CN107475118A - A kind of coal geomicrobiology flora passes on method for preserving - Google Patents

A kind of coal geomicrobiology flora passes on method for preserving Download PDF

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CN107475118A
CN107475118A CN201710600504.0A CN201710600504A CN107475118A CN 107475118 A CN107475118 A CN 107475118A CN 201710600504 A CN201710600504 A CN 201710600504A CN 107475118 A CN107475118 A CN 107475118A
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culture
coal
days
culture medium
aerogenesis
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陈林勇
任恒星
关嘉栋
郭鑫
王江泽
赵晗
王美林
刘健
赵娜
苗彪
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Shanxi Jincheng Anthracite Mining Group Co Ltd
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Shanxi Jincheng Anthracite Mining Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention belongs to microbial preservation technical field, to overcome existing culture collection process to carry out preservation, the vigor of coal geomicrobiology flora methane phase gas after activation seriously reduces, the problem of can not meeting to test the wilderness demand to bacterium source, a kind of coal geomicrobiology flora passage method for preserving, including sampling inoculation are provided, 30 DEG C incubated, a cycle aerogenesis curve map is drawn according to pressure change, an aerogenesis cycle is divided into three phases：First stage, for second stage from 10 12 days to 38 42 days, the phase III was after 42 days to 8 12 days since culture；To the phase III, methane content is stable for culture, and extraction falls half zymotic fluid, adds equivalent sterilising medium and continue to cultivate, and cultivates to phase III supplemented medium again；Cyclic culture strain successively.The good flora of aerogenesis effect can be preserved, especially faces the strain of the coal bed gas well of low water, without activating the bacterium source directly as other experiments, avoids sampling of frequently going up a hill.

