CN104560824A - Extraction method and analysis method of microbial thallus in oil reservoir - Google Patents
Extraction method and analysis method of microbial thallus in oil reservoir Download PDFInfo
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- CN104560824A CN104560824A CN201410855816.2A CN201410855816A CN104560824A CN 104560824 A CN104560824 A CN 104560824A CN 201410855816 A CN201410855816 A CN 201410855816A CN 104560824 A CN104560824 A CN 104560824A
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Abstract
The invention provides an extraction method and an analysis method of microbial thallus in an oil reservoir. The extraction method comprises the steps that a microbial extract is prepared, wherein the extract per liter comprises 0.3-0.5g K2HPO4, 0.25-0.4g KH2PO4, 0.1-0.3g MgSO4, 1.5-2.5mg MnSO4, 2.0-3.0mg Fe2(SO4)3, 0.1-0.2g NaCl, 0.3-0.6g sodium citrate, 0.8-1.2g glucose and the balance of water; the extract and an oil reservoir sample are mixed at a volume ratio of 1:(0.5-3); sufficient vibration contact is performed; standing for 10-30min at 50-60 DEG C is performed; a water sample is separated; repeating is performed for many times; the water samples are merged; and the microbial thallus is extracted. The method can completely extract microbial total 16S rDNA from the oil reservoir with very low water content, and can keep the distribution state of the original flora.
Description
Technical field
The invention belongs to oil extraction in oil field technical field, be specifically related to extracting method and the analytical method of microbial cells in a kind of oil reservoir, directly can be abstracted from crude oil microorganism, ensures still can fully extract the microorganism in oil sample and analyze in the oil reservoir low containing water electrode.
Background technology
Microbial carbonates correlation technique, proposed so far from 1976, through the development of 30 years, had become the 4th kind of displacement of reservoir oil after the chemical displacement of reservoir oil, the gas displacement of reservoir oil, thermal drive, had improved the method for oil recovery factor.Microorganism in oil reservoir is greatly mainly with the nutrients that carbon source (honey, alkane) is its Growth and reproduction.As long as these nutriments are sufficient, microorganism just can continuously produce the metabolite being conducive to improving oil recovery.The mechanism of microbial carbonates technology mainly contains following five kinds:
1) microorganism produces organic acid in metabolic process: these acid can dissolve carbonate rock in oil reservoir, improves stratum osmosis;
2) microorganism produces gaseous material in metabolic process: mainly comprise methane, hydrogen carbon dioxide, hydrogen sulfide etc., and these gases are assembled and improved strata pressure in oil reservoir, increase oil-displacement capacity, serve gas mixed phase and drive or the effect of non-phase-mixing driving;
3) microorganism produces biosurfactant and biological emulsifier in metabolic process: main component is aliphatic acid and lipid thereof, reduces viscous crude molecular viscosity, increases crude oil fluidity;
4) microbial reproduction produces biopolymer: the polymer mainly polysaccharide that microorganism produces, and they can optionally block stratum macropore, improves floood conformance volume factor, improves oil displacement efficiency;
5) microorganism produces the solvent dissolving in-place oil: include propyl alcohol, butanols, acetone, formaldehyde etc., in-place oil can dilute by these solvents, improves the oil production rate of unit interval.
In Microbial Enhanced Oil Recovery, mainly by the screening of microorganism, screen and judge the beneficial bacterium in oil reservoir exploitation and harmful bacteria.To beneficial bacterium, according to its functional study and understanding, it is improving the coagulation efficiency and mechanism in recovery ratio; And for harmful bacteria, then formulated by correlative study and take precautions against and control measure.But all these relevant research, the most initial and most essential steps is exactly the extraction of flora in oil reservoir.The difficulty of microorganism and the diversity of microbe species is extracted, by direct relation and the correlative study having influence on some row microorganisms of later stage in different sample.
At present in domestic and international research in microbial carbonates, to the extraction of flora in oil reservoir be all based on in oil sample moisture in microorganism extract.And oil field sample is oil water mixture mostly, a lot of suspension is created after adding polymer displacement of reservoir oil, or when naturally dividing the water yield less in crude oil to be analyzed (such as heavy crude reservoir), capital occurs that sample is difficult to carry out the situation of water-oil separating, and this just limits the extraction of microorganism completely.In addition, in reservoir sample, bacterium is dense lower, and a lot of bacterial classification is all be distributed on oil-water interfaces, and oil sample directly carries out water-oil separating can make a part of bacterial classification lose, thus affects Analysis of Microbial Diversity result.
