CN101481676A - Preparation of composite bacteria - Google Patents
Preparation of composite bacteria Download PDFInfo
- Publication number
- CN101481676A CN101481676A CNA2009101030855A CN200910103085A CN101481676A CN 101481676 A CN101481676 A CN 101481676A CN A2009101030855 A CNA2009101030855 A CN A2009101030855A CN 200910103085 A CN200910103085 A CN 200910103085A CN 101481676 A CN101481676 A CN 101481676A
- Authority
- CN
- China
- Prior art keywords
- accounts
- bacterial strain
- cellulose
- dsm
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses a method for preparing composite bacterium, comprising the following steps: inoculating microorganism of zymogenous bacteria, hydrogen-producing acetogenic bacteria and methanogen respectively belonging to facultative anaerobic bacteria and strict anaerobic bacteria which are mixed according to a certain proportion into a culture solution containing cellulose, sealing and keeping standing to culture at a temperature of between 30 to 50 DEG C and pH value of 3 to 10, wherein the culture solution containing cellulose contains CaCO3, 0.3 to 0.7 percent of NaCl, 0.5 to 1 percent of cellulose, 0.1 to 0.4 percent of urea, 0.1 to 0.3 percent of peptone and 0.05 to 0.15 percent of yeast powder, and water is used as a solvent. The composite bacterium having effective cellulose degrading property and capable of generating methane is prepared by the method.
Description
Technical field
The present invention relates to cellulose degradation and recovery energy technical field, particularly a kind of making method of composite bacteria of cellulose-degrading methane producing.
Background technology
Natural fiber class material is biomass the abundantest on the earth, and except that small part was utilized, most forms with waste were present in the physical environment.In order fully to develop the abundant renewable resources of this class, scholar's cellulosic biological degradation that begins one's study was just arranged as far back as 1883 and 1886, particularly because fossil energy such as oil, coal price is constantly soaring in recent years, the problem that the Mierocrystalline cellulose biology is converted into new forms of energy obtains the extensive concern of Chinese scholars especially.
Under the state of nature, cellulosic thorough degraded is the long-time results of interaction of multiple microorganism in microorganism system, and this process only depends on a kind of microorganism to realize.Because pure growth has following shortcoming: it is single promptly to produce enzyme, and cellulosic degradation production cellobiose and glucose can suppress the active of cellulase and check the synthetic of cellulase; The accumulation of volatile acid can make nutrient solution pH drop to very low easily and can't return to the condition that is fit to the culture growth, thereby makes the transformation efficiency of fiber substance low.And, utilize pure growth and pure enzyme liberating lignocellulose to have certain degree of difficulty under the situation of pre-treatment and/or sterilization.Therefore, in carrying out cellulose macromolecule Research on degradation process, to consider synergy between the microorganism.
Many bacterial strains mixed culture is not only produced enzyme and is had diversity, can remove the feedback inhibition of product simultaneously, regulates the pH of nutrient solution, thereby improves cellulosic transformation efficiency.Compare with pure growth, stable conspiracy relation between the microorganism in the mixed bacterial is to decomposing the incomparable advantage that has of biomacromolecule as the Mierocrystalline cellulose.Many bacterial strains mixed culture, plurality of enzymes synergy can be comparatively fast and remove the feedback inhibition of product more up hill and dale, regulates the pH of nutrient solution, improves cellulosic transformation efficiency.Chinese patent specification sheets CN 1335288A discloses a kind of bacteria preparation for organic matter to become decomposed fast, the bacteria preparation for organic matter to become decomposed fast that this invention is made has the ability of degraded cellulose preferably, it makes used substratum is peptone fiber substratum, substratum consist of CaCO
3, NaCl, Mierocrystalline cellulose, peptone and yeast powder, its used nitrogenous source is an organic nitrogen source, cultural method is that static cultivation or concussion are cultivated.
