CN104926062A - Enrichment and domestication method of hydrogen nutrition type bacteria groups producing methane - Google Patents
Enrichment and domestication method of hydrogen nutrition type bacteria groups producing methane Download PDFInfo
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- CN104926062A CN104926062A CN201510393213.XA CN201510393213A CN104926062A CN 104926062 A CN104926062 A CN 104926062A CN 201510393213 A CN201510393213 A CN 201510393213A CN 104926062 A CN104926062 A CN 104926062A
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- biogas fermentation
- enrichment
- gas
- producing methane
- hydrogen
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
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Abstract
The invention relates to an enrichment and domestication method of hydrogen nutrition type bacteria groups producing methane. The domestication method that a marsh gas fermentation system runs at constant temperature and constant pressure, H2 and CO2 are added through an external source, and gas circulation is conducted is designed, and the growth and enrichment of the hydrogen nutrition type bacteria groups producing methane in marsh gas fermentation activated sludge are promoted. By means of the method, the content of the hydrogen nutrition type bacteria groups producing methane in methanogens can be effectively increased, and the marsh gas fermentation activated sludge rich in the hydrogen nutrition type bacteria groups producing methane can be obtained.
Description
Technical field
The present invention relates to a kind of enrichment and acclimation method of the hydrogen nutritional type methanogen floras for the production of methane, belong to methane fermentating microorganism technical field.
Background technology
In marsh gas fermentation processes, the hydrogen (H that organism produces in hydrolysis and two stages such as fermentation stage, hydrogenesis and acetogenesis stage
2) and carbonic acid gas (CO
2), methane (CH can be generated by the metabolism of hydrogen nutritional type methanogen floras
4), CH in biogas
4generation have 28% to derive from this approach, i.e. H
2reduction CO
2generate CH
4.
CH in biogas
4content is about 60%, CO
2content is about 35%, CO
2content is more than CH
4the half of content, causes the bottleneck of biogas higher value application, simultaneously CO
2be not utilized effectively, waste the carbon source needed for methane fermentating microorganism growth; From H
2reduction CO
2generate CH
4this approach gains enlightenment, if by the CO in biogas
2pass through H
2carry out micro-reduction, will effectively promote CH in biogas
4content, exploitation bio-natural gas, promotes biogas higher value application; Methanogen can be categorized as acetic acid nutritional type methanogen (metabolism matrix is acetic acid), (metabolism matrix is H to hydrogen nutritional type methanogen
2and CO
2) and Methylotrophic methanogen (metabolism matrix is formic acid, methyl alcohol and methylamine etc.), and tracer experiment shows that the methanogen in methane fermentating microorganism system has more than 70% to be acetic acid nutritional type methanogen, in order to effective implemention H in biogas fermentation system
2reduction CO
2generate CH
4, be necessary to take method to promote the growth of hydrogen nutritional type methanogen floras in microorganism system; Therefore, this experimental design runs biogas fermentation system under normal temperature and pressure conditions, adds H by external source
2and CO
2the acclimation method of matrix, promote the metabolism growth of hydrogen nutritional type methanogen floras in biogas fermentation active sludge, to promoting the content of hydrogen nutritional type methanogen floras in methanogen, and obtain the biogas fermentation active sludge being enriched hydrogen nutritional type methanogen floras.
Summary of the invention
The object of the present invention is to provide a kind of enrichment and acclimation method of the hydrogen nutritional type methanogen floras for the production of methane.
The present invention completes by the following technical programs:
(1) biogas fermentation temperature, pressure: normal temperature, normal pressure
(2) biogas fermentation active sludge consumption: biogas fermentation active sludge consumption is 50% of biogas fermentation reactor volume, residue reactor body product space is filled by natural pond liquid.
(3) metabolism matrix batching: according to H
2reduction CO
2generate CH
4biochemical equation 4H
2+ CO
2→ CH
4+ 2H
2o, with H
2: CO
2volume ratio 4:1 prepares burden, and joins the N of 1 volume
2as the nitrogenous source needed for microorganism growth, often group experiment mixed gas batching 3000mL, wherein H
2there are 2000mL, CO
2there are 500mL, N
2there is 500mL.
(4) acclimation method: the mixed gas prepared is passed in wet storage holders, now gas holder floats the position that cover rises to 3000mL, then gas recycle pump is utilized to be evacuated to by mixed gas in biogas fermentation reactor, get back to again in gas holder after reaction in biogas fermentation active sludge bed, so repeatedly carry out gas circulation, along with reaction is carried out, the floating cover of gas holder constantly declines, and shows that reaction terminates when floating cover no longer declines.
(5) the extraction of biogas fermentation activated sludge sample STb gene, detection and order-checking
1. the extraction of genomic dna: adopt environment DNA to extract test kit Fast DNA spin kit for soil (MP Biomedicals, LLC; Www.mpbio.com) sample is carried out to the extraction of STb gene, adopt agarose gel electrophoresis to detect purity and the concentration of DNA afterwards, get appropriate sample in centrifuge tube, use sterilized water dilute sample to 1ng/ μ l.
