CN109161512B - Enrichment culture method of methane anaerobic oxidizing bacteria flora - Google Patents
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Abstract
The invention provides an enrichment culture method of methane anaerobic oxidation bacteria flora, which comprises the following steps: A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d; B) mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid; the improved NMS culture solution comprises NMS culture solution and an improver; the modifier comprises one or more of sulfate, nitrate and iron salt; the temperature of the anaerobic culture is 32-40 ℃. In the enrichment culture method provided by the invention, the bacterial liquid has an obvious methane oxidation rate within 10 days, the methane oxidation rate at 10 days exceeds 50%, and the culture period is short.
Description
Technical Field
The invention relates to the technical field of environmental protection, in particular to an enrichment culture method of methane anaerobic oxidation bacteria flora.
Background
CH in the atmosphere4Is second to CO only2Is one of the important factors causing global warming. The biological oxidation of methane has important significance for reducing the man-made methane emission, so that the biological oxidation of methane becomes a research hotspot for methane emission reduction. People start to research methane aerobic oxidizing bacteria earlier, and research is relatively deep, so that the methane aerobic oxidizing bacteria have been successfully applied to environmental management. With the progress of research, methane originally considered to be consumed by aerobic methane oxidation in some environments has proven to be actually consumed by microbial anaerobic oxidation processes (AOMs). Compared with aerobic methane oxidation, the research on anaerobic methane oxidation is just started, and the metabolic mechanism, the participating microorganisms and the like of the anaerobic methane oxidation are not completely determined.
Anaerobic Oxidation of Methane (AOM) is an important route to reduce methane. The AOM reaction was first discovered in marine sediments and was mainly accomplished by anaerobic methane-oxidizing bacteria in combination with sulfate-reducing bacteria, a process that consumes most of the seabed-produced methane. More studies have shown that the AOM phenomenon is ubiquitous and has been detected in marine sediments, cold spring areas, above mud volcanic and natural gas hydrate reservoirs, and in locations where methane gas leaks and escapes, such as in human-moving rice fields, landfills, and the like. In 2006, Raghoebarsing et al obtained enriched cultures of methane-oxidizing microorganisms that could utilize nitrite as an electron acceptor under laboratory conditions, confirming that methane oxidation could couple with the reduction of nitrite. This biological process is known as nitrite-type anaerobic methane oxidation (DAMO). The discovery of DAMO provides a new approach to the treatment of nitrogen-containing wastewater. Methane generated in the anaerobic process of the sewage plant can be used as an electron donor in the denitrification process, so that the cost is saved, and the emission of greenhouse gases can be reduced.
The key to the research of anaerobic oxidation of methane is the enrichment and culture of relevant microorganisms. The existing research shows that the growth conditions of the methane anaerobic oxidation bacteria are harsh, the methane anaerobic oxidation bacteria grow very slowly, and the multiplication period is as long as several weeks to several months, so that the enrichment of related microorganisms is difficult to obtain, and further scientific research and engineering application of the methane anaerobic oxidation process are seriously restricted. The existing anaerobic methane-oxidizing bacteria are cultured, sea, lake water and sediments are inoculated frequently, and long-term purification culture is carried out, the process is very long, which is usually more than 1 year, and the methane oxidation rate in the purification culture process is not high.
Ettwig et al used fresh water sediments to culture anaerobic methane-oxidizing bacteria in an enrichment manner, and the purification and enrichment culture took 16 months. Martinez-Cruz et al enriched cultured anaerobic methane-oxidizing microorganisms with lake bottom sediments for 6 months continuously, and studied their population characteristics. The Chinese patent with the application number of 201310193077.0 discloses a high-efficiency co-culture method of a denitrification anaerobic methane oxidation and anaerobic ammonia oxidation mixed microorganism system, wherein 480 days are enriched in three stages by using an SMBR reactor, and the time consumption is long. In the 'nitrite type methane anaerobic oxidation bacteria culture conditioning and ecological function' literature, the enrichment culture is continued for 90 days by utilizing the prior N-DAMO enrichment culture, and then the enrichment sample for molecular biological analysis is obtained. The Chinese patent with the application number of 201310455062.7 discloses a high-efficiency co-culture method of a denitrification anaerobic methane oxidation and anaerobic ammonia oxidation mixed microorganism system, the culture method needs 3-5 months, the methane oxidation rate achieved in the provided embodiment is about 0.3 mu mol/mL/d, and the methane oxidation rate is obviously low.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an enrichment culture method for methanogen anaerobic oxidation bacteria flora, which has a short culture time and a high methane oxidation rate.
