CN104974970A - Composite microbial inoculant capable of efficiently producing methane by grease anaerobic degradation, and preparation method and application thereof - Google Patents
Composite microbial inoculant capable of efficiently producing methane by grease anaerobic degradation, and preparation method and application thereof Download PDFInfo
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- CN104974970A CN104974970A CN201510476319.6A CN201510476319A CN104974970A CN 104974970 A CN104974970 A CN 104974970A CN 201510476319 A CN201510476319 A CN 201510476319A CN 104974970 A CN104974970 A CN 104974970A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
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- 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
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Abstract
The invention provides a composite microbial inoculant capable of efficiently producing methane by grease anaerobic degradation, which comprises the following strains: Anaerovibrio lipolytica DSM 3074, Syntrophomonas erecta DSM 16215, Syntrophomonas bryantii DSM 3014A, Syntrophomonas palmitatica DSM 18709, Desulfovibrio vulgaris DSM 644, Methanospirillum hungatei DSM 13809, Methanobacterium formicicum DSM 1535, Methanobrevibacter smithii DSM861, and Methanosaeta concilii DSM 2139T. The invention also provides a method for preparing the microbial inoculant and application of the microbial inoculant in producing methane by grease anaerobic degradation. The composite microbial inoculant can obviously shorten the start time, enhance the biogas fermentation efficiency and enhance the stability of the fermentation process. The composite microbial inoculant is suitable for different scales of fermentation systems, and has favorable market value.
Description
Technical field
The present invention relates to recycled wood materials recovery energy technical field, the concrete efficient anaerobic degraded methanogenic composite fungus agent of grease and preparation method and application.
Technical background
The animal-plant oil that can not eat again that recycled wood materials (Waste edible oils, WEOs) is catering trade, food-processing industry produces in production and operating activities.It is reported that the amount of China's recycled wood materials accounts for edible oil total quantity consumed
, produce every year
.As direct, the undressed waste water containing recycled wood materials is entered water drain, not only can cause the wasting of resources, but also serious environment pollution.Particularly because the fossil energy such as oil, coal price in recent years constantly rises, the bio-transformation of waste grease is the extensive concern that the problem of new forms of energy obtains Chinese scholars especially.Rational disposal and utilization waste grease had both met the requirement of environment protection, was also the demand of Renewable Energy Development.
Current oil-containing waste water process is just for environment protection standard requirement, and technique has aerobic and anaerobism or the two combination process.Adopt anaerobic biological reactor process oil wastewater, longer chain fatty acid accumulation usually can occur, and the longer chain fatty acid of accumulation produces restraining effect to anaerobion, even causes the collapse of anaerobic digestion reaction vessel.The thorough degraded of grease is the long-time interactional result of multiple-microorganism in microorganism system, rely on special anaerobic bacterium and the ancient bacterium of methane phase, the synergy just because of these microorganisms just makes substrate hydrolysis, acidifying and methane phase process carry out smoothly.Therefore, in the synergy of carrying out will needing in the methanogenic process of grease anaerobic degradation between multiple-microorganism.By many bacterial strains mixed culture, multiple enzyme synergy, comparatively fast and more up hill and dale can remove the feedback inhibition of product, regulate the pH of nutrient solution, improve the transformation efficiency of recycled wood materials.Thus, for oil-containing waste water anaerobic digestion process and with waste grease be carbon source anaerobic digestion methane phase in fermentation inhibitory and the low problem of transformation efficiency, invent the methanogenic composite bacteria of a kind of efficient degradation recycled wood materials, the transformation efficiency that longer chain fatty acid is degraded to volatile acid can be improved, and then improve whole efficiency and speed that grease is converted into methane.
Still do not have special in the methanogenic composite fungus agent of recycled wood materials anaerobic degradation at present, conventional composite fungus agent is when utilizing recycled wood materials methane phase, and gas producing efficiency is poor.CN201510037310.5 discloses the composite bacteria that glyceride stock is rich in process, but the gas producing efficiency of this composite bacteria is not high, and the time that accumulative methane production reaches needed for final methane production 80% is at least 36 days, and aerogenesis excessive cycle, does not possess economy.
