CN100513562C - Preparation method of hybrid hydrolase formulation for promoting methane yield from fermentation - Google Patents
Preparation method of hybrid hydrolase formulation for promoting methane yield from fermentation Download PDFInfo
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- CN100513562C CN100513562C CNB2006100110346A CN200610011034A CN100513562C CN 100513562 C CN100513562 C CN 100513562C CN B2006100110346 A CNB2006100110346 A CN B2006100110346A CN 200610011034 A CN200610011034 A CN 200610011034A CN 100513562 C CN100513562 C CN 100513562C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
The method for preparation of hydrolytic enzyme preparation used for improving gas-producing rate of marsh gas fermentation belongs to preparation of supplementary exoenzyme used for marsh gas fermentation. The invention is to provide a method for preparation of hydrolytic enzyme preparation used for improving gas-producing rate of marsh gas fermentation, which has the production bacteria with the following enzymatic vitality index: selecting wheat bran and rice bran as mixed culture medium, obtaining mixed bran seeds by pedigree breed two-stage enlargement culture of the bacteria with solid method, fermenting for 3-4 days at 28 DEG after evenly mixed, sampling and analyzing every enzymatic vitality index, adding commodity enzyme preparation to reach enzymatic vitality index, and airing until the humidity is lower than 5%. The enzymatic vitality index of the prolease in the said mixed bran seeds is not more than 2000+-5% mug tyrosine /g/min. The bacteria can be obtained by bacteria and fungus separated from different environment. The test proves that when hydrolytic enzyme preparation is input into the methane tank fermentation raw materials, the rate of dissolution and degradation of the marsh gas fermentation raw materials can be improve, and the gas-producing rate of marsh gas fermentation, TS and VS gas-production potential and methane content in marsh gas can be increased.
Description
Affiliated technical field
The invention belongs to the preparation method of the supplemented with exogenous enzyme that is used for biogas fermentation.
Background technology
1901
Point out to produce the process that the methane process is a microorganism, O.W.Lawrence in 1967 and P.L.McCarty have proposed the stage theory of anaerobic digestion process, i.e. hydrolysis, produce hydrogen and produce acetate and produce the methane three phases.Biogas fermentation is the symbiosis metabolism under anaerobic of flora more than, make mineralization of organic material is the process of methane and carbonic acid gas.Since 20th century, the small-sized efficient methane-generating pit appearred in the later stage eighties, applying of biogas arrived a higher level, and use and management is very convenient, the pond type is reasonable in design, need not to reload greatly the semicontinuous fermentation method of realization, the pond holds factor of created gase height, aerogenesis equilibrium.But the existing both at home and abroad equal ubiquity of household methane tank problems such as raw material availability is not high, gas production rate is on the low side, and the pig manure raw material factor of created gase of China's methane-generating pit generally is lower than 0.3m
3/ kgTS.Because the low increase fermentation volume of having to of factor of created gase, the pond of small-sized efficient methane-generating pit holds all at 6m
3More than, some areas also have 8m
3, even 10m
3More than, however, the pond of these methane-generating pits holds factor of created gase and also has only 0.3m
3/ m
3About d.Studies show that the lytic enzyme enzyme is lived and there is proportional relation in biogas output, lytic enzyme live all appear at the aerogenesis peak value near, and lytic enzyme is lived similar to the change curve of biogas output.Domestic and international many scholars attempt to improve with the water-disintegrable bacterium of high yield lytic enzyme the hydrolytic process of biogas fermentation, improve the utilization ratio of fermentation raw material, and then improve the methane gas producing rate, but in fact, the microorganism of seeking the high yield lytic enzyme is difficult to big breakthrough so far.Studies show that, add cellulolytic enzyme in right amount and can accelerate cellulosic degradation speed in the fermentation raw material, improve biogas output.Before this, we obtain ZL01129073.0 " bio-active additive for fermentation of marsh gas " patent for invention, and the number of filing an application 200510010991.2 " the application zytase improves the method for biogas fermentation factor of created gase " application for a patent for invention, these two inventions show: a certain amount of zytase cooperates with bio-active additive for fermentation of marsh gas can improve fermentation appearance, can improve to continuously ferment or the starting process factor of created gase of semicontinuous biogas fermentation.But because the hydrolysis object is different with product, fermentation system causes that mixed bacterial produces superior microorganism lifetime difference, zytase and active additive making time need certain intervals, and the enzyme activity index request is higher, do not relate to the production method of mixing enzyme preparation as yet.
