CN109161512A - A kind of Enrichment culture method of dependent anaerobic methane oxidation bacteria flora - Google Patents
A kind of Enrichment culture method of dependent anaerobic methane oxidation bacteria flora Download PDFInfo
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Abstract
The present invention provides a kind of Enrichment culture methods of dependent anaerobic methane oxidation bacteria flora, comprising: A) deposit in percolate is mixed with NMS culture solution, aerobic preculture is carried out under conditions of methane, obtains preculture bacterium solution；The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；B the deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement), Anaerobic culturel is carried out under conditions of methane, obtains dependent anaerobic methane oxidation bacteria bacterium solution；The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent includes one or more of sulfate, nitrate and molysite；The temperature of the Anaerobic culturel is 32~40 DEG C.In Enrichment culture method provided by the invention, bacterium solution just has an apparent methane oxidation rate in 10d, and methane oxidation rate when 10d is more than 50%, and cultivation cycle is short.
Description
Technical field
The present invention relates to environmental protection technical field more particularly to a kind of enrichment culture sides of dependent anaerobic methane oxidation bacteria flora
Method.
Background technique
CH in atmosphere4It is to be only second to CO2Important greenhouse gases, be one of an important factor for leading to global warming.Methane
Biological oxidation is significant to the discharge of methane for reducing artificial source, therefore, becomes the research hotspot of methane emission reduction.People are to first
The research starting of alkane aerobic oxidation bacterium is more early, also relatively deep, the existing successfully application in environmental improvement of research.With
Going deep into for research, be originally considered as that the methane of aerobic methane oxidation consumption is proved to really by microorganism in some environment
Anaerobic oxidation process (AOM) consumption.Compared with aerobic methane oxidation, just start to walk to the research of methane anaerobic oxidized, it is right
Its metabolic mechanism, microorganism of participation etc. are not very determining.
Methane anaerobic oxidized (Aerobic oxidation of methane, AOM) is the important channel for reducing methane.
AOM reaction is found in marine sediment earliest, is mainly completed jointly by anaerobic methane oxidation bacterium and sulfate reducing bacteria,
This process consumes the methane that most of seabed generates.It is more research shows that AOM phenomenon is generally existing, marine sediment,
The methane gas such as paddy field, the refuse landfill of cold spring area, mud volcano and gas hydrates reservoir top and mankind's activity
The place of leakage escape has detected that.2006, Raghoebarsing etc. is obtained in laboratory conditions can be using Asia
Nitrate is the methane oxidizing microorganism enrichment culture object of electron acceptor, it was confirmed that methane oxidation can couple going back for nitrite
It is former.The bioprocess is referred to as nitrous acid salt form methane anaerobic oxidized (Denitrifying anaerobic methane
Oxidation, DAMO).The processing nitrogenous effluent that is found to be of DAMO provides new method.It can use the production of sewage plant anaerobic processes
Raw methane is as the electron donor in denitrification process, not only save the cost, but also can reduce the discharge of greenhouse gases.
Key to methane anaerobic oxidized research is the enrichment and culture to related microorganisms.Existing research shows that methane is detested
Oxygen oxidizing bacteria growth condition is harsh, and growth is extremely slow, and the multiplication phase, this led to the richness of related microorganisms up to several weeks to some months
Collection object is difficult to obtain, the serious further scientific research for restricting methane anaerobic oxidized process and engineer application.Existing anaerobic methane
The culture of oxidation bacteria is often inoculated with ocean, Lake Water and deposit, carries out long-term purifying culture, process is very very long, often wants
1 year or more, and the methane oxidation rate for purifying incubation is not also high.
For Ettwig et al. with fresh water deposit come enrichment culture anaerobic methane oxidation bacterium, purification enrichment culture have passed through 16
Month.Martinez-Cruz et al. studies its kind with the continuous 6 months enrichment cultures anaerobic methane oxidation microorganism of lake bed sediment
Group character.It discloses a kind of efficient denitrification anaerobic methane oxidation application No. is 201310193077.0 Chinese patent and detests
The co-culture method of anaerobic ammonium oxidation mixed microorganism system is enriched with 480 days in three stages with SMBR reactor, takes a long time totally.