Summary of the invention
For the shortcoming of prior art, the object of the present invention is to provide the methanogenic composite fungus agent of a kind of efficient anaerobic degraded grease, this microbial inoculum comprises following bacterial classification:
Anaerovibrio lipolytica DSM 3074
Syntrophomonas erecta DSM 16215
Syntrophomonas bryantii DSM 3014A
Syntrophomonas palmitatica DSM 18709
Desulfovibrio vulgaris DSM 644
Methanospirillum hungatei DSM 13809
Methanobacterium formicicum DSM 1535
Methanobrevibacter smithii DSM861
Methanosaeta concilii DSM 2139
T。
The essential information of the present invention's bacterial classification used is as follows:
anaerovibrio lipolyticasteatolysis anaerobism vibrios, bracket fungus, the raw flagellum of single end, energy decompose lipide, only utilizes glycerine, bacterial strain deposit number DSM 3074, ATCC 33276;
syntrophomonas erecta,syntrophism Zymomonas mobilis
,rod-short, the straight chain fatty acid decomposing 4 to 18 carbon produces acetic acid and H
2, with methanogen syntrophism, bacterial strain deposit number DSM 16215;
syntrophomonas bryantii,bu Shi syntrophism clostridium, shuttle shape, does not produce gemma, and the lipid acid of energy anaerobic oxidation even carbon to 10 carbon produces acetic acid and H
2, with methanogen syntrophism, bacterial strain deposit number DSM3014A;
syntrophomonas palmitatica,syntrophism Zymomonas mobilis, straight-bar, slight curvature, does not produce gemma, and syntrophism degraded longer chain fatty acid produces acetic acid and H
2, with methanogen syntrophism, bacterial strain deposit number DSM18709;
desulfovibrio vulgariscommon desulphurization vibrios, arcuation, using vitriol as electron acceptor(EA), all right lipid acid such as metabolize lactate, pyruvic acid, produces volatile acid, bacterial strain deposit number DSM 644, ATCC 29579;
methanospirillum hungatei, Heng Shi methanospirillum, knee or length waveform filament not etc., utilizes H
2/ CO
2with formate growth methane phase, bacterial strain deposit number DSM 13809;
methanobacterium formicicum, formic acid methagen, rod bacterium, utilizes formic acid or H
2/ CO
2as carbon source and energy production methane, bacterial strain deposit number DSM 1535, ATCC 33274;
methanobrevibacter smithiishi Shi methane tyrothricin, form is tyrothricin, does not form gemma, H2/CO2 and formic acid can be utilized as carbon source and energy production methane, strictly anaerobic.Bacterial strain deposit number DSM861, ATCC 35061.
methanosaeta conciliimethane mane bacterium, strictly anaerobic, cell is shaft-like, Gram-negative, optimum growth temperature 35 DEG C, growth pH6.5 ~ 7.8, and only utilize acetate to grow methane phase, bacterial strain deposit number is DSM 2139
t.
Above bacterial strain all obtains by German Culture Collection (DSMZ) or American Type culture center (ATCC).
The present invention contains the ancient bacterium of fat splitting bacterium, hydrogen-producing acetogenic bacteria, sulphate reducing bacteria and methane phase; After each for the present invention bacterial classification mixed culture, produce enzyme and enrich, the feedback inhibition of meta-bolites longer chain fatty acid can be removed, ensure the stability of anaerobic digestion environment.When using it for waste grease anaerobic digestion reaction vessel, significantly can shorten start time, improve biogas fermentation efficiency, strengthen the stability of fermenting process.
Load can be added according to fermentation raw material as the ratio of the ancient bacterium of the fat splitting bacterium of inoculum, hydrogen-producing acetogenic bacteria, sulphate reducing bacteria and methane phase in microbial inoculum not coexist in described scope and adjust, make it to reach better result of use.
Microbial inoculum of the present invention is utilizing effect in recycled wood materials methane phase very remarkable, and as shown in one embodiment of the present of invention, fermentation period can be as short as 35 days, and 80% time used that cumulative methane output reaches final methane production can be as short as 19 days.
It is worthy of note, the basis of microbial inoculum of the present invention is added other microbial inoculum any, to utilize for the purpose of grease methane phase, and obtain expected effect, all do not deviate from spirit of the present invention, all belong to protection scope of the present invention.
Preferably, described microbial inoculum is number meter by volume, comprising:
anaerovibrio lipolyticadSM 3074 bacterium liquid 25-35 part
syntrophomonas erectadSM 16215 bacterium liquid 5-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid 8-10 part
syntrophomonas palmitaticadSM 18709 bacterium liquid 2-5 part
desulfovibrio vulgarisdSM 644 bacterium liquid 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid 5-8 part
methanobacterium formicicumdSM 1535 bacterium liquid 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid 2-5 part
methanosaeta conciliidSM 2139
tbacterium liquid
5-10 part;
Described bacterium liquid comprises the one in ferment product, bacterium re-suspension liquid.
