CN104312962A - Separation and purification method for sulfate reducing bacteria in sewage of oil field - Google Patents
Separation and purification method for sulfate reducing bacteria in sewage of oil field Download PDFInfo
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Abstract
The invention discloses a separation and purification method for sulfate reducing bacteria in sewage of an oil field. The separation and purification method comprises the following specific steps of: (1) preparation of an SRB enriching medium; (2) preparation of an SRB separation medium; (3) enriching culture of the SRB; (4) separated culture of the SRB; and (5) purified culture of the SRB. The separation and purification method disclosed by the invention has the advantages that the speed for separation and purification is high, the process from preparation of the SRB enriching medium and the SRB separation medium to purification of a cultured matter only needs 3-5 days, the success rate of separation and purification is high, the purified cultured matter can be obtained by one-time separation and purification, the operation is simple, the requirement for operators is low, and the targeting performance is strong. Due to only suitability for separation and purification of the SRB, the separation and purification method can be widely applied in separation and purification of the sulfate reducing bacteria in the sewage of the oil field.
Description
One, technical field
The invention belongs to petroleum microorganism technical field, particularly the separation purification method of sulphate reducing bacteria in a kind of oilfield sewage.
Two, background technology
Sulphate reducing bacteria (SRB) is the microorganism utilizing the vitriol in oilfield produced fluid or other oxidation state sulfide to carry out alienation organic substance as electron acceptor(EA), and it is by SO
4 2-be reduced into H
2s, and then the pH value and the corrosion of metal state that change oil field water quality, be the important factor that rate of metal corrosion significantly increases, oilfield tube network brought to many deleterious effects such as burn into blocking.
Abstraction and purification SRB obtains purebred SRB single strain, can provide important theoretical foundation for the evaluation of the colony structure analysis of oil field environment and Corrosion Status and water quality situation.
Existing SRB separation purification method has following three kinds: dilution is shaken tube method, repeat dish sandwish method and Hungate and rolled tube method, but current method also exists following problem: tube method observation is shaken in (1) dilution and picking colony is more difficult, time-consuming, effort efficiency is low; (2) the repeat dish sandwish method scope of application is little, and the success ratio of separation and purification is low, often needs the separation and purification repeated for 3 times ~ 6 times just can obtain relative purification culture; (3) Hungate roll pipe to equipment operator require higher, complicated operation, separation and purification effect is low; (4) three kinds of method separation method times all need more than 10d.
Three, summary of the invention
The object of the invention is the separation purification method that sulphate reducing bacteria in a kind of oilfield sewage is provided for the deficiencies in the prior art.The method has that technique is simple, easy to operate, material is easy to get, with strong points, to operator and equipment requirements is lower, economical and practical, separation and purification speed is fast and success ratio is high advantage.
In oilfield sewage, a separation purification method for sulphate reducing bacteria, is characterized in that, specifically comprises the following steps:
1, the preparation of SRB enrichment medium
Be dissolved in by enrichment medium in distilled water, it is in the anaerobism pipe of 25ml that anaerobism is dispensed into volume, often pipe packing 9ml, 121 DEG C of high pressure steam sterilization 30min.
Wherein, enrichment culture based formulas is Sodium.alpha.-hydroxypropionate mass concentration 0.4% ~ 0.6%, ferrous ammonium sulphate mass concentration 0.05% ~ 0.2%, yeast leaching powder mass concentration 0.05% ~ 0.1%, sodium-chlor mass concentration 0.5% ~ 2.0%, magnesium chloride mass concentration 0.01% ~ 0.03%.
2, the preparation of SRB isolation medium
Be dissolved in by isolation medium in distilled water, be heated to abundant dissolving, then anaerobism is dispensed into volume is in the anaerobism pipe of 25ml, often pipe 15ml, 121 DEG C of high pressure steam sterilization 30min, and the complete rear Temperature fall to 50 of sterilizing DEG C ~ 55 DEG C is for subsequent use.
Wherein, separation and Culture based formulas is peptone mass concentration 0.8% ~ 1.2%, meat leaching powder mass concentration 0.9% ~ 1.1%, Sodium.alpha.-hydroxypropionate mass concentration 0.4% ~ 0.6%, ferrous ammonium sulphate mass concentration 0.05% ~ 0.2%, yeast leaching powder mass concentration 0.05% ~ 0.1%, sodium-chlor mass concentration 0.5% ~ 2.0%, magnesium chloride mass concentration 0.01% ~ 0.03%, agar powder mass concentration 0.8% ~ 1.5%.