Summary of the invention
The purpose of this invention is to provide and be used to add the preparation method who enters methane fermentation system, improves the mixed hydrolysis zymin of biogas fermentation factor of created gase.
Above-mentioned purpose realizes by following approach:
A, have the mixed hydrolysis zymin of the index of enzyme activity shown in the following table:
| Hydrolase | Enzyme unit alive | The enzyme activity value |
| Saccharifying amylase | Mg glucose/gh | 50000±1％ |
| Liquefaction type amylase | Mg Zulkovsky starch/gh | 600±10％ |
| Cellulase | μ g glucose/gmin | 1000±10％ |
| Hemicellulase | μ g wood sugar/g.h | 8000±5％ |
B, preparation are produced with planting: with wheat bran is substratum, add water furnishing pulpous state, be sub-packed in the test tube or the triangular flask of 250mL in, sterilized 30 minutes for 121 ℃, insert pure inclined-plane kind after the cooling respectively with bacterial classification of hydrolase in the tabulation of going up, 28 ℃ of constant temperature culture to each bacterial classifications cover with wheat bran, again with bigger container respectively enlarged culturing prepare wheat bran production with kind, each bacterium wheat bran kind mixed obtaining to mix the wheat bran kind by the amount of equal volume or weight;
C, solid state process mixed fermentation: with wheat bran 80%～100%, auxiliary material rice bran 0～20% is a fermentation raw material, adding water is 60～70% of raw material volume, and fermentation raw material is mixed the back thoroughly and inserted about 5% mixing wheat bran kind, mixes the dustpan of packing into thoroughly, the dustpan of the identical size of buckle closure is put on the shelf and is put, in 28 ℃ of constant temperature culture 3～4 days, carry out stirring therebetween 1～2 time, fermentation finishes, air-dryly be lower than 5% to moisture content, each dustpan tunning is concentrated and is mixed the back pack;
D, each enzyme activity value of sampling analysis are replenished the enzyme activity value that enzyme activity reaches tabular with the commercial goods zymin.
The enzyme activity value of proteolytic enzyme is no more than 2000 ± 5% μ g tyrosine/gmin in the described mixing wheat bran kind, and too high proteolytic enzyme can degrade saccharifying enzyme and other lytic enzyme make the inactivation of hydrolytic enzyme of adding.
Described fermentation raw material is a wheat bran 90%+ rice bran 10%.
In the described solid state process mixed fermentation, the material amount of the dustpan of packing into is advisable with 5～7cm, ferments to take off lid cooling in 24 hours, becomes block stirring cooling in 48 hours, treats that mycelia covers with and produce spore, air-dryly is lower than 5% to moisture content.
The present invention produces with bacterial classification and can obtain from the bacterium and the fungi of different environment separation, and source and kind are widely arranged, and has not a particular requirement.The aspergillus niger or the Rhizopus class that have higher saccharifying enzyme type and liquefaction type amylase activity as generation; Trichoderma or aspergillus niger with higher hemicellulase and xylanase activity; Has the aspergillus oryzae of higher protease activity etc.But can not sneak into mixing wheat bran kind based on the bacterial classification that produces proteolytic enzyme.