Document " the multifarious Progress in molecular ecology of oil pool microorganisms " (Li Hui etc., " microbiology circular ", in May, 2008,35th volume the 5th phase, 803-808 page) disclose a kind of collection method of oil pool microorganisms, wherein describe first more weak by polarity organic solvent washing extraction oily substance, cell body is proceeded in aqueous phase as far as possible, most cells body is collected, to collect the microbial cell in sample to greatest extent again with freezing Ultracentrifugation Method.But the method is with organic solvent extraction (be not particularly extraction, but enrichment), the a small amount of bacterium in crude oil can be proposed DNA like this, but organic solvent is nutriment, can grow after bacterium contact, this will destroy distribution situation and the distribution proportion of whole bacterium colony.Utilize the oil pool microorganisms that the method is collected, can be used for analyzing oil reservoir bacterium colony diversity, but be destroyed by late growing stage because oil reservoir primary colony distributes, and the distribution proportion of primary colony cannot be analyzed.
Therefore, if effectively fully extract flora from oil reservoir and keep original distribution of flora, the research and development of promotion oil pool microorganisms is significant.
Summary of the invention
Main purpose of the present invention is, for being difficult in prior art effectively extract microorganism from the crude oil of oil reservoir particularly moisture content less and the disappearance keeping original distribution of flora comprehensively, providing a kind of microorganism that can directly abstract from crude oil, ensure still can carry out more comprehensively fully extracting to the microorganism in oil sample and can keep the method for original distribution of flora in the oil reservoir low containing water electrode.
Another object of the present invention is to the analytical method that microbial cells in a kind of oil reservoir is provided.
For achieving the above object, the invention provides the extracting method of microbial cells in a kind of oil reservoir, the method comprising the steps of:
1) preparing microorganism extract, consisting of of often liter of this extract: K
2hPO
40.3 ~ 0.5g, KH
2pO
40.25 ~ 0.4g, MgSO
40.1 ~ 0.3g, MnSO
41.5 ~ 2.5mg, Fe
2(SO
4)
32.0 ~ 3.0mg, NaCl 0.1 ~ 0.2g, natrium citricum 0.3 ~ 0.6g, glucose 0.8 ~ 1.2g, water surplus;
2) microorganism extract is mixed with the volume ratio of reservoir sample according to 1:0.5 ~ 3, abundant oscillating contact; Extract can well be contacted with microorganism;
3) mixture of mentioned microorganism extract and reservoir sample is placed in 50-60 DEG C and leaves standstill 10 ~ 30 minutes, mixture is divided into oil reservoir and water layer, isolates water sample; In this process, set temperature is applicable to the activity activating and maintain microorganism in most oil reservoir, the activity recovery gradually of the microorganism in oil reservoir, and starts to migrate transfer to (microorganism extract) in the environment of applicable existence simultaneously;
4) oil reservoir is separated with water layer, in isolated oil sample, again adds 0.5 ~ 3 times of microorganism extract mentioned, be placed in 50-60 DEG C and leave standstill 10 ~ 30 minutes, make mixture be divided into oil reservoir and water layer, isolate water sample;
5) above-mentioned extraction step 3 ~ 7 times repeatedly, merges isolated water sample, therefrom extracts microbial cells.
The extracting method of microbial cells in oil reservoir of the present invention, mainly use inorganic extract, and with the addition of certain density natrium citricum and glucose especially, directly can extract microorganism from reservoir sample such as crude oil, extract of the present invention can provide good living environment for microorganism in oil reservoir, microorganism is allowed to sustain life, but can not breed under extraction conditions of the present invention, thus, method of the present invention can fully be extracted the microorganism in reservoir sample to greatest extent, and can original distribution of flora be kept, the microbial cells extracted both can be used for analyzing the diversity of bacterium, also can be used for analyzing Multiple drug resistance and grow ratio.
According to specific embodiment of the invention scheme, in oil reservoir of the present invention microbial cells extracting method in, described reservoir sample can for crude oil (comprising viscous crude etc.), greasy filth or oil mixing with water sample.