2008, " the screening of efficient anaerobic cellulose-degrading bacteria of the Master's thesis of the Luo Hui of Methane Scientific Research Inst., Ministry of Agriculture, the making of composite microbial system and applied research " in two natural composite microbial systems with cellulose-degrading methane producing ability are disclosed, these two natural composite microbial systems are formed by aerobic bacteria and anerobe, comprise the big major function flora of three in the biogas fermentation, be cellulose degradation flora, product hydrogen acetogen group and methane flora, be typical, have the natural composite microbial system of comparatively complete each functional microorganism of biogas based on cellulose degradation.Though these two natural composite microbial systems all have stronger cellulose degradation ability and produce the methane ability, but contained bacterial classification is many bacterium association that unordered set forms in the passive fermenting process of nature in this spontaneous fermentation thing microbial inoculum, can't grasp its bacterial classification and form, result of use is difficult to control; And this natural composite microbial system passive follow the natural circulation rule, its decomposition efficiency is not high, effect is also unstable.The substratum that screens and tame above-mentioned composite microbial system contains 0.5% peptone, 0.1% yeast powder, 0.2% CaCO
3, 0.5% NaCl, carbon source is 0.5% stalk, used nitrogenous source is an organic nitrogen source.
Summary of the invention
It can efficient anaerobic conversion Mierocrystalline cellulose be methane and effect stability that problem to be solved by this invention provides a kind of, the composite bacteria making method that decomposition efficiency is high.
In order to address the above problem, composite bacteria making method of the present invention is: will adhere to being inoculated in the nutrient solution of cellulose after flora that the Institute of Micro-biology of zymogenic bacteria, hydrogen-producing acetogenic bacteria and the methanogen of amphimicrobian and strictly anaerobic forms mixes separately, sealing and standing is cultivated, culture temperature is 30-50 ℃, and pH is 3-10.In containing cellulosic substratum, above-mentioned composite bacteria synergy in degraded cellulose, generates methane.Be exactly the Mierocrystalline cellulose in anaerobic cellulose decomposer and the amphimicrobian cellulose-decomposing bacterium decomposition substratum specifically, obtain glucose and organic acid substance, quilts such as glucose and organic acid produce the hydrogen acid formers and further are degraded to H
2, CO
2With acetate etc., protein in the substratum and other macromolecule substrates are decomposed into amino acid and other organic acids by Desulfovibrio vulgaris and Acetobacterium woodii, amino acid further is decomposed as the nitrogenous source of composite bacteria growth, and other organic acids are produced the hydrogen acid formers and further are degraded to H
2, CO
2With acetate etc.Above-mentioned not methanogenic bacterium can be decomposed generation H to the organism of various complexity as carbohydrate, protein etc.
2, CO
2, NH
3, methyl alcohol, acetate, propionic acid, butyric acid etc., propionic acid, butyric acid also can be become H with the acetic acid bacteria decomposition and inversion by hydrogen bacterium
2, CO
2And acetate, for methanogen provides synthetic cell matter and the precursor that forms methane, electron donor---hydrogen donor and nitrogenous source make methanogen utilize these materials finally to form methane.Above-mentioned several bacterial strains synergy through propagation after a while, forms the composite bacteria system of a kind of composition and stable performance.
Use method of the present invention and can make composite bacteria with strong cellulose degradation ability, its degradation rate to straw fibrid element reaches more than 80% under amphimicrobian and strictly anaerobic condition, in degraded cellulose, this composite bacteria can also obtain methane with cellulosic degraded product metabolism, this composite bacteria went down to posterity through 64 generations, performance remains unchanged substantially, cellulosic degradation capability and methanogenic ability are not obviously changed, be enough to illustrate that it has stability and practical value preferably.
The nutrient solution of the used cellulose of making composite bacteria method of the present invention contains CaCO
3, 3-7 ‰ NaCl, 5-10 ‰ Mierocrystalline cellulose, urea 1-4 ‰, peptone 1-3 ‰, yeast powder 0.5-1.5 ‰ is a solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
In the above-mentioned substratum, CaCO
3Effect be with reaction process in the organic acid reaction that may accumulate, the pH of substratum is stabilized in more than the pH3.Urea and peptone are the required nitrogenous source of composite bacteria growth, present research is thought for promoting microorganism growth and propagation, organic nitrogen source is better than inorganic nitrogen-sourced, and composite bacteria utilization of the present invention is when making nitrogenous source with urea, not only can stable growth, quick degradation of fibers cellulosic material, and compare with organic nitrogen source, production cost greatly reduced.
