 0.2g, (NH4)6Mo7O24·4H2O0.002g, MnSO4·H2O0.001g,
ZnSO40.001g, H3BO30.002g, EDTA0.05g, CuSO4·5H2O0.001g, CaCl2·2H2O0.02g, distilled water
100mL；
The vitamin solution includes: niacin 0.2g, biotin 0.008g, niacinamide 0.3g, p-aminobenzoic acid 0.2g, hydrochloric acid
Thiamine 0.5g, distilled water 1000mL.
4. the culture medium of Rhodopseudomonas palustris according to claim 1 to 3, it is characterized in that: the culture medium at
Point further include: pork liver extracting solution 2-2.5mL.
5. the culture medium of Rhodopseudomonas palustris according to claim 4, it is characterized in that: the ingredient of the culture medium also wraps
It includes: ennel oil 1-1.5mL.
6. a kind of cultural method of the Rhodopseudomonas palustris using any culture medium of claim 1-5, it is characterized in that:
The following steps are included:
A, preparation of culture medium: each component of culture medium is dissolved in distilled water one by one, adjust pH value to 6.8-7.2, through high pressure sterilization
And after cooling down, culture medium is moved into sterilized closed container, immediately closed container, is placed in shady place storage, it is spare；
B, prepared by bacterium solution: taking the Rhodopseudomonas palustris 10-20g that culture presevation number is M209077 that 100-200mL sterile water is added
In, 30min is sufficiently vibrated after standing 20min, forms bacterium solution；
C, Spawn incubation: preparing resulting bacterium solution for B A be added and prepare in resulting culture medium, then that culture medium is placed in illumination is strong
It is cultivated 10-15 days under conditions of degree is 500-600lx, temperature is 20-30 DEG C, can cultivate to obtain Rhodopseudomonas palustris；
Step A and step B is operated before step C, and the operation order of step A and step B does not limit.
7. the cultural method of Rhodopseudomonas palustris according to claim 6, it is characterized in that: control is high in the step A
The temperature of pressure sterilizing is 113 DEG C -122 DEG C, and controlling the autoclaved time is 10-15min.
8. according to the cultural method of any Rhodopseudomonas palustris of claim 6-7, it is characterized in that: in the step B,
Sterile water uses the sterile water with bead.
9. according to the cultural method of any Rhodopseudomonas palustris of claim 6-7, it is characterized in that: in the step C,
The intensity of illumination control of culture is 550-560lx, and temperature control is 28 DEG C -30 DEG C, and incubation time is 10-12 days.
10. the cultural method of Rhodopseudomonas palustris according to claim 9, it is characterized in that: will be trained in the step C
Feeding base is placed under anoxia condition and cultivates.
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