CN107099604A - Method for detecting microorganism in underground water of coal bed methane field - Google Patents
Method for detecting microorganism in underground water of coal bed methane field Download PDFInfo
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- CN107099604A CN107099604A CN201710408122.8A CN201710408122A CN107099604A CN 107099604 A CN107099604 A CN 107099604A CN 201710408122 A CN201710408122 A CN 201710408122A CN 107099604 A CN107099604 A CN 107099604A
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Abstract
The invention belongs to groundwater microbial detection technique field, a kind of method for detecting microorganism in underground water of coal bed methane field is specifically disclosed.This method includes extracting after the genomic DNA in underground water, enters performing PCR amplification to the ancient bacterium and bacterium in underground water respectively using specific primer, carries out Nested PCR Amplification using 16S rDNA v3 areas universal primer, amplified production is separated using DGGE；DGGE glue is digitized using Image J, obtains after the position of band and its information of brightness, data is done with PCA analyses using Matlab, for analytically descending microbial population information.This method obtains groundwater sample microorganism finger-print using 16S rDNA gene libraries, primer PCR amplification and the DGGE technology being combined, biological community structure in underground water can be accurately and efficiently analyzed, is laid a good foundation to study the biological constructive ways of coal bed gas, mechanism.
Description
Technical field
The invention belongs to groundwater microbial detection technique field, and in particular to microorganism in underground water of coal bed methane field detection side
Method.
Background technology
Coal bed gas (coal bed methane, CBM) is commonly called as gas, refers to based on ADSORPTION STATE, supplemented by free state, preservation
It is a kind of from the Unconventional gas for being born from storage by main component of methane in coal seam.The development of coal gas industry, is not only
The inexorable trend that new energy development is utilized, can improve energy resource supply structure, effectively alleviate the insufficient energy of fossil energy
Crisis, while being also the objective requirement of safety of coal mines exploitation and environmental protection, with great economy, society and Significance for Environment.Closely
Nian Lai, in the world each big coal country be sequentially added cbm development using central, the U.S. has realized the extensive business of coal bed gas
Industry melts hair.The underground extraction and surface mining of Coalbed Methane In China utilize also very active, and methane output increases year by year.Coal seam
Gas exploitation all successively obtains important breakthrough on Liulin County, Shanxi Province, Jincheng, Shanxi, Hebei big city, Hancheng Region, Shaanxi, E Nan and other places, particularly
The hot zones Ordos Basin and Qinshui basin of cbm exploration.
Coal bed gas does not only exist Thermal origin, equally exists biogenesis.Biogenetic gas is typically considered to microorganism
In the presence of, produced by degrading coal.The proposition of biogenetic gas serves huge impetus to coal-bed gas exploitation,
It can promote the regeneration of coal bed gas by activating coal seam in-situ microorganism, so that increase coal bed gas reserves, extension coal bed gas field
Exploit the time limit；Microbial degradation coal can increase the permeability in coal seam simultaneously, improve the rate of extraction of coal bed gas；And the micro- life in coal seam
The carbon dioxide conversion of re-injection can be used again by thing for methane so that heat recovery is possibly realized.Thus, grind
Study carefully, detect that microorganism in underground water of coal bed methane field tool has very important significance.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of method for detecting microorganism in underground water of coal bed methane field, the detection
Method specifically includes following steps：
(1) collection coal bed gas field 710~800m of underground or so underground water, is contained in the anaerobism bottle full of nitrogen, will
Anaerobism bottle is positioned over preservation in -22~-25 DEG C of ice chests；
(2) under anaerobic environment, by the above-mentioned underground water filtering collected to 0.22 μm of filter membrane, extract in underground water
Genomic DNA；
(3) performing PCR amplification is entered using the bacterium in primer pair underground water, and entered using 16S rDNA v3 areas universal primer
Row Nested PCR Amplification, is then separated using DGGE to amplified production；
(4) performing PCR amplification is entered using the ancient bacterium in primer pair underground water, and entered using 16S rDNA v3 areas universal primer
Row Nested PCR Amplification, is then separated using DGGE to amplified production；
(5) DGGE glue is digitized using Image J, obtains after the position of band and its information of brightness, use
Matlab does PCA analyses to data, for analytically descending microbial population information.
Wherein, primer described in method for detecting microorganism in underground water of coal bed methane field step (3) is 27F and 1492R.
Wherein, primer described in method for detecting microorganism in underground water of coal bed methane field step (4) is 8F and 1958R.
Wherein, the PCR amplifications described in method for detecting microorganism in underground water of coal bed methane field step (3) and (4), refer to：Enter
Row Nested PCR Amplification and PCR amplifications, respectively obtain the 16S rDNA v3 fragments and pcr amplification product of bacterium and ancient bacterium.