The composite bacteria that method of the present invention is made adheres to zymogenic bacteria, hydrogen-producing acetogenic bacteria and methanogen four quasi-microorganisms of amphimicrobian and strictly anaerobic separately, and the bacterial strain of forming this composite bacteria specifically can be following bacterial strain:
The Cytophaga fermentans Cytophaga that ferments, it is flexible shaft-like that cell is, amphimicrobian, decomposition of cellulose produces acetate, propionic acid, succsinic acid, H under the aerobic conditions
2And CO
2Deng, can produce yellow pigment simultaneously, bacterial strain deposit number ATCC 19072, DSM 9555;
Acetobacterium woodii Wu Shi bacillus aceticus, cell ellipse or quarter butt, become short chain in pairs once in a while at Dan Sheng, strictly anaerobic, hydrogen oxide, reduction CO
2Generate acetate, also ferment fructose and other substrates produce acetate, bacterial strain deposit number ATCC 29683, and DSM 1030;
The common desulfovibrio of Desulfovibrio vulgaris, arcuation, as electron acceptor(EA), all right metabolize carbohydrates, peptone and metabolic intermediate thereof produce volatile acid with vitriol, bacterial strain deposit number ATCC 29579, DSM 644;
Cellulomonas flavigena produces the yellowish fiber Zymomonas mobilis, elongated rod-shaped, and the energy decomposition of cellulose can also utilize glucose and other carbohydrate to produce acid under aerobic and anaerobic condition, bacterial strain deposit number ATCC 482, DSM 20109;
Butyrivibrio fibrisolvens Butyrivibrio fibrisolvens, arcuation, strictly anaerobic, decomposition of cellulose produces organic acid, can produce the hemicellulase of degradation of hemicellulose simultaneously, bacterial strain deposit number ATCC19171, DSM 3071;
Clostridium papyrosolvens Clostridium papyrosolvens, straight-bar, strictly anaerobic, hydrocellulose produces acetate, ethanol and H
2Deng, bacterial strain deposit number ATCC 35413, DSM 2782;
Clostridium cellobioparum clostridium cellobioparum, bending or direct rod shape, hydrocellulose produces cellobiose etc., and ATCC 15832 is compiled in the bacterial strain preservation, and DSM 1351;
Clostridium termitidis bending or direct rod shape, hydrocellulose produces acetate, ethanol and H
2Deng, bacterial strain deposit number ATCC 51846, DSM 5398;
Syntrophomonas Wolfei Wolf association Zymomonas mobilis, slight screw shaped is not produced gemma, decomposes butyric acid and produces acetate and H
2, with the methanogen syntrophism, bacterial strain deposit number DSM 4212;
The asymmetric sphere of Methanosarcina barkeri Pasteur sarcina methanica, can irregular gathering agglomerating, utilize methyl alcohol, Trimethylamine 99, acetate and H
2/ CO
2Methane is produced in growth, bacterial strain deposit number ATCC43241, and DSM 1538;
Methanobrevibacter arboriphilicus has a liking for trees methane tyrothricin, tyrothricin, and Dan Sheng, paired or bunchiness only utilize H
2/ CO
2Methane is produced in growth, bacterial strain deposit number ATCC 33747, and DSM 1125;
Methanosphaera stadtmaniae Si Shi methane ball bacteria is irregular spherical, and single living or paired, extreme strictly anaerobic utilizes H
2/ CO
2Produce methane with formate growth, require acetate etc. as the organic growth factor, bacterial strain deposit number ATCC 43021, DSM 3091;
Methanococcus vannielii Wan Shi methane coccus, regular or irregular sphere, extreme strictly anaerobic utilizes H
2/ CO
2Methane is produced in growth with formate, bacterial strain deposit number ATCC 35089, and DSM 1224.