Description

A kind of coal geomicrobiology flora passes on method for preserving
Technical field
The invention belongs to microbial preservation technical field, and in particular to a kind of coal geomicrobiology flora passes on preservation side Method, especially can long term storage at normal temperatures.
Background technology
Carry out the experiment of coal biogasification, strain is one of key factor, because the bacterium source that coal biogasification uses is One mixed bacterial, it is various micro- including at least the quasi-microorganism of hydrolytic bacteria, zymophyte, hydrogen-producing acetogens and methanogen etc. four The physio-biochemical characteristics of biology differ, and result caused by general low temperature preservation is that the bacterium source after activation is reused for coal life Thing gasifies, and there was only hydrogen in caused gas, without methane, is not suitable for the preservation of coal geomicrobiology flora.
Coal biogasification, which is one, to need to carry out the problem of long-term many experiments, it is necessary to carry out some row experiments, every time Field sampling both wasting manpower and material resources are all arrived in experiment, and are difficult to ensure that the Bacterial community of every sub-sampling is all consistent, cause different batches Variable between secondary experiment increases, and experimental result is difficult to comparative analysis.
In addition, with the growth of the exploitation time limit, the aquifer yield of many coal bed gas wells is more and more lower, and some is exhausted, contains There is the coal bed gas well of preferable aerogenesis flora once stopping production water, will result directly in the loss of microbial resources, it is difficult to which sampling is carried out Subsequent experimental.
The content of the invention
The present invention is in order to overcome existing culture collection process to carry out preservation to coal geomicrobiology flora, coal after activation The vigor of matter microorganism species methane phase gas seriously reduces, and can not meet the problem of experiment is to the wilderness demand of bacterium source, there is provided A kind of coal geomicrobiology flora passage method for preserving, this method can not only realize flora preservation function, and can meet The wilderness demand to bacterium source is tested, the strain that the method preserves is used directly for being inoculated with, carries out other experiments without activation.
What the present invention was realized by following technical scheme：A kind of coal geomicrobiology flora passes on method for preserving, including as follows Step：
（1）Sampling：The coal bed gas well of high yield methane gas is selected, collection well head water outlet utilizes nitrogen as bacterium source, the bacterium source of collection Tank is aerated 5-10min at the scene, and laboratory is taken back in then sealing；
（2）Inoculation：2 times of the culture medium of 10kg anthracites lump coal and 40L concentration for former culture medium is added in fermentation tank in advance, is exposed Nitrogen 25-35min, the water 40L as bacterium source that scene is taken back are pumped into fermentation tank, are aerated 30min again；
（3）Culture：Fermentation jar temperature control is incubated for 30 DEG C, daily recording manometer reading, is drawn according to pressure change A cycle aerogenesis curve map, fermentation tank headspace gas measurement methane content is extracted weekly, an aerogenesis cycle is divided into three ranks Section：First stage starts to 8-12 days since culture, and second stage is from cultivating 10-12 days to 38-42 days, phase III 40-42 After it；
(4) feed supplement：Culture to phase III, pressure change tends towards stability, and methane content is stable, and the fermentation in half tank is fallen in extraction Liquid, add the former culture medium of equivalent sterilizing so that culture medium final concentration is consistent with former culture medium concentration in tank, then repeat step （3）Continue to cultivate strain, treat culture to phase III supplemented medium again；Cyclic culture strain successively；
Wherein：The former culture medium prescription is：Ultra-pure water 1L, dusty yeast 2g, K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g。
The zymotic fluid inoculation for gathering any one aerogenesis cycle second stage carries out the experiment of strain.
The present invention is by periodically releasing zymotic fluid-add fresh culture, it is possible to achieve coal geomicrobiology produces first The long term storage of alkane flora.Lump coal is added in fermentation tank, can be provided for the strain to be cultivated and grow necessary carbon source, simultaneously The substrate of methane phase is also served as, strain adhering medium can also be used as；Zymotic fluid-benefit is released using periodicity of the present invention Add fresh culture to carry out culture strain, the good flora of aerogenesis effect can be preserved, especially face the coal bed gas well of low water Strain, the strain of preservation directly can be as the bacterium source of other experiments without activation.The relatively stable of bacterium source can be ensured, it is long-term to protect The bacterium source of the coal bed gas well of no longer water outlet is deposited, avoids sampling of frequently going up a hill.
Brief description of the drawings
Fig. 1 is the flow chart that coal geomicrobiology flora of the present invention passes on method for preserving；Fig. 2 is the first aerogenesis of culture Gas pressure change figure in cycle fermentation tank, it is methane content that percentage is marked in figure；Fig. 3 is the fermentation of the 5th aerogenesis cycle of culture Gas pressure change figure in tank, it is methane content that percentage is marked in figure；Fig. 4 is gas in the tenth aerogenesis cycle fermentation tank of culture Pressure variation, it is methane content that percentage is marked in figure；Fig. 5 is the tenth aerogenesis cycle second stage strain of culture as bacterium source Gas production result figure is tested, it is methane content to mark percentage in figure.
Embodiment
Embodiment 1：A kind of coal geomicrobiology flora passes on method for preserving, comprises the following steps：
（1）Sampling：State key experiment is adopted according to Shanxi Jincheng Anthracite Mining Industry Group Co., Ltd.'s coal and coal bed gas altogether Room previous experiments experience, selects the abundant coal bed gas well SHX121 of methane phase gas, and well head water outlet 40L is gathered using 10L Plastic Drums As bacterium source, every barrel of the bacterium source of collection is utilized respectively nitrogen pot and is aerated 5-min at the scene, and laboratory is taken back in then rapid sealing；
（2）Inoculation：10kg Jin Mei groups temple river mine coal lump, coal cinder a diameter of 5cm, Ran Houjia are added in 200L fermentation tank in advance Enter 2 times of the culture medium that 40L concentration is former culture medium, expose nitrogen 25min, the rapid pumps of water 40L as bacterium source that scene is taken back Enter in fermentation tank, be aerated 30min again, now culture medium concentration is reduced to former culture medium concentration in fermentation tank；
（3）Culture：Fermentation jar temperature control is incubated for 30 DEG C, daily recording manometer reading, is drawn according to pressure change A cycle aerogenesis curve map, fermentation tank headspace gas measurement methane content is extracted weekly, an aerogenesis cycle is divided into three ranks Section：First stage, for second stage to cultivating the 38th day, the phase III was after cultivating 40 days to 8 days since culture；First rank Section methanogen is slowly bred, and gas production slowly increases；Second stage methanogen reaches higher level, and gas production significantly increases It is long；Phase III methanogen suppresses its growth because reaction product can not be excluded effectively, and gas production tends to slow down；