What is fine to fly in " the methane anaerobic oxidized Bacteria Culture conditioning of nitrous acid salt form and its ecological functions " text, using early period
After some N-DAMO enrichment culture objects continue 90d enrichment culture, the enriched sample for molecular biological analysis is just obtained.Application
Number for 201310455062.7 Chinese patent disclose a kind of efficient denitrification anaerobic methane oxidation with it is anaerobic ammoxidation mixed
The co-culture method of microorganism system is closed, the cultural method needs 3~5 months, the methane oxygen reached in the embodiment provided
About 0.3 μm of ol/mL/d of rate, methane oxidation rate are obviously very low.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of enrichment training of dependent anaerobic methane oxidation bacteria flora
The method of supporting, the Enrichment culture method incubation time of dependent anaerobic methane oxidation bacteria flora provided by the invention is short, and methane oxidation rate
It is high.
The present invention provides a kind of Enrichment culture methods of dependent anaerobic methane oxidation bacteria flora, comprising the following steps:
A the deposit in percolate is mixed with NMS culture solution), aerobic preculture is carried out under conditions of methane, is obtained
To preculture bacterium solution；The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；
B) deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement, under conditions of methane into
Row Anaerobic culturel obtains dependent anaerobic methane oxidation bacteria bacterium solution；
The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent includes sulfate, nitrate
One or more of with molysite；
The temperature of the Anaerobic culturel is 32~40 DEG C.
Preferably, step A) in, the volume ratio of the percolate and NMS culture solution is 1~10:10~15；
The NMS culture solution includes: KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·
7H2O and trace element solution；
The trace element solution includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·
6H2O, KI and CaCl2·2H2O。
Preferably, step A) in, in the system of the aerobic preculture, the volumn concentration of methane is 15~100%；
The aerobic preculture carries out in shaking table, and the revolving speed of the shaking table is 120~150rpm.
Preferably, step B) in, the NMS culture solution includes: KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、
MgSO4·7H2O、FeSO4·7H2O and trace element solution；
The trace element solution includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·
6H2O, KI and CaCl2·2H2O。
Preferably, step B) in,
The amount ratio of sulfate radical and NMS culture solution in the sulfate is 1.15~1.75g:1L；
The amount ratio of nitrate anion and NMS culture solution in the nitrate is 0.90~1.25g:1L；
The amount ratio of iron ion and NMS culture solution in the molysite is 0.25~0.80g:1L.
Preferably, step B) in, the modifying agent includes K2SO4、Na2SO4、KNO3、NaNO3、FeC6H5O7·5H2O and
One or more of EDTA-Fe (III).
Preferably, step B) in, the volume ratio of the NMS culture solution of the preculture bacterium solution and improvement is 3~12:15.
Preferably, step B) in, after the mixing, further includes: carry out deoxygenation to the cultivating system being mixed to get.
Preferably, step B) in, in the system of the Anaerobic culturel, the volumn concentration of methane is 15~100%.
Preferably, step B) in, the Anaerobic culturel carries out under conditions of being protected from light；
The Anaerobic culturel carries out in shaking table, and the revolving speed of the shaking table is 120~150rpm.
The present invention provides a kind of Enrichment culture methods of dependent anaerobic methane oxidation bacteria flora, comprising the following steps: A) it will seep
Deposit in filtrate is mixed with NMS culture solution, and aerobic preculture is carried out under conditions of methane, obtains preculture bacterium solution；
The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；B) by the preculture bacterium solution
In deposit mixed with the NMS culture solution of improvement, Anaerobic culturel is carried out under conditions of methane, obtains methane anaerobic oxidized
Bacterium bacterium solution；The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent include sulfate, nitrate and
One or more of molysite；The temperature of the Anaerobic culturel is 32~40 DEG C.Dependent anaerobic methane oxidation bacteria bacterium provided by the invention
In the Enrichment culture method of group, bacterium solution just has an apparent methane oxidation rate in 10d, and methane oxidation rate when 10d is more than 50%,
Cultivation cycle is short.The Enrichment culture method of dependent anaerobic methane oxidation bacteria flora provided by the invention helps to study in landfill yard environment
Methane anaerobic oxidized mechanism and related microorganisms, it helps methane anaerobic oxidized is in terms of water process Anaerobic Methods in Treating
Using.