3, the enrichment culture of SRB
Get oilfield sewage 1ml and inject the above-mentioned enrichment medium prepared, 30 DEG C ~ 60 DEG C constant temperature culture, to nutrient solution blackening, obtain enrichment culture thing.
4, the separation and Culture of SRB
Get the above-mentioned enrichment culture thing of the 1ml enrichment medium prepared and be diluted to 10 according to 10 times of serial dilutions
-4, by 10
-3with 10
-4the diluent 1ml asepsis injector of gradient gets 0.1ml, slowly be injected in above-mentioned 50 DEG C ~ 55 DEG C isolation mediums for subsequent use respectively, mix, the vertical placement 0.5h ~ 1h that leaves standstill obtains transparent semisolid medium, then be placed in constant incubator to cultivate, culture temperature is 30 DEG C ~ 60 DEG C, is cultured to till there is stain in substratum, obtains isolated culture.
5, the purifying of SRB is cultivated
Get the above-mentioned enrichment medium of 1 pipe, 0.2ml enrichment medium is extracted with 1ml asepsis injector, then the stain of clear-cut in isolated culture is sucked with this syringe, then this pipe enrichment medium is injected, be placed in constant incubator and cultivate, culture temperature 30 DEG C ~ 60 DEG C, cultivates 1d ~ 3d until nutrient solution blackening, obtains pure culture.
Wherein, the complete rear Temperature fall of the sterilizing in described step 2, for guaranteeing lowering temperature uniform, slow rolling anaerobism pipe in temperature-fall period.
Oilfield sewage in described step 3, carries out encapsulation process preservation at 4 DEG C and is no more than 7d after its collection in worksite.
The present invention compared with prior art tool has the following advantages:
(1) simple to operate, lower to operator's requirement;
(2) with strong points, present method is only suitable for carrying out SRB strain separating purifying;
(3) speed of separation and purification is fast, only needs 3d ~ 5d from the pure culture that is prepared into of SRB enrichment and isolation medium;
(4) success ratio of separation and purification is high, and flash liberation purifying both can obtain pure culture.
Four, Figure of description
Accompanying drawing 1 is the isolated culture of block A sulphate reducing bacteria;
Accompanying drawing 2 is block A vitriol pure culture Photomicrograph;
Accompanying drawing 3 is the isolated culture of block B sulphate reducing bacteria;
Accompanying drawing 4 is block B vitriol pure culture Photomicrograph;
Accompanying drawing 5 is block C vitriol pure culture Photomicrograph.
Five, embodiment:
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1
Utilize method of the present invention to carry out separation and purification to the SRB in Shengli Oil Field block A production water, the temperature of block A production water is 30 DEG C, and concrete steps are as follows:
1, the preparation of SRB enrichment medium
Be dissolved in by enrichment medium in distilled water, it is in the anaerobism pipe of 25ml that anaerobism is dispensed into volume, often pipe packing 9ml, 121 DEG C of high pressure steam sterilization 30min.
Wherein, enrichment culture based formulas is Sodium.alpha.-hydroxypropionate mass concentration 0.4%, ferrous ammonium sulphate mass concentration 0.05%, yeast leaching powder mass concentration 0.07%, sodium-chlor mass concentration 1.0%, magnesium chloride mass concentration 0.03%.
2, the preparation of SRB isolation medium
Be dissolved in by isolation medium in distilled water, be heated to abundant dissolving, then anaerobism is dispensed into volume is in the anaerobism pipe of 25ml, often pipe 15ml, 121 DEG C of high pressure steam sterilization 20min, the complete rear Temperature fall to 50 of sterilizing is DEG C for subsequent use, for guaranteeing lowering temperature uniform, slow rolling anaerobism pipe in temperature-fall period.
Wherein, separation and Culture based formulas is peptone mass concentration 0.8%, meat leaching powder mass concentration 0.9%, Sodium.alpha.-hydroxypropionate mass concentration 0.5%, ferrous ammonium sulphate mass concentration 0.1%, yeast leaching powder mass concentration 0.1%, sodium-chlor mass concentration 2.0%, magnesium chloride mass concentration 0.02%, agar powder mass concentration 1.2%.
3, the enrichment culture of SRB
Carry out encapsulation process after collection in worksite block A oilfield sewage sample, and at 4 DEG C preservation 2d, get oilfield sewage 1ml inject the above-mentioned enrichment medium prepared, 30 DEG C of constant temperature culture, to nutrient solution blackening, obtain enrichment culture thing.
4, the separation and Culture of SRB
Get the above-mentioned enrichment culture thing of the 1ml enrichment medium prepared and be diluted to 10 according to 10 times of serial dilutions
-4, by 10
-3with 10
-4the diluent 1ml asepsis injector of gradient gets 0.1ml, slowly be injected in above-mentioned 50 DEG C of isolation mediums for subsequent use respectively, mix, the vertical placement 0.5h that leaves standstill obtains transparent semisolid medium, then be placed in constant incubator to cultivate, culture temperature is 30 DEG C, is cultured to till there is stain in substratum, obtain isolated culture, see accompanying drawing 1.
5, the purifying of SRB is cultivated
Get the above-mentioned enrichment medium of 1 pipe, 0.2ml enrichment medium is extracted with 1ml asepsis injector, then the stain of clear-cut in isolated culture is sucked with this syringe, then this pipe enrichment medium is injected, be placed in constant incubator and cultivate, culture temperature 30 DEG C, cultivate 1d until nutrient solution blackening, obtain pure culture, accompanying drawing 2 is shown in by the Photomicrograph of pure culture.
Embodiment 2
Utilize method of the present invention to carry out separation and purification to the SRB in Shengli Oil Field block B production water, the temperature of block B production water is 48 DEG C, and concrete steps are as follows:
1, the preparation of SRB enrichment medium
Be dissolved in by enrichment medium in distilled water, it is in the anaerobism pipe of 25ml that anaerobism is dispensed into volume, often pipe packing 9ml, 121 DEG C of high pressure steam sterilization 20min.
Wherein, enrichment culture based formulas is Sodium.alpha.-hydroxypropionate mass concentration 0.5%, ferrous ammonium sulphate mass concentration 0.2%, yeast leaching powder mass concentration 0.1%, sodium-chlor mass concentration 0.5%, magnesium chloride mass concentration 0.01%.
2, the preparation of SRB isolation medium
Be dissolved in by isolation medium in distilled water, be heated to abundant dissolving, then anaerobism is dispensed into volume is in the anaerobism pipe of 25ml, often pipe 15ml, 121 DEG C of high pressure steam sterilization 25min, the complete rear Temperature fall to 52 of sterilizing is DEG C for subsequent use, for guaranteeing lowering temperature uniform, slow rolling anaerobism pipe in temperature-fall period.
Wherein, separation and Culture based formulas is peptone mass concentration 1.0%, meat leaching powder mass concentration 1.0%, Sodium.alpha.-hydroxypropionate mass concentration 0.4%, ferrous ammonium sulphate mass concentration 0.2%, yeast leaching powder mass concentration 0.05%, sodium-chlor mass concentration 1.0%, magnesium chloride mass concentration 0.03%, agar powder mass concentration 0.8%.
3, the enrichment culture of SRB
Carry out encapsulation process after collection in worksite block B oilfield sewage, and at 4 DEG C preservation 5d, get oilfield sewage 1ml inject the above-mentioned enrichment medium prepared, 48 DEG C of constant temperature culture, to nutrient solution blackening, obtain enrichment culture thing.
4, the separation and Culture of SRB
Get the above-mentioned enrichment culture thing of the 1ml enrichment medium prepared and be diluted to 10 according to 10 times of serial dilutions
-4, by 10
-3with 10
-4the diluent 1ml asepsis injector of gradient gets 0.1ml, slowly be injected in above-mentioned 52 DEG C of isolation mediums for subsequent use respectively, mix, the vertical placement 0.7h that leaves standstill obtains transparent semisolid medium, then be placed in constant incubator to cultivate, culture temperature is 48 DEG C, is cultured to till there is stain in substratum, obtain isolated culture, see accompanying drawing 3.
5, the purifying of SRB is cultivated
Get the above-mentioned enrichment medium of 1 pipe, 0.2ml enrichment medium is extracted with 1ml asepsis injector, then the stain of clear-cut in isolated culture is sucked with this syringe, then this pipe enrichment medium is injected, be placed in constant incubator and cultivate, culture temperature 48 DEG C, cultivate 2d until nutrient solution blackening, obtain pure culture, accompanying drawing 4 is shown in by the Photomicrograph of pure culture.
Embodiment 3
Utilize method of the present invention to carry out separation and purification to the SRB in Shengli Oil Field block C production water, the temperature of block C production water is 60 DEG C, and concrete steps are as follows:
1, the preparation of SRB enrichment medium
Be dissolved in by enrichment medium in distilled water, it is in the anaerobism pipe of 25ml that anaerobism is dispensed into volume, often pipe packing 9ml, 121 DEG C of high pressure steam sterilization 25min.
Wherein, enrichment culture based formulas is Sodium.alpha.-hydroxypropionate mass concentration 0.6%, ferrous ammonium sulphate mass concentration 0.1%, yeast leaching powder mass concentration 0.05%, sodium-chlor mass concentration 0.08%, magnesium chloride mass concentration 0.02%.
2, the preparation of SRB isolation medium
Be dissolved in by isolation medium in distilled water, be heated to abundant dissolving, then anaerobism is dispensed into volume is in the anaerobism pipe of 25ml, often pipe 15ml, 121 DEG C of high pressure steam sterilization 30min, the complete rear Temperature fall to 55 of sterilizing is DEG C for subsequent use, for guaranteeing lowering temperature uniform, slow rolling anaerobism pipe in temperature-fall period.
Wherein, separation and Culture based formulas is peptone mass concentration 1.2%, meat leaching powder mass concentration 1.1%, Sodium.alpha.-hydroxypropionate mass concentration 0.6%, ferrous ammonium sulphate mass concentration 0.05%, yeast leaching powder mass concentration 0.08%, sodium-chlor mass concentration 0.5%, magnesium chloride mass concentration 0.01%, agar powder mass concentration 1.5%.
3, the enrichment culture of SRB
Carry out encapsulation process, preservation 1d at 4 DEG C after collection in worksite block block C oilfield sewage sample, get oilfield sewage 1ml and inject the above-mentioned enrichment medium prepared, 60 DEG C of constant temperature culture, to nutrient solution blackening, obtain enrichment culture thing.
4, the separation and Culture of SRB
Get the above-mentioned enrichment culture thing of the 1ml enrichment medium prepared and be diluted to 10 according to 10 times of serial dilutions
-4, by 10
-3with 10
-4the diluent 1ml asepsis injector of gradient gets 0.1ml, slowly be injected in above-mentioned 55 DEG C of isolation mediums for subsequent use respectively, mix, the vertical placement 1h that leaves standstill obtains transparent semisolid medium, then be placed in constant incubator to cultivate, culture temperature is 60 DEG C, is cultured to till there is stain in substratum, obtains isolated culture.
5, the purifying of SRB is cultivated
Get the above-mentioned enrichment medium of 1 pipe, 0.2ml enrichment medium is extracted with 1ml asepsis injector, then the stain of clear-cut in isolated culture is sucked with this syringe, then this pipe enrichment medium is injected, be placed in constant incubator and cultivate, culture temperature 60 DEG C, cultivate 3d until nutrient solution blackening, obtain pure culture, accompanying drawing 5 is shown in by the Photomicrograph of pure culture.
Claims (8)
1. the separation purification method of sulphate reducing bacteria in oilfield sewage, it is characterized in that, the method specifically comprises the following steps:
(1) preparation of SRB enrichment medium
Enrichment medium is dissolved in distilled water, it is in the anaerobism bottle of 7.8ml that anaerobism is dispensed into volume, every bottle of packing 7ml, 121 DEG C of high pressure steam sterilizations, wherein, enrichment culture based formulas is Sodium.alpha.-hydroxypropionate mass concentration 0.4% ~ 0.6%, ferrous ammonium sulphate mass concentration 0.05% ~ 0.2%, yeast leaching powder mass concentration 0.05% ~ 0.1%, sodium-chlor mass concentration 0.5% ~ 2.0%, magnesium chloride mass concentration 0.01% ~ 0.03%;
(2) preparation of SRB isolation medium
Isolation medium is dissolved in distilled water, be heated to abundant dissolving, then anaerobism is dispensed into volume is in the anaerobism bottle of 7.8ml, often pipe 7ml, 121 DEG C of high pressure steam sterilizations, the complete rear Temperature fall to 50 of sterilizing DEG C ~ 55 DEG C is for subsequent use, wherein, separation and Culture based formulas is peptone mass concentration 0.8% ~ 1.2%, meat leaching powder mass concentration 0.9% ~ 1.1%, Sodium.alpha.-hydroxypropionate mass concentration 0.4% ~ 0.6%, ferrous ammonium sulphate mass concentration 0.05% ~ 0.2%, yeast leaching powder mass concentration 0.05% ~ 0.1%, sodium-chlor mass concentration 0.5% ~ 2.0%, magnesium chloride mass concentration 0.01% ~ 0.03%, agar powder mass concentration 0.8% ~ 1.5%,
(3) enrichment culture of SRB
Get oilfield sewage 0.2ml and inject the above-mentioned enrichment medium prepared, constant temperature culture, to nutrient solution blackening, obtains enrichment culture thing;
(4) separation and Culture of SRB
Get the above-mentioned enrichment culture thing of the 0.77ml enrichment medium prepared and be diluted to 10 according to 10 times of serial dilutions
-4, by 10
-3with 10
-4the diluent 1ml asepsis injector of gradient gets 0.1ml, slowly be injected in above-mentioned 50 DEG C ~ 55 DEG C isolation mediums for subsequent use respectively, mix, the vertical placement 0.5h ~ 1h that leaves standstill obtains transparent semisolid medium, then be placed in constant incubator to cultivate, be cultured to till there is stain in substratum, obtain isolated culture;
(5) purifying of SRB is cultivated
Get the above-mentioned enrichment medium of 1 pipe, extract 0.2ml enrichment medium with 1ml asepsis injector, then suck the stain of clear-cut in isolated culture with this syringe, then this pipe enrichment medium is injected, be placed in constant incubator and cultivate, until nutrient solution blackening, obtain pure culture.
2. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1, it is characterized in that, 121 DEG C of described high pressure steam sterilizations, sterilization time is 20min ~ 30min.
3. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1 and 2, is characterized in that, the complete rear Temperature fall of described sterilizing, for guaranteeing lowering temperature uniform, and slow rolling anaerobism pipe in temperature-fall period.
4. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1 and 2, is characterized in that, described in get oilfield sewage collection in worksite after carry out encapsulation process preservation at 4 DEG C and be no more than 7d.
5. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1, it is characterized in that, described constant temperature culture, to nutrient solution blackening, obtains enrichment culture thing, and constant temperature culture temperature is 30 DEG C ~ 60 DEG C.
6. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1 or 5, it is characterized in that, the described constant incubator that is placed in is cultivated, and is cultured to till there is stain in substratum, obtain isolated culture, constant temperature culture temperature is 30 DEG C ~ 60 DEG C.
7. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 1 or 5, it is characterized in that, described being placed in constant incubator is cultivated, until nutrient solution blackening, obtains pure culture, and constant temperature culture temperature is 30 DEG C ~ 60 DEG C.
8. the separation purification method of sulphate reducing bacteria in oilfield sewage according to claim 7, it is characterized in that, described being placed in constant incubator is cultivated, until nutrient solution blackening, obtains pure culture, and incubation time is 1d ~ 3d.
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| CN107058108A (en) * | 2017-06-15 | 2017-08-18 | 武汉理工大学 | A kind of method using improved folded ware flat band method screening separation sulfate reducing bacteria |
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| CN109439579A (en) * | 2018-11-14 | 2019-03-08 | 浙江海洋大学 | A kind of petroleum microorganism detection instrument case culture medium group |
| CN110699284A (en) * | 2019-10-22 | 2020-01-17 | 中国石油化工股份有限公司 | Method for separating and purifying anaerobic ammonia oxidation red bacteria in sewage |
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| CN109439579A (en) * | 2018-11-14 | 2019-03-08 | 浙江海洋大学 | A kind of petroleum microorganism detection instrument case culture medium group |
| CN110699284A (en) * | 2019-10-22 | 2020-01-17 | 中国石油化工股份有限公司 | Method for separating and purifying anaerobic ammonia oxidation red bacteria in sewage |
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Application publication date: 20150128 |