CN101705199B - Methane-producing composite microbial inoculum and preparation method thereof - Google Patents
Methane-producing composite microbial inoculum and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a methane-producing composite microbial inoculum and a preparation method thereof, which can solve the long-standing difficult problems that the existing biogas digester is lack of high-efficient methane-producing bacteria, thereby resulting in long initial start-up time of biogas production and poor stability of the biogas production. Methanosarcina acetoacidophilum DSM-N0.2834, methanobacterium formicicum DSM-No.1535, methanobrevibacter arboriphilicus DSM-No.1125, methanolobus tindarius DSM-No.2278 and methanothrix concilii DSM-No.6752 collected in German collection of microorganisms and cell cultures are adopted and inoculated in a liquid methane bacterial culture medium under the anaerobic condition, the independent enlarged culture is carried out grade by grade respectively, and bacterial liquid after the cultured methane bacteria is mixed together according to the volume ratio, thereby preparing a liquid methane-producing composite microbial inoculum; and the liquid methane-producing composite microbial inoculum can also be prepared into a solid methane-producing composite microbial inoculum. The method has reasonable design and adopts the high-efficient strains with the optimal combination and the rapid propagation technology, and the developed high-efficient methane-producing composite microbial inoculum can significantly accelerate the start-up time of the biogas production when in new construction of the biogas digester and large material change, and improve the efficiency and the stability of the biogas production of the biogas digester.
Description
Technical field
The present invention relates to a kind of methane producing composite bacterium agent and preparation method thereof, the methane producing composite bacterium agent is mainly used in the startup agent of new construction of marsh gas tank and lacks the methane-generating pit of methanogen, can accelerate the startup that new construction of marsh gas tank produces biogas, improves gas production rate.
Background technology
At present, according to the pertinent literature report, the inoculum of biogas fermentation startup mainly is the fermented liquid of old methane-generating pit, cloaca mud, pond mud, old manure pit mud etc., wherein contains a large amount of methanogens.In wideling popularize the process of biogas; A ubiquitous outstanding hard nut to crack is that the product biogas initial start time is long; Particularly the place of new construction of marsh gas tank especially in the north, is difficult to find suitable cloaca mud, pond sludge etc. as the inoculum that produces biogas; Leaning on nature to cultivate needs 2-3 month time, and it is long start time to cause producing biogas.Last effect and stable aerogenesis that the quality and quantity of inoculum produces methane phase to anaerobic digestion are extremely important; If the few microbe population of the amount of inoculum is not enough; Especially methanogen is few, and the fermentation starting time is increased, and methanogenic speed is slow; Cause simultaneously the accumulation of acid easily, thereby make the biogas fermentation failure.The generation of biogas is a kind of anaerobic fermentation process, and methanobacteria is as one type of important flora in the biogas production process, and its research receives widely and paying attention to.Methanogen be one type can be with inorganic (H
2/ CO
2) or low-carbon (LC) organism (like formic acid, acetate etc.) anaerobically fermenting change into the archeobacteria of methane and carbonic acid gas, be last member that anaerobic digestion produces the biogas process.Methanobacteria is as the crucial flora in the marsh gas fermentation processes, and its growth metabolism situation directly has influence on the efficient that methane produces.For this reason; (publish in September, 1997 for Zhao Yizhang chief editor, press of Chengdu Univ. of Science & Technology at " methane-producing bacteria and research method " book; 182nd, 184,193-195 page or leaf); Respectively to the research method of the taxonomy of methanogen, ecology, physiology, biological chemistry, micromorphology, molecular genetics, methanogen, and the anaerobic bacterium under the extreme environment etc. all carried out detailed elaboration, and the representative species of existing methanogen is introduced.Growth that has in these representative species and methanogenic TR thereof and pH scope are than broad, and the substrate that various bacterium utilize when producing methane is also different.But because the methanogen growth is slow; The breeding epoch are long, and belong to strict anerobes, and the stripping technique and the equipment thereof of China's methanogen strain are relatively backward; The preservation of high-efficiency high-quality bacterial strain is less, so adopt common method to be difficult to be separated to individual plant and methanogen efficiently generally speaking.
Staple in the biogas is a methane, and its generation receives the influence of multiple envrionment conditions, and the pH value is one of important factor that influences this anaerobic fermentation process.Fermentation substrate in the methane-generating pit is various, and these substrates can be formic acid, acetate, methylamine, the H that methanobacteria utilizes by the anaerobic bacteria flora metabolism in the methane-generating pit
2/ CO
2Etc. multiple product, but the methanobacteria kind in the methane-generating pit is comparatively single, cause produce biogas start slow or gas production rate lower.In addition, the TR that most of methanobacterias adapt to is narrower, and variation of temperature has influenced product biogas amount and start time.