Detailed description of the invention
Fig. 1 is the methane oxidation rate in the bacterium solution that Examples 1 to 2 and comparative example 1 obtain.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Every other embodiment obtained by those of ordinary skill in the art without making creative efforts, belongs to this hair
The range of bright protection.
The present invention provides a kind of Enrichment culture methods of dependent anaerobic methane oxidation bacteria flora, comprising the following steps:
A the deposit in percolate is mixed with NMS culture solution), aerobic preculture is carried out under conditions of methane, is obtained
To preculture bacterium solution；The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；
B) deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement, under conditions of methane into
Row Anaerobic culturel obtains dependent anaerobic methane oxidation bacteria bacterium solution；
The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent includes sulfate, nitrate
One or more of with molysite；
The temperature of the Anaerobic culturel is 32~40 DEG C.
The present invention is using percolate as dependent anaerobic methane oxidation bacteria strain source.The percolate is preferably consumer waste filling and embedding
Field percolate.In certain embodiments of the present invention, the percolate is seven sub- mountain landfill percolates.The percolate can be with
Aged percolate, fresh leachate or both mixed percolate are chosen, which is not limited by the present invention.
Deposit in the percolate is preferably obtained by percolate by centrifugation.Parameter of the present invention to the centrifugation
Special limitation is had no with equipment, using the parameter and equipment of centrifugation well known to those skilled in the art.
The volume ratio of the percolate and NMS culture solution is preferably 1~10:10~15.In certain embodiments of the present invention
In, the volume ratio of the percolate and NMS culture solution is 10:15 or 8:10.
The NMS culture solution preferably includes: KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、
FeSO4·7H2O and trace element solution；
The trace element solution preferably includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、
CoCl2·6H2O, KI and CaCl2·2H2O。
The solvent of the NMS culture solution is preferably water.The pH value of the NMS culture solution is preferably 7.0.
In one embodiment of the invention, in the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·
12H2The content of O is 4.34g/L, NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2O's contains
Amount is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；NMS culture solution
Solvent is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Deposit in percolate is mixed with NMS culture solution, aerobic preculture is carried out under conditions of methane, is obtained
Preculture bacterium solution.
The mixed container is preferably serum bottle.
In the system of the aerobic preculture, the volumn concentration of methane is preferably 15~100%.In certain of the invention
In a little embodiments, the volumn concentration of the methane is 33.3% or 40%.
The temperature of the aerobic preculture is 32~40 DEG C.In certain embodiments of the present invention, the aerobic preculture
35 DEG C or 36 DEG C of temperature.The time of the aerobic preculture is 4~7d.In certain embodiments of the present invention, described aerobic
The time of preculture is 5d.
The aerobic preculture carries out preferably in shaking table.The revolving speed of the shaking table is preferably 120~150rpm.In this hair
In bright some embodiments, the revolving speed of the shaking table is 130rpm or 140rpm.
After obtaining preculture bacterium solution, the deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement,
Anaerobic culturel is carried out under conditions of methane, obtains dependent anaerobic methane oxidation bacteria bacterium solution.
Deposit in the preculture bacterium solution is preferably obtained by preculture bacterium solution by centrifugation.The present invention to it is described from
The parameter and equipment of the heart have no special limitation, using the parameter and equipment of centrifugation well known to those skilled in the art.
The preculture bacterium solution and the volume ratio of the NMS culture solution of improvement are preferably 3~12:15.Of the invention certain
In embodiment, the volume ratio of the NMS culture solution of the preculture bacterium solution and improvement is 10:15 or 12:15.
The NMS culture solution of the improvement includes NMS culture solution and modifying agent.
