CN103740616A - Biological compound fertilizer - Google Patents
Biological compound fertilizer Download PDFInfo
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- CN103740616A CN103740616A CN201310743713.2A CN201310743713A CN103740616A CN 103740616 A CN103740616 A CN 103740616A CN 201310743713 A CN201310743713 A CN 201310743713A CN 103740616 A CN103740616 A CN 103740616A
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- subtilis
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- 239000003337 fertilizer Substances 0.000 title claims abstract description 56
- 150000001875 compounds Chemical class 0.000 title abstract 4
- 241000894006 Bacteria Species 0.000 claims abstract description 57
- 239000000463 material Substances 0.000 claims abstract description 16
- 241001513093 Aspergillus awamori Species 0.000 claims abstract description 15
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 13
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000011591 potassium Substances 0.000 claims abstract description 10
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims description 26
- 241000186660 Lactobacillus Species 0.000 claims description 20
- 229940039696 lactobacillus Drugs 0.000 claims description 20
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 14
- 230000003381 solubilizing effect Effects 0.000 claims description 14
- 239000002131 composite material Substances 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000000084 colloidal system Substances 0.000 claims description 9
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- 239000010452 phosphate Substances 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 230000007480 spreading Effects 0.000 claims description 6
- 238000003892 spreading Methods 0.000 claims description 6
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- 235000019425 dextrin Nutrition 0.000 claims description 4
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- 235000010216 calcium carbonate Nutrition 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000010563 solid-state fermentation Methods 0.000 claims description 3
- 239000002689 soil Substances 0.000 abstract description 23
- 239000000126 substance Substances 0.000 abstract description 11
- 244000063299 Bacillus subtilis Species 0.000 abstract description 6
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- 229930191978 Gibberellin Natural products 0.000 abstract description 2
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- 238000001704 evaporation Methods 0.000 abstract description 2
- 230000008020 evaporation Effects 0.000 abstract description 2
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- 230000002708 enhancing effect Effects 0.000 abstract 1
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 abstract 1
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- 238000011068 loading method Methods 0.000 description 8
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 5
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000194107 Bacillus megaterium Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
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- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 206010010254 Concussion Diseases 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
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- 108010051696 Growth Hormone Proteins 0.000 description 1
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- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
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- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
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Landscapes
- Fertilizers (AREA)
Abstract
The invention discloses a biological compound fertilizer, belonging to the technical field of agricultural fertilizers. The biological compound fertilizer comprises the folllowing components in parts by weight: 5-18 parts of Bacillus mucilaginosus, 2-6 parts of phosphate-solubilizing bacterium, 5-10 parts of potassium bacterium, 18-30 parts of Bacillus subtilis nutrient material, 5-10 parts of Aspergillus awamori nutrient material and 3-8 parts of lactobacillus plantarum microbial inoculum. The fertilizer can enhance the drought resistance of the crops; the Bacillus mucilaginosus in the compound strain is prepared by industrially fermenting high-potassium-solubilizing-capacity silicate bacterium screened from crop roots in a specific culture medium, and can secrete auxin substances, gibberellin substances and other growth promoting substances to reinforce the roots and seedlings; the silicon in the soil is decomposed and supplied to the plant, so that the waxy layer of the plant is thickened, thereby enhancing the water retention capacity of the plant; and the fertilizer can promote the formation of the soil aggregate from, keep soil from packing together, destroy the soil capillarity and prevent the soil moisture from evaporation.
Description
Technical field
The present invention relates to a kind of bio-fertilizer, belong to agricultural fertilizer technical field, particularly a kind of composite bio-fertilizer.
Background technology
Pollution by chemical fertilizer has become the large public hazards in the world today one.Agricultural experts unanimously appeal, administer environment, must reduce fertilizer amount.Expert, the new academician of the Chen Wen of China Agricultural University think, the excessive chemical fertilizer of using of China has arrived the limit at present.Take nitrogenous fertilizer as example, and the average amount of application of China is nearly 3 times of the U.S., more than 8 times of Australia, a series of serious problems such as this has caused, and Soil structure is destroyed, nature biotechnology diversity and ecosystem stability reduction.Agricultural microbiology Professional Committee of Chinese Society for Microbiology director doctor Li Jun points out, wideling popularize microorganism fertilizer, to coordinate chemical fertilizer application be exactly one of solution to this problem.