Above bacterial strain all can obtain by German microbial strains preservation center (DSMZ) or U.S. typical case culture center (ATCC).
The bacterial strain of forming the composite bacteria of method making of the present invention has 13 strains, the per-cent that the cell count of each bacterial strain accounts for the total cell count of composite bacteria is: Cytophaga fermentans accounts for 5-10%, Clostridiumpapyrosolvens accounts for 25-35%, Desulfovibrio vulgaris accounts for 1-3%, Butyrivibriofibrisolvens 2.5-5%, Acetobacterium woodii accounts for 5-10%, Cellulomonasflavigena accounts for 1-5%, Clostridium termitidis accounts for 1-5%, Clostridiumcellobioparum accounts for 2.5-5%, Syntrophomonas Wolfei 5-10%, Methanosarcinabarkeri accounts for 10-20%, Methanobrevibacter arboriphilicus accounts for 10-15%, Methanosphaera stadtmaniae 5-8%, Methanococcus vannielii accounts for 2.5-5%.
Embodiment
Mode below by embodiment further specifies the present invention, does not therefore limit the present invention among the described scope of embodiments." methane production (m among the embodiment
3/ day kgTS) " solid substance that refers to every kg cellulose is after composite bacteria is handled, the methane-containing gas that produce every day under a normal atmosphere, the volume (m in the time of 25 ℃
3), " methane content (%) " is meant that the volume of methane gas accounts for the per-cent of solid substance methane-containing gas cumulative volume of generation after composite bacteria is handled of cellulose, carbon dioxide content (%) is meant that solid substance that the volume of carbon dioxide accounts for cellulose handles the per-cent of the methane-containing gas cumulative volume that the back produces through composite bacteria, and degradation rate is meant that weight that the solid substance of cellulose is reduced accounts for the per-cent of its initial weight after the composite bacteria processing.
Embodiment 1:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 10%, Clostridium papyrosolvens accounts for 25%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 1%, Clostridiumtermitidis accounts for 1%, Clostridium cellobioparum accounts for 2.5%, SyntrophomonasWolfei 5%, and Methanosarcina barkeri accounts for 19%, and Methanobrevibacterarboriphilicus accounts for 15%, Methanosphaera stadtmaniae 8%, Methanococcusvannielii accounts for 2.5%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 3 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 1 ‰, peptone 1 ‰, yeast powder 0.5 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 2:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 7.5%, Clostridium papyrosolvens accounts for 35%, Desulfovibrio vulgaris accounts for 3%, Butyrivibrio fibrisolyens 4%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 3%, Clostridium cellobioparum accounts for 5%, Syntrophomonas Wolfei5%, Methanosarcina barkeri accounts for 10%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae5%, Methanococcus vannielii accounts for 2.5%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 5 ‰, Mierocrystalline cellulose (straw) 8 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 3:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 7 ‰, Mierocrystalline cellulose (straw) 10 ‰, urea 4 ‰, peptone 3 ‰, yeast powder 1.5 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 4:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 20 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 8%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermi tidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 17%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 5:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 50 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 7.5%, Clostridium papyrosolvens accounts for 35%, Desulfovibrio vulgaris accounts for 3%, Butyrivibrio fibrisolvens 4%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 3%, Clostridium cellobioparum accounts for 5%, Syntrophomonas Wolfei5%, Methanosarcina barkeri accounts for 10%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 2.5%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 6:
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 3.0, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 10%, Clostridium papyrosolvens accounts for 25%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas fiavigena accounts for 1%, Clostridiumtermitidis accounts for 1%, Clostridium cellobioparum accounts for 2.5%, SyntrophomonasWolfei 5%, and Methanosarcina barkeri accounts for 19%, and Methanobrevibacterarboriphilicus accounts for 15%, Methanosphaera stadtmaniae 8%, Methanococcusvannieiii accounts for 2.5%.The nutrient solution prescription of above-mentioned cellulose is as follows: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 7
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 7.0, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%.The nutrient solution prescription of above-mentioned cellulose is as follows:: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 8
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 10.0, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%;
The nutrient solution of cellulose is prepared by the following method: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
The gas yield of composite bacteria and cellulose degradation rate see the following form under above-mentioned culture condition:
Embodiment 9
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%.