The invention provides an enrichment culture method of methane anaerobic oxidation bacteria flora, which comprises the following steps:
A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d;
B) mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid;
the improved NMS culture solution comprises NMS culture solution and an improver; the modifier comprises one or more of sulfate, nitrate and iron salt;
the temperature of the anaerobic culture is 32-40 ℃.
Preferably, in the step a), the volume ratio of the leachate to the NMS culture solution is 1-10: 10-15;
the NMS culture solution comprises: KH (Perkin Elmer)2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and a trace element solution;
the microelement solution comprises ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O, KI and CaCl2·2H2O。
Preferably, in the step A), the volume percentage of methane in the aerobic pre-culture system is 15-100%;
the aerobic pre-culture is carried out in a shaking table, and the rotating speed of the shaking table is 120-150 rpm.
Preferably, in step B), the NMS culture solution comprises: KH (Perkin Elmer)2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and a trace element solution;
the microelement solution comprises ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O, KI and CaCl2·2H2O。
Preferably, in step B),
the dosage ratio of sulfate radicals in the sulfate to NMS culture solution is 1.15-1.75 g: 1L;
the dosage ratio of nitrate in the nitrate to NMS culture solution is 0.90-1.25 g: 1L;
the dosage ratio of iron ions in the iron salt to NMS culture solution is 0.25-0.80 g: 1L of the compound.
Preferably, in step B), the modifier comprises K2SO4、Na2SO4、KNO3、NaNO3、FeC6H5O7·5H2O and one or more of EDTA-Fe (III).
Preferably, in the step B), the volume ratio of the pre-culture bacterial liquid to the improved NMS culture liquid is 3-12: 15.
preferably, step B) further comprises, after the mixing: and deoxidizing the culture system obtained by mixing.
Preferably, in the step B), the volume percentage of methane in the anaerobic culture system is 15-100%.
Preferably, in step B), the anaerobic culture is performed under conditions of protection from light;
the anaerobic culture is carried out in a shaking table, and the rotating speed of the shaking table is 120-150 rpm.
The invention provides an enrichment culture method of methane anaerobic oxidation bacteria flora, which comprises the following steps: A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d; B) mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid; the improved NMS culture solution comprises NMS culture solution and an improver; the modifier comprises one or more of sulfate, nitrate and iron salt; the temperature of the anaerobic culture is 32-40 ℃. According to the enrichment culture method of the methane anaerobic oxidation bacteria flora, the bacteria liquid has an obvious methane oxidation rate within 10 days, the methane oxidation rate at 10 days exceeds 50%, and the culture period is short. The enrichment culture method of the methane anaerobic oxidation bacteria flora provided by the invention is beneficial to researching a methane anaerobic oxidation mechanism and related microorganisms in a landfill environment, and is also beneficial to the application of methane anaerobic oxidation in the aspect of water treatment anaerobic treatment technology.
Drawings
FIG. 1 shows the methane oxidation rate of the bacterial solutions obtained in examples 1-2 and comparative example 1.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides an enrichment culture method of methane anaerobic oxidation bacteria flora, which comprises the following steps:
A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d;
B) mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid;
the improved NMS culture solution comprises NMS culture solution and an improver; the modifier comprises one or more of sulfate, nitrate and iron salt;
the temperature of the anaerobic culture is 32-40 ℃.
The invention takes the percolate as the source of methane anaerobic oxidizing bacteria. The percolate is preferably domestic garbage landfill percolate. In certain embodiments of the present invention, the leachate is a heptanes landfill leachate. The percolate can be old percolate, fresh percolate or mixed percolate of the old percolate and the fresh percolate, and the invention is not limited to the method.
The precipitated substances in the percolate are preferably obtained from the percolate by centrifugation. The parameters and equipment of the centrifugation are not particularly limited in the present invention, and those known to those skilled in the art can be used.