Utilizing organic its raw material sources of produce biogas many, mainly is some macromolecular organism, and the speed of its decomposition also has influence on the productive rate of biogas.Organism is converted into the biogas process and mainly contains hydrolysis stage, souring stage, product hydrogen product acetate stage and produce methane phase under the effect of anaerobion, this four-stage is accomplished by the enzyme of different mikrobes or its generation respectively.In order to accelerate organic rate of decomposition (hydrolysis and acidifying), improve biogas yield, the mikrobe of adding decomposing organic matter or enzyme are in methane-generating pit.For example patent publication No. is in " a kind of used for methane tank complex micro organism fungicide and preparation method thereof " of CN101372666A; A kind of aerobic and double oxygen bacterium that utilizes is disclosed: the complex micro organism fungicide that the fermented liquid of Pseudomonas fluorescence, wax bacillus, Bacillus subtillis, bacillus circulans is prepared into; It is added in the methane-generating pit, accelerate the gas production rate and quickening aerogenesis speed that organic rate of decomposition (hydrolysis and acidifying) promotes methane-generating pit to reach.Patent announcement number for CN100513562C " be used for improving the mixed hydrolysis zymin of biogas fermentation factor of created gase the preparation method ", utilize aerobic bacteria and fungi to carry out plate method and be prepared into zymin.It also is to accelerate organic rate of decomposition (hydrolysis and acidifying) to improve biogas yield that this zymin adds in the methane-generating pit.Above-mentioned complex micro organism fungicide or zymin add the opportunity in the methane-generating pit, only normally produce under the biogas situation and could add at methane-generating pit; If produce under the biogas abnormal conditions or add under the pH situation on the low side; Organism decomposes (hydrolysis and acidifying) soon; Producing methane phase utilizes the product methane number of substrates such as formic acid, acetate not enough; A large amount of accumulation of the acid meeting that hydrolysis and souring stage produce and cause the acidifying phenomenon cause aerogenesis aerogenesis not even less.If when starting, add, at first to have quality good and measure more inoculum, otherwise organism decompose (hydrolysis and acidifying) fast, produce the methane number deficiency and also can cause the acidifying phenomenon, can cause producing biogas and start and fail.
Summary of the invention
The purpose of this invention is to provide a kind of methane producing composite bacterium agent and preparation method thereof; It has solved existing methane-generating pit and has lacked methanogen efficiently; It is long to cause producing the biogas initial start time; The hard nut to crack of aerogenesis poor stability, this method is reasonable in design, adopts the efficient bacterial strain of optimum combination, the technology of breeding fast; The efficient methane producing composite bacterium agent of developing can significantly be accelerated new construction of marsh gas tank and the start time of the product biogas when reloading greatly, and the gas producing efficiency that improves methane-generating pit is stable with aerogenesis.
The technical scheme that the present invention adopted is: the technical essential of this methane producing composite bacterium agent is: adopt be deposited in German DSMZ be numbered DSM-No.2834 have a liking for acetate sarcina methanica (Methanosarcina acetivorans), be numbered DSM-No.1535 formic acid methagen (Methanobacterium formicicum), be numbered the having a liking for tree methane tyrothricin (Methanobrevibacter arboriphilicus), be numbered the Ting Daer methane leaf bacterium (Methanolobus tindarius) of DSM-No.2278 of DSM-No.1125, the Ken Shi hair on the neck that is numbered DSM-No.6752 is sent out methanobacteria (Methanosaeta concilii)) as the inoculation methanobacteria; Under anaerobic be inoculated in the liquid methane bacterium culture medium; Enlarged culturing step by step separately respectively; The bacterium liquid of the said methanobacteria after the cultivation is mixed together by volume, is the agent of liquid methane producing composite bacterium.
The preparation method of above-mentioned methane producing composite bacterium agent is: at first send out methanobacteria DSM-No.6752 as the inoculation methanobacteria with above-mentioned through the having a liking for acetate sarcina methanica DSM-No.2834, formic acid methagen DSM-No.1535, have a liking for tree methane tyrothricin DSM-No.1125 of preservation, Ting Daer methane leaf bacterium DSM-No.2278, Ken Shi hair on the neck; Be inoculated in said liquid methane bacterium culture medium; Distinguish separately enlarged culturing step by step then; Each level was under anaerobic cultivated 5-8 days through 35 ℃; Be prepared into the bacterium liquid of said methanobacteria respectively, the bacterium liquid with said methanobacteria is mixed into the agent of liquid methane producing composite bacterium by volume at last; Be inoculated in the independent said inoculation methanobacteria of enlarged culturing step by step of said liquid methane bacterium culture medium, the volume ratio of the inoculum size of each grade is: methanobacteria liquid: the liquid methane bacterium culture medium=(0.5-2): (10-30); The volume ratio that the bacterium liquid of said methanobacteria is mixed is: the bacterium liquid of having a liking for the acetate sarcina methanica: the bacterium liquid of formic acid methagen: the bacterium liquid of having a liking for tree methane tyrothricin: the bacterium liquid of Ting Daer methane leaf bacterium: the Ken Shi hair on the neck is sent out the bacterium liquid of methanobacteria=(0.5-4.0): (0.5-3.5): (0.6-2.5): (0.4-2.0): (0.2-1.5); Contain sodium formiate 3.0-5.0g in every liter of said liquid methane bacterium culture medium, sodium acetate 5.0-8.0g, methyl alcohol 2.0-4.0ml, ethanol (95%) 3.0-5.0ml, MgCl
20.1-0.5g, NHCl 0.5-1.0g, yeast powder 0.5-1.0g, surplus is a water.