Wherein, the amplification journey of the PCR amplifications described in method for detecting microorganism in underground water of coal bed methane field step (3) and (4)
Sequence is：A, 96 DEG C of 4.5min of pre-degeneration；B, 96 DEG C of 20s, 65 DEG C of 25s, 70 DEG C of 30s, it is rapid to circulate 40 times；C, 70 DEG C of extension 8min.
Wherein, the use DGGE described in method for detecting microorganism in underground water of coal bed methane field step (3) and (4) is produced to amplification
Thing carries out separation and referred to：The polyacrylamide gel (concentration is 6.8~7.8%) that denaturing agent gradient is 45~60% is used for 16S
RDNA v3 fragments and pcr amplification product are analyzed.
The beneficial effects of the invention are as follows：This method is combined using 16S rDNA gene libraries, primer PCR amplification and DGGE
Technology obtain coal bed gas field groundwater sample microorganism finger-print there is provided can not still cultivate being survived in groundwater sample
Microorganism detection method, the structure of community of bacterium in underground water and ancient bacterium can be accurately and efficiently analyzed, for research coal seam
The biological constructive ways of gas, mechanism are laid a good foundation.
Embodiment
The invention provides method for detecting microorganism in underground water of coal bed methane field, the detection method specifically includes following steps：
(1) collection coal bed gas field 710~800m of underground or so underground water, is contained in the anaerobism bottle full of nitrogen, will
Anaerobism bottle is positioned over preservation in -22~-25 DEG C of ice chests；
(2) under anaerobic environment, by the above-mentioned underground water filtering collected to 0.22 μm of filter membrane, extract in underground water
Genomic DNA；
(3) performing PCR amplification is entered using the bacterium in 27F, 1492R primer pair underground water, and led to using 16S rDNA v3 areas
Nested PCR Amplification is carried out with primer, the 16S rDNA v3 fragments and pcr amplification product of bacterium is obtained, then using DGGE to expanding
Volume increase thing is separated；
(4) performing PCR amplification is entered using the ancient bacterium in 8F, 1958R primer pair underground water, and led to using 16S rDNA v3 areas
Nested PCR Amplification is carried out with primer, the 16S rDNA v3 fragments and pcr amplification product of ancient bacterium is obtained, then using DGGE to expanding
Volume increase thing is separated；
(5) DGGE glue is digitized using Image J, obtains after the position of band and its information of brightness, use
Matlab does PCA analyses to data, for analytically descending microbial population information.
Wherein, the amplification journey of the PCR amplifications described in method for detecting microorganism in underground water of coal bed methane field step (3) and (4)
Sequence is：A, 96 DEG C of 4.5min of pre-degeneration；B, 96 DEG C of 20s, 65 DEG C of 25s, 70 DEG C of 30s, it is rapid to circulate 40 times；C, 70 DEG C of extension 8min.
Wherein, the use DGGE described in method for detecting microorganism in underground water of coal bed methane field step (3) and (4) is produced to amplification
Thing carries out separation and referred to：The polyacrylamide gel (concentration is 6.8~7.8%) that denaturing agent gradient is 45~60% is used for 16S
RDNA v3 fragments and pcr amplification product are analyzed.
The present invention is made further explanation and description below in conjunction with specific embodiment, but and is not so limited the present invention
Protection domain.
Embodiment
Method for detecting microorganism in underground water of coal bed methane field, the detection method specifically includes following steps：
(1) by taking the peak colliery of Yichang five as an example, collection coal bed gas field underground 750m or so underground water is contained in and is full of
In the anaerobism bottle of nitrogen, anaerobism bottle is positioned in -25 DEG C of ice chests and preserved；
(2) under anaerobic environment, by the above-mentioned underground water filtering collected to 0.22 μm of filter membrane, extract in underground water
Genomic DNA；
(3) performing PCR amplification is entered using the bacterium in 27F, 1492R primer pair underground water, and led to using 16S rDNA v3 areas
Nested PCR Amplification is carried out with primer, PCR amplification programs are：A, 96 DEG C of 4.5min of pre-degeneration；b、96℃20S、65℃25S、70℃
30s, it is rapid to circulate 40 times；C, 70 DEG C of extension 8min；The 16S rDNA v3 fragments and pcr amplification product of bacterium are obtained, is then used
DGGE is separated to amplified production：Denaturing agent gradient is used for 16S for 55% polyacrylamide gel (concentration is 7.0%)
RDNA v3 fragments and pcr amplification product are analyzed；
(4) performing PCR amplification is entered using the ancient bacterium in 8F, 1958R primer pair underground water, and led to using 16S rDNA v3 areas
Nested PCR Amplification is carried out with primer, PCR amplification programs are：A, 96 DEG C of 4.5min of pre-degeneration；b、96℃20s、65℃25s、70℃
30s, it is rapid to circulate 40 times；C, 70 DEG C of extension 8min；The 16S rDNA v3 fragments and pcr amplification product of ancient bacterium are obtained, is then used
DGGE is separated to amplified production：Denaturing agent gradient is used for 16S for 55% polyacrylamide gel (concentration is 7.0%)
RDNA v3 fragments and pcr amplification product are analyzed；
(5) DGGE glue is digitized using Image J, obtains after the position of band and its information of brightness, use
Matlab does PCA analyses to data, for analytically descending microbial population information.