The volume ratio of the percolate to the NMS culture solution is preferably 1-10: 10 to 15. In certain embodiments of the invention, the volume ratio of the leachate to NMS culture is 10: 15 or 8: 10.
the NMS culture preferably comprises: KH (Perkin Elmer)2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and a trace element solution;
the trace element solution preferably comprises 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, KH is present in the NMS culture medium2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And mixing the precipitate in the percolate with an NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain a pre-culture bacterial solution.
The mixing container is preferably a serum bottle.
In the aerobic pre-culture system, the volume percentage of methane is preferably 15-100%. In certain embodiments of the invention, the methane is present in an amount of 33.3% or 40% by volume.
The temperature of the aerobic pre-culture is 32-40 ℃. In certain embodiments of the invention, the aerobic pre-incubation is at a temperature of 35 ℃ or 36 ℃. The time of the aerobic pre-culture is 4-7 days. In certain embodiments of the invention, the aerobic pre-incubation time is 5 days.
The aerobic pre-cultivation is preferably carried out in a shaker. The rotating speed of the shaking table is preferably 120-150 rpm. In certain embodiments of the invention, the shaker is rotated at 130rpm or 140 rpm.
And after obtaining the pre-culture bacterial liquid, mixing the sediment in the pre-culture bacterial liquid with the improved NMS culture liquid, and carrying out anaerobic culture under the condition of methane to obtain the methane anaerobic oxidation bacterial liquid.
The sediment in the pre-cultured bacterial liquid is preferably obtained by centrifuging the pre-cultured bacterial liquid. The parameters and equipment of the centrifugation are not particularly limited in the present invention, and those known to those skilled in the art can be used.
The volume ratio of the pre-culture bacterial liquid to the improved NMS culture liquid is preferably 3-12: 15. in some embodiments of the invention, the volume ratio of the preculture bacterial liquid to the improved NMS culture liquid is 10: 15 or 12: 15.
the improved NMS culture solution comprises NMS culture solution and an improver.
The NMS culture preferably comprises: KH (Perkin Elmer)2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and a trace element solution;
the trace element solution preferably comprises 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, KH is present in the NMS culture medium2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
The modifier comprises one or more of sulfate, nitrate and iron salt; preferably comprising K2SO4、Na2SO4、KNO3、NaNO3、FeC6H5O7·5H2O and one or more of EDTA-Fe (III).
In the invention, the dosage ratio of sulfate in the sulfate to NMS culture solution is preferably 1.15-1.75 g: 1L of the compound. In certain embodiments of the invention, the sulfate in the sulfate is used in an amount of 1.6 g: 1L of the compound.
The dosage ratio of nitrate in the nitrate to NMS culture solution is preferably 0.90-1.25 g: 1L of the compound. In certain embodiments of the invention, the nitrate in the nitrate salt is used in an amount of 0.98 g: 1L of the compound.
The dosage ratio of the iron ions in the iron salt to the NMS culture solution is preferably 0.25-0.80 g: 1L of the compound. In certain embodiments of the invention, the ratio of iron ions in the iron salt to NMS culture is 0.75 g: 1L and 0.45 g: 1L of the compound.
And mixing the sediment in the pre-culture bacterial liquid with the improved NMS culture liquid, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidizing bacteria bacterial liquid.
The mixing container is preferably a serum bottle.
After the mixing, preferably, the method further comprises the following steps: and deoxidizing the culture system obtained by mixing. The specific steps of oxygen removal are preferably as follows: the culture system was purged with nitrogen.
In the anaerobic culture system, the volume percentage of methane is preferably 15-100%. In certain embodiments of the invention, the methane is present in an amount of 33.3% by volume.
The temperature of the anaerobic culture is 32-40 ℃. In certain embodiments of the invention, the temperature of the anaerobic culture is 35 ℃ or 36 ℃. The time of anaerobic culture is preferably 4-10 days. In certain embodiments of the invention, the anaerobic culture is for a period of 5 days.
The anaerobic cultivation is preferably carried out in a shaker. Specifically, it is preferable that: and placing the serum bottle in a shaking table for anaerobic culture. The rotating speed of the shaking table is preferably 120-150 rpm. In certain embodiments of the invention, the shaker is rotated at 130rpm or 140 rpm.
The anaerobic cultivation is preferably carried out under conditions protected from light.