The agent of aforesaid liquid methane producing composite bacterium can be used as inoculum and joins as being prepared into the agent of solid methane producing composite bacterium in the mud of sorbent material or the pig manure; Its operation steps is following: the agent of said liquid methane producing composite bacterium is joined in the above-mentioned mud or pig manure that water ratio is 70-75%, add aforesaid liquid methanobacteria substratum simultaneously; The weight ratio that adds is: liquid methane producing composite bacterium agent: mud or pig manure: the liquid methane bacterium culture medium=(0.5-3.5): (6.0-8.0): (0.5-2.5), mixing is back in 35 ℃ of cultivations 7-10 days, cultivates to be the agent of solid methane producing composite bacterium.
The agent of above-mentioned solid methane producing composite bacterium also can be used as the inoculum of next time cultivating the agent of solid methane producing composite bacterium; With above-mentioned mud or pig manure as sorbent material; Add aforesaid liquid methanobacteria substratum simultaneously; The weight ratio that adds is: solid methane producing composite bacterium agent: mud or pig manure: liquid methane bacterium culture medium=(0.5-4.0): (6.5-8.5): (0.5-2.5), after mixing, in 35 ℃ of cultivations 8-12 days, cultivate and be the agent of solid methane producing composite bacterium.
Advantage and positively effect that the present invention has are: because the inventive method is reasonable in design, adopt the efficient bacterial strain of optimum combination, the technology of breeding fast, so developed efficient methane producing composite bacterium agent.Each strain methanogen in this methane producing composite bacterium agent has following biology, physiology characteristic:
Have a liking for acetate sarcina methanica (Methanosarcina acetivorans): the irregular sphere of diameter 1.5～2.5um, form microsporangium, Gram-positive is not moved, and bacterium colony is yellowish, 10～50 ℃ of growth temperatures, pH5.5～9.0.Utilize acetate, methyl alcohol, methylamine, n n dimetylaniline to produce methane.
Formic acid methagen (Methanobacterium formicicum): elongated rod shape, wide 0.4～0.8um, variable-length, the bacterium colony pearl, 37～45 ℃ of growth temperatures, ph optimum 6.6～7.8 utilizes H
2/ CO
2, formic acid produces methane.
Have a liking for tree methane tyrothricin (Methanobrevibacter arboriphilicus): lack and microbacterium (microbacteria), 0.5-0.7 * 1.8-3.5um, Dan Sheng, paired or chaining, Gram-positive, bacterium colony is little, and faint yellow circle is utilized H
2/ CO
2, formic acid produces methane.37～39 ℃ of growth temperatures, pH6.9～7.4.
Ting Daer methane leaf bacterium (Methanolobus tindarius): diameter is 0.8～1.25um, irregular sphere, and Dan Sheng forms loose aggregates sometimes, the green white of bacterium colony, 10～45 ℃ of growth temperatures, growth pH5.8～8.0 utilize methyl alcohol, methylamine to produce methane.
The Ken Shi hair on the neck is sent out methanobacteria (Methanosaeta concilii): shaft-like, it is thread that liquid culture becomes, 0.8 * 1.8～2um, and Gram-negative fragments into shaft-like during vibration.PH6.8～8.2,30～45 ℃ of growth temperatures. only utilize acetic acid and methane producing.
This shows that the present invention can make full use of the combination and compatibility of above high-quality methanogen strain, these intermediate products of producing in the marsh gas fermentation processes are produced methane as the substrate of methanobacteria.Cause is produced biogas in marsh gas fermentation processes pH scope is generally at 6.5-8.5, if it is few and slow to be lower than this scope product biogas; Particularly in the start-up course when newly-built pond and existing methane-generating pit reload greatly, very slow if pH is lower than 6.5 startup meetings.In addition, present most methane-generating pit, particularly methane-generating pit for farm household produce the temperature of biogas all by natural temperature, and variation of temperature is also big to the gas production rate influence, and is produced from newly-built pond can influence start time when biogas starts.Therefore; The present invention is preferred growth methanotrophic bacterial strain pH scope and TR adapt to the methanogen strain than broad; As the growth pH scope of having a liking for the acetate sarcina methanica is at 5.5-9.0, and growth temperature range is at 10-50 ℃, and the growth pH scope of Ting Daer methane leaf bacterium is 5.5-8.0; The growth temperature range of having a liking for tree methane tyrothricin and Ting Daer methane leaf bacterium is at 10-45 ℃, and this had not only reached many bacterium complementary effect, but also has played the effect of quick breeding.The invention solves existing methane-generating pit and lack methanogen efficiently, it is long to cause producing the biogas initial start time, the hard nut to crack of aerogenesis poor stability.This methane producing composite bacterium agent can be used as the inoculum that new construction of marsh gas tank and the existing methane-generating pit product biogas when reloading greatly starts; Particularly be difficult to find the place of producing the biogas inoculum, the start time of the product biogas when accelerating new construction of marsh gas tank and existing methane-generating pit and reloading greatly; It also can add in the pH of the biogas fermentation methane-generating pit on the low side, increases the quantity of the methanogen that produces methane phase, and the gas producing efficiency that improves methane-generating pit is stable with aerogenesis.