(4) feed supplement：Culture to phase III, pressure change tends towards stability, and methane content is stable, illustrates that methanogen stops production Gas, pressure gauge reading stabilization is shown as, the zymotic fluid in half tank is fallen in extraction, adds the former culture medium of equivalent sterilizing so that tank Interior culture medium final concentration is consistent with former culture medium concentration, then repeat step（3）Continue to cultivate strain, treat culture to the phase III Supplemented medium again；Cyclic culture strain successively；
（5）Repeat step（4）And step（3）, in first aerogenesis cycle, the 5th aerogenesis cycle, the tenth aerogenesis cycle inspection Survey gas pressure and methane content change in fermentation tank.
Wherein：The former culture medium prescription is：Ultra-pure water 1L, dusty yeast 2g, K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g。
Embodiment 2：A kind of coal geomicrobiology flora passes on method for preserving, comprises the following steps：
（1）Sampling：State key experiment is adopted according to Shanxi Jincheng Anthracite Mining Industry Group Co., Ltd.'s coal and coal bed gas altogether Room previous experiments experience, selects the abundant coal bed gas well SHX121 of methane phase gas, and well head water outlet 40L is gathered using 10L Plastic Drums As bacterium source, every barrel of the bacterium source of collection is utilized respectively nitrogen pot and is aerated 8min at the scene, and laboratory is taken back in then rapid sealing；
（2）Inoculation：10kg Jin Mei groups temple river mine coal lump, coal cinder a diameter of 8cm, Ran Houjia are added in 200L fermentation tank in advance Enter 2 times of the culture medium that 40L concentration is former culture medium, expose nitrogen 30min, the rapid pumps of water 40L as bacterium source that scene is taken back Enter in fermentation tank, be aerated 30min again, now culture medium concentration is reduced to former culture medium concentration in fermentation tank；
（3）Culture：Fermentation jar temperature control is incubated for 30 DEG C, daily recording manometer reading, is drawn according to pressure change A cycle aerogenesis curve map, fermentation tank headspace gas measurement methane content is extracted weekly, an aerogenesis cycle is divided into three ranks Section：First stage, to 40 days, the phase III was after 41 days for second stage culture to 10 days since culture；First stage produces Methane backeria is slowly bred, and gas production slowly increases；Second stage methanogen reaches higher level, and gas production significantly increases；The Three stage methanogens suppress its growth because reaction product can not be excluded effectively, and gas production tends to slow down；
(4) feed supplement：Culture to phase III, pressure change tends towards stability, and methane content is stable, illustrates that methanogen stops production Gas, pressure gauge reading stabilization is shown as, the zymotic fluid in half tank is fallen in extraction, adds the former culture medium of equivalent sterilizing so that tank Interior culture medium final concentration is consistent with former culture medium concentration, then repeat step（3）Continue to cultivate strain, treat culture to the phase III Supplemented medium again；Cyclic culture strain successively；
（5）Repeat step（4）And step（3）, in first aerogenesis cycle, the 5th aerogenesis cycle, the tenth aerogenesis cycle inspection Gas pressure and methane content change in fermentation tank are surveyed, as a result sees Fig. 2-4.
Wherein：The former culture medium prescription is：Ultra-pure water 1L, dusty yeast 2g, K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g。
Embodiment 3：A kind of coal geomicrobiology flora passes on method for preserving, comprises the following steps：
（1）Sampling：State key experiment is adopted according to Shanxi Jincheng Anthracite Mining Industry Group Co., Ltd.'s coal and coal bed gas altogether Room previous experiments experience, selects the abundant coal bed gas well SHX121 of methane phase gas, and well head water outlet 40L is gathered using 10L Plastic Drums As bacterium source, every barrel of the bacterium source of collection is utilized respectively nitrogen pot and is aerated 10min at the scene, and laboratory is taken back in then rapid sealing；
（2）Inoculation：Add 10kg Jin Mei groups temple river mine coal lump in 200L fermentation tank in advance, a diameter of 10cm of coal cinder, then 2 times of the culture medium that 40L concentration is former culture medium is added, exposes nitrogen 35min, the water 40L as bacterium source that scene is taken back is rapid It is pumped into fermentation tank, is aerated 30min again, now culture medium concentration is reduced to former culture medium concentration in fermentation tank；
（3）Culture：Fermentation jar temperature control is incubated for 30 DEG C, daily recording manometer reading, is drawn according to pressure change A cycle aerogenesis curve map, fermentation tank headspace gas measurement methane content is extracted weekly, an aerogenesis cycle is divided into three ranks Section：First stage, for second stage to 42 days, the phase III was after 42 days to 12 days since culture；First stage methane phase Bacterium slowly breeds, and gas production slowly increases；Second stage methanogen reaches higher level, and gas production significantly increases；3rd rank Section methanogen suppresses its growth because reaction product can not be excluded effectively, and gas production tends to slow down；
(4) feed supplement：Culture to phase III, pressure change tends towards stability, and methane content is stable, illustrates that methanogen stops production Gas, pressure gauge reading stabilization is shown as, the zymotic fluid in half tank is fallen in extraction, adds the former culture medium of equivalent sterilizing so that tank Interior culture medium final concentration is consistent with former culture medium concentration, then repeat step（3）Continue to cultivate strain, treat culture to the phase III Supplemented medium again；Cyclic culture strain successively；
（5）Repeat step（4）And step（3）, in first aerogenesis cycle, the 5th aerogenesis cycle, the tenth aerogenesis cycle inspection Survey gas pressure and methane content change in fermentation tank.
Wherein：The former culture medium prescription is：Ultra-pure water 1L, dusty yeast 2g, K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g。
Experimental example 1：Bacterium source utilizes experiment：Shanxi Jincheng Anthracite Mining Industry Group Co., Ltd.'s coal is adopted altogether with coal bed gas National Key Laboratory is tested, and takes 10g Yima, henan Province pit coal powder（Below 80 mesh）It is added in 500ml bottle,suctions, then adds Enter 300ml original culture mediums, formula is identical with former culture medium prescription described in embodiment 1, and deoxygenation in anaerobic box, oxygen are sent into after sterilizing The cycle second stage of 10ml the tenth is added after eliminating（20th day）Zymotic fluid as bacterium source, 30 DEG C are incubated, draining gas collection Gas caused by method collection.Methane phase gas curve such as Fig. 5.Illustrate that the passage preserving process can ensure the production first of bacterium source with reference to Fig. 5 The activity of alkane gas.