Microorganism fertilizer is a kind ofly with microbial life activity and product thereof, to cause farm crop to obtain the microorganism live body goods of specific fertilizer effect, it at culture fertility, improve chemical fertilizer utilization ratio, suppress farm crop there is to irreplaceable effect the aspects such as utilizations of becoming thoroughly decomposed of the absorption of the objectionable impuritiess such as heavy metal and agricultural chemicals, purification and rehabilitating soil, promotion agricultural crop straw and municipal wastes, raising quality of agricultural product.The efficacy exertion of microbial fertilizer is mainly by the synergism to traditional fertilizer, fertilizer, improvement activation to soil, and the modes such as physiological action of microorganism realize.
Therefore, microbial fertilizer has just had multiple fertility and the effect of chemical fertilizer, fertilizer, beneficial organism bacterium, is activated fertilizer nutrient, improves effect of fertilizer, Crop Improvement quality, promotes the desirable fertilizer of agricultural produce synergy.Microbial fertilizer is live body fertilizer, and a large amount of beneficial microorganism vital movements that its effect mainly contains by it complete.Only have when these beneficial microorganisms are in vigorous breeding and metabolic situation, Substance Transformation and useful meta-bolites could constantly form.Therefore, beneficial microorganism kind in microbial fertilizer, whether vital movement is vigorous is the basis of its validity, and is to take the form of the principal elements such as nitrogen, phosphorus, potassium and how many for basic unlike other fertilizer.Just because of microbial fertilizer is the preparation of living, so its fertilizer efficiency and number of viable, intensity and ambient environmental conditions are closely related, comprise temperature, moisture, potential of hydrogen, nutritional condition and formerly live in indigenous microorganism repulsive interaction in soil and have certain influence.
For many years, most processing modes of microbial fertilizer only adopt finished product microbial inoculum to mix with filler, lack fermentating metabolism product, had a strong impact on the fertilizer efficiency of microbial fertilizer product, the patent application that for example publication No. is CN103396252A, disclose a kind of amino acid whose composite microbiological fertilizer that contains, the finished product microbial inoculum wherein adding is mixed and forms according to a certain percentage by subtilis, Bacillus licheniformis and bacillus megaterium.
And agricultural microorganism microbial inoculum is in the market mainly single microbial strains, be difficult to reduce micro-ecologic structure, simultaneously a little less than the synergy of common bacterial classification and farm crop, unfavorable as improved stress resistance of plant and root system nutrition absorption, the patent application that for example publication No. is CN102888356A, discloses a kind of method and application thereof that utilizes subtilis to prepare microbial fertilizer.Although there are in the market some complex micro organism fungicides, but itself or function singleness, or its using method is restricted, crop yield effect after use can't reach satisfactory, so still need to develop complex micro organism fungicide or the fertilizer that is applicable to multiple route of administration, has comprehensive function and good effect of increasing production at present.
Because China lack to drop in research aspect microbial fertilizer for a long time, make that the microbial fertilizer industry of China still exists that integral level is not high, technological innovation is not enough, quality product and effect show understable problem.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, a kind of composite bio-fertilizer is provided, parts by weight consist of: colloid bud pole bacterium 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, plant lactobacillus agent 3-8 part.
Described subtilis culture preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, the complete fermented liquid that ferments obtains subtilis culture through Plate Filtration, after dry.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.6:1, and vehicle group becomes: CaCO3 20-30 part, dextrin 10-15 part.Fluidised bed drying, 50 ℃ of drying temperatures.
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-33 ℃ is cultured to mycelia and covers with culture material, and low temperature fluidized-bed dry, pulverize dry thing.
Fertile preparation method is as follows for described high-effective microorganism: according to aforementioned proportion, various microbial inoculums are mixed, or granulate after mixing.
The bacterial classification that the present invention adopts is as follows:
Subtilis (Bacillus subtilis subsp) CGMCC 7926
Plant lactobacillus (Lactobacillus plantarum) CGMCC 7928
Aspergillus awamori (Aspergillus awamori) CGMCC3.6484
The depositary institution of above-mentioned three kinds of bacterial classifications is Chinese common micro-organisms culture presevation administrative centers.Address: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute; Postcode: 100101.
Phosphate solubilizing bacteria is phosphorus decomposing bacillus megaterium, and phosphorus decomposing microbial inoculum is provided by Cangzhou prosperous generation thing technical institute, address: liberation West Road, Canal Zone, Hebei China Cangzhou City chin or cheek and Building A, international business affairs center 1 district 807-812.