Embodiment
Below in conjunction with embodiment the present invention is elaborated.This methane producing composite bacterium agent is in the representative species according to the existing methanogen of " methane-producing bacteria and research method " book introduction; Optimum combination through different good quality and high output methane bacterial strains and correlation function flora; Select growth and methanogenic TR thereof and pH scope bacterial strain than broad; Through introducing this type of fine methanogen strain of Germany, utilize quick breeding technology to cultivate enough product methane inoculums in the short period of time.The concrete practice is: adopt from Germany and purchase the next preferred bacterium that is deposited in German DSMZ (DSMZ); Promptly be numbered the having a liking for acetate sarcina methanica (Methanosarcina acetivorans), be numbered the formic acid methagen (Methanobacterium formicicum) of DSM-No.1535 of DSM-No.2834, have a liking for the tree methane tyrothricin (Methanobrevibacter arboriphilicus), the Ting Daer methane leaf bacterium (Methanolobus tindarius) that is numbered DSM-No.2278, the Ken Shi hair on the neck that is numbered DSM-No.6752 that is numbered DSM-No.1125 sends out methanobacteria (Methanosaetaconcilii)) as the inoculation methanobacteria; Under anaerobic be inoculated in the liquid methane bacterium culture medium of preparation in advance; Respectively separately by the step of anaerobism pipe → anaerobism bottle → anaerobic jar enlarged culturing step by step; Each bacterium liquid of above-mentioned methanobacteria after the cultivation is mixed together by volume, promptly becomes the methane producing composite bacterium agent with the bigger liquid of the pH scope utilizing the substrate broad, adapt to produce biogas and TR.
According to actual needs, the preparation method of above-mentioned methane producing composite bacterium agent can prepare agent of liquid methane producing composite bacterium and the agent of solid methane producing composite bacterium, and its concrete operations step is following:
The agent of preparation liquid methane producing composite bacterium
Step 1, preparation liquid methane bacterium culture medium
The component and the content thereof of liquid methane bacterium culture medium are following, weigh every liter of needed raw material of substratum of preparation in proportion to be: sodium formiate 3.0-5.0g, sodium acetate 5.0-8.0g, methyl alcohol 2.0-4.0ml, ethanol (95%) 3.0-5.0ml, MgCl
20.1-0.5g, NHCl 0.5-1.0g, yeast powder 0.5-1.0g, surplus is a water.Prepare above-mentioned raw materials, substratum is boiled 15 minutes after, feed the anaerobic high pure nitrogen and prevent air admission; Under oxygen free condition, be sub-packed in anaerobism pipe, anaerobism bottle or the anaerobic jar; Cover sealing cover, and in 121 ℃ the sterilization 30 minutes after, the cooling substratum to room temperature subsequent use.
The bacterium liquid of step 2, preparation methanobacteria
At first send out methanobacteria DSM-No.6752 as the inoculation methanobacteria through the having a liking for acetate sarcina methanica DSM-No.2834, formic acid methagen DSM-No.1535, have a liking for tree methane tyrothricin DSM-No.1125 of preservation, Ting Daer methane leaf bacterium DSM-No.2278, Ken Shi hair on the neck, be inoculated in subsequent use liquid methane bacterium culture medium above-mentioned.
Distinguish separately enlarged culturing step by step then, promptly enlarge step by step by anaerobism pipe (1 grade) → anaerobism bottle (2 grades) → anaerobic jar, the volume ratio of the inoculum size of each grade is: methanobacteria liquid: the liquid methane bacterium culture medium=(0.5-2): (10-30).Each level promptly is prepared into the bacterium liquid of methanobacteria respectively under anaerobic through 35 ℃ of cultivations 5-8 days.
At last with the bacterium liquid of above-mentioned each methanobacteria; Be mixed by following volume ratio: the bacterium liquid of having a liking for the acetate sarcina methanica: the bacterium liquid of formic acid methagen: the bacterium liquid of having a liking for tree methane tyrothricin: the bacterium liquid of Ting Daer methane leaf bacterium: the Ken Shi hair on the neck is sent out the bacterium liquid of methanobacteria=(0.5-4.0): (0.5-3.5): (0.6-2.5): (0.4-2.0): (0.2-1.5), be the agent of liquid methane producing composite bacterium.