In the present invention, the source of the raw material used is not particularly limited, and may be generally commercially available.
The invention provides an enrichment culture method of methane anaerobic oxidation bacteria flora, which comprises the following steps: A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d; B) mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid; the improved NMS culture solution comprises NMS culture solution and an improver; the modifier comprises one or more of sulfate, nitrate and iron salt; the temperature of the anaerobic culture is 32-40 ℃. According to the enrichment culture method of the methane anaerobic oxidation bacteria flora, the bacteria liquid has an obvious methane oxidation rate within 10 days, the methane oxidation rate at 10 days exceeds 50%, and the culture period is short. The enrichment culture method of the methane anaerobic oxidation bacteria flora provided by the invention is beneficial to researching a methane anaerobic oxidation mechanism and related microorganisms in a landfill environment, and is also beneficial to the application of methane anaerobic oxidation in the aspect of water treatment anaerobic treatment technology.
In order to further illustrate the present invention, the following examples are provided to describe the enrichment culture method of methanogen-anaerobic bacteria flora according to the present invention, but should not be construed as limiting the scope of the present invention.
The starting materials used in the following examples are all generally commercially available.
Example 1
10mL of mixed percolate (including old percolate and fresh percolate) of a Hemsleya amabilis landfill is taken for centrifugation, the centrifuged precipitate and 15mL of MS culture solution are filled into a 300mL serum bottle, 100mL of methane is filled into the serum bottle and is put into a shaking table, and aerobic pre-culture is carried out for 5d under the conditions of 35 ℃ and 130rpm, so as to obtain pre-culture bacterial liquid. In the aerobic pre-culture system, the volume percentage of methane is 33.3 percent.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
10mL of the preculture broth was centrifuged, and the resulting pellet and 15mL of modified NMS culture broth were placed in a 300mL serum bottle.
The improved NMS culture solution comprises NMS culture solution and Na2SO4. The Na is2SO4The dosage ratio of the sulfate radical in the NMS culture solution is 1.6 g: 1L of the compound.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) purging the serum bottle with nitrogen, sealing, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing anaerobic culture for 5d in a dark place at 35 ℃ and 130rpm to obtain the methane anaerobic oxidation bacteria liquid. In the anaerobic culture system, the volume percentage of methane is 33.3%.
The concentration of methane in the serum bottle is respectively measured before and after the culture, the ratio of the reduction amount of the concentration of methane to the initial concentration of methane is the methane oxidation rate, and the result of detecting the methane oxidation rate in the bacterial liquid is shown in figure 1. FIG. 1 shows the methane oxidation rate of the bacterial solutions obtained in examples 1-2 and comparative example 1. In fig. 1, graph a shows the methane oxidation rate in the bacterial suspension obtained in comparative example 1, graph b shows the methane oxidation rate in the bacterial suspension obtained in example 1, and graph c shows the methane oxidation rate in the bacterial suspension obtained in example 2. As can be seen from the graph b in FIG. 1, the methane oxidation rate was 58.5% or more than 50% in the case of the aerobic pre-culture 5d and the anaerobic re-culture 5 d.
Example 2
Centrifuging 10mL of fresh percolate of a Heptazi mountain landfill, filling the centrifuged precipitate and 15mL of MS culture solution into a 300mL serum bottle, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing aerobic pre-culture for 5d under the conditions of 35 ℃ and 130rpm to obtain pre-culture bacterial solution. In the aerobic pre-culture system, the volume percentage of methane is 33.3 percent.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
10mL of the preculture broth was centrifuged, and the resulting pellet and 15mL of modified NMS culture broth were placed in a 300mL serum bottle.
The improved NMS culture solution comprises NMS culture solution and Na2SO4. The Na is2SO4The dosage ratio of the sulfate radical in the NMS culture solution is 1.6 g: 1L of the compound.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) purging the serum bottle with nitrogen, sealing, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing anaerobic culture for 5d in a dark place at 35 ℃ and 130rpm to obtain the methane anaerobic oxidation bacteria liquid. In the anaerobic culture system, the volume percentage of methane is 33.3%.