2. pcr amplification: the genomic dna after dilution is template; According to the selection in order-checking region, use the special primer of band Barcode; Use the Phusion High-Fidelity PCR Master Mix with GC Buffer of New England Biolabs company.Use enzyme that is efficient and high-fidelity to carry out PCR, guarantee amplification efficiency and accuracy.Primer corresponding zone: 16S V4 district primer is 515F-806R; 18S V4 district primer is 528F-706R; 18S V9 district primer is 1380F-1510R; ITS1 district primer is ITS1-5F--ITS2; ITS2 district primer is: ITS2-3F--ITS2-4R.
3. the mixed sample of PCR primer and purifying: PCR primer uses the sepharose of 2% concentration to carry out electrophoresis detection; Carry out isoconcentration according to PCR primer concentration and mix sample, fully after mixing, use the agarose gel electrophoresis of 2 % to detect PCR primer, use the GeneJET glue of Thermo Scientific company to reclaim test kit and reclaim product.
4. library construction and the order-checking of upper machine: use the NEB Next Ultra DNA Library Prep Kit for Illumina of New England Biolabs company to build the structure that storehouse test kit carries out library, the library built is quantitative and library detection through Qubit, after qualified, MiSeq is used to carry out upper machine order-checking.
The mechanism of action of the present invention: the present invention's substrate passed in biogas fermentation reactor is H
2, CO
2and N
2, H
2and CO
2as the metabolism matrix of methane fermentating microorganism, N
2as the nitrogenous source of methane fermentating microorganism; In gas recycle process, hydrogen nutritional type methanogen is owing to there being sufficient metabolism matrix H
2and CO
2, thus in marsh gas fermentation processes, constantly can grow enrichment, and acetic acid nutritional type methanogen and Methylotrophic methanogen be not owing to obtaining metabolism matrix, will constantly wither away in marsh gas fermentation processes.
Beneficial effect of the present invention: (1) the present invention has both CO
2the environment protection significance, the biosynthesizing CH that reduce discharging
4the advantage such as renewable energy resources meaning and potential economic return, extensive application prospect.(2) by not easily by H that highly effective and safe utilizes
2, pass in biogas fermentation system, with the CO of accounting in biogas about 35%
2there is biochemical reaction, be converted into another kind of clean energy CH
4, effectively promote the CH of traditional biogas fermentation
4content, makes bio-natural gas, realizes the higher value application of biogas.
accompanying drawing illustrates:
Fig. 1 is structural representation of the present invention;
Fig. 2 is embodiment of the present invention H
2reduction CO
2generate CH
4experimental result.
embodiment:
Embodiment:
(1) H
2reduction CO
2generate CH
4experimental result
At the normal operating phase of experiment, the acclimating of hydrogen nutritional type methanogen has carried out 5 groups altogether and has effectively tested, these 5 groups of CH effectively tested
4growing amount the results are shown in Figure 2.
As can be seen from Figure 2, the CH of the 1 to 5 group gas circulation experiment
4output is respectively 185mL, 258mL, 166mL, 136mL, 140mL, shows that the microorganism in biogas fermentation system can by H
2and CO
2convert CH to preferably
4; According to H
2reduction CO
2conversion generates CH
4biochemical equation 4H
2+ CO
2→ CH
4+ 2H
2o, the 2000mL H that external source adds
2with 500mL CO
2convertible in theory generation 500mL CH
4, from experimental result, the actual CH of the 1 to 5 group gas circulation experiment
4output accounts for 37%, 51.6%, 33.2%, 27.2%, 28% of theoretical yield respectively, wherein the CH of the 2nd group
4output is the highest, reaches 51.6% of theoretical value.
N
2the situation reducing (or utilization) illustrates: the N of the 1 to 5 group gas circulation experiment
2consumption is respectively 151mL, 44mL, 210mL, 157mL, 45mL, shows under this experiment condition, by the mode of gas circulation, and the N that external source adds
2nitrogenous source can be used as by methane fermentating microorganism directly to utilize, also show to also exist with N in the methane fermentating microorganism that enrichment obtains simultaneously
2for the microorganism of nitrogenous source.
(2) methanogen group and mutation analysis
From the gas circulation experimental result of Fig. 2, the H that external source can add by the present invention
2and CO
2good conversion generates CH
4, from microbiological angle, should be that hydrogen nutritional type methanogen in system has played vital role, therefore, respectively total DNA extraction is carried out to the biogas fermentation active sludge after the biogas fermentation active sludge before startup and the 5th group of gas circulation experiment terminate, application 16S rRNA gene engineering is studied the prokaryotic micro-organisms group in biogas fermentation active sludge before and after gas circulation experiment, the front and back changing conditions of comparative analysis prokaryotic micro-organisms group, the content situation of methanogen in ancient bacterium in the biogas fermentation active sludge of front and back of testing sees table (in table ↑ represent growth, ↓ represent and reduce).