The agent of preparation solid methane producing composite bacterium
Because the agent of liquid methane producing composite bacterium is not easy to transportation, and the preservation time is shorter, so can the agent of aforesaid liquid methane producing composite bacterium be joined sorbent material as inoculum, is prepared into the agent of solid methane producing composite bacterium.Sorbent material adopts both cheap, again can be as the part nutrition of methane producing composite bacterium agent, and the methanobacteria in the methane producing composite bacterium agent is had sewage work's water ratio of proliferation function is mud or the pig manure of 70-75%.Its concrete operations step is following:
The agent of aforesaid liquid methane producing composite bacterium is joined in the mud or pig manure that water ratio is 70-75%, add the liquid methane bacterium culture medium simultaneously; The weight ratio that adds is: liquid methane producing composite bacterium agent: mud or pig manure: the liquid methane bacterium culture medium=(0.5-3.5): (6.0-8.0): (0.5-2.5), mix the back in 35 ℃ of cultivations 7-10 days, i.e. cultivation is the agent of solid methane producing composite bacterium.
Because of above-mentioned each methanobacteria is cultivated into bacterium liquid respectively; Remix becomes the agent of liquid methane producing composite bacterium, operates cumbersomely, and the time is longer; So can be with the part (about 20%) of above-mentioned solid methane producing composite bacterium agent as the inoculum of cultivating the agent of solid methane producing composite bacterium next time; As sorbent material, add the liquid methane bacterium culture medium with mud or pig manure simultaneously, the weight ratio of adding is: solid methane producing composite bacterium agent: mud or pig manure: the liquid methane bacterium culture medium=(0.5-4.0): (6.5-8.5): (0.5-2.5); In 35 ℃ of cultivations 8-12 days, promptly cultivating was the agent of solid methane producing composite bacterium after mixing.
To collect the biogas of generation in the culturing process of above-mentioned solid methane producing composite bacterium agent.Solid methane producing composite bacterium agent after the cultivation reserves about 20% as inoculum by the total amount of the solid methane producing composite bacterium agent that will prepare next time, and remaining packs.
Embodiment one
The agent of preparation liquid methane producing composite bacterium
The preparation method one
The component of liquid methane bacterium culture medium and content thereof (g or ml/ liter): sodium formiate 3g, sodium acetate 8g, methyl alcohol 2ml, ethanol (95%) 5ml, MgCl 0.1g, NHCl 1g, yeast powder 0.5g adds water to 1 liter.
Prepare desired raw material and add institute's water requirement, the liquid methane bacterium culture medium is boiled feed the anaerobic high pure nitrogen after 15 minutes and prevent air admission, under oxygen free condition, be sub-packed in anaerobism pipe and the anaerobism bottle, cover sealing cover and sterilized 30 minutes in 121 ℃.Went out the liquid methane bacterium culture medium cooling back of bacterium by above-mentioned methanobacteria liquid through preservation: the volume ratio of liquid methane bacterium culture medium=0.5: 10 is inoculated in separately every strain bacterium and is equipped with in the liquid methane bacterium culture medium anaerobism test tube; In 35 ℃ of cultivations 8 days, the bacterium liquid that this anaerobism test tube is cultivated was 1 grade of kind.Then by methanobacteria liquid: the volume ratio of liquid methane bacterium culture medium=0.5: 10 inserts 1 grade of kind respectively and is equipped with in the liquid methane bacterium culture medium anaerobism bottle, cultivates 5 days in 35 ℃, and the nutrient solution of cultivating in this anaerobism bottle is 2 grades of kinds.Liquid methane bacterium culture medium component and content with anaerobic jar is cultivated are the same, and every strain bacterium is also distinguished single culture.Different is earlier with required water ebuillition of heated 5 minutes, adds the liquid methane bacterium culture medium and continues to boil 15 minutes, packs in the anaerobic jar by the liquid methane bacterium culture medium method of dress anaerobism test tube and anaerobism bottle then and seals, and 121 ℃ went out 30 minutes.Liquid methane bacterium culture medium cooling back is by methanobacteria liquid: the volume ratio of substratum=0.5: 10 is inoculated in 2 grades of kinds of cultivating in the anaerobism bottle in the anaerobic jar separately every strain bacterium, after 8 days, becomes the bacterium liquid of respectively inoculating methanobacteria in 35 ℃ of cultivations.
The bacterium liquid of the methanobacteria of cultivating is respectively mixed by volume; Promptly have a liking for acetate sarcina methanica liquid: formic acid methagen liquid: have a liking for tree methane tyrothricin liquid: Ting Daer methane leaf bacterium liquid: Ken Shi mane methanobacteria liquid=0.5: 3.5: 2.5: 0.4: 0.2, be mixed into the agent of liquid methane producing composite bacterium.