Claims (2)

1. a kind of coal geomicrobiology flora passes on method for preserving, it is characterised in that：Comprise the following steps：
（1）Sampling：The coal bed gas well of high yield methane gas is selected, collection well head water outlet utilizes nitrogen as bacterium source, the bacterium source of collection Tank is aerated 5-10min at the scene, and laboratory is taken back in then sealing；
（2）Inoculation：2 times of the culture medium of 10kg anthracites lump coal and 40L concentration for former culture medium is added in fermentation tank in advance, is exposed Nitrogen 25-35min, the water 40L as bacterium source that scene is taken back are pumped into fermentation tank, are aerated 30min again；
（3）Culture：Fermentation jar temperature control is incubated for 30 DEG C, daily recording manometer reading, is drawn according to pressure change A cycle aerogenesis curve map, fermentation tank headspace gas measurement methane content is extracted weekly, an aerogenesis cycle is divided into three ranks Section：First stage starts to 8-12 days since culture, and second stage is from cultivating 10-12 days to 38-42 days, phase III 40-42 After it；
(4) feed supplement：Culture to phase III, pressure change tends towards stability, and methane content is stable, and the fermentation in half tank is fallen in extraction Liquid, add the former culture medium of equivalent sterilizing so that culture medium final concentration is consistent with former culture medium concentration in tank, then repeat step （3）Continue to cultivate strain, treat culture to phase III supplemented medium again；Cyclic culture strain successively；
Wherein：The former culture medium prescription is：Ultra-pure water 1L, dusty yeast 2g, K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g。
A kind of 2. coal geomicrobiology flora passage method for preserving according to claim 1, it is characterised in that：Collection is any The zymotic fluid inoculation of one aerogenesis cycle second stage carries out the experiment of strain.
CN201710600504.0A 2017-07-21 2017-07-21 A kind of coal geomicrobiology flora passes on method for preserving Pending CN107475118A (en)

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Publication number Priority date Publication date Assignee Title
US20160108435A1 (en) * 2010-06-16 2016-04-21 Taxon Biosciences Inc Compositions and methods for identifying and modifying carbonaceous compositions
CN104295276B (en) * 2014-07-29 2016-07-06 太原理工大学 A kind of method improving coal bed gas recovery ratio
CN106285581A (en) * 2016-08-23 2017-01-04 中国矿业大学(北京) A kind of method utilizing origin bacterium to improve methane output
CN106405043A (en) * 2016-08-31 2017-02-15 太原理工大学 Device for coal-derived bio-methane and method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160108435A1 (en) * 2010-06-16 2016-04-21 Taxon Biosciences Inc Compositions and methods for identifying and modifying carbonaceous compositions
CN104295276B (en) * 2014-07-29 2016-07-06 太原理工大学 A kind of method improving coal bed gas recovery ratio
CN106285581A (en) * 2016-08-23 2017-01-04 中国矿业大学(北京) A kind of method utilizing origin bacterium to improve methane output
CN106405043A (en) * 2016-08-31 2017-02-15 太原理工大学 Device for coal-derived bio-methane and method thereof

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