CN108624504A - A method of detaching nitrogen-fixing bacteria from petroleum pollution - Google Patents
A method of detaching nitrogen-fixing bacteria from petroleum pollution Download PDFInfo
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Abstract
The present invention provides a kind of methods detaching nitrogen-fixing bacteria from petroleum pollution, belong to microorganism and are separately cultured technical field.The method of the present invention includes the following steps：(1) petroleum pollution is acquired, aqueous suspension, suspension is added to carry out gradient dilution；(2) nitrogen-free solid medium is prepared, the suspension of different dilutions is separately added into culture medium melting state, stationary culture after mixing, if there are a bacterium colony growth in media surface or inside, the nitrogen-fixing bacteria in isolated petroleum pollution.The present invention can make up that conventional method can only obtain can be with the bacterium of fixed nitrogen under aerobic condition, it is easy to omit micro- aerobic or anaerobic condition fixed nitrogen bacterium in screening, meet diversity of the nitrogen-fixing bacteria to oxygen demand, to improve the accuracy rate and separative efficiency that detach nitrogen-fixing bacteria in petroleum pollution, have a extensive future.
Description
Technical field
The present invention relates to microorganisms to be separately cultured technical field, more particularly to one kind from oil-polluted soils or petroleum wastewater
Contaminate the method that nitrogen-fixing bacteria are detached in water body.
Background technology
Biological nitrogen fixation refers to that nitrogen-fixing microorganism converts the nitrogen in air to can be by the nitrogen source process that organism utilizes, can
It is divided into symbiotic nitrogen fixation, association nitrogen fixation and growing nitrogen-fixing.Symbiotic nitrogen fixation is usually that microorganism is closely living together with other biological,
And it forms special symbiotic structure and carries out nitrogen fixation；Association nitrogen fixation refers to the root that some azotobacters can be colonized in plant
Border is internal, and there is mutualities of interest between host, and have certain specificity；Azotobacter usually in natural environment or
Live on one's own life in culture medium can fixed member state nitrogen, and be reduced to ammonia.
Nitrogen-fixing bacteria are widely distributed in nature, and for petroleum-contaminated soil microorganism analysis shows, the number of nitrogen-fixing bacteria
There are positive correlations with the oil pollution of soil a certain concentration for amount.With the raising of soil petroleum pollution concentration, there are nitrogenase activities
High expression and nitrogen-fixing bacteria enrichment, thus it is speculated that the reason is that since the content of contaminated soil petrochina is higher, in the micro- life of degraded oil
Under object effect, the content in carbon in soil source is increased, under conditions of nitrogen source content relative reduction, causes the enrichment of nitrogen-fixing bacteria.
The presence of nitrogen-fixing bacteria can enhance the ability of crude oil pollution biological treating, some bacterial strains even concentration is relatively low can also to increase stone
Oil degradation capabilities.Ikechukwu et al. is by the pseudomonad of nitrogen-fixing bacteria (Azotobacter vinelandii) and degraded oil
In contaminated soil, petroleum hydrocarbon degradation rate is increased to 66.83- from 23.2-44.45% for (Pseudomonas sp) combined inoculation
69.6%.Nitrogen-fixing bacteria are detached from oil-polluted soils, are provided important bacterial strain is provided for the biological treating of oil-polluted soils
Source.However the presence of each ingredient influences and inhibits the growth of the most of microbe including nitrogen-fixing bacteria in oil, therefore
Separation nitrogen-fixing bacteria are not easy to isolated from oil-polluted soils.
Common nitrogen-fixing bacteria separation method mainly uses Ashby nitrogen-free agars, by diluting sample gradient to be separated
Afterwards, surface is coated in nitrogen-free agar, after being cultivated 3-5 days at 30 DEG C, is observed and is screened purifying bacterial strain.Subsequently through azotase
Whether the amplification and sequence analysis of structural gene nifH is that nitrogen-fixing bacteria are determined to it in conjunction with nitrogenase activity analysis.However
Azotobacter wide variety, including aerobic, anaerobism, micro- aerobic, amphimicrobian and photosynthetic bacteria etc., since azotase is to oxygen height
Degree is sensitive, many nitrogen-fixing bacteria can only under anaerobism or micro- aerobic condition could fixed nitrogen, be easy to be missed in conventional separation methods.