The NMS culture solution preferably includes: KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、
FeSO4·7H2O and trace element solution；
The trace element solution preferably includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、
CoCl2·6H2O, KI and CaCl2·2H2O。
The solvent of the NMS culture solution is preferably water.The pH value of the NMS culture solution is preferably 7.0.
In one embodiment of the invention, in the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·
12H2The content of O is 4.34g/L, NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2O's contains
Amount is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；NMS culture solution
Solvent is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
The modifying agent includes one or more of sulfate, nitrate and molysite；Preferably include K2SO4、Na2SO4、
KNO3、NaNO3、FeC6H5O7·5H2One or more of O and EDTA-Fe (III).
In the present invention, the amount ratio of the sulfate radical in the sulfate and NMS culture solution is preferably 1.15~1.75g:
1L.In certain embodiments of the present invention, the amount ratio of the sulfate radical in the sulfate and NMS culture solution is 1.6g:1L.
The amount ratio of nitrate anion and NMS culture solution in the nitrate is preferably 0.90~1.25g:1L.In the present invention
Some embodiments in, the amount ratio of nitrate anion in the nitrate and NMS culture solution is 0.98g:1L.
The amount ratio of iron ion and NMS culture solution in the molysite is preferably 0.25~0.80g:1L.Of the invention
In some embodiments, the amount ratio of iron ion and NMS culture solution in the molysite is 0.75g:1L and 0.45g:1L.
Deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement, is carried out under conditions of methane
Anaerobic culturel obtains dependent anaerobic methane oxidation bacteria bacterium solution.
The mixed container is preferably serum bottle.
After the mixing, it is also preferable to include: deoxygenation is carried out to the cultivating system being mixed to get.The deoxygenation it is specific
It is preferably a step: purging the cultivating system with nitrogen.
In the system of the Anaerobic culturel, the volumn concentration of methane is preferably 15~100%.Of the invention certain
In embodiment, the volumn concentration of the methane is 33.3%.
The temperature of the Anaerobic culturel is 32~40 DEG C.In certain embodiments of the present invention, the temperature of the Anaerobic culturel
Degree is 35 DEG C or 36 DEG C.The time of the Anaerobic culturel is preferably 4~10d.In certain embodiments of the present invention, the anaerobism
The time of culture is 5d.
The Anaerobic culturel carries out preferably in shaking table.It is particularly preferred as: the serum bottle being placed in shaking table, is detested
Oxygen culture.The revolving speed of the shaking table is preferably 120~150rpm.In certain embodiments of the present invention, the revolving speed of the shaking table
For 130rpm or 140rpm.
The Anaerobic culturel preferably carries out under conditions of being protected from light.
The present invention has no special limitation to the source of above-mentioned used raw material, can be general commercially available.
The present invention provides a kind of Enrichment culture methods of dependent anaerobic methane oxidation bacteria flora, comprising the following steps: A) it will seep
Deposit in filtrate is mixed with NMS culture solution, and aerobic preculture is carried out under conditions of methane, obtains preculture bacterium solution；
The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；B) by the preculture bacterium solution
In deposit mixed with the NMS culture solution of improvement, Anaerobic culturel is carried out under conditions of methane, obtains methane anaerobic oxidized
Bacterium bacterium solution；The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent include sulfate, nitrate and
One or more of molysite；The temperature of the Anaerobic culturel is 32~40 DEG C.Dependent anaerobic methane oxidation bacteria bacterium provided by the invention
In the Enrichment culture method of group, bacterium solution just has an apparent methane oxidation rate in 10d, and methane oxidation rate when 10d is more than 50%,
Cultivation cycle is short.The Enrichment culture method of dependent anaerobic methane oxidation bacteria flora provided by the invention helps to study in landfill yard environment
Methane anaerobic oxidized mechanism and related microorganisms, it helps methane anaerobic oxidized is in terms of water process Anaerobic Methods in Treating
Using.
In order to further illustrate the present invention, with reference to embodiments to a kind of dependent anaerobic methane oxidation bacteria bacterium provided by the invention
The Enrichment culture method of group is described in detail, but cannot be understood as limiting the scope of the present invention.