The preparation method two
The raw material of liquid methane bacterium culture medium and content thereof (g or ml/ liter): sodium formiate 5g, sodium acetate 5g, methyl alcohol 4ml, ethanol (95%) 3ml, MgCl 0.3g, NHCl 0.8g, yeast powder 0.8g adds water to 1 liter.Liquid methane bacterium culture medium preparation method and culture condition that anaerobism test tube, anaerobism bottle and anaerobic jar are cultivated are identical with method one, and difference is inoculum size and incubation time.The inoculum size that is inoculated in the anaerobism test tube is above-mentioned methanobacteria liquid through preservation: liquid methane bacterium culture medium=1: 20 (volume ratio), cultivated 6 days in 35 ℃; The inoculum size that is inoculated into the anaerobism bottle from the anaerobism test tube is a methanobacteria liquid: liquid methane bacterium culture medium=1.5: 30 (volume ratio), cultivated 7 days in 35 ℃; The inoculum size that is inoculated into anaerobic jar from the anaerobism bottle is a methanobacteria liquid: liquid methane bacterium culture medium=1.5: 30 (volume ratio) in 35 ℃ of cultivations 6 days, becomes the bacterium liquid of each methanobacteria.
The bacterium liquid of the methanobacteria of cultivating is respectively mixed by volume; Promptly have a liking for acetate sarcina methanica liquid: formic acid methagen liquid: have a liking for tree methane tyrothricin liquid: Ting Daer methane leaf bacterium liquid: Ken Shi mane methanobacteria liquid=4.0: 0.5: 1.5: 2.0: 2.0, be mixed into the agent of liquid methane producing composite bacterium.
The preparation method three
The raw material of liquid methane bacterium culture medium and content thereof (g or ml/ liter): sodium formiate 4g, sodium acetate 6g, methyl alcohol 3ml, ethanol (95%) 4ml, MgCl 0.5g, NHCl 1.0g, yeast powder 1.0g adds water to 1 liter.Liquid methane bacterium culture medium preparation method and culture condition that anaerobism test tube, anaerobism bottle and anaerobic jar are cultivated are identical with method one, and difference also is inoculum size and incubation time.The inoculum size that is inoculated in the anaerobism test tube is above-mentioned methanobacteria liquid through preservation: liquid methane bacterium culture medium=2.0: 30 (volume ratio), cultivated 6 days in 35 ℃; The inoculum size that is inoculated into the anaerobism bottle from the anaerobism test tube is a methanobacteria liquid: liquid methane bacterium culture medium=2.0: 20 (volume ratio), cultivated 5 days in 35 ℃; The inoculum size that is inoculated into anaerobic jar from the anaerobism bottle is a methanobacteria liquid: liquid methane bacterium culture medium=2.0: 20 (volume ratio) in 35 ℃ of cultivations 5 days, becomes the bacterium liquid of each methanobacteria.
The bacterium liquid of the methanobacteria of cultivating is respectively mixed by volume; Promptly have a liking for acetate sarcina methanica liquid: formic acid methagen liquid: have a liking for tree methane tyrothricin liquid: Ting Daer methane leaf bacterium liquid: Ken Shi mane methanobacteria liquid=2.0: 1.5: 0.6: 1.0: 1.0, be mixed into the agent of liquid methane producing composite bacterium.
The preparation method four
The raw material of liquid methane bacterium culture medium and content thereof (g or ml/ liter): sodium formiate 5g, sodium acetate 5g, methyl alcohol 3ml, ethanol (95%) 4ml, MgCl 0.5g, NHCl 1.0g, yeast powder 1.0g adds water to 1 liter.Liquid methane bacterium culture medium preparation method and culture condition that anaerobism test tube, anaerobism bottle and anaerobic jar are cultivated are identical with method one, and difference is inoculum size and incubation time.The inoculum size that is inoculated in the anaerobism test tube is above-mentioned methanobacteria liquid through preservation: liquid methane bacterium culture medium=2.0: 20 (volume ratio), cultivated 5 days in 35 ℃; The inoculum size that is inoculated into the anaerobism bottle from the anaerobism test tube is a methanobacteria liquid: liquid methane bacterium culture medium=1.5: 30 (volume ratio), cultivated 8 days in 35 ℃; The inoculum size that is inoculated into anaerobic jar from the anaerobism bottle is a methanobacteria liquid: liquid methane bacterium culture medium=2.0: 20 (volume ratio) in 35 ℃ of cultivations 7 days, becomes the bacterium liquid of each methanobacteria.
The bacterium liquid of the methanobacteria of cultivating is respectively mixed by volume; Promptly have a liking for acetate sarcina methanica liquid: formic acid methagen liquid: have a liking for tree methane tyrothricin liquid: Ting Daer methane leaf bacterium liquid: Ken Shi mane methanobacteria liquid=2.0: 2.0: 1.5: 1.0: 1.0, be mixed into the agent of liquid methane producing composite bacterium.
