CN104560824B - The extracting method and analysis method of microbial cells in oil reservoir - Google Patents
The extracting method and analysis method of microbial cells in oil reservoir Download PDFInfo
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Abstract
The invention provides the extracting method and analysis method of microbial cells in a kind of oil reservoir.The extracting method includes：Preparing microorganism extract, every liter of extract composition：K2HPO40.3~0.5g, KH2PO40.25~0.4g, MgSO40.1~0.3g, MnSO41.5~2.5mg, Fe2(SO4)30.1~0.2g of 2.0~3.0mg, NaCl, 0.3~0.6g of sodium citrate, 0.8~1.2g of glucose, water surplus；By extract and reservoir sample according to 1:0.5~3 volume ratio mixes, abundant oscillating contact, and 50 60 DEG C stand 10~30 minutes, separate water sample, repeatedly, merges water sample, therefrom extracts microbial cells.The method of the present invention can comprehensively be extracted to microbial total 16S rDNA from aqueous extremely low oil reservoir and be kept original distribution of flora.
Description
Technical field
The invention belongs to oil extraction in oil field technical field, and in particular in a kind of oil reservoir the extracting method of microbial cells and point
Analysis method, can directly abstract from crude oil microorganism, it is ensured that in the aqueous extremely low oil reservoir still can to oil sample in micro- life
Thing fully extract and analyze.
Background technology
Microbial carbonates correlation technique, proposed so far, by the development of 30 years, it has also become after chemistry from 1976
The 4th kind of displacement of reservoir oil, the method for raising oil recovery factor after the displacement of reservoir oil, the gas displacement of reservoir oil, thermal drive.Microorganism in oil reservoir is most
With the nutrients that carbon source (honey, alkane) is its growth and breeding.As long as these nutriments are sufficient, microorganism just can in a steady stream not
The metabolite for being conducive to improving oil recovery is produced disconnectedly.The mechanism of microbial carbonates technology mainly has following five
Kind：
1) microorganism produces organic acid in metabolic process：These acid can dissolve carbonate rock in oil reservoir, improve stratum
Osmosis；
2) microorganism produces gaseous material in metabolic process：Mainly comprising methane, hydrogen carbon dioxide, hydrogen sulfide etc.,
These gases assemble raising strata pressure in oil reservoir, increase oil-displacement capacity, serve the work of the drive of gas mixed phase or non-phase-mixing driving
With；
3) microorganism produces biosurfactant and biological emulsifier in metabolic process：Main component be aliphatic acid and
Its lipid, reduces viscous crude molecular viscosity, increases crude oil fluidity；
4) microbial reproduction produces biopolymer：The polymer that microorganism produces is mainly polysaccharide, and they can be selective
Ground blocks stratum macropore, improves floood conformance volume factor, improves oil displacement efficiency；
5) microorganism produces the solvent of dissolving in-place oil：Include propyl alcohol, butanol, acetone, formaldehyde etc., these solvents can
Diluted with by in-place oil, improve the oil production rate of unit interval.
In Microbial Enhanced Oil Recovery, mainly by the screening of microorganism, having in oil reservoir is exploited is screened and judged
Beneficial bacteria and harmful bacteria.To beneficial bacterium is according to its functional study and understands its effect and mechanism on recovery ratio is improved；And for
Harmful bacteria, then formulated by correlative study and taken precautions against and control measure.But all these related researchs, most initial and most critical
Step is exactly the extraction of flora in oil reservoir.The difficulty of microorganism and the diversity of microbe species are extracted in different samples,
By direct relation and the correlative study for having influence on some row microorganisms of later stage.
At present in research in terms of microbial carbonates both at home and abroad, it is right that the extraction of flora in oil reservoir is all based on
In oil sample it is aqueous in microorganism extracted.And oil field sample is mostly oil water mixture, produced after adding polymer displacement of reservoir oil
Many suspensions, or when dividing water less in crude oil to be analyzed naturally (such as heavy crude reservoir) are given birth to, sample can have all occurred
Product are difficult to carry out the situation of water-oil separating, and this has just been entirely limited the extraction of microorganism.Additionally, bacterium is dense relatively low in reservoir sample,
And many strains are all distributed across on oil-water interfaces, oil sample directly carries out water-oil separating can lose a part of strain, so that
Influence Analysis of Microbial Diversity result.