The bacterium of every part of each bacterial classification is dense is 1-4 × 10
8individual/ml.
It will be apparent to those skilled in the art that described bacterium re-suspension liquid for utilizing any liquid that can be used for resuspended bacterium, can comprise physiological saline, PBS etc.
When each bacterial classification mixes with aforementioned proportion, very outstanding technique effect can be obtained.
Second object of the present invention is the preparation method providing the methanogenic composite fungus agent of efficient anaerobic degraded grease, and the method comprises the steps:
By bacteria liquid product, by 25-35 part
anaerovibrio lipolyticadSM 3074 bacterium liquid, 5-10 part
syntrophomonas erectadSM 16215 bacterium liquid, 8-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid, 2-5 part
syntrophomonas palmitaticadSM 18709 bacterium liquid, 5-8 part
desulfovibrio vulgarisdSM 644 bacterium liquid, 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid, 2-5 part
methanobacterium formicicumdSM 1535 bacterium liquid, 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid, 5-10 part
methanosaeta conciliidSM 2139
tbe inoculated in the nutrient solution of grease after the mixing of bacterium liquid, sealing and standing cultivates 10-20 days, and culture temperature is 30 DEG C-40 DEG C, and pH is 4-8;
Described nutrient solution, by mass percentage, containing CaCl
21-2 ‰, MgCl
22-8 ‰, CaCO
31-5 ‰, NaCl 4-8 ‰, urea 4-8 ‰, KH
2pO
410 ‰, grease 1-2%, all the other are water.
Preferably, the bacterium of every part of bacterium liquid is dense is 1-4 × 10
8individual/ml.
3rd object of the present invention is to provide the application of above-mentioned microbial inoculum on anaerobic degradation grease methane phase, and the method for this application comprises the steps:
By bacteria liquid product, by 25-35 part
anaerovibrio lipolyticadSM 3074 bacterium liquid, 5-10 part
syntrophomonas erectadSM 16215 bacterium liquid, 8-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid, 2-5 part
syntrophomonas palmitaticadSM 18709 bacterium liquid, 5-8 part
desulfovibrio vulgarisdSM 644 bacterium liquid, 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid, 2-5 part
methanobacterium formicicumdSM 1535 bacterium liquid, 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid, 5-10 part
methanosaeta conciliidSM 2139
tbe inoculated in recycled wood materials after the mixing of bacterium liquid, grease concentration is 5-20 gVS/L, at 30 DEG C of-40 DEG C of temperature, carry out gassy fermentation.
Preferably, the bacterium of every part of bacterium liquid is dense is 1-4 × 10
8individual/ml.
Beneficial effect of the present invention:
1, the present invention significantly can shorten start time, improves biogas fermentation efficiency, strengthens the stability of fermenting process;
2, the present invention is suitable for the fermentation system of different scales, has good marketable value;
3, the preparation method of microbial inoculum of the present invention is simple, and microbial inoculum is easy to obtain;
4, the present invention is when utilizing the product Gas application of grease, and technique is simple, easy to implement.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that following examples are just for being further detailed the present invention; limiting the scope of the invention can not be interpreted as; some nonessential improvement and adjustment that the person skilled in the art in this field makes according to foregoing invention content, still belong to protection scope of the present invention.
Embodiment 1
Number is amassed, containing, for example lower bacterial classification in composite fungus agent by bacteria liquid:
anaerovibrio lipolyticadSM 3,074 35 parts
syntrophomonas erectadSM 16,215 10 parts
syntrophomonas bryantiidSM 3014A 10 parts
syntrophomonas palmitaticadSM 18,709 5 parts
desulfovibrio vulgarisdSM 644 8 parts
methanospirillum hungateidSM 13,809 8 parts
methanobacterium formicicumdSM 1,535 5 parts
methanobrevibacter smithiidSM861 5 parts
methanosaeta conciliidSM 2139
t10 parts.
The bacterium of every part of each bacterial classification is dense is 4 × 10
8individual/ml.
Embodiment 2
Number is amassed, containing, for example lower bacterial classification in composite fungus agent by bacteria liquid:
anaerovibrio lipolyticadSM 3,074 25 parts
syntrophomonas erectadSM 16,215 5 parts
syntrophomonas bryantiidSM 3014A 8 parts
syntrophomonas palmitaticadSM 18,709 2 parts
desulfovibrio vulgarisdSM 644 5 parts
methanospirillum hungateidSM 13,809 5 parts
methanobacterium formicicumdSM 1,535 2 parts
methanobrevibacter smithiidSM861 2 parts
methanosaeta conciliidSM 2139
t5 parts.
The bacterium of every part of each bacterial classification is dense is 1 × 10
8individual/ml.
Embodiment 3
Number is amassed, containing, for example lower bacterial classification in composite fungus agent by bacteria liquid:
anaerovibrio lipolyticadSM 3,074 30 parts
syntrophomonas erectadSM 16,215 8 parts
syntrophomonas bryantiidSM 3014A 9 parts
syntrophomonas palmitaticadSM 18,709 3 parts
desulfovibrio vulgarisdSM 644 7 parts
methanospirillum hungateidSM 13,809 7 parts
methanobacterium formicicumdSM 1,535 4 parts
methanobrevibacter smithiidSM861 4 parts
methanosaeta conciliidSM 2139
t8 parts.
The bacterium of every part of each bacterial classification is dense is 2 × 10
8individual/ml.
Embodiment 4
Example 1 gained microbial inoculum is inoculated in the nutrient solution of grease, and sealing and standing is cultivated, and culture temperature is 30 DEG C, and incubation time is 20 days, and pH is 4;
Described nutrient solution, by mass percentage, containing CaCl
21 ‰, MgCl
22 ‰, CaCO
31 ‰, NaCl 4 ‰, urea 4 ‰, KH
2pO
410 ‰, grease 2%, all the other are water.
Embodiment 5
Example 2 gained microbial inoculum is inoculated in the nutrient solution of grease, and sealing and standing is cultivated, and culture temperature is 40 DEG C, and incubation time is 10 days, and pH is 8;
Described nutrient solution, by mass percentage, containing CaCl
22 ‰, MgCl
28 ‰, CaCO
35 ‰, NaCl 8 ‰, urea 8 ‰, KH
2pO
410 ‰, grease 1%, all the other are water.
Embodiment 6
Example 3 gained microbial inoculum is inoculated in the nutrient solution of grease, and sealing and standing is cultivated, and culture temperature is 35 DEG C, and incubation time is 15 days, and pH is 5;
Described nutrient solution, by mass percentage, containing CaCl
21.5 ‰, MgCl
24 ‰, CaCO
33 ‰, NaCl 6 ‰, urea 5 ‰, KH
2pO
410 ‰, grease 1.5 %, all the other are water.
Experimental example
Be that raw material carries out mesophilic digestion with recycled wood materials, arrange A, B, C, D two batching formula biogas fermentation experiment, initial charge grease concentration is 10gVS/L.Wherein experimental group A, when biogas fermentation starts, uses anaerobic sludge as inoculum; Wherein experimental group B, when biogas fermentation starts, uses embodiment 1 to be inoculum; Wherein experimental group C, when biogas fermentation starts, uses embodiment 2 to be inoculum; Wherein experimental group D, when biogas fermentation starts, uses embodiment 3 to be inoculum.Whole experiment is to accumulation aerogenesis stops.
Experimental group A, raw material factor of created gase is 730L/kgVS, whole fermentation period 50 days, and 80% time used that cumulative methane output reaches final methane production is 35d.
Experimental group B, raw material factor of created gase is 1150L/kgVS, whole fermentation period 35 days, and 80% time used that cumulative methane output reaches final methane production is 19d.
Experimental group C, raw material factor of created gase is 1110L/kgVS, whole fermentation period 35 days, and 80% time used that cumulative methane output reaches final methane production is 20d.
Experimental group D, raw material factor of created gase is 11
30L/kgVS, whole fermentation period 35 days, 80% time used that cumulative methane output reaches final methane production is 20d.
Claims (8)
1. the methanogenic composite fungus agent of efficient anaerobic degraded grease, is characterized in that described microbial inoculum comprises following bacterial classification:
Anaerovibrio lipolytica DSM 3074
Syntrophomonas erecta DSM 16215
Syntrophomonas bryantii DSM 3014A
Syntrophomonas palmitatica DSM 18709
Desulfovibrio vulgaris DSM 644
Methanospirillum hungatei DSM 13809
Methanobacterium formicicum DSM 1535
Methanobrevibacter smithii DSM861
Methanosaeta concilii DSM 2139
T。
2. microbial inoculum according to claim 1, is characterized in that, described microbial inoculum is number meter by volume, comprising:
anaerovibrio lipolyticadSM 3074 bacterium liquid 25-35 part
syntrophomonas erectadSM 16215 bacterium liquid 5-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid 8-10 part
syntrophomonas palmitaticadSM 18709 bacterium liquid 2-5 part
desulfovibrio vulgarisdSM 644 bacterium liquid 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid 5-8 part
methanobacterium formicicumdSM 1535 bacterium liquid 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid 2-5 part
methanosaeta conciliidSM 2139
tbacterium liquid
5-10 part;
Described bacterium liquid comprises the one in ferment product, bacterium re-suspension liquid.
3. microbial inoculum according to claim 2, is characterized in that, the bacterium of every part of bacterium liquid is dense is 1-4 × 10
8individual/ml.
4. the preparation method of the methanogenic composite fungus agent of efficient anaerobic degraded grease, it is characterized in that, described method comprises the steps:
By bacteria liquid product, by 25-35 part
anaerovibrio lipolyticadSM 3074 bacterium liquid, 5-10 part
syntrophomonas erectadSM 16215 bacterium liquid, 8-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid, 2-5 part
syntrophomonas palmitaticadSM 18709 bacterium liquid, 5-8 part
desulfovibrio vulgarisdSM 644 bacterium liquid, 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid, 2-5 part
methanobacterium formicicumdSM 1535 bacterium liquid, 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid, 5-10 part
methanosaeta conciliidSM 2139
tbe inoculated in the nutrient solution of grease after the mixing of bacterium liquid, sealing and standing cultivates 10-20 days, and culture temperature is 30 DEG C-40 DEG C, and pH is 4-8;
Described nutrient solution, by mass percentage, containing CaCl
21-2 ‰, MgCl
22-8 ‰, CaCO
31-5 ‰, NaCl 4-8 ‰, urea 4-8 ‰, KH
2pO
410 ‰, grease 1-2%, all the other are water.
5. method according to claim 4, is characterized in that, the bacterium of every part of bacterium liquid is dense is 1-4 × 10
8individual/ml.
6. the application of the microbial inoculum described in any one of claim 1-3 on anaerobic degradation grease methane phase.
7. application according to claim 5, is characterized in that, the method for described application comprises the steps:
By bacteria liquid product, by 25-35 part
anaerovibrio lipolyticadSM 3074 bacterium liquid, 5-10 part
syntrophomonas erectadSM 16215 bacterium liquid, 8-10 part
syntrophomonas bryantiidSM 3014A bacterium liquid, 2-5 part
syntrophomonas palmitaticadSM 18709 bacterium liquid, 5-8 part
desulfovibrio vulgarisdSM 644 bacterium liquid, 5-8 part
methanospirillum hungateidSM 13809 bacterium liquid, 2-5 part
methanobacterium formicicumdSM 1535 bacterium liquid, 2-5 part
methanobrevibacter smithiidSM861 bacterium liquid, 5-10 part
methanosaeta conciliidSM 2139
tbe inoculated in recycled wood materials after the mixing of bacterium liquid, grease concentration is 5-20 gVS/L, at 30 DEG C of-40 DEG C of temperature, carry out gassy fermentation.
8. application according to claim 7, is characterized in that, the bacterium of every part of bacterium liquid is dense is 1-4 × 10
8individual/ml.
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Cited By (2)
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|---|---|---|---|---|
| WO2021046857A1 (en) * | 2019-09-14 | 2021-03-18 | 南京大学（溧水）生态环境研究院 | Mixed bacteria for use in anaerobic treatment of kitchen waste |
| WO2021046859A1 (en) * | 2019-09-14 | 2021-03-18 | 南京大学（溧水）生态环境研究院 | Mixed bacteria for anaerobic treatment of kitchen waste |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021046857A1 (en) * | 2019-09-14 | 2021-03-18 | 南京大学（溧水）生态环境研究院 | Mixed bacteria for use in anaerobic treatment of kitchen waste |
| WO2021046859A1 (en) * | 2019-09-14 | 2021-03-18 | 南京大学（溧水）生态环境研究院 | Mixed bacteria for anaerobic treatment of kitchen waste |
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