The nutrient solution of cellulose is prepared by the following method: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
Above-mentioned culture with in the fresh medium that went to the identical cellulose of prescription in 7 days, is cultivated under identical condition, constantly gone down to posterity, each gets part in 4 ℃ of preservations for culture.
Be taken at 4 ℃ of preservations the 64th generation complex bacteria culture be inoculated in to cultivate in the nutrient solution of the identical cellulose of prescription and bring back to life, sealing and standing is cultivated, 40 ℃ of culture temperature, pH7.
The gas yield and the cellulose degradation rate of the 26th generation culture composite bacteria under above-mentioned culture condition see the following form:
Embodiment 10
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermen tans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Celiulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilicus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%;
The nutrient solution of cellulose is prepared by the following method: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
Above-mentioned culture with in the fresh medium that went to the identical cellulose of prescription in 7 days, is cultivated under identical condition, constantly gone down to posterity, each gets part in 4 ℃ of preservations for culture.
Be taken at 4 ℃ of preservations the 64th generation complex bacteria culture be inoculated in to cultivate in the nutrient solution of the identical cellulose of prescription and bring back to life, sealing and standing is cultivated, 40 ℃ of culture temperature, pH7.
The gas yield and the cellulose degradation rate of the 46th generation culture composite bacteria under above-mentioned culture condition see the following form:
Embodiment 11
To be inoculated in the nutrient solution of cellulose after the following 13 strain bacterium mixing, sealing and standing is cultivated, culture temperature is 40 ℃, pH is 6.5, the ratio that the cell count of each bacterial strain accounts for the total cell count of composite bacteria in the 13 strain bacterium is respectively: Cytophaga fermentans accounts for 5%, Clostridium papyrosolvens accounts for 30%, Desulfovibrio vulgaris accounts for 1%, Butyrivibrio fibrisolvens 5%, Acetobacterium woodii accounts for 5%, Cellulomonas flavigena accounts for 5%, Clostridiumtermitidis accounts for 2%, Clostridium cellobioparum accounts for 3%, Syntrophomonas Wolfei6%, Methanosarcina barkeri accounts for 20%, and Methanobrevibacter arboriphilieus accounts for 10%, Methanosphaera stadtmaniae 5%, Methanococcus vannielii accounts for 3%;
The nutrient solution of cellulose is prepared by the following method: NaCl 5 ‰, Mierocrystalline cellulose (straw) 5 ‰, urea 2 ‰, peptone 2 ‰, yeast powder 1 ‰, CaCO
32 ‰, be solvent with water, above-mentioned content is meant CaCO
3, NaCl, Mierocrystalline cellulose, urea, peptone, yeast powder quality account for the ratio of substratum total mass.
Above-mentioned culture with in the fresh medium that went to the identical cellulose of prescription in 7 days, is cultivated under identical condition, constantly gone down to posterity, each gets part in 4 ℃ of preservations for culture.
Be taken at 4 ℃ of preservations the 64th generation complex bacteria culture be inoculated in to cultivate in the nutrient solution of the identical cellulose of prescription and bring back to life, sealing and standing is cultivated, 40 ℃ of culture temperature, pH7.
The gas yield and the cellulose degradation rate of the 64th generation culture composite bacteria under above-mentioned culture condition see the following form:
Claims (4)
1. the making method of a composite bacteria, after mixing with certain proportion, the microorganism that the steps include: to adhere to separately zymogenic bacteria, hydrogen-producing acetogenic bacteria and the methanogen of amphimicrobian and strictly anaerobic is inoculated in the nutrient solution of cellulose, sealing and standing is cultivated, culture temperature is 30-50 ℃, and pH is 3-10.