The concentration of methane in the serum bottle is respectively measured before and after the culture, the ratio of the reduction amount of the concentration of methane to the initial concentration of methane is the methane oxidation rate, and the result of detecting the methane oxidation rate in the bacterial liquid is shown in figure 1. FIG. 1 shows the methane oxidation rate of the bacterial solutions obtained in examples 1-2 and comparative example 1. As can be seen from FIG. 1, the methane oxidation rate in the aerobic preculture 5d and the anaerobic culture 5d was 50.4% or more, which was more than 50%.
Comparative example 1
10mL of mixed percolate (including old percolate and fresh percolate) of a Hemsleya amabilis landfill is taken for centrifugation, the centrifuged precipitate and 15mL of MS culture solution are filled into a 300mL serum bottle, 100mL of methane is filled into the serum bottle and is put into a shaking table, and aerobic pre-culture is carried out for 5d under the conditions of 35 ℃ and 130rpm, so as to obtain pre-culture bacterial liquid. In the aerobic pre-culture system, the volume percentage of methane is 33.3 percent.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) centrifuging 10mL of the preculture bacterial liquid, and filling the obtained precipitate and 15mL of the MS culture solution into a 300mL serum bottle.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) purging the serum bottle with nitrogen, sealing, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing anaerobic culture for 5d in a dark place at 35 ℃ and 130rpm to obtain the methane anaerobic oxidation bacteria liquid. In the anaerobic culture system, the volume percentage of methane is 33.3%.
The concentration of methane in the serum bottle is respectively measured before and after the culture, the ratio of the reduction amount of the concentration of methane to the initial concentration of methane is the methane oxidation rate, and the result of detecting the methane oxidation rate in the bacterial liquid is shown in figure 1. As can be seen from FIG. 1, the methane oxidation rate was 17.2% in the case of aerobic pre-culture for 5d and anaerobic re-culture for 5d, and the methane oxidation rate was remarkably poor.
Example 3
Taking 8mL of mixed percolate (including old percolate and fresh percolate) of a Qizi mountain landfill, centrifuging, filling the centrifuged precipitate and 10mL of MS culture solution into a 300mL serum bottle, filling 120mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing aerobic pre-culture for 5d at 40 ℃ and 130rpm to obtain pre-culture bacterial liquid. In the aerobic pre-culture system, the volume percentage of methane is 40%.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
10mL of the preculture broth was centrifuged, and the resulting pellet and 15mL of modified NMS culture broth were placed in a 300mL serum bottle.
The improved NMS culture solution comprises NMS culture solution and EDTA-Fe (III). The dosage ratio of the iron ions in the EDTA-Fe (III) to the NMS culture solution is 0.75 g: 1L of the compound.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2Containing of OThe amount is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) purging the serum bottle with nitrogen, sealing, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing anaerobic culture for 5 days at 40 ℃ and 140rpm in the dark to obtain methane anaerobic oxidation bacteria liquid. In the anaerobic culture system, the volume percentage of methane is 33.3%.
The method comprises the steps of respectively measuring the methane concentration in a serum bottle before and after culture, wherein the ratio of the reduction amount of the methane concentration to the initial methane concentration is the methane oxidation rate, and detecting the methane oxidation rate in the bacterial liquid, wherein the experimental result shows that the methane oxidation rate is 61.8% and exceeds 50% when the bacteria liquid is subjected to aerobic pre-culture for 5 days and then anaerobic culture for 5 days.
Example 4
10mL of mixed percolate (including old percolate and fresh percolate) of a Hemsleya amabilis landfill is taken for centrifugation, the centrifuged precipitate and 10mL of LNMS culture solution are filled into a 300mL serum bottle, 120mL of methane is filled into the serum bottle and is put into a shaking table, and aerobic pre-culture is carried out for 5d under the conditions of 36 ℃ and 140rpm, so as to obtain pre-culture bacterial liquid. In the aerobic pre-culture system, the volume percentage of methane is 40%.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
12mL of the preculture broth was centrifuged, and the resulting pellet and 15mL of modified NMS culture broth were placed in a 300mL serum bottle.
Improved NMS culture solution comprises NMS culture solution and FeC6H5O7·5H2And O. The FeC6H5O7·5H2The dosage ratio of the iron ions in the O to the NMS culture solution is 0.45 g: 1L of the compound.