As a result show, the ancient bacterium of five peak coal bed gas fields only has the ancient bacterium of methane phase, with Methanosaeta and
Based on Methanosarcina；Abundant bacterium is detected simultaneously by, based on Rheinheimera and Hydrogenophaga.
Methanosaeta and Methanosarcina are common two kinds of methanogens in nature, can utilize a variety of simple substrates
Produce methane, such as acetate, formates, methanol, H2.The environment of coal bed gas field underground anaerobism provides for the existence of methanogen
Advantage.Meanwhile, abundant fermentation class bacterium is also detected in bacterium, the macromolecular compound in coal seam can be degraded
For simple small molecule, substrate is provided for the metabolism of methanogen.
Described above is the preferred embodiments of the present invention, it is noted that for those skilled in the art, do not taken off
On the premise of structure of the present invention, various modifications and improvements can be made, these should also be as being considered as protection scope of the present invention,
The practicality of these effects implemented all without the influence present invention and this patent.
Claims (7)
1. method for detecting microorganism in underground water of coal bed methane field, it is characterised in that the detection method specifically includes following steps：
(1) collection coal bed gas field 710~800m of underground or so underground water, is contained in the anaerobism bottle full of nitrogen, by anaerobism
Bottle is positioned over preservation in -22~-25 DEG C of ice chests；
(2) under anaerobic environment, by the above-mentioned underground water filtering collected to 0.22 μm of filter membrane, the base in underground water is extracted
Because of a group DNA；
(3) performing PCR amplification is entered using the bacterium in primer pair underground water, and it is embedding using the universal primer progress of 16S rDNA v3 areas
PCR amplifications are covered, then amplified production is separated using DGGE；
(4) performing PCR amplification is entered using the ancient bacterium in primer pair underground water, and it is embedding using the universal primer progress of 16S rDNA v3 areas
PCR amplifications are covered, then amplified production is separated using DGGE；
(5) DGGE glue is digitized using Image J, obtains after the position of band and its information of brightness, use
Matlab does PCA analyses to data, for analytically descending microbial population information.
2. method for detecting microorganism in underground water of coal bed methane field according to claim 1, it is characterised in that institute in step (3)
Primer is stated for 27F and 1492R.
3. method for detecting microorganism in underground water of coal bed methane field according to claim 1, it is characterised in that institute in step (4)
Primer is stated for 8F and 1958R.
4. method for detecting microorganism in underground water of coal bed methane field according to claim 1, it is characterised in that step (3) and (4)
Described in PCR amplification, refer to：Nested PCR Amplification and PCR amplifications are carried out, the 16S rDNA v3 of bacterium and ancient bacterium are respectively obtained
Fragment and pcr amplification product.
5. method for detecting microorganism in underground water of coal bed methane field according to claim 4, it is characterised in that described PCR expands
The amplification program of increasing is：A, 96 DEG C of 4.5min of pre-degeneration；B, 96 DEG C of 20s, 65 DEG C of 25s, 70 DEG C of 30s, it is rapid to circulate 40 times；c、70℃
Extend 8min.
6. method for detecting microorganism in underground water of coal bed methane field according to claim 1, it is characterised in that step (3) and (4)
Described in use DGGE to amplified production carry out separation refer to：Denaturing agent gradient for 45~60% polyacrylamide gel by with
Analyzed in 16S rDNA v3 fragments and pcr amplification product.
7. method for detecting microorganism in underground water of coal bed methane field according to claim 6, it is characterised in that the polyacrylamide
The concentration of amine glue is 6.8~7.8%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111041118A (en) * | 2019-12-25 | 2020-04-21 | 山西晋城无烟煤矿业集团有限责任公司 | Method for rapidly detecting flora structure in drainage and production water of coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) |
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| CN103981259A (en) * | 2014-05-06 | 2014-08-13 | 山西晋城无烟煤矿业集团有限责任公司 | Analysis method for diversity of microbes and abundance of species in coal seam water |
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Application publication date: 20170829 |