2. the preparation method of composite bacteria according to claim 1, it is characterized in that: the nutrient solution of described cellulose contains CaCO
3, 3-7 ‰ NaCl, 5-10 ‰ Mierocrystalline cellulose, urea 1-4 ‰, peptone 1-3 ‰, yeast powder 0.5-1.5 ‰ is a solvent with water.
3. the preparation method of composite bacteria according to claim 1 and 2, it is characterized in that: the described flora of being made up of the zymogenic bacteria that adheres to amphimicrobian and strictly anaerobic separately, hydrogen-producing acetogenic bacteria and methanogen Institute of Micro-biology is made up of following bacterial strain:
The Cytophaga fermentans Cytophaga that ferments, it is flexible shaft-like that cell is, amphimicrobian, decomposition of cellulose produces acetate, propionic acid, succsinic acid, H under the aerobic conditions
2And CO
2Deng, can produce yellow pigment simultaneously, bacterial strain deposit number ATCC 19072, DSM 9555;
Acetobacterium woodii Wu Shi bacillus aceticus, cell ellipse or quarter butt, become short chain in pairs once in a while at Dan Sheng, strictly anaerobic, hydrogen oxide, reduction CO
2Generate acetate, also ferment fructose and other substrates produce acetate, bacterial strain deposit number ATCC 29683, and DSM 1030;
The common desulfovibrio of Desulfovibrio vulgaris, arcuation, as electron acceptor(EA), all right metabolize carbohydrates, peptone and metabolic intermediate thereof produce volatile acid with vitriol, bacterial strain deposit number ATCC 29579, DSM 644;
Cellulomonas flavigena produces the yellowish fiber Zymomonas mobilis, elongated rod-shaped, and the energy decomposition of cellulose can also utilize glucose and other carbohydrate to produce acid under aerobic and anaerobic condition, bacterial strain deposit number ATCC 482, DSM 20109;
Butyrivibrio fibrisolvens Butyrivibrio fibrisolvens, arcuation, strictly anaerobic, decomposition of cellulose produces organic acid, can produce the hemicellulase of degradation of hemicellulose simultaneously, bacterial strain deposit number ATCC19171, DSM 3071;
Clostridium papyrosolvens Clostridium papyrosolvens, straight-bar, strictly anaerobic, hydrocellulose produces acetate, ethanol and H
2Deng, bacterial strain deposit number ATCC 35413, DSM 2782;
Clostridium cellobioparum clostridium cellobioparum, bending or direct rod shape, hydrocellulose produces cellobiose etc., and ATCC 15832 is compiled in the bacterial strain preservation, and DSM 1351;
Clostridium termitidis bending or direct rod shape, hydrocellulose produces acetate, ethanol and H
2Deng, bacterial strain deposit number ATCC 51846, DSM 5398;
Syntrophomonas Wolfei Wolf association Zymomonas mobilis, slight screw shaped is not produced gemma, decomposes butyric acid and produces acetate and H
2, with the methanogen syntrophism, bacterial strain deposit number DSM 4212;
The asymmetric sphere of Methanosarcina barkeri Pasteur sarcina methanica, can irregular gathering agglomerating, utilize methyl alcohol, Trimethylamine 99, acetate and H
2/ CO
2Methane is produced in growth, bacterial strain deposit number ATCC43241, and DSM 1538;
Methanobrevibacter arboriphilicus has a liking for trees methane tyrothricin, tyrothricin, and Dan Sheng, paired or bunchiness only utilize H
2/ CO
2Methane is produced in growth, bacterial strain deposit number ATCC 33747, and DSM 1125;
Methanosphaera stadtmaniae Si Shi methane ball bacteria is irregular spherical, and single living or paired, extreme strictly anaerobic utilizes H
2/ CO
2Produce methane with formate growth, require acetate etc. as the organic growth factor, bacterial strain deposit number ATCC 43021, DSM 3091;
Methanococcus vannielii Wan Shi methane coccus, regular or irregular sphere, extreme strictly anaerobic utilizes H
2/ CO
2Methane is produced in growth with formate, bacterial strain deposit number ATCC 35089, and DSM 1224.