The present invention adopts the purebred two-stage enlarged culturing of the solid state process of bacterial classification process to produce and uses kind of an acquisition mixing wheat bran kind, and promptly the microorganism with common lytic enzyme is a bacterial classification, obtains wheat bran production with planting through wheat bran rice bran mixed culture medium; Each enzyme activity value of sampling analysis is added the commercial enzyme preparation in right amount and is reached the enzyme activity index after the solid-state mixed fermentation.During use, mixed hydrolysis zymin input is normally produced in the methane-generating pit fermentation raw material of biogas, improve the hydrolysis rate of biogas fermentation raw material in anaerobic digestion process, promote the anaerobion activity, improve the degradation rate and the raw material availability of fermentation raw material, shorten the new pond starting time, increase methane content in biogas fermentation gas production rate, TS and VS production potential and the biogas.
Description of drawings
Fig. 1 cultivates the wheat bran production of hybrid seeds process flow sheet of planting for adopting mixed strains.
Fig. 2 is employing wheat bran raw material fermentation, and suitably adds the commercial enzyme preparation, the product processes schema of mixed hydrolysis zymin.
Embodiment
(1) preparation of mixed hydrolysis zymin
The preparation of mixed hydrolysis zymin of the present invention relates to the production of hybrid seeds and two parts of products production, and first production of hybrid seeds process using pure strain solid state process sterilization grog enlarges, and cultivates wheat bran production with planting; It two is that product processes adopts the wheat bran raw material fermentation, and suitably adds the commercial enzyme preparation.
As Fig. 1, with wheat bran is substratum, add water to wheat bran water content 60～70% furnishing pulpous states, be sub-packed in the test tube or the triangular flask of 250mL in, 121 ℃ of sterilizations 30 minutes insert the pure inclined-plane kind of required each bacterial classification respectively after the cooling, 28 ℃ of constant temperature culture to each bacterial classifications cover with wheat bran, use larger container again, some as the triangular flask of 1000mL or common Cans, correspondence respectively enlarged culturing be prepared as wheat bran production with kind.Mixing promptly by the amount of about 1:1 each bacterium wheat bran kind, acquisition mixes the wheat bran kind.Can not sneak into based on the bacterial classification that produces proteolytic enzyme, need single culture and production.
As Fig. 2, fermentation raw material is a wheat bran 90%+ rice bran 10%, and it is moisture 60～70% to raw material to add water, and fermentation raw material is mixed the back thoroughly and inserted about 5% mixing wheat bran kind, mixes thoroughly in the dustpan of packing into, and the material amount of the dustpan of packing into is advisable with 6～8cm winter, and be advisable with 4～6cm summer; Put on the shelf and put, the dustpan of the identical size of last buckle closure in incubated at room temperature 3～4 days, carries out stirring therebetween 1～2 time; During stirring, should not break material into pieces, should become bulk to carry out the stirring cooling; Fermentation finishes, and air-dryly is lower than 5% to moisture content.After each dustpan tunning was concentrated and mixed, each enzyme of sampling analysis was lived and is worth.If enzyme activity does not reach the enzyme of enzyme activity value shown in the following table 1, after the additional corresponding enzyme of fair commercial enzyme preparation index alive, the pack sealing is located storage in shady and cool drying.
Table 1 mixed hydrolysis zymin enzyme index alive
| Hydrolase | Enzyme unit alive | The requirement alive of product enzyme |
| Saccharifying amylase | Mg glucose/gh | 50000±1％ |
| Liquefaction type amylase | Mg Zulkovsky starch/gh | 600±10％ |
| Cellulase | μ g glucose/gmin | 1000±10％ |
| Proteolytic enzyme | μ g tyrosine/gmin | 2000±5％ |
| Hemicellulase | μ g wood sugar/gh | 8000±5％ |
(2) effect and the dose-effect relationship of mixed hydrolysis zymin in biogas fermentation
In the following term,
Total solid TS (Total Solid) refers to that a certain amount of fermentation materials dries to constant weight gained net weight under 105 ℃.
Volatile matter VS (Volatility Substrate) refers to TS 550 ℃ of volatility parts that calcination is fallen in retort furnace.