Potassium solubilizing bacteria is provided by infinitesimal bio tech ltd, Guangzhou, address: No. 101 506, science main road, Luogang District, Guangzhou.
Rui Gu bio tech ltd, Baoding provides colloid bacillus cereus bacterium powder.
Beneficial effect
Fertilizer of the present invention can strengthen the drought-resistant ability of crop.Colloid bud pole bacterium in composite bacteria of the present invention is the silicate bacteria that ability of dissolving potassium is very strong, adopts specific substratum, through industrial fermentation, develops.This bacterium can secrete the growth-promoting substances such as growth hormone material and Plant hormones regulators,gibberellins material, extraneous root strong sprout; Decompose element silicon in soil and, for plant utilization, the wax layer of plant is thickened, improve plant water keeping ability; Can promote soil aggregate to form, keep soil from packing together; Spoiled soil capillarity, prevents soil water evaporation.
Fertilizer of the present invention can be improved soil.In fertilizer, beneficial microorganism can produce glucide and plant mucilage, mineral idiosome and organic colloid combine, and can improve soil aggregate, strengthen the physicals of soil and the loss of minimizing soil particle, under certain conditions, can also participate in soil ulmin forms.So use microbial fertilizer, can improve soil physical property, be conducive to increase soil fertility.
This product using method is simple, every mu of ground usage quantity 0.3-1 kilogram, and cost is only 80-100 unit/mu, spray on seed dressing or the front earth's surface of pouring water vegetative period.
The conversion of soil organisms enzyme, reducing agriculture production cost, reducing use, the recovery soil ecology soil fertility of chemical fertilizer and effectively improve the aspects such as crop yield and all can play significant effect, gives full play to the organic usefulness of soil.
Embodiment
Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope, the various changes that the reaction conditions in these embodiments, separation and Extraction condition are carried out or change also belong to protection scope of the present invention.
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.Embodiment 1
Subtilis (Bacillus subtilis) Li-2013-02, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 15th, 2013 and (is called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute), preserving number is CGMCC No.7926.
Described strain characteristic is that the enzyme activity of the high temperature resistant α-amylase of product is high, heat-resisting, acid resistance is strong.
High temperature resistant α-amylase enzyme activity prepared by described bacterial strain is 30000-35000u/ml; Applicable temperature scope is 105-115 ℃, and 110 ℃ of optimal reactive temperatures, at 110 ℃ of enzymes complete stability alive; Applicable pH value in reaction scope is 3.0-7.0, in pH value, is 3.0 o'clock enzyme complete stabilities alive, and optimal reaction pH value is 4.2.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, and neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into positive.
Described subtilis (Bacillus subtilis) Li-2013-02 is produced high temperature resistant α-amylase subtilis Li-2013 by a strain obtains through UV-LiCl-ethyl sulfate Mutation screening, specifically screens step as follows:
(1) preparation of bacteria suspension
By in the mono-bacterium colony of the Li-2013 growing after plate streaking separation access seed culture medium, 100r/min, cultivates after 12h for 40 ℃, uses physiological saline washed twice after getting 1mL medium centrifugal, and in resuspended and 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in to aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.To after gradient dilution, coat lithium chloride flat board through the bacterium liquid irradiating, and contrast with the bacterium liquid dilution painting flat board without uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, with cloth or the newspaper of black, wrap, put 40 ℃ and cultivate 48h, on the flat board of bacterium colony, filter out hydrolysis circle and choose to inclined-plane and preserve with colony diameter ratio the maximum growing, after purifying, be mixed with bacteria suspension, after gradient dilution, fully mix with ethyl sulfate stoste, and process 40min in 40 ℃ of concussions, the bacterium liquid of processing is coated to lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, put 40 ℃ and cultivate 48h, go out hydrolysis circle and choose to inclined-plane and preserve with colony diameter ratio the greater growing on the flat board of bacterium colony primary dcreening operation, after purifying, obtain three strain bacterium Li-2013-01, Li-2013-02, Li-2013-03.
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-01 that obtain, Li-2013-02, Li-2013-03 carries out shake flask fermentation in the 250mL shaking flask that contains 30mL fermention medium, seed inoculum size 10% (V/V), 40 ℃, 100r/min are cultivated 72h, and centrifuging and taking fermented supernatant fluid makes crude enzyme liquid.
(5) enzyme activity determination
The definition of Mei Huo unit: 1mL crude enzyme liquid, under 105 ℃, pH4.2 condition, 1min liquefaction 1mg Zulkovsky starch,
Be 1 enzyme activity unit, with U/mL, represent.
After measured, bacterial strain Li-2013-02, be stable superior strain, and enzyme work reaches 30000U/mL.
Described lithium chloride is dull and stereotyped: starch 1%, and peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
Described seed culture medium: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl 1%.
Described fermention medium: Semen Maydis powder 5%-15%, soybean cake powder 4%-10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake-flask culture condition: this bacterium in the 250mL shaking flask that contains 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 ℃ of fermentation culture 72h.
Described high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is between 100-110 ℃, and 110 ℃ of following preservations, temperature stability is better, and 110 ℃ above to preserve long-time temperature stabilities poor.
(2) this enzyme optimal reaction pH value is 4.2.Between pH value 3.0-7.0, all having high enzyme vigor, is 3.0 o'clock enzyme complete stabilities alive in pH value.
(3) enzymic activity: by mutant strain Li-2013-02 provided by the present invention, the high temperature resistant α-amylase enzyme activity of preparation is 30000-35000U/ml.
Plant lactobacillus provided by the present invention (Lactobacillus plantarum) Li-2013-01, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.7928, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on July 15th, 2013.This bacterial strain feature is as follows: examine under a microscope, this bacterial strain is rod-short, and gramstaining is positive, and boundless hair, does not produce gemma; On solid medium, this bacterium bacterium colony is white, smooth surface, and densification, form is circular, edge is more neat.Physicochemical characteristics is: catalase (-), gelatine liquefication (-), indoles experiment (+), mobility (-), fermentation gas (-), nitrite reduction (-), fermentation gas (-), produces hydrogen sulfide (-), growth (+) in pH4.5 MRS substratum.
Plant lactobacillus of the present invention adopts following flow process to carry out seed selection:
Original sieve again → mitotic stability of bacterial classification → test tube activation → ethyl sulfate (DES) mutagenesis → dull and stereotyped primary dcreening operation → nitrosoguanidine (NTG) mutagenesis → dull and stereotyped primary dcreening operation → shaking flask test of setting out.
The original bacterial classification that sets out is CICC20242, is purchased from Chinese industrial microbial strains preservation administrative center.
Original strain of the present invention is in xylan substratum, and the output of lactic acid is 12.5g/L.In order to improve its lactic acid production, adopt successively DES and NTG to carry out mutagenesis to this bacterial classification, mutagenesis adopts MRS calcium carbonate flat board to carry out primary dcreening operation, then adopt 500mL shake flask fermentation, biosensor analysis instrument carries out multiple sieve to Producing Strain, the lactobacterium plantarum strain that seed selection is good, the experiment of then going down to posterity, evaluates its genetic stability.
Bacterial strain CGMCC No.7928 genetic stability result shows: through continuous passage ten times, property indices is all more stable, and heredity is better, and proterties is not replied, the object bacterial strain therefore bacterial strain CGMCC No.7928 being obtained as seed selection.
Object bacterial strain CGMCC No.7928 is done to the experiment of 10L fermentor tank, and result shows: after fermentation 72h, take xylan as carbon source, the lactic acid concn of plant lactobacillus CGMCC No.7928 can reach 57g/L, compares and has improved 356% with starting strain.
Object bacterial strain CGMCC No.7928 is done to the experiment of 10L fermentor tank, and result shows: after fermentation 72h, take glucose as carbon source, the lactic acid concn of plant lactobacillus CGMCC No.7928 can reach 68g/L.
Detailed process is as follows:
Substratum:
Liquid MRS xylan substratum (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 20g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulates pH7.0-7.2); MRS xylan screening solid medium (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 90g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulate pH7.0-7.2, add 20g agar).
1. ethyl sulfate (DES) mutagenic and breeding
1) on super clean bench, get plant lactobacillus one ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 12h left and right for 40 ℃, makes thalline in logarithmic growth in earlier stage.
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with physiological saline washing 2 times.
3) with pH7.0 phosphoric acid buffer, be diluted to 107/mL bacteria suspension.
4) potassium phosphate buffer, 8mL bacteria suspension, 0.4mL DES of getting 32mL pH7.0 fully mixes at the 150mL triangular flask of putting in advance rotor, and making DES ultimate density is 1%(v/v).
5) 150rpm reaction 30min in 30 ℃ of shaking tables, gets 1mL mixed solution, adds 0.5mL 25%Na
2s
2o
3solution stopped reaction.
6) dilution spread is in the MRS xylan screening solid medium plate containing 90g/L xylan.At the bacterial strain of 40 ℃ of cultivations picking transparent circle/colony diameter maximum after 2～3 days, label is DES bacterium.
2. nitrosoguanidine mutagenesis
1) on super clean bench, get plant lactobacillus DES mono-ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 12h left and right for 40 ℃, makes thalline in logarithmic growth in earlier stage.
2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with physiological saline washing 2 times.
3) with pH6.0 phosphoric acid buffer, be diluted to 107/mL bacteria suspension.
4) get 10mL bacteria suspension and be transferred in 100mL triangular flask, add the NTG of 10mg, being mixed with final concentration is the NTG solution of 10mg/mL, and adds 4-5 to drip acetone, is beneficial to NTG and dissolves.
5) 200rpm oscillatory reaction 30min at 30 ℃, the centrifugal 10min of 5000rpm collects thalline, with stroke-physiological saline solution washing for several times, stopped reaction.
6) suitably dilution, gets last dilution bacterium liquid 0.2mL, and dilution spread is in the MRS xylan screening solid medium plate containing 90g/L xylan.150 of 40 ℃ of cultivation bacterial strains that after 2～3 days, picking transparent circle/colony diameter is larger.
3. shaking flask is sieved again
1) on super clean bench, get respectively plant lactobacillus one ring on each test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 3-4 days, detects glucose concn and Pfansteihl change in concentration every day for 40 ℃.After fermentation ends, relatively the xylan wear rate of 150 strain bacterial classifications and lactic acid produce transformation efficiency and the heteroacid content of speed, lactic acid.
2) selection xylan metabolic rate is fast, lactic acid concn is high, transformation efficiency is high and the poor bacterial classification of heteroacid is final bacterial classification, called after Li bacterium.
4. genetic stability test
Li-2013-01 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and the method for sieving again by shaking flask detects the fermentation situation after at every turn going down to posterity.Experiment discovery is gone down to posterity for continuous ten times on inclined-plane, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
The preparation method of Aspergillus awamori microbial inoculum:
Slant strains activation culture: Aspergillus awamori slant strains is transferred on slant medium, cultivates 3 days for 27 ℃.
Solid first order seed is cultivated: picking Aspergillus awamori slant strains accesses in 500 milliliters of triangular flasks that 100 grams of substratum are housed carries out seed culture, cultivates 3 days for 30 ℃.
Solid secondary seed is cultivated: above-mentioned cultured solid first order seed is stirred as adding after fragment and in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed, carries out seed culture, culture condition: cultivate 3 days for 30 ℃.
Solid fermentation is cultivated: secondary shake-flask seed is pulverized, added to be equipped with in the fermentation vat of sterilising medium or pallet to mix rear cultivation, bent material culture temperature is controlled at 26-35 ℃, humidity 80-90%, every 10 hours stirrings once, incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat that culture material covers with mycelia and can finish to cultivate, substratum is in advance through high temperature steaming sterilising treatment, and sterilising conditions is controlled 121 ℃ of temperature, 1 hour time.
Drying and crushing: fermentation ends culture material is dried on fluidized-bed or other drying plants, and drying temperature is controlled at 60 ℃, is dried to moisture content below 10%, then solid culture medium is pulverized, and crushing material aperture is more than 60 orders.
Substratum forms: solid material: wheat bran 80, and soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initially
ph nature.
Embodiment 2
A composite bio-fertilizer, parts by weight consist of: 12 parts of colloid bud pole bacterium, 5 parts of phosphate solubilizing bacterias, 8 parts of potassium solubilizing bacterias, 25 parts of subtilis cultures, 7 parts of Aspergillus awamori cultures, 6 parts of plant lactobacillus agent.