4. the preparation method of composite bacteria according to claim 2, it is characterized in that: the described zymogenic bacteria that adheres to amphimicrobian and strictly anaerobic separately, the per-cent that the cell count of each bacterial strain of hydrogen-producing acetogenic bacteria and methanogen accounts for the total cell count of composite bacteria flora is: Cytophaga fermentans accounts for 5-10%, Clostridium papyrosolvens accounts for 25-35%, Desulfovibrio vulgaris accounts for 1-3%, Butyrivibrio fibrisolvens 2.5-5%, Acetobacterium woodii accounts for 5-10%, Cellulomonas flavigena accounts for 1-5%, Clostridium termitidis accounts for 1-5%, Clostridium cellobioparum accounts for 2.5-5%, Syntrophomonas Wolfei 5-10%, Methanosarcina barkeri accounts for 10-20%, Methanobrevibacter arboriphilicus accounts for 10-15%, Methanosphaera stadtmaniae 5-8%, Methanococcus vannielii accounts for 2.5-5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910103085 CN101481676B (en) | 2009-01-19 | 2009-01-19 | Preparation of composite bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910103085 CN101481676B (en) | 2009-01-19 | 2009-01-19 | Preparation of composite bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101481676A true CN101481676A (en) | 2009-07-15 |
| CN101481676B CN101481676B (en) | 2011-01-19 |
Family
ID=40878966
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910103085 Expired - Fee Related CN101481676B (en) | 2009-01-19 | 2009-01-19 | Preparation of composite bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101481676B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559499A (en) * | 2012-01-31 | 2012-07-11 | 农业部沼气科学研究所 | Preparation method of methane dry fermentation compound bacterial preparation |
| CN103555638A (en) * | 2013-11-15 | 2014-02-05 | 河北省科学院生物研究所 | Ethanol-tolerant acetic acid-producing microbial complex bacteria and use thereof |
| CN103725638A (en) * | 2014-01-20 | 2014-04-16 | 农业部沼气科学研究所 | Compound bacterium agent for preparing fertilizer |
| CN104878066A (en) * | 2015-06-26 | 2015-09-02 | 农业部沼气科学研究所 | Fertilizer making agent and preparation method and application thereof |
| CN107326060A (en) * | 2017-04-06 | 2017-11-07 | 甘肃省科学院生物研究所 | A kind of method that anaerobic fermentation maize straw produces acetic acid |
| CN107674906A (en) * | 2017-11-16 | 2018-02-09 | 农业部沼气科学研究所 | A kind of biogas fermentation promotes the preparation method of microbial inoculum |
| CN110616163A (en) * | 2018-06-20 | 2019-12-27 | 南方医科大学珠江医院 | Enterococcus faecalis capable of reducing concentration of trimethylamine and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1158225C (en) * | 2000-07-26 | 2004-07-21 | 崔宗均 | Bacteria prepn for organic matter to become decomposed fast |
-
2009
- 2009-01-19 CN CN 200910103085 patent/CN101481676B/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559499A (en) * | 2012-01-31 | 2012-07-11 | 农业部沼气科学研究所 | Preparation method of methane dry fermentation compound bacterial preparation |
| CN102559499B (en) * | 2012-01-31 | 2014-07-30 | 农业部沼气科学研究所 | Preparation method of methane dry fermentation compound bacterial preparation |
| CN103555638B (en) * | 2013-11-15 | 2016-01-13 | 河北省科学院生物研究所 | A kind of alcohol patience produces acetic acid microorganism composite bacteria and uses thereof |
| CN103555638A (en) * | 2013-11-15 | 2014-02-05 | 河北省科学院生物研究所 | Ethanol-tolerant acetic acid-producing microbial complex bacteria and use thereof |
| CN103725638B (en) * | 2014-01-20 | 2016-03-16 | 农业部沼气科学研究所 | Composting composite fungus agent |
| CN103725638A (en) * | 2014-01-20 | 2014-04-16 | 农业部沼气科学研究所 | Compound bacterium agent for preparing fertilizer |
| CN104878066A (en) * | 2015-06-26 | 2015-09-02 | 农业部沼气科学研究所 | Fertilizer making agent and preparation method and application thereof |