Raw material used in following embodiment is general commercially available.
Embodiment 1
Seven sub- mountain landfill yard mixing percolate (including aged percolate and fresh leachate) 10mL centrifugations are taken, after centrifugation
Precipitating and 15mLNMS culture solution be packed into 300mL serum bottle, 100mL methane is filled with into serum bottle and is put into shaking table, 35 DEG C,
Aerobic preculture 5d, obtains preculture bacterium solution under the conditions of 130rpm.In the system of the aerobic preculture, the volume basis of methane
Content is 33.3%.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Preculture bacterium solution described in 10mL is centrifuged, the NMS culture solution of obtained deposit and 15mL improvement is packed into 300mL
Serum bottle.
The NMS culture solution of improvement includes NMS culture solution and Na2SO4.The Na2SO4In sulfate radical and the NMS cultivate
The amount ratio of liquid is 1.6g:1L.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Serum bottle seals after being purged with nitrogen, and 100mL methane is filled with into serum bottle and is put into shaking table, in 35 DEG C, 130rpm
Under the conditions of be protected from light Anaerobic culturel 5d, obtain dependent anaerobic methane oxidation bacteria bacterium solution.In the system of the Anaerobic culturel, the volume hundred of methane
Dividing content is 33.3%.
The present invention measures the methane concentration in serum bottle respectively before and after culture, and methane concentration reduction amount and initial methane are dense
The ratio of degree is methane oxidation rate, detects the methane oxidation rate in above-mentioned bacterium solution, as a result as shown in Figure 1.Fig. 1 is embodiment 1
~2 and the obtained bacterium solution of comparative example 1 in methane oxidation rate.Wherein, the first in bacterium solution that the figure a in Fig. 1 obtains for comparative example 1
Alcoxyl rate, the figure b in Fig. 1 are the methane oxidation rate in the bacterium solution that embodiment 1 obtains, and the figure c in Fig. 1 is that embodiment 2 obtains
Bacterium solution in methane oxidation rate.Methane oxygen when can be seen that aerobic preculture 5d, again Anaerobic culturel 5d from the figure b in Fig. 1
Rate is 58.5%, has been more than 50%.
Embodiment 2
Seven sub- mountain landfill yard fresh leachate 10mL centrifugations are taken, by the precipitating and the loading of 15mLNMS culture solution after centrifugation
300mL serum bottle is filled with 100mL methane into serum bottle and is put into shaking table, aerobic preculture 5d under the conditions of 35 DEG C, 130rpm,
Obtain preculture bacterium solution.In the system of the aerobic preculture, the volumn concentration of methane is 33.3%.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Preculture bacterium solution described in 10mL is centrifuged, the NMS culture solution of obtained deposit and 15mL improvement is packed into 300mL
Serum bottle.
The NMS culture solution of improvement includes NMS culture solution and Na2SO4.The Na2SO4In sulfate radical and the NMS cultivate
The amount ratio of liquid is 1.6g:1L.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Serum bottle seals after being purged with nitrogen, and 100mL methane is filled with into serum bottle and is put into shaking table, in 35 DEG C, 130rpm
Under the conditions of be protected from light Anaerobic culturel 5d, obtain dependent anaerobic methane oxidation bacteria bacterium solution.In the system of the Anaerobic culturel, the volume hundred of methane
Dividing content is 33.3%.
The present invention measures the methane concentration in serum bottle respectively before and after culture, and methane concentration reduction amount and initial methane are dense
The ratio of degree is methane oxidation rate, detects the methane oxidation rate in above-mentioned bacterium solution, as a result as shown in Figure 1.Fig. 1 is embodiment 1
~2 and the obtained bacterium solution of comparative example 1 in methane oxidation rate.It can be seen that aerobic preculture 5d, again anaerobism from the figure c in Fig. 1
Methane oxidation rate when cultivating 5d is 50.4%, has been more than 50%.
Comparative example 1
Seven sub- mountain landfill yard mixing percolate (including aged percolate and fresh leachate) 10mL centrifugations are taken, after centrifugation
Precipitating and 15mLNMS culture solution be packed into 300mL serum bottle, 100mL methane is filled with into serum bottle and is put into shaking table, 35 DEG C,
Aerobic preculture 5d, obtains preculture bacterium solution under the conditions of 130rpm.In the system of the aerobic preculture, the volume basis of methane
Content is 33.3%.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Preculture bacterium solution described in 10mL is centrifuged, obtained deposit and 15mLNMS culture solution are packed into 300mL serum
Bottle.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Serum bottle seals after being purged with nitrogen, and 100mL methane is filled with into serum bottle and is put into shaking table, in 35 DEG C, 130rpm
Under the conditions of be protected from light Anaerobic culturel 5d, obtain dependent anaerobic methane oxidation bacteria bacterium solution.In the system of the Anaerobic culturel, the volume hundred of methane
Dividing content is 33.3%.
The present invention measures the methane concentration in serum bottle respectively before and after culture, and methane concentration reduction amount and initial methane are dense
The ratio of degree is methane oxidation rate, detects the methane oxidation rate in above-mentioned bacterium solution, as a result as shown in Figure 1.From the figure a in Fig. 1
Methane oxidation rate when can be seen that aerobic preculture 5d, again Anaerobic culturel 5d is 17.2%, and methane oxidation rate is obviously very poor.
Embodiment 3
Seven sub- mountain landfill yard mixing percolate (including aged percolate and fresh leachate) 8mL centrifugations are taken, after centrifugation
Precipitating and 10mLNMS culture solution be packed into 300mL serum bottle, 120mL methane is filled with into serum bottle and is put into shaking table, 40 DEG C,
Aerobic preculture 5d, obtains preculture bacterium solution under the conditions of 130rpm.In the system of the aerobic preculture, the volume basis of methane
Content is 40%.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Preculture bacterium solution described in 10mL is centrifuged, the NMS culture solution of obtained deposit and 15mL improvement is packed into 300mL
Serum bottle.
The NMS culture solution of improvement includes NMS culture solution and EDTA-Fe (III).Iron ion in the EDTA-Fe (III)
Amount ratio with the NMS culture solution is 0.75g:1L.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Serum bottle seals after being purged with nitrogen, and 100mL methane is filled with into serum bottle and is put into shaking table, in 40 DEG C, 140rpm
Under the conditions of be protected from light Anaerobic culturel 5d, obtain dependent anaerobic methane oxidation bacteria bacterium solution.In the system of the Anaerobic culturel, the volume hundred of methane
Dividing content is 33.3%.
The present invention measures the methane concentration in serum bottle respectively before and after culture, and methane concentration reduction amount and initial methane are dense
The ratio of degree is methane oxidation rate, detects the methane oxidation rate in above-mentioned bacterium solution, the experimental results showed that, aerobic preculture 5d,
Methane oxidation rate when Anaerobic culturel 5d is 61.8% again, has been more than 50%.
Embodiment 4
Seven sub- mountain landfill yard mixing percolate (including aged percolate and fresh leachate) 10mL centrifugations are taken, after centrifugation
Precipitating and 10mLNMS culture solution be packed into 300mL serum bottle, 120mL methane is filled with into serum bottle and is put into shaking table, 36 DEG C,
Aerobic preculture 5d, obtains preculture bacterium solution under the conditions of 140rpm.In the system of the aerobic preculture, the volume basis of methane
Content is 40%.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Preculture bacterium solution described in 12mL is centrifuged, the NMS culture solution of obtained deposit and 15mL improvement is packed into 300mL
Serum bottle.
The NMS culture solution of improvement includes NMS culture solution and FeC6H5O7·5H2O.The FeC6H5O7·5H2Iron in O from
The sub amount ratio with the NMS culture solution is 0.45g:1L.