Document " the multifarious Progress in molecular ecology of oil pool microorganisms " (Li Hui etc.,《Microbiology is circulated a notice of》, 2008
May in year, volume 35 the 5th phase, 803-808 pages) a kind of collection method of oil pool microorganisms is disclosed, wherein describe first using polarity
Weaker organic solvent washing extraction oily substance, makes cell body try one's best and is transferred in water phase, then received with freezing Ultracentrifugation Method
Collect most cells body, with the microbial cell being collected into greatest extent in sample.However, the method is extracted with organic solvent
Take (it is not particularly extraction, but enrichment), so it is that a small amount of bacterium in crude oil can be proposed DNA, but organic solvent is
Nutriment, can grow after bacterium contact, and this will destroy the distribution situation and distribution proportion of whole bacterium colony.It is collected into using the method
Oil pool microorganisms, can be used to analyze oil reservoir bacterium colony diversity, but because the distribution of oil reservoir primary colony is destroyed by late growing stage
, and the distribution proportion of primary colony cannot be analyzed.
Therefore, if effectively fully extracting flora from oil reservoir and keeping original distribution of flora, for promoting oil
Microbe research development is hidden to be significant.
The content of the invention
It is a primary object of the present invention to be directed to the original being difficult in the prior art effectively from oil reservoir particularly moisture content less
Microorganism is extracted in oil comprehensively and the missing of original distribution of flora is kept, there is provided one kind can directly abstract from crude oil micro- life
Thing, ensure can still to be carried out the microorganism in oil sample in aqueous extremely low oil reservoir more comprehensively fully to extract and can protect
The method for holding original distribution of flora.
Analysis method another object of the present invention is to provide microbial cells in a kind of oil reservoir.
To achieve the above object, the invention provides a kind of extracting method of microbial cells in oil reservoir, the method includes
Step：
1) preparing microorganism extract, the composition of every liter of extract is：K2HPO40.3~0.5g, KH2PO40.25~
0.4g, MgSO40.1~0.3g, MnSO41.5~2.5mg, Fe2(SO4)30.1~0.2g of 2.0~3.0mg, NaCl, sodium citrate
0.3~0.6g, 0.8~1.2g of glucose, water surplus；
2) by microorganism extract and reservoir sample according to 1:0.5~3 volume ratio mixing, abundant oscillating contact；Make extraction
Liquid is taken to can be very good to be contacted with microorganism；
3) mixture of mentioned microorganism extract and reservoir sample is placed in 50-60 DEG C and stands 10~30 minutes, mixing
Thing is divided into oil reservoir and water layer, isolates water sample；During being somebody's turn to do, set temperature is adapted in activation and the most oil reservoirs of maintenance
The activity of microorganism, microorganism in oil reservoir gradually activity recovery, and start simultaneously in the environment to suitable existence that (microorganism extracts
Take liquid) migrate transfer；
4) oil reservoir is separated with water layer, adds 0.5~3 times of microorganism for referring to extract again in the oil sample isolated
Liquid, is placed in 50-60 DEG C and stands 10~30 minutes, mixture is divided into oil reservoir and water layer, isolates water sample；
5) above-mentioned extraction step 3~7 times repeatedly, merge it is separated go out water sample, therefrom extract microbial cells.
The extracting method of microbial cells in oil reservoir of the invention, mainly using inorganic extract, and is especially added with
Certain density sodium citrate and glucose, can directly extract microorganism, extraction of the invention from reservoir sample such as crude oil
Liquid can in oil reservoir microorganism good living environment is provided, allow microorganism to sustain life, but in extraction conditions of the invention
Under will not breed so that, the method for the present invention can be extracted fully to the microorganism in reservoir sample to greatest extent, and
Original distribution of flora can be kept, the microbial cells for being extracted both can be used to analyze the diversity of bacterium, can be provided for
Analysis Multiple drug resistance development ratio.
Specific embodiment of the invention, in oil reservoir of the invention in the extracting method of microbial cells, the oil
It can be crude oil (including viscous crude etc.), greasy filth or oil mixing with water sample to hide sample.
Specific embodiment of the invention, it is described micro- in oil reservoir of the invention in the extracting method of microbial cells
The dispensing of biological extract can be with K2HPO4、KH2PO4、MgSO4、MnSO4、Fe2(SO4)3, NaCl, sodium citrate, glucose etc.