Embodiment two preparation solid methane producing composite bacterium agent
The preparation method one
The agent of liquid methane producing composite bacterium is joined in the mud that water ratio is 70-75% and adds the liquid methane bacterium culture medium; Its ratio (weight ratio) is the agent of liquid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=0.5: 6.0: 2.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 10 days.This solid methane producing composite bacterium agent with 80% is packed, and remaining about 20% as the inoculum of cultivating the agent of solid methane producing composite bacterium next time; Its ratio (weight ratio) is the agent of solid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=4: 8.5: 0.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 8 days.
The preparation method two
The agent of liquid methane producing composite bacterium is joined in the mud that water ratio is 70-75% and adds the liquid methane bacterium culture medium; Its ratio (weight ratio) is the agent of liquid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=3.5: 8.0: 0.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 8 days.This solid methane producing composite bacterium agent with 80% is packed, and remaining about 20% as the inoculum of cultivating the agent of solid methane producing composite bacterium; Its ratio (weight ratio) is the agent of solid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=0.5: 6.5: 2.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 12 days.
The preparation method three
The agent of liquid methane producing composite bacterium is joined in the mud that water ratio is 70-75% and adds the liquid methane bacterium culture medium; Its ratio (weight ratio) is the agent of liquid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=2.0: 7.0: 1.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 7 days.This solid methane producing composite bacterium agent with 80% is packed, and remaining about 20% as the inoculum of cultivating the agent of solid methane producing composite bacterium; Its ratio (weight ratio) is the agent of solid methane producing composite bacterium: water ratio is the mud of 70-75%: liquid methane bacterium culture medium=2.5: 7.5: 1.5; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 10 days.
The preparation method four
To join water ratio be in 75% the pig manure and add the liquid methane bacterium culture medium with the agent of liquid methane producing composite bacterium; Its ratio (weight ratio) is the agent of liquid methane producing composite bacterium: water ratio is the pig manure of 70-75%: liquid methane bacterium culture medium=1.5: 7.5: 1.0; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 9 days.This solid methane producing composite bacterium agent with 80% is packed, and remaining about 20% as the inoculum of cultivating the agent of solid methane producing composite bacterium; Its ratio (weight ratio) is the agent of solid methane producing composite bacterium: water ratio is 75% pig manure: liquid methane bacterium culture medium=3.0: 7.0: 1.0; 35 ℃ of cultivations under anaerobic after the mixing; Collect the biogas that produces in the culturing process, cultivate and be the agent of solid methane producing composite bacterium in 9 days.
The effect test of embodiment three solid methane producing composite bacterium agent
(1) with inoculating anaerobic sludge as contrast
By the agent of solid methane producing composite bacterium: Ts content is that the agent of solid methane producing composite bacterium is inserted Ts content is in 5% the pig manure to the inoculum size (volume ratio) of 5% pig manure=1: 9; Use in addition anaerobic sludge: Ts content is that the inoculum size (volume ratio) of 5% pig manure=1: 9 is as contrast, respectively at 20 ℃, 50 ℃ and normal temperature condition cultivation down.Under 20 ℃ of conditions, the gas production rate of the methane composite fungus agent of practicing midwifery after the 15th day is 2.5 times of contrast; Under 50 ℃ of conditions, the gas production rate of the 2nd day methane composite fungus agent of practicing midwifery is 2 times of contrast, and the gas production rate of the methane composite fungus agent of practicing midwifery after the 7th day rises very fast, and gas production rate reached peak in the 10th day.Just begin to rise and contrast the 16th day gas production rate, the 19th talent reaches peak, and the total gas production of the methane composite fungus agent of in 20 days, practicing midwifery is 2.7 times of contrast; Under normal temperature condition, the total gas production of the methane composite fungus agent of practicing midwifery in three weeks is 1.6 times of contrast
(2) use no inoculum as contrast
By solid methane producing composite bacterium agent: Ts is that respectively the agent of solid methane producing composite bacterium to be inserted Ts be in 5% the pig manure for the inoculum size (volume ratio) of 5% pig manure=1: 49,1: 19,1: 9; And, divide under 20 ℃, 50 ℃ conditions and cultivate with using no inoculum as contrast.Under 20 ℃ of conditions, inoculum size be 1: 49,1: 19,1: 9 o'clock respectively at the 8th day, 5 days, 2 days beginning aerogenesis, methane concentration reached 46%, 51%, 58.9% respectively in the 14th day, and contrasted the 14th day beginning aerogenesis, methane concentration just 4.3%.Under 50 ℃ of bars, inoculum size be 1: 49,1: 19,1: 9 o'clock all the 2nd day the beginning aerogenesis, methane concentration reached 32%, 39%, 51.2% respectively in the 7th day; Begin aerogenesis, methane concentration ability 13.7% and contrast the 4th day.