Carry out the biogas fermentation experiment with mixed hydrolysis zymin of the present invention:
Fermentation raw material: fresh pig dung, TS=34.96%, VS=47.07%
Inoculum: this chamber enrichment, TS=7.28%, VS=79.17%
Fermentation process: 500ml is fermentation unit in batches, and carry out normal temperature and ferment in batches,
The fermented feed liquid proportioning: inoculum 2400ml+ fresh pig dung 644.2g+ adds natural pond liquid=8000ml
After fermented feed liquid stirs, its TS of sampling and measuring, VS, pH, its Mierocrystalline cellulose of mensuration, xylogen, lipid content saw Table 2 after oven dry was pulverized.
Every index of table 2 fermentation raw material
Adopt fermentation process fermentation in batches, concrete operations are as follows: get the good batch fermentation unit of 16 cover 500ml resistance to air loss, numbering is respectively 1-No. 16, the above mixed solution 400ml that shakes up respectively packs into, ferment in advance, treat normal gas producing after, be divided into 4 groups at random, be control group, test 1 group, test 2 groups, test 3 groups.Get biogas fermentation mixed hydrolysis zymin 2.0g respectively, 4.0g, 6.0g are mixed with the 50ml aqueous solution, respectively get 5ml, add 1 group of experiment, 2 groups of experiments respectively and test 3 groups, control group respectively adds the 5ml tap water, fermenting experiment totally 17 days, carry out the aerogenesis quantitative statistics every day, and statistics sees Table 3.PH and room temperature are more stable in the whole fermentation process, pH between 7.2-7.3, room temperature is between 23 ℃ one 25 ℃.
Table 3 bio-active additive for fermentation of marsh gas is to the influence of gas production rate
Fermentation ends, TS, VS, pH are surveyed in sampling respectively, and feed liquid is measured the content of Mierocrystalline cellulose, xylogen, fat in drying in oven, pulverizing, and the result adds up as table 4.The result shows, the mixed hydrolysis zymin shows that to the influence of biogas fermentation the gas production rate that improves biogas fermentation improves TS, the VS factor of created gase of biogas fermentation raw material more than 15%, improved the degradation rate of Mierocrystalline cellulose in the biogas fermentation raw material, fat, xylogen.
Table 4 bio-active additive for fermentation of marsh gas is to the influence of biogas fermentation
(3) using method of mixed hydrolysis enzyme agent
When new pond starts,, promptly add biogas fermentation mixed hydrolysis zymin when being dosed to about half, specific practice be with the mixed hydrolysis zymin with 10～20 kg water or liquid dung, natural pond liquid all can, dissolve and pour in the pond after mixing thoroughly, be dosed in the same day and seal the pond.Afterwards in service adds mixed once lytic enzyme preparation annual winter, and specific practice is for after fully mixing mixed hydrolysis zymin and 10～20 kilograms of fermentation raw materials thoroughly, pours into the pond from opening for feed to get final product.
The mixed hydrolysis enzyme preparation amount determines with the enzyme value of living, and packing has about 400 grams, 600 grams one bag two kinds, each every mouthful of 4m
3Biomass pool built from glass fiber reinforced plastic uses one bag of 400 gram packing; 6m
3Biomass pool built from glass fiber reinforced plastic uses one bag of 600 gram packing; 8m
3Biomass pool built from glass fiber reinforced plastic uses two bags of 400 gram packings.The mixed hydrolysis zymin easily places storage under lucifuge, drying and the low temperature, preservation period 2 years.
(4) mixed hydrolysis zymin biogas fermentation experiment effect
Utilize biogas fermentation mixed hydrolysis zymin to 4m
3The biogas fermentation test in 1 year has by a definite date been carried out with biomass pool built from glass fiber reinforced plastic in the commercialization family, and the result shows that the mixed hydrolysis zymin can improve the biogas output of pig manure fermentation, produces biogas ultimate production 604.3m per year
3, than the voluminous biogas 100m of routine
3More than; With temperature normal temperature fermentation between 19～24 ℃, the raw material factor of created gase of pig manure is 0.377m
3/ KgTS is than the 0.30m of routine
3/ KgTS improves 25.67%; The pond holds factor of created gase whole year all at 0.4～0.5m3/m
3About d, hold factor of created gase 0.3m than conventional pond
3/ m
3D improves more than 33%; The mixed hydrolysis zymin can also shorten the start time in new pond except that the factor of created gase that improves biogas fermentation, biogas output, raw material availability; Fire damp content is brought up to about 70%.
Claims (4)
1, be used to improve the preparation method of the mixed hydrolysis zymin of biogas fermentation factor of created gase, it is characterized in that:
A, have the mixed hydrolysis zymin of the index of enzyme activity shown in the following table:
B, preparation are produced with planting: with wheat bran is substratum, add water furnishing pulpous state, be sub-packed in the test tube or the triangular flask of 250mL in, sterilized 30 minutes for 121 ℃, insert pure inclined-plane kind after the cooling respectively with bacterial classification of hydrolase in the tabulation of going up, 28 ℃ of constant temperature culture to each bacterial classifications cover with wheat bran, again with bigger container respectively enlarged culturing prepare wheat bran production with kind, each bacterium wheat bran kind mixed obtaining to mix the wheat bran kind by the amount of equal volume or weight;
C, solid state process mixed fermentation: with wheat bran 80%～100%, auxiliary material rice bran 0～20% is a fermentation raw material, adding water is 60%～70% of raw material volume, and fermentation raw material is mixed the back thoroughly and inserted about 5% mixing wheat bran kind, mixes the dustpan of packing into thoroughly, the dustpan of the identical size of buckle closure is put on the shelf and is put, in 28 ℃ of constant temperature culture 3～4 days, carry out stirring therebetween 1～2 time, fermentation finishes, air-dryly be lower than 5% to moisture content, each dustpan tunning is concentrated and is mixed the back pack;
D, each enzyme activity value of sampling analysis are replenished the enzyme activity value that enzyme activity reaches tabular with the commercial goods zymin.
2, the preparation method who is used to improve the mixed hydrolysis zymin of biogas fermentation factor of created gase according to claim 1 is characterized in that the enzyme activity value of proteolytic enzyme in mixing the wheat bran kind is no more than 2000 ± 5% μ g tyrosine/gmin.
3, the preparation method who is used to improve the mixed hydrolysis zymin of biogas fermentation factor of created gase according to claim 1 is characterized in that fermentation raw material is a wheat bran 90%+ rice bran 10%.
4, the preparation method who is used to improve the mixed hydrolysis zymin of biogas fermentation factor of created gase according to claim 1, it is characterized in that in solid state process mixed fermentation, the material amount thickness of dustpan of packing into is 5～7cm, ferment and took off the lid cooling in 24 hours, became block stirring cooling in 48 hours, treat that mycelia covers with and produce spore, air-dryly be lower than 5% to moisture content.
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| CN102206620B (en) * | 2011-03-09 | 2013-02-13 | 枣庄市杰诺生物酶有限公司 | Compound enzyme used for methane gas fermentation and capable of supplementing trace elements |
| CN102719483B (en) * | 2012-05-02 | 2016-02-03 | 中国热带农业科学院热带生物技术研究所 | A kind of method improving plant fermentation biogas output |
| CN103146760A (en) * | 2013-03-18 | 2013-06-12 | 张其标 | High-efficiency, energy-saving and environment-friendly technology for fermentation and purification of biogas |
| CN110066831B (en) * | 2019-03-13 | 2021-03-09 | 杭州电子科技大学 | Method for quickly preparing biogas from kitchen waste |
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Non-Patent Citations (4)
| Title |
|---|
| 产沼气微生物促进剂的研制与应用. 叶发斌等.中国沼气，第17卷第1期. 1999 |
| 产沼气微生物促进剂的研制与应用. 叶发斌等.中国沼气，第17卷第1期. 1999 * |
| 水解酶提高猪粪沼气发酵产气率. 张无敌等.太阳能学报，第23卷第5期. 2002 |
| 水解酶提高猪粪沼气发酵产气率. 张无敌等.太阳能学报，第23卷第5期. 2002 * |
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