According to specific embodiment of the invention scheme, in oil reservoir of the present invention microbial cells extracting method in, the batching of described microorganism extract can with K
2hPO
4, KH
2pO
4, MgSO
4, MnSO
4, Fe
2(SO
4)
3, NaCl, natrium citricum, the reagent such as glucose and water formulated, wherein said MgSO
4, MnSO
4, Fe
2(SO
4)
3can adopt the reagent of anhydrous reagent or the band crystallization water, in the present invention, in aforementioned extract, each constituent content is in pure active principle, is appreciated that its consumption should suitably convert when adopting the reagent of the band crystallization water.In of the present invention one preferred specific embodiment, the composition of described microorganism extract is: in often liter of extract, K
2hPO
40.4g, KH
2pO
40.3 ~ 0.35g, MgSO
40.15 ~ 0.25g, MnSO
41.5 ~ 2.5mg, Fe
2(SO
4)
32.0 ~ 3.0mg, NaCl 0.13g, natrium citricum 0.4 ~ 0.5g, glucose 1.0g, water surplus.
According to specific embodiment of the invention scheme, in oil reservoir of the present invention microbial cells extracting method in, step 2) in the cumulative volume of mixture of microorganism extract and reservoir sample be 100ml ~ 1000ml.Sample or extract are unfavorable for effective extraction very little, are too much unfavorable for operation.Preferably, step 2) in the mixed proportion of microorganism extract and reservoir sample be 80ml ~ 500ml:80ml ~ 200ml.Common lab operates usual microorganism extract and reservoir sample respectively gets 80ml ~ 120ml.
According to specific embodiment of the invention scheme, in oil reservoir of the present invention microbial cells extracting method in, step 3), step 4) in, the mixture of microorganism extract and crude oil is placed in 50 DEG C to leave standstill 18 ~ 25 minutes, 55 DEG C and leave standstill 18 ~ 22 minutes or 60 DEG C and leave standstill 15 ~ 20 minutes, is preferably 50 DEG C ~ 55 DEG C and leaves standstill 18 ~ 22 minutes.More preferably, the mixture of microorganism extract and crude oil controls at 90 ~ 110 minutes at total extraction time of 50-60 DEG C.The control of temperature and time is mainly extracted out for making microorganism in oil reservoir effectively recover motoricity, and is unlikely and occurs breeding; Can ensure that low temperature group can not lose free active, mesophile group can be avoided again to enter growth period and make the bacterium colony separated and primary colony distribution situation occur the situation of deviation.
According to specific embodiment of the invention scheme, according to the method described above, in crude oil sample, most microorganism is all extracted in obtained water sample, then routine operation conventionally therefrom extracts microbial cells, namely, in oil reservoir of the present invention microbial cells extracting method in, step 5) in any possible technique in affiliated field can be utilized from water sample to extract microbial cells, such as, business-like DNA extraction kit is normally utilized to extract microbial cells in the present invention from the water sample merged, associative operation can carry out according to the manual of kit or manufacturer's suggestion.
On the other hand, present invention also offers the analytical method of microbial cells in a kind of oil reservoir, the method comprises:
From reservoir sample, microbial cells is extracted according to preceding method of the present invention;
Extracted microbial cells is analyzed.
Particularly, described analysis can comprise: bacterium colony diversity analysis, or the original microbe colony distribution situation of oil reservoir and distribution proportion analysis etc.
In a specific embodiment of the present invention, utilize method of the present invention successfully from the target reservoir crude oil of low moisture full-bodied Jilin, to be extracted microorganism, and tree phenogram is grown in the distribution depicting oil pool microorganisms.Experimental result shows, utilize method of the present invention, the large microbe species of two in oil reservoir that is present in that bacterium and Archimycetes are known at present is all extracted preferably, and there is a considerable amount of diversity, and through and withdrawal well water sample in bacterium colony diversity contrast, utilize method of the present invention to extract oil sample microorganism, bacterium class comparatively has more a kind in water sample, can illustrate that extraction of the present invention is relatively fully comprehensively.Generally speaking, Advantageous Effects of the present invention comprises:
1) microorganism extract is rich in microorganism and sustains life and active necessary mineral ion, and cost is very low, and preparation easily;
2) equipment simple (water bath containers), operation is succinct convenient;
3) method of microbial growth of allowing is not adopted, the microorganism in oil sample effectively fully can be extracted in the oil reservoir low containing water electrode, and can original distribution of flora be kept, for the microbe research in most oil reservoir provides technical guarantee and support.
Accompanying drawing explanation
Fig. 1 is for extracting set up fungi development tree graph to Jilin large peace oil sample in the embodiment of the present invention 1.
Fig. 2 is that method of the present invention and art methods carry out the result of microorganism enrichment experiment to primary sample.
Detailed description of the invention
In order to there be understanding clearly to technical characteristic of the present invention, object and beneficial effect, existing following detailed description is carried out to technical scheme of the present invention, but can not be interpreted as to of the present invention can the restriction of practical range.
Embodiment 1
Oil reservoir 1, oil reservoir degree of depth 980m; Core intersection 3m; Reservoir permeability 180 × 10
-3μm
2; Reservoir temperature ~ 90 DEG C; Viscosity of crude 267mPa.s; Oil density 0.8980g/cm
3; Oil saturation 72%; The content of wax 22.3%; Containing colloid 14.3%; 27.5 DEG C, average freezing point;
Crude oil denseness is higher, and natural separation profit liquid level is very difficult, arrives natural separation water scarcely ever, cannot extract microorganism from water.
In the present embodiment, adopt and from above-mentioned oil reservoir, extract microorganism with the following method:
1) preparing microorganism extract (through autoclave sterilization after extract preparation, preventing external microbial contamination) is formed according to following table 1:
Table 1 microorganism extract is filled a prescription
| Chemical composition | Content in every premium on currency |
| K 2HPO 4 | 0.4g |
| KH 2PO 4 | 0.31g |
| MgSO 4·7H 2O | 0.25g |
| MnSO 4·4H 2O | 2.5mg |
| Fe(SO 4) 3·H 2O | 2.5mg |
| NaCl | 0.13g |
| Natrium citricum (Sodium citrate) | 0.5g |
| Glucose (Glucose) | 1.0g |
2) extract and crude oil are respectively got 100ml to mix, be placed in the aseptic pure glass flask of 500ml.
3) by step 2) flask that has a mixed liquor is positioned in 50 DEG C of hot baths and leaves standstill 20 minutes, extracted out by the water liquid level of separation and be put in clean tube;
4) 100ml bacterium extract is rejoined, by above-mentioned steps 3) repeat five times, collect 500ml altogether containing bacteria liquid;
5) extract by DNA extraction kit the gene that total flora of extracting falls, and carry out the reaction of bacterial 16 S rDNA PCR chain amplification subsequently;
6) by the case study to Jilin target reservoir, this method is successfully extracted microorganism from low moisture high viscosity oil, and tree phenogram is grown in the distribution depicting oil pool microorganisms.Specifically see Fig. 1.From experimental result, in the oil reservoir of Jilin, proteus is the main same clan of bacterium class; And methane germ is the main same clan of Archimycetes.The large microbe species of two in oil reservoir that is present in that bacterium and Archimycetes are known at present is all extracted preferably, and has a considerable amount of diversity.Through and withdrawal well water sample in bacterium colony diversity contrast, bacterium class comparatively has more a kind in water sample.Fully confirm some conjectures of this area, i.e. some specified microorganisms hobby crude oil, be difficult in water sample find.Therefore, this invention can break through currently available technology bottleneck (can only carry bacterium from water sample), makes those skilled in the art can understand distribution and the formation of microorganism in oil reservoir more accurately.
Separately, inventor's part test under study for action shows, the pure inorganic salts extract (other components of extract formula and table 1 and consumption substantially identical) not adding natrium citricum and glucose is adopted to extract oil sample of originating same with above-described embodiment 1, extracting method and above-described embodiment 1 basically identical, after measured, extract 2827B29, HE565361Arcobacter (arch bar Pseudomonas) of not finding as shown in underscore in Fig. 1 in the microorganism of qualification.
In addition, for verifying the concentration effect of method of the present invention to extracted microorganism, microorganism enrichment Experimental comparison has been targetedly in the present invention, wherein, utilize conventional microbiological and crude oil preparation primary sample, adopt table 1 extract formula and method in the embodiment of the present invention 1 respectively, prior art document D1: " the multifarious Progress in molecular ecology of oil pool microorganisms " (Li Hui etc., biological journal, in May, 2008, 35th volume the 5th phase, 803-808 page) culture medium recorded is (containing yeast, glucose and peptone) and correlation method microorganism enrichment experiment is carried out to primary sample, result is see Fig. 2.
As can be seen from Fig. 2 result, cultivated through 48 hours, total bacterium of D1 is dense has reached extremely enrichment state, original microbe species changes completely, and analyze its condition of culture, temperature and aeration condition are the best enrichment state of aerobic microbiological, do not meet oil reservoir anaerobic condition.Therefore, D1 analyzes from technical standpoint, is conventional aerobic microbiological standard culture scheme.Data analyze (two-tail T test) through statistics standard variance, and P<0.001 (contrasting with primary sample), has reached statistics standard deviation distance.Illustrate that microbial bacteria is dense already far away higher than reset condition, and the factor of condition of culture in addition, microbe species thoroughly changes after cultivating.