Claims (4)
1. methane producing composite bacterium agent; It is characterized in that: adopt be deposited in German DSMZ be numbered DSM-No.2834 have a liking for acetate sarcina methanica (Methanosarcina acetivorans), be numbered DSM-No.1535 formic acid methagen (Methanobacterium formicicum), be numbered having a liking for tree methane tyrothricin (Methanobrevibacter arboriphilicus), being numbered the Ting Daer methane leaf bacterium (Methanolobus tindarius) of DSM-No.2278 and the Ken Shi hair on the neck that is numbered DSM-No.6752 is sent out methanobacteria (Methanosaetaconcilii) of DSM-No.1125) as inoculating methanobacteria; Under anaerobic be inoculated in the liquid methane bacterium culture medium; Enlarged culturing step by step separately respectively; The bacterium liquid of the said methanobacteria after the cultivation is mixed together by volume, is the methane producing composite bacterium agent of liquid.
2. the preparation method of methane producing composite bacterium agent according to claim 1; It is characterized in that: at first send out methanobacteria DSM-No.6752 as the inoculation methanobacteria through the having a liking for acetate sarcina methanica DSM-No.2834, formic acid methagen DSM-No.1535, have a liking for tree methane tyrothricin DSM-No.1125 of preservation, Ting Daer methane leaf bacterium DSM-No.2278, Ken Shi hair on the neck said; Be inoculated in the liquid methane bacterium culture medium; Distinguish separately enlarged culturing step by step then; Each level is prepared into the bacterium liquid of said methanobacteria respectively under anaerobic through 35 ℃ of cultivations 5-8 days, and the bacterium liquid with said methanobacteria is mixed into the agent of liquid methane producing composite bacterium by volume at last; Be inoculated in the independent said methanobacteria of enlarged culturing step by step of said liquid methane bacterium culture medium, the volume ratio of the inoculum size of each grade is: methanobacteria liquid: the liquid methane bacterium culture medium=(0.5-2): (10-30); The volume ratio that the bacterium liquid of said methanobacteria is mixed is: the bacterium liquid of having a liking for the acetate sarcina methanica: the bacterium liquid of formic acid methagen: the bacterium liquid of having a liking for tree methane tyrothricin: the bacterium liquid of Ting Daer methane leaf bacterium: the Ken Shi hair on the neck is sent out the bacterium liquid of methanobacteria=(0.5-4.0): (0.5-3.5): (0.6-2.5): (0.4-2.0): (0.2-1.5); Contain sodium formiate 3.0-5.0g in every liter of said liquid methane bacterium culture medium, sodium acetate 5.0-8.0g, methyl alcohol 2.0-4.0ml, 95% ethanol 3.0-5.0ml, MgCl
20.1-0.5g, NHCl 0.5-1.0g, yeast powder 0.5-1.0g, surplus is a water.
3. the preparation method of methane producing composite bacterium agent according to claim 2; It is characterized in that: the agent of said liquid methane producing composite bacterium is joined as being prepared into the agent of solid methane producing composite bacterium in the mud of sorbent material or the pig manure as inoculum; Its operation steps is following: the agent of said liquid methane producing composite bacterium is joined in the said mud or pig manure that water ratio is 70-75%, add said liquid methane bacterium culture medium simultaneously; The weight ratio that adds is: liquid methane producing composite bacterium agent: mud or pig manure: the liquid methane bacterium culture medium=(0.5-3.5): (6.0-8.0): (0.5-2.5), mixing is back in 35 ℃ of cultivations 7-10 days, cultivates to be the agent of solid methane producing composite bacterium.
4. the preparation method of methane producing composite bacterium agent according to claim 3; It is characterized in that: with the agent of said solid methane producing composite bacterium as the inoculum of cultivating the agent of solid methane producing composite bacterium next time; With said mud or pig manure as sorbent material; Add said liquid methane bacterium culture medium simultaneously; The weight ratio that adds is: solid methane producing composite bacterium agent: mud or pig manure: liquid methane bacterium culture medium=(0.5-4.0): (6.5-8.5): (0.5-2.5), after mixing, in 35 ℃ of cultivations 8-12 days, cultivate and be the agent of solid methane producing composite bacterium.
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| CN105802868A (en) * | 2014-12-27 | 2016-07-27 | 甘肃新德燃气有限公司 | Mixed methane bacteria and culture method thereof |
| CN104862258A (en) * | 2015-06-11 | 2015-08-26 | 中国科学院成都生物研究所 | Complex microbial agent for high-organic-load and high-temperature biogas fermentation as well as preparation method and application thereof |
| CN106948797B (en) * | 2017-04-07 | 2020-02-11 | 中国地质大学（北京） | Method for increasing production of coal bed gas |
| US20210079340A1 (en) | 2019-09-17 | 2021-03-18 | Indian Oil Corporation Limited | Microbial consortium for enhancing the methane production from feedstock |
| CN114933996B (en) * | 2022-06-08 | 2023-10-27 | 四川剑南春（集团）有限责任公司 | Pit mud synthesis microbiome and preparation and application thereof |
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