In the NMS culture solution, KH2PO4Content be 1.06g/L, Na2HPO4·12H2The content of O be 4.34g/L,
NaNO3Content be 1.70g/L, K2SO4Content be 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·
7H2The content of O is 22.4mg/L, the content of trace element solution is 2mL/L；The solvent of NMS culture solution is water, pH value 7.0；
In the trace element solution, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is
0.446mg/L、H3BO3Content be 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2O's contains
Amount is that the content of 0.096mg/L, KI are 0.166mg/L, CaCl2·2H2The content of O is 7.0mg/L.
Serum bottle seals after being purged with nitrogen, and 100mL methane is filled with into serum bottle and is put into shaking table, in 36 DEG C, 120rpm
Under the conditions of be protected from light Anaerobic culturel 5d, obtain dependent anaerobic methane oxidation bacteria bacterium solution.In the system of the Anaerobic culturel, the volume hundred of methane
Dividing content is 33.3%.
The present invention measures the methane concentration in serum bottle respectively before and after culture, and methane concentration reduction amount and initial methane are dense
The ratio of degree is methane oxidation rate, detects the methane oxidation rate in above-mentioned bacterium solution, the experimental results showed that, aerobic preculture 5d,
Methane oxidation rate when Anaerobic culturel 5d is 65.3% again, has been more than 50%.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of Enrichment culture method of dependent anaerobic methane oxidation bacteria flora, comprising the following steps:
A the deposit in percolate is mixed with NMS culture solution), aerobic preculture is carried out under conditions of methane, is obtained pre-
Cultivate bacterium solution；The temperature of the aerobic preculture is 32~40 DEG C；The time of the aerobic preculture is 4~7d；
B the deposit in the preculture bacterium solution is mixed with the NMS culture solution of improvement), is detested under conditions of methane
Oxygen culture obtains dependent anaerobic methane oxidation bacteria bacterium solution；
The NMS culture solution of the improvement includes NMS culture solution and modifying agent；The modifying agent includes sulfate, nitrate and iron
One or more of salt；
The temperature of the Anaerobic culturel is 32~40 DEG C.
2. Enrichment culture method according to claim 1, which is characterized in that step A) in, the percolate and NMS are cultivated
The volume ratio of liquid is 1~10:10~15；
The NMS culture solution includes: KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and
Trace element solution；
The trace element solution includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O、
KI and CaCl2·2H2O。
3. Enrichment culture method according to claim 1, which is characterized in that step A) in, the body of the aerobic preculture
In system, the volumn concentration of methane is 15~100%；
The aerobic preculture carries out in shaking table, and the revolving speed of the shaking table is 120~150rpm.
4. Enrichment culture method according to claim 1, which is characterized in that step B) in, the NMS culture solution includes:
KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and trace element solution；
The trace element solution includes ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O、
KI and CaCl2·2H2O。
5. Enrichment culture method according to claim 1, which is characterized in that step B) in,
The amount ratio of sulfate radical and NMS culture solution in the sulfate is 1.15~1.75g:1L；
The amount ratio of nitrate anion and NMS culture solution in the nitrate is 0.90~1.25g:1L；
The amount ratio of iron ion and NMS culture solution in the molysite is 0.25~0.80g:1L.
6. Enrichment culture method according to claim 1, which is characterized in that step B) in, the modifying agent includes K2SO4、
Na2SO4、KNO3、NaNO3、FeC6H5O7·5H2One or more of O and EDTA-Fe (III).
7. Enrichment culture method according to claim 1, which is characterized in that step B) in, the preculture bacterium solution with change
The volume ratio of good NMS culture solution is 3~12:15.
8. Enrichment culture method according to claim 1, which is characterized in that step B) in, after the mixing, further includes:
Deoxygenation is carried out to the cultivating system being mixed to get.
9. Enrichment culture method according to claim 1, which is characterized in that step B) in, the system of the Anaerobic culturel
In, the volumn concentration of methane is 15~100%.
10. Enrichment culture method according to claim 1, which is characterized in that step B) in, the Anaerobic culturel is being protected from light
Under conditions of carry out；
The Anaerobic culturel carries out in shaking table, and the revolving speed of the shaking table is 120~150rpm.
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