Reagent is formulated with water, wherein the MgSO4、MnSO4、Fe2(SO4)3Anhydrous reagent or the reagent with the crystallization water can be used,
Each component content is in terms of pure active principle in foregoing extract in the present invention, it will be understood that when using the reagent with the crystallization water
When, its consumption should suitably be converted.In a preferred embodiment of the invention, the ingredient groups of the microorganism extract
Turn into：In terms of every liter of extract, K2HPO40.4g, KH2PO40.3~0.35g, MgSO40.15~0.25g, MnSO41.5~
2.5mg, Fe2(SO4)32.0~3.0mg, NaCl 0.13g, 0.4~0.5g of sodium citrate, glucose 1.0g, water surplus.
Specific embodiment of the invention, in oil reservoir of the invention in the extracting method of microbial cells, step 2)
Middle microorganism extract is 100ml~1000ml with the cumulative volume of the mixture of reservoir sample.Sample or extract are unfavorable very little
In effectively extraction, excessively it is unfavorable for operation.Preferably, step 2) in microorganism extract be with the mixed proportion of reservoir sample
80ml~500ml：80ml~200ml.Common lab operate usual microorganism extract and reservoir sample respectively take 80ml~
120ml。
Specific embodiment of the invention, in oil reservoir of the invention in the extracting method of microbial cells, step 3),
Step 4) in, microorganism extract is placed in 50 DEG C of standings and stands 18~22 points in 18~25 minutes, 55 DEG C with the mixture of crude oil
Clock or 60 DEG C stand 15~20 minutes, and preferably 50 DEG C~55 DEG C stand 18~22 minutes.It is highly preferred that microorganism extracts
The mixture of liquid and crude oil is controlled at 90~110 minutes in 50-60 DEG C of total extraction time.The control of temperature and time is mainly
It is extracted out to make microorganism in oil reservoir effectively recover motoricity, and is not breeding occur；Both can guarantee that pyschrophile
Group will not lose free activity, and mesophile group is avoided that again into growth period so that the bacterium colony separated and primary colony point
There is the situation of deviation in cloth situation.
Specific embodiment of the invention, according to the method described above, most microorganisms are all extracted in crude oil sample
In water sample obtained by getting, then microbial cells are therefrom extracted according to the routine operation of prior art, i.e. the present invention
Oil reservoir in microbial cells extracting method in, step 5) in carried from water sample using any possible technique of art
Microbial cells are taken, for example, typically extracting micro- from the water sample for merging using commercialized DNA extraction kit in the present invention
Biomass, associative operation can be carried out according to the specification of kit or manufacturer's recommendations.
On the other hand, present invention also offers a kind of analysis method of microbial cells in oil reservoir, the method includes：
Microbial cells are extracted from reservoir sample according to preceding method of the present invention；
Microbial cells to being extracted are analyzed.
Specifically, the analysis can include：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 invention, using the method for the present invention successfully from low aqueous high-viscosity Jilin mesh
Microorganism is extracted in mark Reservoir Crude Oil, and depicts the distribution development tree phenogram of oil pool microorganisms.Test result indicate that, profit
With the method for the present invention, the two big microbe species being present in oil reservoir that bacterium and Archimycetes are currently known all preferably are carried
Take out, and with a considerable amount of diversity, and contrasted by with the bacterium colony diversity in withdrawal well water sample, using this
The method of invention extracts oil sample microorganism, and bacterium class is compared with having more a species in water sample, it may be said that bright of the invention extraction is to compare
Fully comprehensively.Generally speaking, Advantageous Effects of the invention include：
1) microorganism extract sustains life and mineral ion necessary to activity rich in microorganism, and cost is very low,
Prepare easy；
2) equipment is simple (water bath containers), and operation is succinct convenient；
3) method of microbial growth of allowing is not used, you can effectively fully extract oil in aqueous extremely low oil reservoir
Microorganism in sample, and original distribution of flora can be kept, for the microbe research in most oil reservoirs provides skill
Art is ensured and supported.
Brief description of the drawings
Fig. 1 by the embodiment of the present invention 1 it is big to Jilin peace oil sample extract the fungi development tree graph set up.
Fig. 2 is the result that the method for the present invention carries out microorganism enrichment experiment with art methods to primary sample.
Specific embodiment
In order to be more clearly understood to technical characteristic of the invention, purpose and beneficial effect, now to skill of the invention
Art scheme carry out it is described further below, but it is not intended that to it is of the invention can practical range restriction.
Embodiment 1
Oil reservoir 1, oil reservoir depth 980m；Core intersection 3m；Reservoir permeability 180 × 10-3μm2；Reservoir temperature~90 DEG C；It is former
Oil viscosity 267mPa.s；Oil density 0.8980g/cm3；Oil saturation 72%；The content of wax 22.3%；Containing colloid 14.3%；It is flat
Equal 27.5 DEG C of freezing point；
Crude oil denseness is higher, and natural separation profit liquid level is extremely difficult, it is extremely difficult to obtain natural separation water, it is impossible to carried from water
Take microorganism.
In the present embodiment, adopt and extract microorganism from above-mentioned oil reservoir with the following method：
1) (through autoclave sterilization after extract preparation, prevent external micro- according to table 1 below composition preparing microorganism extract
Biological pollution)：
The microorganism of table 1 extracts formula of liquid
| Chemical composition | Content in every liter of water |
| K2HPO4 | 0.4g |
| KH2PO4 | 0.31g |
| MgSO4·7H2O | 0.25g |
| MnSO4·4H2O | 2.5mg |
| Fe(SO4)3·H2O | 2.5mg |
| NaCl | 0.13g |
| Sodium citrate (Sodium citrate) | 0.5g |
| Glucose (Glucose) | 1.0g |
2) extract is respectively taken into 100ml with crude oil to mix, is placed in the aseptic pure glass flasks of 500ml.
3) by step 2) flask that has a mixed liquor is positioned in 50 DEG C of hot baths and stands 20 minutes, the water level that will be separate
Extraction is put in clean tube；
4) 100ml bacterium extracts are rejoined, by above-mentioned steps 3) repeat five times, 500ml is collected altogether contains bacteria liquid；
5) gene that the total flora for extracting falls is extracted with DNA extraction kit, and then carries out bacterial 16 S rDNA PCR
Chain amplification reacts；
6) by the case study to Jilin target reservoir, this method is successfully extracted from low aqueous high viscosity oil
Microorganism, and depict the distribution development tree phenogram of oil pool microorganisms.Referring specifically to Fig. 1.From the point of view of experimental result, Jilin oil
Proteus is the main same clan of bacterium class in Tibetan；And methane germ is the main same clan of Archimycetes.Bacterium is current with Archimycetes
The two big microbe species being present in oil reservoir known all preferably are extracted, and with a considerable amount of diversity.Through
Cross and withdrawal well water sample in bacterium colony diversity contrasted, bacterium class is compared with having more a species in water sample.Fully confirm ability
Some conjectures in domain, i.e. some specified microorganisms hobby crude oil, are difficult to find in water sample.Therefore, this invention can break through at present
Prior art bottleneck (can only carry bacterium) from water sample so that those skilled in the art can more accurately understand micro- life in oil reservoir
The distribution of thing and composition.
Separately, inventor's part test under study for action shows, using the purely inorganic salt for being not added with sodium citrate and glucose
The oil sample in extract (extraction formula of liquid is essentially identical with the other components of table 1 and consumption) pair source same with above-described embodiment 1 enters
Row is extracted, and extracting method is basically identical with above-described embodiment 1, after measured, extracts discovery such as Fig. 1 in the microorganism of identification
2827B29, HE565361Arcobacter (arch Bacillus) shown in middle underscore.
Additionally, the concentration effect of the microorganism for the checking method of the present invention to being extracted, in the present invention targetedly
Microorganism enrichment Experimental comparison has been, wherein, primary sample is prepared using conventional microbiological and crude oil, it is respectively adopted of the invention real
Apply the extraction of table 1 formula of liquid and method, prior art literature D1 in example 1：" the multifarious molecular ecology of oil pool microorganisms study into
The culture medium that exhibition " (Li Hui etc., biological journal, in May, 2008, volume 35 the 5th phase, the 803-808 pages) is recorded (contains yeast, Portugal
Grape sugar and peptone) and correlation method microorganism enrichment experiment is carried out to primary sample, as a result referring to Fig. 2.
Be can be seen that from Fig. 2 results and cultivated by 48 hours, total bacterium of D1 is dense to have reached extreme enrichment state, original micro-
Biological species change completely, and analyze its condition of culture, and temperature and aeration condition are aerobic microbiological and are most preferably enriched with
State, and it is unsatisfactory for oil reservoir anaerobic condition.Therefore, D1 is analyzed from technical standpoint, is conventional aerobic microbiological standard culture side
Case.Data analyze (two-tail T test) through statistics standard variance, P<0.001 (being contrasted with primary sample), has reached system
Meter learn standard deviation away from.Illustrate after culture, microbial bacteria is dense to be significantly larger than reset condition already, in addition the factor of condition of culture,
Microbe species thoroughly change.
And the bacterium method that carries of the invention, there is provided be that original microbe colony is directly extracted from oil sample.With inorganic ions
Based on, microorganism recovers biochemical activity wherein, but does not grow, and still keeps original distribution state.Surveyed through statistics variance
Examination, P>0.05 (being contrasted with primary sample).Illustrate that microorganism is basically identical with concentration in primary sample, and appearance breeding useless is rich
Collection state.Using the method for the present invention, microorganism original distribution state can be reflected, not only solve heavy crude reservoir microorganism point
The Pinch technology of analysis, and cause microbial profile analysis result closer to oil reservoir reality.
Claims (8)
1. in a kind of oil reservoir microbial cells extracting method, the method comprises the following steps：
1) preparing microorganism extract, the composition of every liter of extract is：K2HPO40.3~0.5g, KH2PO40.25~0.4g,
MgSO40.1~0.3g, MnSO41.5~2.5mg, Fe2(SO4)30.1~0.2g of 2.0~3.0mg, NaCl, sodium citrate
0.3~0.6g, 0.8~1.2g of glucose, water surplus；
2) by microorganism extract and reservoir sample according to 1:0.5~3 volume ratio mixing, abundant oscillating contact；
3) mixture of mentioned microorganism extract and reservoir sample is placed in 50-60 DEG C and stands 10~30 minutes, mixture point
It is oil reservoir and water layer, isolates water sample；
4) oil reservoir is separated with water layer, adds 0.5~3 times of microorganism extract of volume again in the oil sample isolated, put
10~30 minutes are stood in 50-60 DEG C, mixture is divided into oil reservoir and water layer, isolate water sample；
5) above-mentioned extraction step 3~7 times repeatedly, merge it is separated go out water sample, therefrom extract microbial cells.
2. method according to claim 1, wherein, the reservoir sample is crude oil, greasy filth or oil mixing with water sample.
3. method according to claim 1, wherein, the composition of the microorganism extract is：In terms of every liter of extract, its
Include：K2HPO40.4g, KH2PO40.3~0.35g, MgSO40.15~0.25g, MnSO41.5~2.5mg, Fe2
(SO4)32.0~3.0mg, NaCl 0.13g, 0.4~0.5g of sodium citrate, glucose 1.0g, water surplus.
4. method according to claim 1, wherein, step 2) in microorganism extract it is total with the mixture of reservoir sample
Volume is 100ml~1000ml.
5. the method according to claim 1 or 4, wherein, step 2) in microorganism extract and reservoir sample mixing ratio
Example is meeting 1:On the premise of 0.5~3, microorganism extract respectively takes 80ml~120ml with reservoir sample.
6. method according to claim 1, wherein, step 3), step 4) in, microorganism extract is mixed with reservoir sample
Compound be placed in 50 DEG C stand 18~25 minutes, 55 DEG C stand 18~22 minutes or 60 DEG C stand 15~20 minutes.
7. method according to claim 1, wherein, the mixture of microorganism extract and reservoir sample is at 50-60 DEG C
Total extraction time control was at 90~110 minutes.
8. method according to claim 1, wherein, step 5) in be using DNA extraction kit from the water sample for merging
Extract microbial cells.
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| 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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| 大庆油田油藏采出水的细菌菌落结构;艾明强 等;《应用生态学报》;20100430;第21卷(第4期);第1015页 * |
| 胜利油田纯梁采油厂某区块油藏中微生物菌群分析;张强 等;《西安石油大学学报（自然科学版）》;20130531;第28卷(第3期);83-87 * |
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