As seen from the above table, the methane mane Pseudomonas content of obligate acetic acid type is rear on pretreatment obviously to be reduced, and be reduced to 64% from 84%, decrease by 23.8%, this is because this experiment does not provide acetic acid matrix, and methane mane Pseudomonas can not utilize H
2and CO
2matrix, methane mane Pseudomonas causes content significantly to reduce owing to lacking metabolism matrix; Methanosarcina not only can utilize acetic acid matrix but also can utilize H
2and CO
2matrix, this experiment does not provide acetic acid matrix, but the content of Methanosarcina in ancient bacterium bring up to experiment by 8% before testing after 16%, content increases one times, and this is the H because external source adds
2and CO
2as the growth matrix of Methanosarcina, make caused by its continuous enrichment and growth; Belong to the methane grain Pseudomonas of hydrogen nutritional type, Methanoculleus, methanospirillum belong to, Methanobacterium on pretreatment after content there occurs equally and rise appreciably, rate of increase is respectively 16.7%, 80%, 150%, 11.1%, indicates the H that external source in this experiment adds
2and CO
2fully facilitate the growth of these hydrogen nutritional type methanogens; It should be noted that, the hydrogen nutritional type methanogens such as the methane rope Pseudomonas do not detected on pretreatment, methane Peziza, methane bubble Pseudomonas, all detect the existence of these methanogens in biogas fermentation active sludge after experiment terminates, describe and add H by external source
2and CO
2matrix, effective stimulus can utilize H
2and CO
2the enrichment of methanogen.
Claims (3)
1. the enrichment of a hydrogen nutritional type methanogen floras and acclimation method, it is characterized in that: at normal temperatures and pressures, the mixed gas prepared is passed in wet storage holders, then gas recycle pump is utilized to be evacuated to by mixed gas in biogas fermentation reactor, get back to again in gas holder after reaction in biogas fermentation active sludge bed, so repeatedly carry out gas circulation.
2., according to the mixed gas described in claims 1, it is characterized in that: mixed gas is by H
2, CO
2and N
2form, according to H
2reduction CO
2generate CH
4biochemical equation 4H
2+ CO
2→ CH
4+ 2H
2o, with H
2: CO
2volume ratio 4:1 prepares burden, and joins the N of 1 volume
2, often group experiment mixed gas batching 3000mL, wherein H
2there are 2000mL, CO
2there are 500mL, N
2there is 500mL.
3. according to the biogas fermentation active sludge bed described in claims 1, it is characterized in that: biogas fermentation active sludge consumption is 50% of biogas fermentation reactor volume.
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Cited By (5)
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|---|---|---|---|---|
| CN106434518A (en) * | 2016-12-09 | 2017-02-22 | 中国科学院广州能源研究所 | Domestication method of acid-resistant syntrophic propionic acid methane-producing bacterium strain |
| CN106701617A (en) * | 2016-12-09 | 2017-05-24 | 中国科学院广州能源研究所 | Method for domesticating auto-aggregation propionic acid methanogenesis microbial agent |
| CN106754611A (en) * | 2016-12-09 | 2017-05-31 | 中国科学院广州能源研究所 | A kind of acclimation method of the propionic acid of resistance to ammonia nitrogen methane phase fungus strain |
| CN108265081A (en) * | 2018-01-24 | 2018-07-10 | 中国石油大学(北京) | A kind of simple method for producing high quality biological methane |
| CN111088967A (en) * | 2018-10-24 | 2020-05-01 | 中国石油化工股份有限公司 | Method for improving microbial methane production yield of oil reservoir |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434518A (en) * | 2016-12-09 | 2017-02-22 | 中国科学院广州能源研究所 | Domestication method of acid-resistant syntrophic propionic acid methane-producing bacterium strain |
| CN106701617A (en) * | 2016-12-09 | 2017-05-24 | 中国科学院广州能源研究所 | Method for domesticating auto-aggregation propionic acid methanogenesis microbial agent |
| CN106754611A (en) * | 2016-12-09 | 2017-05-31 | 中国科学院广州能源研究所 | A kind of acclimation method of the propionic acid of resistance to ammonia nitrogen methane phase fungus strain |
| CN106754611B (en) * | 2016-12-09 | 2019-11-29 | 中国科学院广州能源研究所 | A kind of acclimation method of resistance to ammonia nitrogen propionic acid methane phase fungus strain |
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| CN108265081A (en) * | 2018-01-24 | 2018-07-10 | 中国石油大学(北京) | A kind of simple method for producing high quality biological methane |
| CN111088967A (en) * | 2018-10-24 | 2020-05-01 | 中国石油化工股份有限公司 | Method for improving microbial methane production yield of oil reservoir |
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| CN104926062B (en) | 2018-09-28 |
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