And bacterium method of carrying of the present invention, provide and directly from oil sample, extract original microbe colony.Based on inorganic ions, microorganism recovers biochemical activity wherein, but does not grow, and still keeps original distribution state.Through the test of statistics variance, P>0.05 (contrasting with primary sample).Illustrate that in microorganism and primary sample, concentration is basically identical, and appearance useless breeding enrichment state.Use method of the present invention, microorganism original distribution state can be reflected, not only solve the Pinch technology of heavy crude reservoir microbiological analysis, and make microbial profile analysis result closer to oil reservoir reality.
Claims (10)
1. the extracting method of microbial cells in oil reservoir, the method comprises the following steps:
1) preparing microorganism extract, consisting of of often liter of this extract: K
2hPO
40.3 ~ 0.5g, KH
2pO
40.25 ~ 0.4g, MgSO
40.1 ~ 0.3g, MnSO
41.5 ~ 2.5mg, Fe
2(SO
4)
32.0 ~ 3.0mg, NaCl 0.1 ~ 0.2g, natrium citricum 0.3 ~ 0.6g, glucose 0.8 ~ 1.2g, water surplus;
2) microorganism extract is mixed with the volume ratio of reservoir sample according to 1:0.5 ~ 3, abundant oscillating contact;
3) mixture of mentioned microorganism extract and reservoir sample is placed in 50-60 DEG C and leaves standstill 10 ~ 30 minutes, mixture is divided into oil reservoir and water layer, isolates water sample;
4) oil reservoir is separated with water layer, in isolated oil sample, again adds 0.5 ~ 3 times of microorganism extract mentioned, be placed in 50-60 DEG C and leave standstill 10 ~ 30 minutes, make mixture be divided into oil reservoir and water layer, isolate water sample;
5) above-mentioned extraction step 3 ~ 7 times repeatedly, merges isolated water sample, therefrom extracts microbial cells.
2. method according to claim 1, wherein, described reservoir sample is crude oil, greasy filth or oil mixing with water sample.
3. method according to claim 1, wherein, consisting of of described microorganism extract: in often liter of extract, comprising: K
2hPO
40.4g, KH
2pO
40.3 ~ 0.35g, MgSO
40.15 ~ 0.25g, MnSO
41.5 ~ 2.5mg, Fe
2(SO
4)
32.0 ~ 3.0mg, NaCl 0.13g, natrium citricum 0.4 ~ 0.5g, glucose 1.0g, water surplus.
4. method according to claim 1, wherein, step 2) in the cumulative volume of mixture of microorganism extract and reservoir sample be 100ml ~ 1000ml.
5. the method according to claim 1 or 4, wherein, step 2) in the mixed proportion of microorganism extract and reservoir sample be 80ml ~ 500ml:80ml ~ 200ml.
6. method according to claim 1, wherein, step 3), step 4) in, the mixture of microorganism extract and crude oil is placed in 50 DEG C to leave standstill 18 ~ 25 minutes, 55 DEG C and leave standstill 18 ~ 22 minutes or 60 DEG C leave standstill 15 ~ 20 minutes.
7. method according to claim 1, wherein, the mixture of microorganism extract and crude oil controls at 90 ~ 110 minutes at total extraction time of 50-60 DEG C.
8. method according to claim 1, wherein, step 5) in be utilize DNA extraction kit to extract microbial cells from the water sample merged.
9. the analytical method of microbial cells in oil reservoir, the method comprises:
From reservoir sample, microbial cells is extracted according to the method described in any one of claim 1 ~ 8;
Extracted microbial cells is analyzed.
10. method according to claim 9, wherein, described analysis comprises: bacterium colony diversity analysis, or the original microbe colony distribution situation of oil reservoir and distribution proportion analysis.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102425398A (en) * | 2011-11-17 | 2012-04-25 | 中国石油化工股份有限公司 | Produced fluid circulating treatment method for microbial flooding |
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2014
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102425398A (en) * | 2011-11-17 | 2012-04-25 | 中国石油化工股份有限公司 | Produced fluid circulating treatment method for microbial flooding |
Non-Patent Citations (2)
| Title |
|---|
| 张强 等: "胜利油田纯梁采油厂某区块油藏中微生物菌群分析", 《西安石油大学学报（自然科学版）》 * |
| 艾明强 等: "大庆油田油藏采出水的细菌菌落结构", 《应用生态学报》 * |
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