Therefore the method for conventional nitrogen-free agar surface coating can only obtain some aerobic nitrogen-fixing bacteria there are Oxygen protection mechanism,
Demand of the nitrogen-fixing bacteria of its type due to azotase to oxygen is different, and nitrogen can not be fixed under aerobic condition, thus loses
The ability grown in nitrogen-free agar, to be missed in screening.
Invention content
The purpose of the present invention is to provide a kind of to detach the method for nitrogen-fixing bacteria to overcome the prior art from petroleum pollution
Deficiency.
Present invention firstly provides a kind of nitrogen-free solid medium improved, the formula of 1L is：Glucose 20-50g；
K2HPO40.5-1.0g；MgSO4·7H2O 0.1-0.2g；Ferric iron citrate【Fe(Ⅲ)-citrate】0.02-0.05g；
NaMoO4·2H2O0.001-0.76g；Agar powder 8-15g；Add water to 1L；pH 7.0.
Preferably, the nitrogen-free solid medium of offer of the invention, the formula of 1L are：Glucose 25-40g；K2HPO4
0.8-1.0g；MgSO4·7H2O 0.15-0.2g；Ferric iron citrate 0.03-0.05g；NaMoO4·2H2O 0.001-
0.05g；Agar powder 8-12g；Add water to 1L；pH 7.0.
It is highly preferred that the nitrogen-free solid medium of the offer of the present invention, the formula of 1L are：Glucose 30g；K2HPO4
1.0g；MgSO4·7H2O 0.2g；Ferric iron citrate 0.05g；NaMoO4·2H2O 0.001g；Agar powder 10g；It adds water to
1L；pH 7.0.
In above-mentioned nitrogen-free solid medium, due to increasing carbon source, energy is provided for the growth of nitrogen-fixing bacteria, convenient for more preferable
Ground culture simultaneously detaches nitrogen-fixing bacteria.Carbon source is less in nitrogen-free solid medium in the prior art, and it is 10g left usually to add glucose
The right side, applicant is had found during nitrogenase activity determination in every liter of culture medium, when glucose reaches 30-50g, nitrogen-fixing bacteria with
Nitrogen is the growth ability highest of only nitrogen source, when more than 80g, then has inhibiting effect to the growth of nitrogen-fixing bacteria, therefore utilize this
Carbon source concentration detaches nitrogen-fixing bacteria, the ability that bacterial strain is grown in nitrogen-free agar is would be even more beneficial to, to improve recall rate.Together
When, the dosage of optimization coagulator agar powder is 1% in separatory nitrogen-free agar, and culture medium can still form solid state,
It does not flow, is conducive to the dispersion and fixation of thalline, while in follow-up bacterial strain screening, since culture medium is relatively soft, being conducive to difference
The picking of single bacterium colony in level.
The present invention provides the nitrogen-free solid mediums in the application for detaching nitrogen-fixing bacteria from oil-polluted soils.
Further, the method that the present invention provides a kind of detaching nitrogen-fixing bacteria from petroleum pollution, includes the following steps：
(1) petroleum pollution is acquired, aqueous suspension, suspension is added to carry out gradient dilution；(2) nitrogen-free solid culture of the present invention is prepared
Base is separately added into the suspension of different dilutions, stationary culture after mixing, if media surface or interior in culture medium melting state
There is a bacterium colony growth in portion, then the nitrogen-fixing bacteria in isolated petroleum pollution.The petroleum pollution be oil-polluted soils or
Petroleum-contaminated water.
In the slave petroleum pollution of the present invention in the method for separation nitrogen-fixing bacteria, culture medium melting state refers in step (2)
The Fusion Strain of culture medium at 50-55 DEG C.
The condition of stationary culture is cultivated 2-4 days for 25-35 DEG C in step (2).
Preferably, the condition of stationary culture is cultivated 2-4 days for 30 DEG C in step (2).
The present invention also provides the purification process using the isolated nitrogen-fixing bacteria of the above method, picking nitrogen-free solid cultures
The bacterium colony grown in base, is purified using GCMY culture mediums, and the formula of the 1L of the GCMY culture mediums is：Glucose 10-30g；
K2HPO40.5-1.0g；MgSO4·7H2O 0.1-0.2g；FeSO4·7H2O 0.02-0.05g；CaCl2·2H2O0.05-
0.1g, NaMoO4·2H2O 0.001-0.76g；Yeast powder 0.5-1g；Caseinhydrolysate 0.5-1g；Brewer's wort 0.5-1g；Agar
Powder 15g；Add water to 1L；pH 7.0.
Preferably, the formula of the 1L of the GCMY culture mediums is：Glucose 10g；K2HPO41.0g；MgSO4·7H2O
0.2g；FeSO4·7H2O 0.05g；CaCl2·2H2O 0.1g；NaMoO4·2H2O 0.001g；Yeast powder 0.5g；Hydrolyze junket egg
White 0.5g；Brewer's wort 0.5g；Agar powder 15g；Add water to 1L；pH 7.0.
The present invention also provides a kind of methods of separation anaerobic nitrogen-fixation bacterium to be chosen using the isolated nitrogen-fixing bacteria of the above method
The bacterium colony grown in nitrogen-free solid medium is taken, is purified using anaerobism pipe rolling tube method, obtains anaerobic nitrogen-fixation bacterium.
Nitrogen-fixing bacteria separative efficiency that the present invention provides the above methods in improving oil-polluted soils or petroleum-contaminated water
Application.
The beneficial effects of the present invention are：The present invention makes up that conventional method can only obtain can be with fixed nitrogen under aerobic condition
Bacterium is easy to omit the deficiency of amphimicrobian or anaerobic bacteria in screening, nitrogen-fixing bacteria is screened using the nitrogen-free agar after improvement, with
Mixed bacterium inocalation method replaces the surface coating in conventional method, optimizes the dosage of coagulator agar powder in separatory nitrogen-free agar
It is 1%, culture medium can still form solid state, not flow, and be conducive to the dispersion and fixation of thalline, while follow-up bacterial strain screening
In, since culture medium is relatively soft, be conducive to the picking of single bacterium colony in different levels.The culture medium of the present invention is in anaerobic or micro- good
The microbe to screen that nitrogen fixation is carried out under the conditions of oxygen provides necessary condition, meets diversity of the nitrogen-fixing bacteria to oxygen demand,
The recall rate for greatly improving the diversity and nitrogen-fixing bacteria of nitrogen-fixing microorganism separation, can not only be from being difficult to be separated to nitrogen-fixing bacteria
Be separated to nitrogen-fixing bacteria in oil-polluted soils or water body, and separated nitrogen-fixing bacteria include aerobic, anaerobism, it is micro- aerobic, facultative
The nitrogen-fixing bacteria such as anaerobism, can be isolated for the nitrogen-fixing bacteria of aerobic or anaerobism spectrum, improves oil-polluted soils or water body
The accuracy rate and separative efficiency of middle separation nitrogen-fixing bacteria, have a good application prospect.
Description of the drawings
Fig. 1 is the growing state that nitrogen-fixing bacteria add different glucose in nitrogen-free agar
Fig. 2 is that petroleum pipeline explosion crude oil leakage contaminated soil in the Qingdao City Huangdao District sinopec Huang Weihe River detaches nitrogen-fixing bacteria 16S
RRNA gene order phylogenetic trees.
Fig. 3 is that Liaoning new people's oil (wax viscous crude) contaminated soil detaches nitrogen-fixing bacteria 16S rRNA gene order systematic growths
Tree.
Fig. 4 is that Liaoning Panjin oil-polluted soils sample 1 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Fig. 5 is that Liaoning Panjin oil-polluted soils sample 2 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Fig. 6 is that Liaoning Panjin oil-polluted soils sample 3 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Fig. 7 is that Liaoning Panjin oil-polluted soils sample 4 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Fig. 8 is that Liaoning Panjin oil-polluted soils sample 5 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Fig. 9 is that Liaoning Panjin oil-polluted soils sample 6 detaches nitrogen-fixing bacteria 16S rRNA gene order phylogenetic trees.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Culture medium used in the present invention is：
(1) nitrogen-free solid medium (nitrogen-free soil improvement)：
Glucose 30g；K2HPO41.0g；MgSO4·7H2O 0.2g；Fe(Ⅲ)-citrate 0.05g；NaMoO4·
2H2O 0.001g；Agar powder 10g；Add water to 1L；PH 7.0 (2) GMCY culture mediums：
Glucose 10g；K2HPO41.0g；MgSO4·7H2O 0.2g；FeSO4·7H2O 0.05g；CaCl2·2H2O
0.1g；NaMoO4·2H2O 0.001g；Yeast powder 0.5g；Caseinhydrolysate 0.5g；Brewer's wort 0.5g；Agar powder 15g.
SQ-204 types gas Chromatographic Determination condition in embodiment：Carrier：N2, amplifier range：10-11A/mV, column temperature：70
DEG C, detector temperature：150℃.
1 nitrogen-fixing bacteria of embodiment add the growing state of different glucose in nitrogen-free agar
(1) culture of thalline：Selection determines the bacterial strain Klebsiella sp PJ35 with nitrogen fixing capacity as experiment material
Material, with LB culture mediums by after strain culturing to logarithmic phase, adjustment initial density is OD600=0.2；
(2) detection of growth ability：By cultured nitrogen-fixing bacteria, it is inoculated into the nitrogen-free training containing different concentration of glucose
It supports in base, concentration of glucose is respectively:10g/L, 30g/L, 50g/L, 80g/L.A thalline OD was surveyed every two hours600Value.Knot
Fruit sees Fig. 1.
Show under 30g/L and 50g/L concentration of glucose, growth ability of the nitrogen-fixing bacteria in nitrogen-free agar is optimal, surpasses
After crossing 80g/L concentration of glucose, there can be inhibition to growth, and 10g/L concentration of glucose, after more than r for 24 hours, growth ability has
Decline.Therefore it is 30g/L to improve concentration of glucose in nitrogen-free agar in the present invention, promotes nitrogen-fixing bacteria in nitrogen-free agar
Growth ability, to improve the detection of isolated strains.
The separation of 2 Qingdao City Huangdao District sinopec Huang Weihe River petroleum pipeline explosion crude oil leakage contaminated soil nitrogen-fixing bacteria of embodiment
(1) sample collection：Acquire the screening that petroleum pipeline explosion oil contaminated soil in the Huang Weihe River, Qingdao carries out nitrogen-fixing bacteria
(2) separation of nitrogen-fixing bacteria：It weighs 2.5g soil samples and the sterile aqueous suspensions of 10ml is added, vortex oscillator shakes 10min, promotees
It is 2-5 minutes static into dispersion, suspension is drawn, is diluted to 10-3, it is kept the temperature for 55 DEG C after nitrogen-free solid medium is melted, it is each dilute
Degree of releasing takes 1ml to be added in 25ml nitrogen-free solid mediums, after mixing well, prepares tablet, 3 weights of each dilution at once
It is multiple.After 30 DEG C are cultivated 2-4 days, can there are bacterium colony growth, i.e. separation to obtain in oil-polluted soils in media surface or inside
Nitrogen-fixing bacteria, picking single bacterium drops down onto GMCY culture mediums after purification, -80 DEG C of preservations.
(3) nitrogen-fixing bacteria Phylogenetic is analyzed：Will after purification, picking colony is suspended in 100 μ l sterile waters, boils
It is centrifuged after 10min, ice bath 5min, takes supernatant as pcr template.The reaction system (50 μ L) of PCR amplification：
PCR reaction conditions：94 DEG C of 4min of pre-degeneration；Be denaturalized 94 DEG C of 30s, anneal 57 DEG C of 30s, extend 72 DEG C of 90s, totally 30
Cycle；72 DEG C of extension 10min.
By pcr amplification product detected through gel electrophoresis, there is specific band in 1.5kb, after recycling, pass through 16S rDNA-
RFLP digestion partings select different digestion partings, expand 16S rDNA again and send Hua Da gene sequencing, utilize MEGA5 softwares
Analysis is based on Tamura-Nei models using Maximum Likelihood (ML) method, and bacterium is calculated using maximum phase likelihood method
The Phylogenetic of strain.
(4) qualitative analysis of nitrogen-fixing bacteria Characteristics of Nitrogen Fixation：Bacterial strain after purification is cultivated in LB liquid medium to logarithm
It after phase, is transferred in 25ml LB liquid mediums according to 1% inoculum concentration, after culture to logarithmic phase, bacterium is collected by centrifugation in 5000rmp
Body suspends again after nitrogen-free agar washing thalline 1-2 times, adjusts OD600=1.0, draw 715 μ l of bacterium solution be injected into containing
It in the anaerobism pipe of 10ml semisolid culturemediums, mixes well, after 30 DEG C of incubator cultures 2 days, is filled with 10% volume acetylene gas
(i.e. 1.4ml), different time points take 100 μ l gases, and in gas spectrum, (SQ-204 type gas-chromatographies, fid detector, 2 meters of packed columns are interior
Fill GDX502) measure gas in ethylene contents (since individual bacterial strain ethylene emanations are less, as qualitative experiment, with ethylene peak
The presence or absence of and ethylene peak area numerical value as primary dcreening operation data).
In the present embodiment, nitrogen-fixing bacteria quantity reaches 103/ g soil samples are chosen by the differentiation of colonial morphology and have purified 22
A bacterium colony, 16S rDNA-RFLP digestion partings obtain 8 different digestion partings, and sequencing result shows that belonging to 5 belongs to 8
Different kinds.
The separation of 3 new people Gao Ning soil contaminated by crude oil nitrogen-fixing bacteria of embodiment
(1) sample collection：Oil-polluted soils are acquired from oil field.
(2) separation of nitrogen-fixing bacteria：It weighs 2.5g soil samples and the sterile aqueous suspensions of 10ml is added, vortex oscillator shakes 10min, promotees
It is 2-5 minutes static into dispersion, suspension is drawn, is diluted to 10-3, it is kept the temperature for 55 DEG C after nitrogen-free solid medium is melted, it is each dilute
Degree of releasing takes 1ml to be added in 25ml nitrogen-free solid mediums, after mixing well, prepares tablet, 3 weights of each dilution at once
It is multiple.After 30 DEG C are cultivated 2-4 days, can there are bacterium colony growth, i.e. separation to obtain in oil-polluted soils in media surface or inside
Nitrogen-fixing bacteria, picking single bacterium drops down onto GMCY culture mediums after purification, -80 DEG C of preservations.
(3) nitrogen-fixing bacteria Phylogenetic is analyzed：Will after purification, picking colony is suspended in 100 μ l sterile waters, boils
It is centrifuged after 10min, ice bath 5min, takes supernatant as pcr template, the reaction system (50 μ L) of PCR amplification：
PCR reaction conditions：94 DEG C of 4min of pre-degeneration；Be denaturalized 94 DEG C of 30s, anneal 57 DEG C of 30s, extend 72 DEG C of 90s, totally 30
Cycle；72 DEG C of extension 10min.
By pcr amplification product detected through gel electrophoresis, there is specific band in 1.5kb, passes through 16S rDNA-RFLP digestions point
Type selects different digestion partings, after expanding 16S rDNA recycling again, send Hua Da gene sequencing, utilizes MEGA5 softwares point
Analysis is based on Tamura-Nei models using Maximum Likelihood (ML) method, and bacterial strain is calculated using maximum phase likelihood method
Phylogenetic, as a result see Fig. 2.
(4) qualitative analysis of nitrogen-fixing bacteria Characteristics of Nitrogen Fixation：Bacterial strain after purification is cultivated in LB liquid medium to logarithm
It after phase, is transferred in 25ml LB liquid mediums according to 1% inoculum concentration, after culture to logarithmic phase, bacterium is collected by centrifugation in 5000rmp
Body suspends again after nitrogen-free agar washing thalline 1-2 times, adjusts OD600=1.0, draw 715 μ l of bacterium solution be injected into containing
It in the anaerobism pipe of 10ml semisolid culturemediums, mixes well, after 30 DEG C of incubator cultures 2 days, is filled with 10% volume acetylene gas
(i.e. 1.4ml), different time points take 100 μ l gases, and in gas spectrum, (SQ-204 type gas-chromatographies, fid detector, 2 meters of packed columns are interior
Fill GDX502) measure gas in ethylene contents (since bacterial strain ethylene emanation out of the ordinary is less, as qualitative experiment, with ethylene peak
The presence or absence of and ethylene peak area numerical value as primary dcreening operation data).
In the present embodiment, nitrogen-fixing bacteria quantity reaches 103/ g soil samples are chosen by the differentiation of colonial morphology and have purified 12
A bacterium colony, 16S rDNA-RFLP digestion partings obtain 7 different digestion partings, and sequencing result shows that belonging to 3 belongs to 7
Different kinds.
The separation of 4 Liaoning Panjin petroleum-contaminated soil nitrogen-fixing bacteria of embodiment
(1) sample collection：6 parts of oil-polluted soils are acquired from oil field, sample is marked according to locality different；
(2) separation of nitrogen-fixing bacteria：It weighs 2.5g soil samples and the sterile aqueous suspensions of 10ml is added, vortex oscillator shakes 10min, promotees
It is 2-5 minutes static into dispersion, suspension is drawn, is diluted to 10-3, it is kept the temperature for 55 DEG C after nitrogen-free solid medium is melted, it is each dilute
Degree of releasing takes 1ml to be added in 25ml nitrogen-free solid mediums, after mixing well, prepares tablet, 3 weights of each dilution at once
It is multiple.After 30 DEG C are cultivated 2-4 days, can there are bacterium colony growth, i.e. separation to obtain in oil-polluted soils in media surface or inside
Nitrogen-fixing bacteria, picking single bacterium drops down onto GMCY culture mediums after purification, -80 DEG C of preservations.
(3) nitrogen-fixing bacteria Phylogenetic is analyzed：Picking colony is suspended in 100 μ l sterile waters, boils 10min, ice bath
It is centrifuged after 5min, takes supernatant as pcr template.PCR reaction systems and reaction condition are referring to Examples 1 and 2.
By pcr amplification product detected through gel electrophoresis, there is specific band in 1.5kb, passes through 16S rDNA-RFLP digestions point
Type selects different digestion partings, after expanding 16S rDNA again, send Hua Da gene sequencing, is analyzed, is made using MEGA5 softwares
With Maximum Likelihood (ML) method, Tamura-Nei models are based on, are using what maximum phase likelihood method calculated bacterial strain
System development status, as a result sees Fig. 3-8.
(4) qualitative analysis of nitrogen-fixing bacteria Characteristics of Nitrogen Fixation：Bacterial strain after purification is cultivated in LB liquid medium to logarithm
It after phase, is transferred in 25ml LB liquid mediums according to 1% inoculum concentration, after culture to logarithmic phase, bacterium is collected by centrifugation in 5000rmp
Body suspends again after nitrogen-free agar washing thalline 1-2 times, adjusts OD600=1.0, draw 715 μ l of bacterium solution be injected into containing
It in the anaerobism pipe of 10ml semisolid culturemediums, mixes well, after 30 DEG C of incubator cultures 2 days, is filled with 10% volume acetylene gas
(i.e. 1.4ml), different time points take 100 μ l gases, and in gas spectrum, (SQ-204 type gas-chromatographies, fid detector, 2 meters of packed columns are interior
Fill GDX502) measure gas in ethylene contents (since bacterial strain ethylene emanation out of the ordinary is less, as qualitative experiment, with ethylene peak
The presence or absence of and ethylene peak area numerical value as primary dcreening operation data).
In the present embodiment, nitrogen-fixing bacteria quantity reaches 103/ g soil samples are chosen by the differentiation of colonial morphology and have purified 126
A bacterium colony, 16S rDNA-RFLP digestions partings and sequencing result show that Panjin oil pollution soil sample 1 has purified 29 bacterium colonies, point
Belong to 9 categories, 16 different kinds；Soil sample 2 has purified 23 bacterium colonies, belongs to 4 categories, 8 different kinds；Win soil sample 3 to purify
11 bacterium colonies belong to 3 categories, 3 different kinds；Soil sample 4 has purified 16 bacterium colonies, belong to 10 belong to 15 it is different
Kind；Soil sample 5 has purified 28 bacterium colonies, belongs to 7 categories, 12 different kinds；Soil sample 6 has purified 19 bacterium colonies, belongs to 5
Belong to 6 different kinds.
The separation of 5 Xinjiang oil reservoir water nitrogen-fixing bacteria of embodiment
(1) sample collection：From oil well acquisition oil reservoir water；
(2) separation of nitrogen-fixing bacteria：It draws 1ml water samples and the dilution of 10ml sterile waters is added, be diluted to 10-3, nitrogen-free solid is trained
55 DEG C of heat preservations after base melts are supported, each dilution takes 1ml to be added in 25ml nitrogen-free agars, after mixing well, prepares at once
Tablet, 3 repetitions of each dilution.After 30 DEG C are cultivated 2-4 days, there can be bacterium colony growth in media surface or inside, that is, detach
The nitrogen-fixing bacteria in oil reservoir water are obtained, picking single bacterium drops down onto GMCY culture mediums after purification, -80 DEG C of preservations.
The present embodiment simultaneously using K.oxytoca nitrogen-frees solid medium as a control group, surface coating and mixed bacterium method into
Row strain isolation purifies bacterial strain using K.oxytoca culture mediums.
K.oxytoca culture mediums：Glucose 50g；NaCl 2g；MgSO4·7H2O 0.2g；Fe(Ⅲ)-citrate
36mg；NaMoO4·2H2O 7.6mg；NH4Ac 1.54g(20mM)；Na2HPO4·12H2O 9.84g；KH2PO41.7g；Agar
Powder 15g；Add water to 1L；pH 7.0.
Note：It is not added with NH4Ac is Koxytoca nitrogen-free solid mediums.
(3) nitrogen-fixing bacteria Phylogenetic is analyzed：By bacterial strain after purification, picking colony is suspended in 100 μ l sterile waters
In, 10min is boiled, is centrifuged after ice bath 5min, takes supernatant as pcr template.PCR reaction systems and reaction condition are referring to embodiment
1 and 2.
By pcr amplification product detected through gel electrophoresis, there is specific band in 1.5kb, after recycling, send Hua Da gene sequencing.
2-5 of the embodiment of the present invention is isolated from 49, area samples obtain 83 plants of nitrogen-fixing bacteria respectively, is belonged to
Paenibacillus bacillus genus, Rhizobium rhizobiums, Pseudomonas pseudomonas, Bacillus buds
Spore Bacillus, Azotobacter growing nitrogen-fixings Pseudomonas, Achromobacter achromobacters, Microbacterium microbacteriums
Category, Sphingobium sphingol Pseudomonas sphingomonas Sphingomonas, caulobacter Caulobacters,
Azospirillum Azospirillums, Arthrobacter Arthrobacters, Pandoraea Pandoras category, Cellulomonas are fine
The plain zygosaccharomyces of dimension, Acidovorax acidophilus Pseudomonas, Streptomyces streptomyces, Devosia wear Butterworth Bordetella,
Klebsiella klebsiellas, Flavobacterium Flavobacteriums, Massilia horse Seeleys subgenus,
Pseudoxanthomonas vacations xanthomonas, Citricoccus citric acids Pseudomonas, the raw Pseudomonas of Salinicola salt,
Paracoccus paracoccus, Aurantimonas oranges zygosaccharomyces, Defluviimonas removing toxic substances zygosaccharomyces 26 are different
Belong to, that is, include aerobic azotobacter, also include the klebsiella of micro- aerobic Azospirillum and amphimicrobian,
Since subsequent purification is purified using rich medium under aerobic condition in the present invention, it does not detach and has obtained
The nitrogen-fixing bacteria of full anaerobism separate the nitrogen-fixing bacteria of anaerobism if step 2) is purified using anaerobism pipe rolling tube method.
Meanwhile in the embodiment of the present invention 5, using Xinjiang region oil well oil reservoir water, same sample is separately cultured using difference
Base and separation method, as a result, it has been found that, using nitrogen-free solid medium provided by the invention and mixed bacterium method, 6 are obtained altogether and belongs to 8 not
Bacterial strain (table 1) of the same race, bacterial number is 103/ ml water samples, and other nitrogen-free agars and surface coating process are used, bacterium
Quantity is 102/ ml water samples, and only obtain a kind of bacterium Salinicola acroporae.It can be seen that for most in the present invention
The nitrogen-fixing bacteria of limits obtained in oil polluted environment have simple and easy to do, the increase multifarious advantage of nitrogen-fixing bacteria.
1 Xinjiang region oil reservoir water nitrogen-fixing bacteria separating resulting of table (nitrogen-free agar mixes bacterium method)
| Bacterial strain name | Bacterial strain kind | 16S rDNA homologys |
| 1--1 | Citricoccus nitrophenolicus | 99.44% |
| 1--3 | Salinicola halophilus | 98.20% |
| 1--4 | Pseudomonas balearica | 99.24% |
| 2--2 | Salinicola salarius | 99.31% |
| 3 | Paracoccus saliphilus | 96.90% |
| 4--1 | Paracoccus saliphilus | 97.26% |
| 4--2 | Paracoccus saliphilus | 97.06% |
| 4--3 | Paracoccus siganidrum | 97.35% |
| 5--1 | Aurantimonas endophytica | 98.91% |
| XJ4 | Defluviimonas alba | 97.18% |
83 plants of nitrogen-fixing bacteria that the present invention detaches embodiment 2-5 select 40 plants of different genera as representative, and it is solid to analyze it
Nitrogen enzymatic activity (although the bacterium having is discretely different, belong to same, thus such bacterial strain just only selected one as generation
Table, although such as D12 and C27 it is discretely different, all with Paenibacillus typhae xj7 homology highests, so just
Select D12 as representative analysis fixed nitrogen enzyme activity), bacterium method is mixed using semisolid, the results are shown in Table 2.
2 oil-polluted soils of table detach the kind homology and nitrogenase activity determination (qualitative experiment) of nitrogen-fixing bacteria
By result it is found that most of bacterial strain that separation obtains has Acetylene Reduction ability, for follow-up screening high-efficiency nitrogen-fixing bacterium
Strain is laid a good foundation.
Although having used general explanation, specific implementation mode and experiment above, the present invention is described in detail,
But some on the basis of the present invention, can be made to it to modify or improve, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of nitrogen-free solid medium, which is characterized in that the formula of its 1L is：Glucose 20-50g；K2HPO40.5-1.0g；
MgSO4·7H2O 0.1-0.2g；Ferric iron citrate 0.02-0.05g；NaMoO4·2H2O 0.001-0.76g；Agar powder
8-15g；Add water to 1L；pH 7.0.
2. nitrogen-free solid medium as described in claim 1, which is characterized in that the formula of its 1L is：Glucose 25-40g；
K2HPO40.8-1.0g；MgSO4·7H2O 0.15-0.2g；Ferric iron citrate 0.03-0.05g；NaMoO4·2H2O
0.001-0.05g；Agar powder 8-12g；Add water to 1L.
3. nitrogen-free solid medium as described in claim 1, which is characterized in that the formula of its 1L is：Glucose 30g；K2HPO4
1.0g；MgSO4·7H2O 0.2g；Ferric iron citrate 0.05g；NaMoO4·2H2O 0.001g；Agar powder 10g；It adds water to
1L。
4. any nitrogen-free solid mediums of claim 1-3 are detached from oil-polluted soils or petroleum-contaminated water
The application of nitrogen-fixing bacteria.
5. a kind of method detaching nitrogen-fixing bacteria from petroleum pollution, which is characterized in that include the following steps：(1) oil is acquired
Pollutant adds aqueous suspension, suspension to carry out gradient dilution；(2) any nitrogen-free solid cultures of claim 1-3 are prepared
Base is separately added into the suspension of different dilutions, stationary culture after mixing, if media surface or interior in culture medium melting state
There is a bacterium colony growth in portion, then the nitrogen-fixing bacteria in isolated petroleum pollution；The petroleum pollution be oil-polluted soils or
Petroleum-contaminated water.
6. method as claimed in claim 4, which is characterized in that culture medium melting state refers to being trained at 50-55 DEG C in step (2)
Support the Fusion Strain of base；The condition of stationary culture is cultivated 2-4 days for 25-35 DEG C in step (2).
7. the purification process of any isolated nitrogen-fixing bacteria of method of claim 5-6, which is characterized in that picking nitrogen-free
The bacterium colony grown in solid medium, is purified using GCMY culture mediums, and the formula of the 1L of the GCMY culture mediums is：Glucose
10-30g；K2HPO40.5-1.0g；MgSO4·7H2O 0.1-0.2g；FeSO4·7H2O 0.02-0.05g；CaCl2·2H2O
0.05-0.1g；NaMoO4·2H2O 0.001-0.76g；Yeast powder 0.5-1g；Caseinhydrolysate 0.5-1g；Brewer's wort 0.5-1g；
Agar powder 15g；Add water to 1L；pH 7.0.
8. purification process as claimed in claim 7, which is characterized in that the formula of the 1L of the GCMY culture mediums is：Glucose
10g；K2HPO41.0g；MgSO4·7H2O 0.2g；FeSO4·7H2O 0.05g；CaCl2·2H2O 0.1g；NaMoO4·2H2O
0.001g；Yeast powder 0.5g；Caseinhydrolysate 0.5g；Brewer's wort 0.5g；Agar powder 15g；Add water to 1L；pH 7.0.
9. a kind of method of separation anaerobic nitrogen-fixation bacterium, which is characterized in that detached using any methods of claim 5-6
To nitrogen-fixing bacteria, the interior bacterium colony grown of picking nitrogen-free solid medium is purified using anaerobism pipe rolling tube method, obtains anaerobic nitrogen-fixation
Bacterium.
10. any methods of claim 5-6 or 9 nitrogen-fixing bacteria point in improving oil-polluted soils or petroleum-contaminated water
Application from efficiency.
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