KH in the NMS culture solution2PO4The content of (A) is 1.06g/L, Na2HPO4·12H2The content of O was 4.34g/L, NaNO3The content of (B) is 1.70g/L, K2SO4The content of (B) is 0.34g/L, MgSO4·7H2The content of O is 0.074g/L, FeSO4·7H2The content of O is 22.4mg/L, and the content of the trace element solution is 2 mL/L; the NMS culture solution adopts water as solvent and has a pH value of 7.0;
in the solution of the trace elements, ZnSO4·7H2The content of O is 0.57mg/L, MnSO4·7H2The content of O is 0.446mg/L, H3BO3The content of (A) is 0.124mg/L, Na2MoO4·2H2The content of O is 0.096mg/L, CoCl2·6H2The content of O is 0.096mg/L, KI, and the content is 0.166mg/L, CaCl2·2H2The O content was 7.0 mg/L.
And (3) purging the serum bottle with nitrogen, sealing, filling 100mL of methane into the serum bottle, putting the serum bottle into a shaking table, and performing anaerobic culture for 5 days at 36 ℃ and 120rpm in the dark to obtain methane anaerobic oxidation bacteria liquid. In the anaerobic culture system, the volume percentage of methane is 33.3%.
The method comprises the steps of respectively measuring the methane concentration in a serum bottle before and after culture, wherein the ratio of the reduction amount of the methane concentration to the initial methane concentration is the methane oxidation rate, and detecting the methane oxidation rate in the bacterial liquid, wherein the experimental result shows that the methane oxidation rate is 65.3% and exceeds 50% when the bacteria liquid is subjected to aerobic pre-culture for 5 days and then anaerobic culture for 5 days.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. An enrichment culture method of methane anaerobic oxidizing bacteria flora comprises the following steps:
A) mixing the precipitate in the percolate with NMS culture solution, and carrying out aerobic pre-culture under the condition of methane to obtain pre-culture bacterial solution; the temperature of the aerobic pre-culture is 32-40 ℃; the aerobic pre-culture time is 4-7 d;
the volume ratio of the percolate to the NMS culture solution is 1-10: 10-15;
the NMS culture solution is KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and trace element solution, wherein the solvent is water;
the microelement solution is ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O, KI and CaCl2·2H2O；
B) Mixing the sediment in the pre-culture bacterial liquid with an improved NMS culture solution, and carrying out anaerobic culture under the condition of methane to obtain methane anaerobic oxidation bacterial liquid;
the improved NMS culture solution is NMS culture solution and modifier; the modifier is Na2SO4、FeC6H5O7·5H2One or more of O and EDTA-Fe (III);
the Na is2SO4Use of sulfate radical in NMS culture solutionThe weight ratio is 1.15-1.75 g: 1L;
the FeC6H5O7·5H2The dosage ratio of the iron ions in O and/or EDTA-Fe (III) to the NMS culture solution is 0.25-0.80 g: 1L;
the NMS culture solution is KH2PO4、Na2HPO4·12H2O、NaNO3、K2SO4、MgSO4·7H2O、FeSO4·7H2O and trace element solution, wherein the solvent is water;
the microelement solution is ZnSO4·7H2O、MnSO4·7H2O、H3BO3、Na2MoO4·2H2O、CoCl2·6H2O, KI and CaCl2·2H2O; the temperature of the anaerobic culture is 32-40 ℃.
2. The enrichment culture method according to claim 1, wherein in the step A), the volume percentage of methane in the aerobic pre-culture system is 15-100%;
the aerobic pre-culture is carried out in a shaking table, and the rotating speed of the shaking table is 120-150 rpm.
3. The enrichment culture method according to claim 1, wherein in the step B), the volume ratio of the preculture bacterial liquid to the improved NMS culture liquid is 3-12: 15.
4. the enrichment culture method according to claim 1, wherein the step B) further comprises, after the mixing: and deoxidizing the culture system obtained by mixing.
5. The enrichment culture method according to claim 1, wherein in the step B), the methane content in the anaerobic culture system is 15-100% by volume.
6. The enrichment culture method according to claim 1, wherein in step B), the anaerobic culture is performed under conditions of light protection;
the anaerobic culture is carried out in a shaking table, and the rotating speed of the shaking table is 120-150 rpm.
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