| CN104878066B (en) * | 2015-06-26 | 2018-08-31 | 农业部沼气科学研究所 | Fertile agent of a kind of system and its preparation method and application |
| CN107326060A (en) * | 2017-04-06 | 2017-11-07 | 甘肃省科学院生物研究所 | A kind of method that anaerobic fermentation maize straw produces acetic acid |
| CN107326060B (en) * | 2017-04-06 | 2019-06-11 | 甘肃省科学院生物研究所 | A kind of method of anaerobic fermentation corn stover production acetic acid |
| CN107674906A (en) * | 2017-11-16 | 2018-02-09 | 农业部沼气科学研究所 | A kind of biogas fermentation promotes the preparation method of microbial inoculum |
| CN107674906B (en) * | 2017-11-16 | 2020-11-03 | 农业部沼气科学研究所 | Preparation method of methane fermentation promoting microbial inoculum |
| CN110616163A (en) * | 2018-06-20 | 2019-12-27 | 南方医科大学珠江医院 | Enterococcus faecalis capable of reducing concentration of trimethylamine and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101481676B (en) | 2011-01-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101475926B (en) | Anaerobic cellulose-degrading methane producing composite bacterium | |
| Saratale et al. | Biohydrogen production from renewable biomass resources | |
| CN102559499B (en) | Preparation method of methane dry fermentation compound bacterial preparation | |
| Zhang et al. | Bioaugmentation with an acetate-type fermentation bacterium Acetobacteroides hydrogenigenes improves methane production from corn straw | |
| Zeikus | Chemical and fuel production by anaerobic bacteria | |
| Laxman Pachapur et al. | Co‐culture strategies for increased biohydrogen production | |
| CN102533624B (en) | Microorganism bacteria mixture and application thereof in promoting production of methane by dregs of traditional Chinese medicine by anaerobic fermentation | |
| Lin et al. | Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures | |
| Argun et al. | Biohydrogen production potential of different biomass sources | |
| Lo et al. | Cellulosic hydrogen production with a sequencing bacterial hydrolysis and dark fermentation strategy | |
| Cheng et al. | Fungal pretreatment enhances hydrogen production via thermophilic fermentation of cornstalk | |
| CN101481676B (en) | Preparation of composite bacteria | |
| Sheng et al. | Lignocellulosic saccharification by a newly isolated bacterium, Ruminiclostridium thermocellum M3 and cellular cellulase activities for high ratio of glucose to cellobiose | |
| CN106148224B (en) | A kind of straw degradative acidification microbial inoculum and its manufacturing method | |
| CN101851656B (en) | Method for producing cellulosic ethanol | |
| CN102174433B (en) | Clostridium beijerinckii with high stress resistance and application thereof | |
| CN106811438B (en) | Straw degradation acidification microbial inoculum and preparation method thereof | |
| CN101289672A (en) | Process for producing hydrogen and/or methane | |
| CN102533609A (en) | Methane dry fermentation compound bacteria | |
| Tran et al. | Developing co-culture system of dominant cellulolytic Bacillus sp. THLA0409 and dominant ethanolic Klebsiella oxytoca THLC0409 for enhancing ethanol production from lignocellulosic materials | |
| Lü et al. | The effect of temperature on the structure and function of a cellulose-degrading microbial community | |
| Seesatat et al. | Biological degradation of rice straw with thermophilic lignocellulolytic bacterial isolates and biogas production from total broth by rumen microorganisms | |
| CN103060245B (en) | Compound microorganism bacterium agent of biological coalbed methane prepared by coal bed organic impurities and application thereof | |
| Sriyod et al. | One-step multi enzyme pretreatment and biohydrogen production from Chlorella sp. biomass | |
| CN102433262B (en) | Complex microbial agent for low-temperature methane fermentation and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110119 Termination date: 20180119 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |