CN106222124A - The culture medium of carbon sequestration nitrogen microorganism and the screening technique of carbon sequestration nitrogen microorganism - Google Patents
The culture medium of carbon sequestration nitrogen microorganism and the screening technique of carbon sequestration nitrogen microorganism Download PDFInfo
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Abstract
The present invention provides culture medium and the screening technique of carbon sequestration nitrogen microorganism of carbon sequestration nitrogen microorganism, belongs to microbial technology field.Screening technique comprises the following steps: the preparation of (1) facultative fixed nitrogen carbon culture medium；(2) selection of microorganism；(3) aerobic domestication enrichment；(4) anaerobic acclimation enrichment；(5) gradient plate separates；Nitrogen-free carbon culture medium is utilized can effectively to filter out the microorganism of facultative fixed nitrogen carbon, utilize aerobic and anaerobic acclimation enrichment process simultaneously, the facultative fixed nitrogen carbon microorganism screened can be made further also to have facultative anaerobic, the microorganism filtered out is made to adapt to the mal-conditions such as oxygen-enriched or anoxia, improve the adaptability of facultative fixed nitrogen carbon microorganism, and utilize electron transit mediator to carry out communication for information, promote breathing and the growth of microorganism, the interpolation of trace element is also greatly shortened the screening cycle, the screening technique of the present invention not only reduces fixed nitrogen carbon cost, reduce the discharge of greenhouse gases simultaneously, turn waste into wealth to reach, recycle.
Description
[technical field]
The present invention relates to microbial technology field, be specifically related to the culture medium of carbon sequestration nitrogen microorganism and carbon sequestration nitrogen microorganism
Screening technique.
[background technology]
At present, known microorganism carbon sequestration bacterium, microorganism nitrogen-fixing bacteria can only fix CO respectively2And N2, academicly and tradition
Fix N in theory2Nitrogen-fixing bacteria and fixing CO2Carbon sequestration bacterium respectively the most studied and find.Existing numerous studies show big from
Especially existing in soil in Ran and much have the most carbon functional microorganism, for microbial evolution theory, microorganism is to ring
The strong adaptability in border, fast, the metabolic pathway that makes a variation and nutrient type variation, fix N simultaneously2And CO2Microorganism existence not without
May, especially it is distributed in leguminous plant (nitrogen fertilizer application is not optimum selection) root soil and (is the most also not excluded for other habitat
This quasi-microorganism may be there is also) very likely there is some in a lot of nitrogen-fixing bacteria and can have the most carbon functional microorganism simultaneously.
It addition, the most very likely there is some in some the carbon sequestration bacterium being distributed in water body especially deep-sea can have fixed nitrogen function simultaneously
Microorganism.
" one can fix CO to " ACTA Scientiae Circumstantiae " interim open paper of volume 33 the 4th simultaneously2And N2Microorganism--
Facultative solid CO2、N2The isolation identification of bacterium and confirmatory experiment thereof ", in the culture medium of this paper fixed nitrogen or carbon sequestration, optimization is facultative solid
Nitrogen carbon microbiological culture media screens facultative fixed nitrogen carbon antibacterial, but does not has electron transit mediator in this electron transmission, is difficult to promote micro-
Biological breathing and growth, although and the facultative nitrogen-free carbon culture medium of this paper can screen antibacterial, can be used for screening fungus and
Some alga microbials, but adaptability is not strong, and under this culture medium, antibacterial, fungus and alga microbial growth are slow, screening week
Phase is long, and antibacterial, fungus and the alga microbial respiratory chain in incubation easily causes problem and cause death.
[summary of the invention]
The goal of the invention of the present invention is: for the problem of above-mentioned existence, it is provided that provide the training of a kind of carbon sequestration nitrogen microorganism
Support base and the screening technique of facultative carbon sequestration nitrogen microorganism, be possible not only to screen the antibacterial of facultative fixed nitrogen carbon, and can screen true
Bacterium and part alga microbial, add electron transit mediator in the facultative nitrogen-free carbon culture medium of the present invention simultaneously and can also promote micro-life
The breathing of thing and growth, the preparation of trace element and add the growth that also can further speed up antibacterial, fungus and alga microbial,
Further increase the efficiency of screening.
To achieve these goals, the technical solution used in the present invention is as follows:
The culture medium of carbon sequestration nitrogen microorganism, the composition of described culture medium and weight is: KH2PO41.0～1.5g, K2HPO4
2.0～2.5g, MgSO4·7H2O 0.2～0.3g, NaCl 20～30g, CaCl20.01～0.02g, FeSO40.01～
0.02g, electron transport substance 2.0～3.0g, trace element mixed liquor 2～3ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.68～1.70mg, H3BO30.4～
0.5mg、ZnSO4·7H2O 1.0～1.5mg, MnSO4·5H2O 1.0～1.5mg, CuSO4·5H2O 7.0～8.0mg,
CoCl2·6H2O 1.0～1.5mg, NiSO4·7H2O 1.0～1.5mg.
The culture medium of the most described carbon sequestration nitrogen microorganism, the composition of described culture medium and weight is: KH2PO41.1～
1.4g、K2HPO42.1～2.4g, MgSO4·7H2O 0.24～0.28g, NaCl 22～28g, CaCl20.012～0.018g,
FeSO40.012～0.018g, electron transport substance 2.2～2.8g, trace element mixed liquor 2.2～2.8ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.685～1.695mg, H3BO30.42～
0.48mg、ZnSO4·7H2O 1.1～1.4mg, MnSO4·5H2O 1.1～1.4mg, CuSO4·5H2O 7.2～7.8mg,
CoCl2·6H2O 1.1～1.4mg, NiSO4·7H2O 1.1～1.4mg.
According to claim 1, the culture medium of carbon sequestration nitrogen microorganism, the composition of described culture medium and weight are: KH2PO4
1.25g、K2HPO4 2.3g、MgSO4·7H2O 0.26g、NaCl 25g、CaCl20.015g、FeSO40.015g, electron transmission
Material 2.5g, trace element mixed liquor 2.5ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.69mg、H3BO3 0.45mg、ZnSO4·
7H2O 1.3mg、MnSO4·5H2O 1.3mg、CuSO4·5H2O7.5mg、CoCl2·6H2O 1.25mg、NiSO4·7H2O
1.3mg。
According to the formula of above culture medium, the screening technique of described a kind of facultative carbon sequestration nitrogen microorganism, comprise the following steps:
(1) selection of microorganism:
The mushroom method of sampling: be sampled as 3 parts of the soil more than 15cm from earth's surface, weigh 1g after three parts of soil are ground mixing
Add to equipped with in the triangular flask of bead, and inject 9～10ml sterilized water wherein, be subsequently placed in shaking table 28～30 DEG C, 120
～150r/min vibration 20～30min, it is sufficiently mixed and is configured to Soil Slurry；
(2) aerobic domestication enrichment: use draining ventilation distribution, after serum bottle is filled water, back-off is in water, by mixing
Gas ratio N2∶CO2∶O2With syringe squeeze into the N of respective amount at=8.5: 0.5: 12、CO2And O2, seal with sealed membrane；Carbon-free nitrogen is trained
Supporting base and inject in serum bottle, another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus；After by the soil in step (2)
Earth suspension joins in the serum bottle equipped with carbon-free nitrogen culture medium, is subsequently placed in shaking table 28～30 DEG C, and 120～150r/min shake
Swinging 20～30min, 2～3d is an enrichment cycle, re-fills gaseous mixture, ratio N after each end cycle2∶CO2∶O2=
8.5: 0.5: 1, repeat enrichment process and obtain bacterium solution totally 2～3 times；
(5) anaerobic acclimation enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off is in water, by aerobic
Atmosphere gaseous mixture i.e. air: CO2The gas syringe of=9: 1 is squeezed into, and seals with sealed membrane, and carbon-free nitrogen culture medium is injected serum
In Ping, another syringe needle is inserted in serum bottle by plug simultaneously, in order to aerofluxus, the bacterium solution of step (3) is transferred to new equipped with
In the serum bottle of carbon-free nitrogen culture medium, it is subsequently placed in shaking table 28～30 DEG C, 120～150r/min vibrations 20～30min, 2～3d
It is an enrichment cycle, cultivates all after date, then by oxygen-free atmosphere gaseous mixture N2∶CO2With syringe squeeze in serum bottle at=9: 1,
It is an enrichment cycle with 2～3d again, cultivates all after date, then repeat 5～6 under the conditions of having oxygen atmosphere and oxygen-free atmosphere
In the cycle, finally obtain purpose bacterium solution；
(6) gradient plate separates: using 10 times of gradient dilution methods that purpose bacterium solution is made suspension concentration is 10-1、10-2、
10-3、10-4、10-5、10-6、10-7, use spread plate culture method, first carbon-free nitrogen culture medium poured in aseptic flat board, wait to coagulate
Numbering 10 after Gu-1、10-2、10-3、10-4、10-5、10-6、10-7, each number arranges three repetitions, draws each gradient dilution respectively
Liquid 0.1ml, in corresponding flat board, coats carbon-free nitrogen culture medium flat plate with spreading rod, lies against on table quiet by coated flat board
Put 5～10min, permeate 30 DEG C of inversions after in culture medium until bacterium solution and cultivate；Picking variform single bacterium colony line training after 48h
Supporting, wherein single bacterium colony is the microorganism screened.
Further, the sampling soil in step (2) is at soil at Semen arachidis hypogaeae rhizosphere, bean rhizosphere in soil
Kind or the mixing of two kinds, a lot of researchs show in the Nature that especially a lot of microorganisms have carbon sequestration function in soil, but differ
Surely there is fixed nitrogen function.Nitrogen-fixing bacteria are distributed in leguminous plant (nitrogen fertilizer application is not optimum selection) root soil mostly, this part
Very likely some can have carbon sequestration function to nitrogen-fixing bacteria simultaneously.
Further, one or both the mixture during described electron transport substance is sodium sulfide or sodium thiosulfate；
Because facultative carbon sequestration nitrogen bacterium carbon-free nitrogen culture medium without electron transit mediator therefore adds electron transit mediator, microorganism utilizes free sulphion to make
Carry out communication for information for electron transit mediator, and promote breathing and the growth of microorganism.
Further, described facultative carbon sequestration nitrogen microorganism carbon-free nitrogen culture medium is to cultivate at carbon sequestration bacterium culture medium and nitrogen-fixing bacteria
Optimum organization on the basis of base and come, be possible not only to screen the antibacterial of facultative fixed nitrogen carbon, and fungus and part can be screened
Alga microbial.
Further, the pH value of described culture medium is 4.5～6.8, not only contributes to antibacterial, is also beneficial to fungus and algae
The growth of microorganism, and adapt to the growth of a greater variety of antibacterial, fungus and algae.
Further, for resistance to CO in step (5)2、N2Bacterium also need with plug test tube replace culture medium flat plate sieve
Choosing.
Further, described method can be also used for filtering out fixed nitrogen carbon alga microbial from water body.
In sum, owing to have employed technique scheme, the invention has the beneficial effects as follows:
(1) present invention uses nitrogen-free, the culture medium of carbon source is possible not only to screen facultative fixed nitrogen carbon antibacterial, and can enter one
Step filters out fungus and the alga microbial of facultative fixed nitrogen carbon, and traditional microbiological nutrition and metabolism theory will be weighed by it
Supplement, and facultative solid CO2、N2Bacterium is not required to additional carbon when carbon sequestration, thus reduces carbon sequestration cost, reduces greenhouse gases simultaneously
Discharge, it will be for realizing CO2Microorganism incubation reclaim with resource provide an important effective way, with reach change give up into
Treasured, recycles.
(2) present invention uses sodium sulfide or sodium thiosulfate as electron transit mediator, microorganism utilize free sulfur from
Son carries out communication for information as electron transit mediator, and promotes breathing and the growth of microorganism.
(3) present invention apply aerobic domestication enrichment and anaerobic acclimation enrichment screen facultative fixed nitrogen carbon microorganism further,
The facultative fixed nitrogen carbon microorganism screened can be made further also to have facultative anaerobic, make the microorganism filtered out to fit
Should the mal-condition such as oxygen-enriched or anoxia, improve the adaptability of facultative fixed nitrogen carbon microorganism.
(4) trace element that the present invention uses can be effectively improved the speed of growth of microorganism, makes enrichment and screening cycle
Significantly shorten, need at least 72h without growth cycle before trace element of the present invention screening, after using trace element of the present invention, raw
Long period foreshortens to 48h, improves screening efficiency.
(5) present invention carbon-free nitrogen Medium's PH Value 4.5～6.8 scope, it is adaptable to more antibacterial, fungus and algae
Survival and screening.
[detailed description of the invention]
Below by way of specific embodiment and verification the verifying results, the invention will be further described.
Embodiment 1
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism, comprises the following steps:
(1) the preparation component of facultative carbon sequestration nitrogen microorganism carbon-free nitrogen culture medium and consumption:
KH2PO4 1.0g、K2HPO4 2.0g、MgSO4·7H2O 0.2g、NaCl 20g、CaCl2 0.01g、FeSO4
0.01g, electron transport substance 2.0g, trace element mixed liquor 2ml, add distilled water to 1000ml, 121 DEG C of sterilizing 20min, pH
Value 4.5；
Wherein trace element mixed liquor (mg/L): Na2MoO4·2H2O 1.68mg、H3BO3 0.4mg、ZnSO4·7H2O
1.0mg、MnSO4·5H2O 1.0mg、CuSO4·5H2O 7.0mg、CoCl2·6H2O 1.0mg、NiSO4·7H2O 1.0mg；
(2) selection of microorganism:
The mushroom method of sampling: be sampled as being more than from earth's surface at Semen arachidis hypogaeae rhizosphere 3 parts of the soil of 15cm, three parts of soil are ground mixed
Weigh 1g after even to add to equipped with in the triangular flask of bead, and inject 9ml sterilized water wherein, be subsequently placed in shaking table 28～30
DEG C, 120r/min vibrates 20min, is sufficiently mixed and is configured to Soil Slurry；
(3) aerobic domestication enrichment: use draining ventilation distribution, after serum bottle is filled water, back-off is in water, by mixing
Gas ratio (N2∶CO2∶O2=8.5: 0.5: 1) N of respective amount is squeezed into syringe2、CO2And O2, seal with sealed membrane；By carbon-free nitrogen
Culture medium is injected in serum bottle, and another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus；After by step (2)
Sample diluting liquid joins in the serum bottle equipped with carbon-free nitrogen culture medium, is subsequently placed in shaking table 28 DEG C, and 120r/min vibrates
20min, 2d are an enrichment cycle, again inflate after each end cycle, repeat enrichment process and obtain purpose bacterium solution totally 2 times；
(4) anaerobic acclimation enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off is in water, by aerobic
The gaseous mixture proportional air of atmosphere: CO2Under conditions of=9: 1, squeeze into syringe, seal with sealed membrane, by carbon-free nitrogen culture medium
Injecting in serum bottle, another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus, the bacterium solution of step (3) is transferred to
In the new serum bottle equipped with carbon-free nitrogen culture medium, being subsequently placed in shaking table 28 DEG C, vibrate 20min, 2d of 120r/min is a richness
In the collection cycle, cultivate all after date, then by oxygen-free atmosphere gaseous mixture N2∶CO2With syringe squeeze in serum bottle at=9: 1, then with 2d is
In one enrichment cycle, cultivate all after date, then repeat 5 cycles under the conditions of having oxygen atmosphere and oxygen-free atmosphere, finally obtain
Purpose bacterium solution；
(5) gradient plate separates: using 10 times of gradient dilution methods that purpose bacterium solution is made suspension concentration is 10-1、10-2、
10-3、10-4、10-5、10-6、10-7, use spread plate culture method, first carbon-free nitrogen culture medium poured in aseptic flat board, wait to coagulate
Numbering 10 after Gu-1、10-2、10-3、10-4、10-5、10-6、10-7, each number arranges three repetitions, draws each gradient dilution respectively
Liquid 0.1ml, in corresponding flat board, coats carbon-free nitrogen culture medium flat plate with spreading rod, lies against on table quiet by coated flat board
Put 5～10min, permeate 30 DEG C of inversions after in culture medium until bacterium solution and cultivate；Picking variform single bacterium colony line training after 48h
Supporting, wherein single bacterium colony is the microorganism screened.
A lot of researchs show in the Nature that especially a lot of microorganisms have carbon sequestration function in soil, but not necessarily have fixed nitrogen
Function.Nitrogen-fixing bacteria are distributed in leguminous plant (nitrogen fertilizer application is not optimum selection) root soil mostly, the nitrogen-fixing bacteria pole of this part
Likely some can have carbon sequestration function simultaneously；And use sodium sulfide to utilize free sulfur as electron transport substance, microorganism
Ion carries out communication for information as electron transit mediator, and promotes breathing and the growth of microorganism；The most facultative carbon sequestration nitrogen microorganism
The optimum organization on the basis of carbon sequestration bacterium culture medium and nitrogen-fixing bacteria culture medium of carbon-free nitrogen culture medium, is possible not only to screening
The antibacterial of facultative fixed nitrogen carbon, and fungus and part alga microbial can be screened；Aerobic and the atmosphere group of anaerobism in step (4)
Becoming to be respectively as follows: oxygen atmosphere is air: CO2=9: 1, anaerobism atmosphere is N2∶CO2=9: 1, can be to facultative fixed nitrogen carbon microorganism
Screen accurately, and the microorganism screened also is amphimicrobe；Meanwhile, for resistance to CO in step (5)2、N2's
Bacterium also needs to replace culture medium flat plate to screen with plug test tube, and the method can be also used for filtering out fixed nitrogen carbon algae from water body
Quasi-microorganism.
Embodiment 2
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism, comprises the following steps:
(1) the preparation component of facultative carbon sequestration nitrogen microorganism carbon-free nitrogen culture medium and consumption:
KH2PO4 1.5g、K2HPO4 2.5g、MgSO4·7H2O 0.3g、NaCl 30g、CaCl2 0.02g、FeSO4
0.02g, electron transport substance 3.0g, trace element mixed liquor 3ml, add distilled water to 1000ml, 125 DEG C of sterilizing 25min, pH
Value 5.5；
Wherein trace element mixed liquor (mg/L): Na2MoO4·2H2O 1.70mg、H3BO3 0.5mg、ZnSO4·7H2O
1.5mg、MnSO4·5H2O 1.5mg、CuSO4·5H2O 8.0mg、CoCl2·6H2O 1.5mg、NiSO4·7H2O 1.5mg；
(2) selection of microorganism:
The mushroom method of sampling: be sampled as being more than from earth's surface at Rhizosphere of Pea 3 parts of the soil of 15cm, three parts of soil are ground mixed
Weigh 1g after even to add to equipped with in the triangular flask of bead, and inject 10ml sterilized water wherein, be subsequently placed in shaking table 30 DEG C,
150r/min vibrates 30min, is sufficiently mixed and is configured to Soil Slurry；
(3) aerobic domestication enrichment: use draining ventilation distribution, after serum bottle is filled water, back-off is in water, by mixing
Gas ratio (N2∶CO2∶O2=8.5: 0.5: 1) N of respective amount is squeezed into syringe2、CO2And O2, seal with sealed membrane；By carbon-free nitrogen
Culture medium is injected in serum bottle, and another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus；After by step (2)
Sample diluting liquid joins in the serum bottle equipped with carbon-free nitrogen culture medium, is subsequently placed in shaking table 30 DEG C, and 150r/min vibrates
30min, 3d are an enrichment cycle, again inflate after each end cycle, repeat enrichment process and obtain purpose bacterium solution totally 3 times；、
(4) anaerobic acclimation enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off is in water, by aerobic
The gaseous mixture proportional air of atmosphere: CO2Under conditions of=9: 1, squeeze into syringe, seal with sealed membrane, by carbon-free nitrogen culture medium
Injecting in serum bottle, another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus, the bacterium solution of step (3) is transferred to
In the new serum bottle equipped with carbon-free nitrogen culture medium, being subsequently placed in shaking table 30 DEG C, vibrate 30min, 3d of 150r/min is a richness
In the collection cycle, cultivate all after date, then by oxygen-free atmosphere gaseous mixture N2∶CO2With syringe squeeze in serum bottle at=9: 1, then with 3d is
In one enrichment cycle, cultivate all after date, then repeat 6 cycles under the conditions of having oxygen atmosphere and oxygen-free atmosphere, finally obtain
Purpose bacterium solution；
(5) gradient plate separates: using 10 times of gradient dilution methods that purpose bacterium solution is made suspension concentration is 10-1、10-2、
10-3、10-4、10-5、10-6、10-7, use spread plate culture method, first carbon-free nitrogen culture medium poured in aseptic flat board, wait to coagulate
Numbering 10 after Gu-1、10-2、10-3、10-4、10-5、10-6、10-7, each number arranges three repetitions, draws each gradient dilution respectively
Liquid 0.1ml, in corresponding flat board, coats carbon-free nitrogen culture medium flat plate with spreading rod, lies against on table quiet by coated flat board
Put 5～10min, permeate 30 DEG C of inversions after in culture medium until bacterium solution and cultivate；Picking variform single bacterium colony line training after 48h
Supporting, wherein single bacterium colony is the microorganism screened.
Other are same as in Example 1.
Embodiment 3
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism, comprises the following steps:
(1) the preparation component of facultative carbon sequestration nitrogen microorganism carbon-free nitrogen culture medium and consumption:
KH2PO4 1.25g、K2HPO4 2.25g、MgSO4·7H2O 0.25g、NaCl 25g、CaCl2 0.015g、FeSO4
0.015g, electron transport substance 2.5g, trace element mixed liquor 2.5ml, add distilled water to 1000ml, 123 DEG C of sterilizings
25min, pH value 6.8；
Wherein trace element mixed liquor (mg/L): Na2MoO4·2H2O 1.70mg、H3BO3 0.45mg、ZnSO4·7H2O
1.25mg、MnSO4·5H2O 1.25mg、CuSO4·5H2O 7.5mg、CoCl2·6H2O 1.25mg、NiSO4·7H2O
1.25mg；
(2) selection of microorganism:
The mushroom method of sampling: sampling takes the water sample 1ml of below water surface 0.5m in lake and adds to the triangle equipped with bead
In Ping, and injecting 9.5ml sterilized water wherein, be subsequently placed in shaking table 29 DEG C, 135r/min vibrates 25min, is sufficiently mixed configuration
Become Soil Slurry；
(3) aerobic domestication enrichment: use draining ventilation distribution, after serum bottle is filled water, back-off is in water, by mixing
Gas ratio (N2∶CO2∶O2=8.5: 0.5: 1) N of respective amount is squeezed into syringe2、CO2And O2, seal with sealed membrane；By carbon-free nitrogen
Culture medium is injected in serum bottle, and another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus；After by step (2)
Sample diluting liquid joins in the serum bottle equipped with carbon-free nitrogen culture medium, is subsequently placed in shaking table 30 DEG C, and 135r/min vibrates
25min, 2d are an enrichment cycle, again inflate after each end cycle, repeat enrichment process and obtain purpose bacterium solution totally 3 times；
(4) anaerobic acclimation enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off is in water, by aerobic
The gaseous mixture proportional air of atmosphere: CO2Under conditions of=9: 1, squeeze into syringe, seal with sealed membrane, by carbon-free nitrogen culture medium
Injecting in serum bottle, another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus, the bacterium solution of step (3) is transferred to
In the new serum bottle equipped with carbon-free nitrogen culture medium, being subsequently placed in shaking table 29 DEG C, vibrate 25min, 2.5d of 135r/min is one
In the enrichment cycle, cultivate all after date, then by oxygen-free atmosphere gaseous mixture N2∶CO2With syringe squeeze in serum bottle at=9: 1, then with 2d
It is an enrichment cycle, cultivates all after date, then repeat 5 cycles under the conditions of having oxygen atmosphere and oxygen-free atmosphere, finally
To purpose bacterium solution；
(5) gradient plate separates: using 10 times of gradient dilution methods that purpose bacterium solution is made suspension concentration is 10-1、10-2、
10-3、10-4、10-5、10-6、10-7, use spread plate culture method, first carbon-free nitrogen culture medium poured in aseptic flat board, wait to coagulate
Numbering 10 after Gu-1、10-2、10-3、10-4、10-5、10-6、10-7, each number arranges three repetitions, draws each gradient dilution respectively
Liquid 0.1ml, in corresponding flat board, coats carbon-free nitrogen culture medium flat plate with spreading rod, lies against on table quiet by coated flat board
Put 5～10min, permeate 30 DEG C of inversions after in culture medium until bacterium solution and cultivate；Picking variform single bacterium colony line training after 48h
Supporting, wherein single bacterium colony is the microorganism screened.
Other are same as in Example 1.
Checking is analyzed
Invention group: the microorganism screened in embodiment 1 is included antibacterial, fungus and alga microbial, respectively at A, B
At CO in two kinds of culture medium2、N2Cultivating under gaseous mixture atmosphere, culture medium A is the carbon-free nitrogen culture medium in embodiment 1；
Culture medium B is that " one can fix CO to paper simultaneously2And N2Microorganism--facultative solid CO2、N2The isolation identification of bacterium and checking thereof
Experiment " facultative carbon sequestration nitrogen culture medium；
Matched group: " one can fix CO simultaneously by paper2And N2Microorganism--facultative solid CO2、N2The isolation identification of bacterium
And confirmatory experiment " antibacterial screened, respectively at CO in two kinds of culture medium of A, B2、N2Cultivate under gaseous mixture atmosphere,
Culture medium A is the carbon-free nitrogen culture medium in embodiment 1；Culture medium B is that " one can fix CO to paper simultaneously2And N2Microorganism--
Facultative solid CO2、N2The isolation identification of bacterium and confirmatory experiment thereof " facultative carbon sequestration nitrogen culture medium；
The results are shown in Table shown in 1,2 of concrete compliance test result.
Table 1 invention group and the contrast of matched group microbe survival situation
Table 2 different culture media is on antibacterial/fungus/algal grown and the impact in screening cycle
As can be seen from Table 1: antibacterial, fungus and the algae in matched group can survive in two kinds of culture medium, and invents
Antibacterial, fungus and the algae of group only have antibacterial can survive in two kinds of culture medium, and fungus and algae can not be in culture medium B
Existence, illustrates that the kind adapting to screening microorganism of culture medium B is less, is unfavorable for generally utilizing；Table 2 illustrates simultaneously, the present invention
Culture medium A can be effectively improved cultivation speed, shorten the screening cycle, substantially increase work efficiency.
Claims (9)
1. the culture medium of carbon sequestration nitrogen microorganism, it is characterised in that: the composition of described culture medium and weight is: KH2PO41.0～
1.5g、K2HPO42.0～2.5g, MgSO47H2O 0.2～0.3g, NaCl 20～30g, CaCl20.01～0.02g, FeSO4
0.01～0.02g, electron transport substance 2.0～3.0g, trace element mixed liquor 2～3ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.68～1.70mg, H3BO30.4～0.5mg,
ZnSO4·7H2O 1.0～1.5mg, MnSO4·5H2O 1.0～1.5mg, CuSO4·5H2O 7.0～8.0mg, CoCl2·6H2O
1.0～1.5mg, NiSO4·7H2O 1.0～1.5mg.
The culture medium of carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: the composition of described culture medium and weight
For: KH2PO41.1～1.4g, K2HPO42.1～2.4g, MgSO4·7H2O 0.24～0.28g, NaCl 22～28g, CaCl2
0.012～0.018g, FeSO40.012～0.018g, electron transport substance 2.2～2.8g, trace element mixed liquor 2.2～
2.8ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.685～1.695mg, H3BO30.42～
0.48mg、ZnSO4·7H2O 1.1～1.4mg, MnSO4·5H2O 1.1～1.4mg, CuSO4·5H2O 7.2～7.8mg,
CoCl2·6H2O 1.1～1.4mg, NiSO4·7H2O 1.1～1.4mg.
The culture medium of carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: the composition of described culture medium and weight
For: KH2PO4 1.25g、K2HPO4 2.3g、MgSO4·7H2O 0.26g、NaCl 25g、CaCl20.015g、FeSO40.015g,
Electron transport substance 2.5g, trace element mixed liquor 2.5ml；
Wherein the solute in trace element mixed liquor is: Na2MoO4·2H2O 1.69mg、H3BO3 0.45mg、ZnSO4·7H2O
1.3mg、MnSO4·5H2O 1.3mg、CuSO4·5H2O7.5mg、CoCl2·6H2O 1.25mg、NiSO4·7H2O 1.3mg。
4. according to the screening technique of facultative carbon sequestration nitrogen microorganism a kind of described in claims 1 to 3 any one, it is characterised in that:
Comprise the following steps:
(1) selection of microorganism:
The mushroom method of sampling: be sampled as 3 parts of the soil more than 15cm from earth's surface, weighs 1g and adds after three parts of soil are ground mixing
To equipped with in the triangular flask of bead, and inject 9～10ml sterilized water wherein, be subsequently placed in shaking table 28～30 DEG C, 120～
150r/min vibration 20～30min, is sufficiently mixed and is configured to Soil Slurry；
(2) aerobic domestication enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off is in water, by gaseous mixture ratio
Example N2∶CO2∶O2With syringe squeeze into the N of respective amount at=8.5: 0.5: 12、CO2And O2, seal with sealed membrane；By carbon-free nitrogen culture medium
Injecting in serum bottle, another syringe needle inserts in serum bottle by plug simultaneously, in order to aerofluxus；After by the soil in step (2) hang
Supernatant liquid joins in the serum bottle equipped with carbon-free nitrogen culture medium, is subsequently placed in shaking table 28～30 DEG C, 120～150r/min vibrations 20
～30min, 2～3d is an enrichment cycle, re-fills gaseous mixture, ratio N after each end cycle2∶CO2∶O2=8.5:
0.5: 1, repeat enrichment process and obtain bacterium solution totally 2～3 times；
(3) anaerobic acclimation enrichment: using draining ventilation distribution, after serum bottle is filled water, back-off, in water, will have oxygen atmosphere
Gaseous mixture i.e. air: CO2The gas syringe of=9: 1 is squeezed into, and seals with sealed membrane, and carbon-free nitrogen culture medium is injected serum bottle
In, another syringe needle is inserted in serum bottle by plug simultaneously, in order to aerofluxus, the bacterium solution of step (3) is transferred to new equipped with nothing
In the serum bottle of carbon nitrogen culture medium, being subsequently placed in shaking table 28～30 DEG C, 120～150r/min vibrations 20～30min, 2～3d are
In one enrichment cycle, cultivate all after date, then by oxygen-free atmosphere gaseous mixture N2∶CO2With syringe squeeze in serum bottle at=9: 1, then
It is an enrichment cycle with 2～3d, cultivates all after date, then repeated for 5～6 week under the conditions of having oxygen atmosphere and oxygen-free atmosphere
Phase, finally obtain purpose bacterium solution；
(4) gradient plate separates: using 10 times of gradient dilution methods that purpose bacterium solution is made suspension concentration is 10-1、10-2、10-3、
10-4、10-5、10-6、10-7, use spread plate culture method, first carbon-free nitrogen culture medium is poured in aseptic flat board, rear volume to be solidified
Numbers 10-1、10-2、10-3、10-4、10-5、10-6、10-7, each number arranges three repetitions, draws each gradient dilution liquid respectively
0.1ml, in corresponding flat board, coats carbon-free nitrogen culture medium flat plate with spreading rod, is lain against by coated flat board and stands on table
5～10min, permeate 30 DEG C of inversions after in culture medium until bacterium solution and cultivate；Picking variform single bacterium colony line training after 48h
Supporting, wherein single bacterium colony is the microorganism screened.
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: in step (1)
Sampling soil be one or both the mixing in soil at soil at Semen arachidis hypogaeae rhizosphere, bean rhizosphere.
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: described electronics passes
Passing material is one or both the mixture in sodium sulfide or sodium thiosulfate.
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: described carbon-free nitrogen
The pH value of culture medium is 4.5～6.8.
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: in step (4)
Bacterium for resistance to CO2, N2 also needs to replace culture medium flat plate to screen with plug test tube.
The screening technique of a kind of facultative carbon sequestration nitrogen microorganism the most according to claim 1, it is characterised in that: described method is also
May be used for the screening of fixed nitrogen carbon alga microbial.
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| CN115820493A (en) * | 2022-12-01 | 2023-03-21 | 安徽农业大学 | Method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose |
| WO2023091349A3 (en) * | 2021-11-22 | 2023-07-27 | Carboniferous Inc. | Carbon sequestration in anoxic zones |
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| CN104204211A (en) * | 2012-03-03 | 2014-12-10 | 科技部生物技术局 | Recombinant nitrogen-fixing microorganism and uses thereof |
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| 周盛 等: "一种能同时固定CO_2和N_2的微生物――兼性固CO_2、N_2菌的分离鉴定及其验证实验", 《环境科学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023091349A3 (en) * | 2021-11-22 | 2023-07-27 | Carboniferous Inc. | Carbon sequestration in anoxic zones |
| CN115820493A (en) * | 2022-12-01 | 2023-03-21 | 安徽农业大学 | Method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose |
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