The preparation method of subtilis culture:
The acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;
(1) first order seed is cultivated: subtilis slant strains is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 180 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: three grades of seeds be take to the first class seed pot that 10% inoculum size access cubic capacity is 150L, fermention medium loading amount 100L, 28 ℃ of culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours;
(5) fermentation culture: it is 1.5 tons of secondary seed tanks that first class seed pot bacterial classification be take to 10% inoculum size access cubic capacity, 1 ton of fermention medium loading amount, 28 ℃ of culture condition culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours.The complete fermented liquid that ferments obtains containing bacillus subtilis microbial inoculum at interior subtilis culture through Plate Filtration, after dry.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
The preparation method of plant lactobacillus agent:
(1) first order seed is cultivated: plant lactobacillus bacterial classification is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 30 ℃ of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 30 ℃ of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: three grades of seeds be take to the first class seed pot that 5% inoculum size access cubic capacity is 150L, fermention medium loading amount 100L, 30 ℃ of culture temperature, tank pressure 0.05MPa, incubation time 18 hours;
(5) fermentor cultivation: it is 3 tons of secondary seed tanks that first class seed pot bacterial classification be take to 5% inoculum size access cubic capacity, 2 tons of fermention medium loading amounts, 30 ℃ of culture condition culture temperature, tank pressure 0.05MPa, incubation time 22 hours.The complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin, fluidised bed drying, 50 ℃ of drying temperatures.
Substratum consists of: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, citric acid diamines 0.2%, Tween 80 0.1%, K
2hPO
40.2%, MgSO
4.7H
2o 0.02%, MnSO
4.H
2o 0.005%, CaCO
32%, agar 1.5%, pH6.8.
Embodiment 3
A composite bio-fertilizer, parts by weight consist of: 10 parts of colloid bud pole bacterium, 6 parts of phosphate solubilizing bacterias, 7 parts of potassium solubilizing bacterias, 27 parts of subtilis cultures, 9 parts of Aspergillus awamori cultures, 7 parts of plant lactobacillus agent.
Described subtilis culture preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, the complete fermented liquid that ferments obtains subtilis culture through Plate Filtration, after dry.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin.Fluidised bed drying, 50 ℃ of drying temperatures.
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 27-33 ℃ is cultured to mycelia and covers with culture material, and low temperature fluidized-bed dry, pulverize dry thing.
Embodiment 4
The experiment of product effect
Selection experimental field and test design: tested 27 days-September 30 April in 2009 and carry out in Ba Bao village, dapple pond town, Yanchi county Ningxia.
Experimental plot reaches 10 mu of field maize plantings, uses 0.5 kilogram every mu of product of the present invention respectively when plantation, emerges and by loose ground mode, uses 0.5 kilogram of invention product about 1 month, and control group is used conventional fertilizer.
Invention product is used corn field corn yield to reach 650 kilograms, and control group reaches 500 kilograms; The use of invention product makes corn per mu yield improve 30%.This plot was at the 2nd year plant spring wheat, and spring wheat production has reached 400 kilograms, than control group per unit area yield, has improved 20%.And experimental plot Soil structure is good, without bulk and hardening.
Claims (6)
1. a composite bio-fertilizer, is characterized in that, parts by weight consist of: colloid bud pole bacterium 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, plant lactobacillus agent 3-8 part; Described subtilis preserving number is CGMCC No.7926, and described plant lactobacillus deposit number is CGMCC No.7928.
2. a kind of composite bio-fertilizer according to claim 1, it is characterized in that, the preparation method of described subtilis culture is as follows: the acquisition of fermented liquid: from inclined-plane switching, cultivate subtilis, seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture through Plate Filtration, after dry.
3. a kind of composite bio-fertilizer according to claim 1, it is characterized in that, the preparation method of described plant lactobacillus agent is as follows: from inclined-plane switching culturing plants Bacterium lacticum, seed liquor after spreading cultivation is step by step transferred in fermentor tank, the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.6:1, and vehicle group becomes: CaCO3 20-30 part, dextrin 10-15 part; Fluidised bed drying, 50 ℃ of drying temperatures.
4. a kind of composite bio-fertilizer according to claim 1, it is characterized in that, the preparation method of described Aspergillus awamori microbial inoculum is as follows: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, 26-33 ℃ is cultured to mycelia and covers with culture material, low temperature fluidized-bed dry, pulverize dry thing.
5. a kind of composite bio-fertilizer according to claim 1, is characterized in that, parts by weight consist of: 12 parts of colloid bud pole bacterium, 5 parts of phosphate solubilizing bacterias, 8 parts of potassium solubilizing bacterias, 25 parts of subtilis cultures, 7 parts of Aspergillus awamori cultures, 6 parts of plant lactobacillus agent.
6. a kind of composite bio-fertilizer according to claim 1, is characterized in that, parts by weight consist of: 10 parts of colloid bud pole bacterium, 6 parts of phosphate solubilizing bacterias, 7 parts of potassium solubilizing bacterias, 27 parts of subtilis cultures, 9 parts of Aspergillus awamori cultures, 7 parts of plant lactobacillus agent.
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